Angiotensin II type 1 receptor antagonists in animal models of vascular, cardiac, metabolic and renal disease

    Angiotensin II type 1 receptor antagonists in animal models of vascular, cardiac, metabolic and renal disease Martin C. Michel, Hans R. Brunner, Carolyn Foster, Yong Huo PII: DOI: Reference:

S0163-7258(16)30041-9 doi: 10.1016/j.pharmthera.2016.03.019 JPT 6892

To appear in:

Pharmacology and Therapeutics

Please cite this article as: Michel, M.C., Brunner, H.R., Foster, C. & Huo, Y., Angiotensin II type 1 receptor antagonists in animal models of vascular, cardiac, metabolic and renal disease, Pharmacology and Therapeutics (2016), doi: 10.1016/j.pharmthera.2016.03.019

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ACCEPTED MANUSCRIPT P&T 23028 Angiotensin II type 1 receptor antagonists in animal models of vascular, cardiac, metabolic and renal disease

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Martin C. Michel1,2, Hans R. Brunner3, Carolyn Foster4, Yong Huo5 1

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Dept. Pharmacology, Johannes Gutenberg University, Mainz, Germany Dept. Translational Medicine & Clinical Pharmacology, Boehringer Ingelheim, Ingelheim, Germany 3 Emeritus of Lausanne University, Riehen, Switzerland 4 Retiree from Dept. of Research Networking, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT, USA 5 Dept. Cardiology & Heart Center, Peking University First Hospital, Beijing, China

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Address for correspondence: Prof. Martin C. Michel Dept. of Pharmacology Johannes Gutenberg University Obere Zahlbacher Str. 67 51101 Mainz, Germany Phone: +49-6132-7799000 Fax: +49-6132-7299000 Mail: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT ............................................................................................................................................ 5 Introduction ..................................................................................................................................... 7 1.1.

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Blood pressure reduction ............................................................................................................... 10 Normotensive animals ........................................................................................................... 10

2.2.

Spontaneously hypertensive rat (SHR) ................................................................................. 12 BP lowering in established hypertension....................................................................... 13

2.2.2.

Effect on hypertension development ............................................................................. 14

2.2.3.

Persistence of BP lowering after end of treatment ........................................................ 14

2.2.4.

Stroke-prone SHR and otherwise aggravated hypertension in SHR ............................. 15

2.2.5.

Combination treatments................................................................................................. 16

2.2.6.

Effects on mortality, particularly in stroke-prone SHR ................................................. 17

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Renal hypertensive animals ................................................................................................... 18 Renovascular hypertension ............................................................................................ 18

2.3.2.

Hypertension associated with reduced renal mass or parenchymal renal disease ......... 19

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2.3.1.

Dahl salt-sensitive rats........................................................................................................... 19

2.5.

Mineralocorticoid-induced hypertension............................................................................... 20

2.6.

Dietary, obesity and diabetes models of hypertension .......................................................... 20

2.7.

Transgenic and knock-out hypertension models ................................................................... 21

2.8.

Other hypertension models .................................................................................................... 22

2.9.

Hypotensive states ................................................................................................................. 24

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2.4.

Mechanistic considerations ............................................................................................... 24

2.10. 2.10.1.

Central nervous contributions to blood pressure control ............................................... 26

Hypertension conclusions .................................................................................................. 28

2.11.

Vascular function and atherosclerosis ........................................................................................... 28 3.1.

Normal vascular function ...................................................................................................... 29

3.2.

Altered endothelial function .................................................................................................. 33

3.3.

Vascular remodeling and atherosclerosis .............................................................................. 34

3.3.1.

In vitro models............................................................................................................... 35

3.3.2.

In vivo models ............................................................................................................... 40

3.4. 4.

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2.2.1.

2.3.

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Literature search strategy ........................................................................................................ 9

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Other effects on vascular function ......................................................................................... 48

Heart function ................................................................................................................................ 49 4.1.

Normal heart function ........................................................................................................... 49

4.1.1.

Signal transduction and cellular function ...................................................................... 49 2

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Heart rate and conduction system.................................................................................. 50

4.1.3.

Inotropy ......................................................................................................................... 51

4.1.4.

Coronary blood flow...................................................................................................... 51

Heart hypertrophy and fibrosis .............................................................................................. 52

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4.2.

In vitro models............................................................................................................... 52

4.2.2.

In vivo models of hypertension- or resistance-associated cardiovascular hypertrophy . 54

4.2.3.

Post-infarction models ................................................................................................... 60

4.2.4.

Blood pressure-independent and other in vivo models .................................................. 62

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4.3.

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4.2.1.

Myocardial infarction ............................................................................................................ 63 Preventive ARB administration ..................................................................................... 64

4.3.2.

Early post-infarction ARB administration..................................................................... 66

4.3.3.

Late post-infarction ARB administration ...................................................................... 68

4.3.4.

Ischemic preconditioning .............................................................................................. 69

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4.4.

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4.3.1.

Heart failure........................................................................................................................... 70

Embolism and stroke ..................................................................................................................... 74

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Metabolic effects ........................................................................................................................... 77

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In vitro studies ....................................................................................................................... 77

6.2.

In vivo studies on body weight and food intake.................................................................... 80 Studies in lean euglycemic animals ............................................................................... 80

6.2.2.

Studies in lean diabetic animals .................................................................................... 81

6.2.3.

Studies in obese animals with and without diabetes .................................................... 81

6.3.2. 6.3.3.

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In vivo studies on glucose homeostasis................................................................................. 85

6.3.1.

6.4.

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6.2.1.

6.3.

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6.1.

Studies in lean euglycemic animals ............................................................................... 85 Studies in lean diabetic animals .................................................................................... 85 Studies in obese animals with and without diabetes .................................................... 87

In vivo studies on lipid metabolism ....................................................................................... 91

6.4.1.

Studies in lean euglycemic animals ............................................................................... 91

6.4.2.

Studies in lean diabetic animals .................................................................................... 92

6.4.3.

Studies in obese animals with and without diabetes .................................................... 92

6.5.

In vivo studies on adipose mass and function ....................................................................... 93

6.6.

Liver morphology and function ............................................................................................. 95

Renal effects and nephroprotection ............................................................................................... 97 7.1.

Normal renal function ........................................................................................................... 97

7.1.1.

Signal transduction and in vitro studies ........................................................................ 97

7.1.2.

Renal blood flow and glomerular filtration ................................................................... 99 3

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Tubular function and diuresis ...................................................................................... 100

Development and progression of nephropathy .................................................................... 102 In vitro studies ............................................................................................................. 102

7.2.2.

Models of primary renal disease.................................................................................. 103

7.2.3.

SHR ............................................................................................................................. 107

7.2.4.

Other hypertension models .......................................................................................... 110

7.2.5.

Ureteral obstruction ..................................................................................................... 113

7.2.6.

Diabetes mellitus and obesity ...................................................................................... 114

7.2.7.

Other models of impaired renal function ..................................................................... 118

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7.2.1.

Conclusions ................................................................................................................................. 119

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Tables........................................................................................................................................... 121

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Figure legends ......................................................................................................................... 127

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References ............................................................................................................................... 129

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ACCEPTED MANUSCRIPT ABSTRACT

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We have reviewed the effects of angiotensin II type 1 receptor antagonists (ARBs) in various animal models of hypertension, atherosclerosis, cardiac function, hypertrophy and fibrosis, glucose and lipid metabolism, and renal function and morphology. Those of azilsartan and telmisartan have been included comprehensively whereas those of other ARBs have been included systematically without intention of completeness. ARBs as a class lower blood pressure in established hypertension and prevent hypertension development in all applicable animal models except those with a markedly suppressed renin-angiotensin system; blood pressure lowering even persists for a considerable time after discontinuation of treatment. This translates into a reduced mortality, particularly in models exhibiting marked hypertension. The retrieved data on vascular, cardiac and renal function and morphology as well as on glucose and lipid metabolism are discussed to address three main questions: 1. Can ARB effects on blood vessels, heart, kidney and metabolic function be explained by blood pressure lowering alone or are they additionally directly related to blockade of the reninangiotensin system? 2. Are they shared by other inhibitors of the renin-angiotensin system, e.g. angiotensin converting enzyme inhibitors? 3. Are some effects specific for one or more compounds within the ARB class? Taken together these data profile ARBs as a drug class with unique properties that have beneficial effects far beyond those on BP reduction and, in some cases distinct from those of angiotensin converting enzyme inhibitors. The clinical relevance of angiotensin receptor-independent effects of some ARBs remains to be determined.

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Abbreviations: ACE, angiotensin converting enzyme AGE, advanced glycosylation end product ApoE, apolipoprotein E1 ARB, angiotensin receptor blocker ANG, angiotensin II AT1R, angiotensin II type 1 receptor AT2R, angiotensin II type 2 receptor BP, blood pressure CHF, congestive heart failure DOCA, deoxycorticosterone acetate ERK, extracellular signal-regulated kinase GFR, glomerular filtration rate HUVEC, human umbilical vein endothelial cell IFNγ, interferon-γ IL, interleukin i.v., intravenous i.c.v., intracerebroventricular L-NAME, L-nitro-arginine methyl ester MCP, monocyte chemoattractant protein PPAR, peroxisome proliferator-activated receptor RAGE, receptor for advanced glycosylation end product RAS, renin-angiotensin system RBF, renal blood flow SHR, spontaneously hypertensive rat siRNA, small interfering RNA STZ, streptozotocin TGF, transforming growth factor TNF, tumor necrosis factor VCAM, vascular cell adhesion molecule VSMC, vascular smooth muscle cell

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ACCEPTED MANUSCRIPT 1. Introduction

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Angiotensin II (ANG) plays an important function in the regulation of the cardiovascular system. This largely occurs via its role as a circulating hormone in the renin-angiotensin system (RAS), but it may also involve locally formed ANG as part of a tissue RAS (Bader, 2010; Fukuda et al., 1999; Siragy & Carey, 2010; Tamura et al., 1997b) or, in the central nervous system, as a neurotransmitter (Culman et al., 2002). Angiotensin II type 1 receptors (AT1Rs) mediate many of the physiological and pathophysiological effects of ANG. Thus, AT1R antagonists, also known as angiotensin receptor blockers (ARBs), have become an important drug class in the treatment of arterial hypertension and congestive heart failure and for nephroprotection. Following the discovery of the first non-peptidic ARB, losartan (also known as DUP-753 or MK-954, or EXP 3174 for the active metabolite) (Chiu et al., 1990b; Wong et al., 1990b), several other representatives of this drug class have become clinically available. These include azilsartan (also known as TAK-491 or as TAK-536 for its prodrug), candesartan (also known as TCV-116 for the prodrug or CV-11974 for the active metabolite), eprosartan (also known as SK&F 108566), irbesartan (also known as SR 47436 or BMS 186295), olmesartan (also known as CS-866 for the prodrug and RNH-6270 for the active metabolite), telmisartan (also known as BIBR 277) and valsartan (also known as CGP 48,933).

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As reviewed previously, the various ARBs have different chemical structures and accordingly use different binding pockets in the receptor, which are associated with differences in dissociation times and, in most cases, apparently insurmountable antagonism (Michel et al., 2013). The physicochemical differences between ARBs also manifest in different tissue penetration, including passage through the blood-brain-barrier. The combination of these factors is associated with differences in pharmacokinetic profile, particularly duration of action. While generally being highly specific for angiotensin II type 1 receptors, some ARBs, particularly telmisartan, are partial agonists at peroxisome proliferator-activated receptor-γ. More recently, it was discovered that ARBs, similar to ligands for many other receptors (Kenakin & Christopoulos, 2013), exhibit a property called ‘biased agonism’ or ‘liganddirected signaling’ (Tilley, 2011; Wilson et al., 2013). This means that some AT1R antagonists can inhibit canonical signaling via the Gq/phospholipase C pathway but may be agonists for other signaling pathways such as β-arrestin pathways. Emerging examples of a potential clinical relevance of this phenomenon are discussed in section 4.3.3. While ARBs are mostly known for their effects in the cardiovascular system, they can also affect the function of other organ systems. Such additional effects may occur as a result of vasodilation, often assessed as blood pressure (BP) lowering, or at least in part, independently of it. All clinically used ARBs have high selectivity for AT1R over angiotensin II type 2 receptors (AT2R) as shown for azilsartan (Ojima et al., 2011), candesartan (Shibouta et al., 1993), eprosartan (Keiser et al., 1995), irbesartan (Cazaubon et al., 1993), losartan (Chiu et al., 1990a), olmesartan (Mizuno et al., 1995), telmisartan (Wienen et al., 1993) or valsartan (Criscione et al., 1993). Most ARBs have also high selectivity for AT1R as compared to molecular targets outside the RAS. Notable exceptions include interaction with some types of potassium channels reported for candesartan, eprosartan, losartan and telmisartan, but all of these occur at much higher concentrations than those needed for AT1R occupancy and in none of these cases clinical correlates have been reported (Michel et al., 2013). Some ARBs, i.e. irbesartan, losartan and telmisartan, can inhibit thromboxane A2 receptor-mediated effects in blood vessels and platelets, and some data suggest that this may be explained by direct interaction with that receptor (Monton et al., 2000). Based on molecular modeling studies, it 7

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has been proposed that some ARBs may also bind to vitamin D receptors and chemokine CCR2 receptors (Marshall et al., 2006) but this has not been substantiated experimentally. Several ARBs are substrates of transporter molecules such as P-glycoprotein or members of the Organic Acid Transporting Polypeptide family, which may play a role in their pharmacokinetic profile including penetration into the brain (Michel et al., 2013). For instance telmisartan may inhibit P-glycoprotein, which may lead to drug-drug-interactions (Stangier et al., 2000), and losartan (but not EXP 3174) and candesartan can interact with uric acid transporters, leading to decreases or increases in serum uric acid levels by losartan and candesartan, respectively (Manolis et al., 2000; Nakashima et al., 1992). Uric acid is a substrate of several transporters including OAT1, OAT3, OAT4, and MRP4 (Sato et al., 2008) and the glucose transporter, GLUT9 (also known as URATv1) (Anzai et al., 2008). Although the results for any given transporter have not been fully consistent across ARBs and individual transporters, effects on uric acid transporters have been shown for candesartan (Nakamura et al., 2010; Sato et al., 2008; Yamashita et al., 2006), eprosartan (Edwards et al., 1996), irbesartan (Nakamura et al., 2010), losartan (Anzai et al., 2008; Edwards et al., 1996; Iwanaga et al., 2007; Sato et al., 2008; Yamashita et al., 2006), olmesartan (Iwanaga et al., 2007; Sato et al., 2008), telmisartan (Iwanaga et al., 2007; Nakamura et al., 2010; Sato et al., 2008) and valsartan (Anzai et al., 2008; Iwanaga et al., 2007; Sato et al., 2008; Yamashita et al., 2006). Such interactions of most members of this drug class apparently contribute to clinical effects of at least some ARBs on serum uric acid (Manolis et al., 2000; Nakashima et al., 1992; Sato et al., 2008).

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The potentially most relevant molecular interaction of some ARBs with a target other than the AT1R is that with peroxisome proliferator-activated receptors (PPARs), specifically PPAR-γ. The most direct evidence for the interaction of some ARBs with PPAR-γ comes from studies demonstrating direct binding of azilsartan, candesartan, irbesartan, losartan and telmisartan to this receptor (Erbe et al., 2006; Kakuta et al., 2014; Storka et al., 2008). This molecular interaction can be associated with partial PPAR-γ agonism, for which telmisartan appears to show the most potent effects (Kakuta et al., 2014; Michel et al., 2013). The potential involvement of PPAR-γ in ARB effects is often studied using inhibition by the PPAR-γ antagonist GW9662 (Willson et al., 2000) or PPAR-γ knock-out (Rong et al., 2010) or knockdown approaches (Nakaya et al., 2007; Scalera et al., 2008; Walcher et al., 2008). Thus, alteration of a given cell or tissue function by an ARB suggests but does not prove AT1R involvement in the regulation of that function, unless such effects have been demonstrated for multiple members of this drug class. On the other hand, lack of effect of an ARB does not necessarily exclude a role for ANG acting via other receptor subtypes such as AT2R. Based on the typically observed increase in ANG upon treatment with an ARB, ARB treatment may actually enhance AT2R activation. Against this background we have aimed to systematically review the effects of ARBs in non-clinical models, particularly related to disease states of the cardiovascular system, metabolism and the kidney. Based on a cataloguing of these effects we wished to address three main questions: 1. Do ARB effects occur secondary to BP lowering, i.e. are mimicked by other BPlowering approaches? 2. If not, are they shared by RAS inhibitors as a class, specific for ARBs as compared to angiotensin-converting enzyme (ACE) inhibitors (Bernstein et al., 2013)? 3. If not, do the effects of some ARBs occur independent of AT1R, i.e. are mediated by other targets? These findings provide insight into the wide-spread pathophysiological role of ANG and AT1R activation and also highlight the therapeutic promise of the ARB drug class via and beyond BP lowering. 8

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In the overall interpretation of the data reviewed here it needs to be considered that not all findings can be extrapolated between species, specifically from experimental animals to humans. Reasons may include a species-dependent pathophysiology of a given condition and a different responsiveness to ARBs in some species. For example rabbits may be more sensitive to BP-lowering effects of ARBs than other species as doses typically used in rats may already cause profound hypotension and even death (Sato et al., 1997). On the other hand, some species may be less responsive to at least some ARBs because the AT1R orthologs in those species have lower affinity for ARBs, e.g. in dogs for losartan and valsartan (de Gasparo & Whitebread, 1995).

Literature search strategy

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We have primarily explored ARB effects in non-clinical models, i.e. studies in experimental animals, with isolated animal or human tissues and with animal or human isolated cells or cell lines. In vivo studies in humans, particularly patient treatment studies, were not systematically considered and only in selected cases examples are referenced to illustrate that a given nonclinical observation indeed has established clinical correlates.

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Our initial search strategy involving all clinically used ARBs indicated that about 16,000 articles have been published in the scientific literature on this drug class as listed in PubMed, of which approximately 4,000 appear to be original full papers on ARB effects in non-clinical models with possible relevance to the topic reviewed here. The enormous wealth of scientific literature documents the interest in this drug class; however, it also led us to conclude that a truly comprehensive review of effects of all clinically used ARBs in non-clinical models was unfeasible. Therefore, we shifted to an alternative strategy focussing on two ARBs as an example. We have picked telmisartan as one example, for which >500 non-clinical articles have been published which were comprehensively catalogued in a proprietary database available to the authors. Moreover, we considered telmisartan very suitable for our third objective, as it has been associated with AT1R-independent effects more often than any other ARB. The second ARB used as an example was azilsartan, as it is the most recent member of this drug class. For each major effect of azilsartan or telmisartan we have identified during our literature search, we performed dedicated searches to explore whether this effect was shared by other ARBs or even BP-lowering drugs in general. Thus, our manuscript represents a comprehensive analysis of fully published non-clinical azilsartan and telmisartan data (systematic search completed in July 2015; data published in abstract form only were not considered), whereas data on other ARBs or anti-hypertensive drugs acting independently of the RAS were only included as examples to document the absence or presence of class effects. This search strategy implies that articles on azilsartan and telmisartan are overrepresented in our analysis. However, as the vast majority of reported azilsartan and telmisartan effects are shared by one or more other ARBs, in the following all reported azilsartan and telmisartan data are assumed to be applicable to ARBs. Thus, mentioning of more studies with azilsartan or telmisartan as compared to other ARBs should not be interpreted as a claim for a larger database for these ARBs or as support for effects unique to these two; support for the latter can only be derived in specifically mentioned instances where this is supported by corresponding data. Studies with overtly unethical designs, such as killing animals by intravenous (i.v.) KCl injection (Gu et al., 2011), were excluded from the analysis. For ease of reading we do not consistently discriminate between parent prodrugs and active metabolites in the case of azilsartan, candesartan and olmesartan, as with oral administration of their prodrugs only active metabolite is detectable in the circulation (Michel et al., 2013); 9

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this means that, unless otherwise specified, the mentioning of a given ARB implies that for in vivo studies with oral administration the respective prodrug had been used (azilsartan has sometimes also been used for oral administration). On the other hand, upon oral administration of losartan, both parent compound and the active metabolite EXP3174 are detectable and contribute to observed effects but the ratio of both differs markedly between species (Stearns et al., 1992; Suzuki et al., 2001). This is important because EXP3174 is not only more potent than losartan (Koike et al., 2001; Lin et al., 1992; Miura et al., 2011; Mizuno et al., 1995; Nishikawa et al., 1994; Noda et al., 1993; Nussberger & Koike, 2004; Sudoh et al., 2003; Tamura et al., 1997a; Vanderheyden et al., 1999; Virone-Oddos et al., 1997; Wienen et al., 1992; Wong et al., 1990e) but also, in contrast to losartan, more consistently exhibits insurmountable antagonism (Cazaubon et al., 1993; Dickinson et al., 1994; Jin et al., 1997; Mizuno et al., 1995; Morsing et al., 1999; Shibouta et al., 1993; Wienen et al., 1992). A comprehensive review of the antagonistic properties of all ARBs at the molecular, cellular, tissue and organism level has been published elsewhere (Michel et al., 2013).

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2. Blood pressure reduction

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Arterial hypertension is a highly prevalent condition which, if untreated or treated insufficiently, can lead to morbidity such as myocardial infarction and stroke, kidney failure and, eventually, premature death, making a profound understanding and effective treatment necessary. There are many different animal models of hypertension which can be used to test the antihypertensive properties of ARBs. Obviously, animal models involving a strong RAS activation, e.g. the classic 2-kidney-1-clip Goldblatt model of renovascular hypertension or the related 1-kidney-1-clip model, are most sensitive in this regard. The spontaneously hypertensive rat (SHR) is the most frequently studied animal model of hypertension, but many other models in various species are available and have been used to study ARBs. In this section we review ARB effects on BP in various animal models of hypertension (for summary see Table 1). ARB effects on end-organ damage associated with such hypertension will be discussed in the sections related to the respective organs. <<>>

An important consideration implies to the interpretation of data in all animal models of hypertension. If treatment starts at an early phase of hypertension or even before BP elevation occurs, e.g. in 4-week old SHR, this implies a prevention and not a treatment approach. While prevention of hypertension is a theoretically attractive concept, it should be noted that neither ARBs nor any other class of anti-hypertensive drugs have been approved for the prevention of hypertension. Use of ARBs and other anti-hypertensive drugs in patients typically starts when elevated BP is already present and, according to guidelines (James et al., 2014; Weber et al., 2014), when non-pharmacological approaches have been insufficiently effective. As presence of hypertension often remains undiagnosed for many years, it can be assumed that treatment often starts in settings where the chronically elevated BP has already structurally affected the cardiovascular system. Obviously, this is only reflected in animal models that have started treatment with ARBs or other drugs in late phases of the condition. Therefore, we specifically highlight studies with such late start of treatment throughout this section, as these studies may be the most representative ones for the clinical situation.

2.1.

Normotensive animals 10

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Normotensive animals do not have the suppressed RAS activity seen in models of mineralocorticoid-induced hypertension but typically exhibit a much lower RAS activity than SHR or models of renal hypertension. Hence, one would expect that ARBs typically have limited BP effects in normotensive animals. Many studies have characterized ARB effects in normotensive animals, often as internal control group in studies of hypertensive animals. Most of these studies have been performed in rats but some have also used other species including mice (Bivalacqua et al., 1999; Bo et al., 2010; Matsumoto et al., 2014; Mazzolai et al., 2000; Nakamura et al., 2013; Ohshima et al., 2012; Penna-de-Carvalho et al., 2014; Sakai et al., 2008), rabbits (Azuma et al., 1992; Hajj-Ali & Wong, 1993), sheep (Peers et al., 2001), dogs (Chan et al., 1992; Hayashi et al., 1997b; Keiser et al., 1992; MacFadyen et al., 1992) and monkeys (Criscione et al., 1993; Lacour et al., 1993; Roccon et al., 1994).

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Studies in normotensive animals were reported for all ARBs including azilsartan (Carroll et al., 2015; Iwai et al., 2007; Kusumoto et al., 2011; Matsumoto et al., 2014; Nakamura et al., 2013; Sueta et al., 2013), candesartan (Bivalacqua et al., 1999; Cervenka et al., 1998; Demeilliers et al., 1999; Inada et al., 1994; Kobayashi et al., 1993; Li & Widdop, 1996; Nishimura et al., 1998b; Shibouta et al., 1993), eprosartan (Edwards et al., 1992a), irbesartan (Soltis et al., 1993), losartan and EXP3174 (Azuma et al., 1992; Criscione et al., 1993; Harada et al., 2009; Inada et al., 1994; Li & Widdop, 1996; MacFadyen et al., 1992; Mazzolai et al., 2000; Mizuno et al., 1992a; Ohshima et al., 2012; Soltis et al., 1993; Wong et al., 1990d; Wong et al., 1990e), olmesartan (DeMarco et al., 2011; Harada et al., 2009; Koike et al., 2001; Mizuno et al., 1995), telmisartan (DeMarco et al., 2011; Harada et al., 2009; MüllerFielitz et al., 2015; Ohshima et al., 2012; Penna-de-Carvalho et al., 2014; Toba et al., 2012; Wienen & Entzeroth, 1994) and valsartan (Criscione et al., 1993; Harada et al., 2009; Hayashi et al., 1997b; Sakai et al., 2008; Siragy et al., 2000; Yamamoto et al., 1997b). They included acute (Bivalacqua et al., 1999; Cervenka et al., 1998; Criscione et al., 1993; Demeilliers et al., 1999; Edwards et al., 1992a; Harada et al., 2009; Hayashi et al., 1997b; Inada et al., 1994; Kusumoto et al., 2011; Li & Widdop, 1996; MacFadyen et al., 1992; Mizuno et al., 1995; Shibouta et al., 1993; Soltis et al., 1993; Wienen & Entzeroth, 1994; Wong et al., 1990d; Wong et al., 1990e) and chronic treatment (Kobayashi et al., 1993; Mizuno et al., 1992a; Mizuno et al., 1995; Müller-Fielitz et al., 2015; Ohshima et al., 2012; Soltis et al., 1993; Toba et al., 2012), and oral (Hayashi et al., 1997b; Inada et al., 1994; Kobayashi et al., 1993; Kusumoto et al., 2011; Matsumoto et al., 2014; Mizuno et al., 1992a; Müller-Fielitz et al., 2015; Nakamura et al., 2013; Ohshima et al., 2012; Toba et al., 2012) and i.v. administration (Bivalacqua et al., 1999; Cervenka et al., 1998; Criscione et al., 1993; Demeilliers et al., 1999; Edwards et al., 1992a; Harada et al., 2009; Hayashi et al., 1997b; Li & Widdop, 1996; MacFadyen et al., 1992; Shibouta et al., 1993; Siragy et al., 2000; Wienen & Entzeroth, 1994; Wong et al., 1990d; Wong et al., 1990e) as well as other forms of parenteral administration (Soltis et al., 1993). While most of these studies were performed in conscious animals, anesthetized animals have also been investigated (Bivalacqua et al., 1999; Cervenka et al., 1998; Demeilliers et al., 1999; Hayashi et al., 1997b; Li & Widdop, 1996; Macari et al., 1993; Wienen & Entzeroth, 1994; Zhuo et al., 1992). In most of these studies with normotensive animals ARBs have shown a small if any effect on BP unless high doses were administered, i.e. doses exceeding those required to yield substantial inhibition of responses to exogenously administered ANG (Cervenka et al., 1998; Yamamoto et al., 1997b). However, in some studies in normotensive rats some degree of blood pressure lowering upon ARB administration has been observed in nomotensive animals, with e.g. candesartan (Inada et al., 1994; Kobayashi et al., 1993; Nishimura et al., 1998b), irbesartan (Soltis et al., 1993), losartan (Mazzolai et al., 2000; Soltis et al., 1993), olmesartan (Koike et al., 2001) or telmisartan (Yoshihara et al., 2013). Of note, a lowered baseline BP was reported in AT1R 11

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knock-out mice, in which telmisartan did not cause any additional change of BP (Rong et al., 2010); a moderate BP lowering upon telmisartan treatment was reported in ApoE/AT1R double knock-out mice (Fukuda et al., 2010). Moderate BP lowering by losartan has been reported in normotensive anesthetized rabbits (Hajj-Ali & Wong, 1993) or guinea pigs (El Amrani et al., 1993), but at least rabbits generally are more sensitive to ARBs than most other species (Sato et al., 1997). Sodium depleted animals have normal to low basal BP but exhibit an activated RAS and hence ARBs have been shown to lower BP in sodium-depleted rats (Jover et al., 1991; Macari et al., 1993; Siragy et al., 2000) and dogs (MacFadyen et al., 1992). BP lowering in normotensive sodium-depleted monkeys has consistently been reported (Criscione et al., 1993; DeGraaf et al., 1993; Lacour et al., 1993; Roccon et al., 1994), but these data are more difficult to interpret as sodium replete monkeys within the same studies had simiar BP lowering upon administration of irbesartan, losartan or valsartan, indicating that normotensive monkeys irrespective of the state of their sodium balance may be more responsive to ARB-induced BP lowering. Greater BP lowering in sodium-depleted animals as a clinical correlate, as ARBs cause greater BP lowering in patients who have been pre-treated with diuretics.

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Wherever hypertensive and normotensive animals were tested within the same study, ARBinduced BP reductions were always greater in hypertensive than in normotensive animals. The latter observation includes studies in which the antagonist potency to inhibit pressor responses to exogenously administered ANG was similar in normotensive and hypertensive animals, demonstrating that any difference between the two was not explained by pharmacokinetic differences or differences in AT1R responsiveness (Li & Widdop, 1996). In addition to BP normalization, ARBs were also reported to normalize the disturbed auto-regulation of cerebral blood flow (Nishimura et al., 1998b).

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In summary, ARBs as a class cause little BP lowering in normotensive animals unless they are sodium-depleted, except in monkeys or when very high doses are administered. Even in conditions where ARBs produce some BP lowering in normotensive animals, the extent of this effect is consistently lower than in ARB-responsive hypertension models. Moreover, it is noteworthy that basically all of the above studies in normotensive animals reported a lack of effect of ARBs on heart rate, regardless whether BP was altered or not (see section 4.1.2). However, when valsartan was administered to normotensive rats on top of an isoprenaline infusion, which already lowered BP and activated the RAS, no futher BP lowering was observed but rather an increase in heart rate became the primary valsartan response (Blanc et al., 2000).

2.2.

Spontaneously hypertensive rat (SHR)

The SHR is the most widely used animal model of arterial hypertension. It is a model of genetically caused hypertension based on many generations of inbreeding. However, genetic drift may have occurred in the inbreeding procedures, resulting in genetical heterogeneity between SHR currently available from multiple suppliers and the emergence of specific substrains of SHR (Nabika et al., 1991). Moreover, in most cases Wistar Kyoto rats are used as the normotensive control for SHR, but the systematic breeding of these started later and has led to even more heterogeneity (Lindpaintner et al., 1992). Moreover, it should be noted that some of the phenotypes displayed by SHR and plausibly linked to the pathophysiology of hypertension do not co-seggregate with hypertension in cross-breeding studies (Katsuya et al., 1992; Kunes et al., 1990; Lodwick et al., 1993; Michel et al., 1992; Orlow et al., 1991; Pravenec et al., 1992; Pravenec et al., 1995), indicating that hypertension and other phenotypes of SHR are genetically determined but not necessarily by the same set of genes. 12

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For instance, the hyperactivity phenotype did not coseggregate with the hypertensive phenotype in such cross-breeding studies. All of these factors need to be considered when comparing results reported with SHR and Wistar Kyoto rats obtained from different suppliers and when trying to understand the pathophysiology of hypertension-related phenotypes in SHR. Therefore, several investigators have used other normotensive rat strains such as Sprague-Dawley rats as (additional) comparators for their findings in SHR.

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2.2.1. BP lowering in established hypertension

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All clinically used ARBs effectively lower BP in SHR, irrespective of source of substrain. This was consistently observed with acute (oral and parenteral) and chronic dosing and in conscious and and anesthetized animals. Moreover, hypertension in SHR can be aggravated genetically, i.e. in the stroke-prone sub-strain of the SHR, or by various other measures, and ARBs also lower BP in these models (see section 2.2.4).

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SHR typically remain normotensive up to an age of 4-6 weeks, and then exhibit increasing BP with time, which typically reaches fairly stable levels at or beyond 12 weeks of age. However, structural consequences of hypertension, which contribute to the maintenance of elevated BP, can become increasingly important in SHR aged 6 months or older. Thus, ARB-induced BP lowering in SHR aged 12 weeks or older can be seen as a model of the clinical situation where a patient presents with established hypertension. In such animals i.v. (Harada et al., 2009; Lee et al., 2010) or intra-peritoneal administration (Grundt et al., 2009) of telmisartan dose-dependently acutely lowers BP. Similar BP effects of acute oral or parenteral ARB administration were reported with azilsartan (Kusumoto et al., 2011; Takai et al., 2013b), candesartan (Inada et al., 1994; Inada et al., 1999; Li & Widdop, 1996; Nishimura et al., 1998b; Takai et al., 2013b; Wada et al., 1996a; Yamada et al., 1995), eprosartan (Inada et al., 1999), irbesartan (Inada et al., 1999; Lacour et al., 1994), losartan (Cachofeiro et al., 1992; Harada et al., 2009; Hashimoto et al., 1997; Inada et al., 1994; Inada et al., 1999; Kumagai et al., 1993; Li & Widdop, 1996; Oddie et al., 1993; Ohlstein et al., 1992; Soltis, 1993; Tofovic et al., 1991; Wong et al., 1990d), olmesartan (Harada et al., 2009; Koike et al., 2001) or valsartan (Harada et al., 2009; Inada et al., 1999; Yamamoto et al., 1997b). This was also observed in a stroke-resistant substrain of SHR following ligation of the middle cerebral artery during a 15 months follow-up (Kurata et al., 2014a; Kurata et al., 2014b). Interestingly, the BP lowering effect of a single dose of ARB measured was correlated to plasma concentration when measured after 2 h but not after 24 h; in contrast, after 24 h BP reduction was correlated to vascular ARB concentration, whereas plasma and vascular concentration were not associated (Takai et al., 2013b), indicating that compartmentalization into target tissue may contribute to effect duration beyond what would be expected based on pharmacokinetics as measured in plasma. Even more relevant for the clinical treatment situation is the chronic administration of ARBs to SHR with established hypertension. This has mostly been done by oral administration, but parenteral administration schemes, e.g. based on osmotic minipumps chronically delivering an ARB subcutaneously have also been used (Nishimura et al., 1998b). Again, all ARBs having been tested effectively lowered BP under these conditions, e.g. azilsartan (Hye Khan et al., 2014a; Isegawa et al., 2015; Sueta et al., 2013), candesartan (Chen et al., 2010; Inada et al., 1994; Isegawa et al., 2015; Ishimitsu et al., 2010; Jones et al., 2004; Jones et al., 2012; Mizuno et al., 1992b; Mizuno et al., 1994; Mori et al., 1995; Nishimura et al., 1998b; Takeda et al., 1994; Tsuchihashi et al., 1999), irbesartan (Lacour et al., 1994), losartan (Dai et al., 2007; Hashimoto et al., 1997; Inada et al., 1994; Li & Widdop, 1996), olmesartan (Koike et al., 2001), telmisartan (Chen et al., 2010; Dai et al., 2007; Khan & Imig, 2011; Kumai et al., 13

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2008; Li et al., 2006; Liu et al., 2009a; Liu et al., 2011; Ma et al., 2010; Mandarim-deLacerda & Pereira, 2004; Müller-Fielitz et al., 2012; Wienen et al., 2001; Xu & Liu, 2013; Zhong et al., 2011; Zhu et al., 2012) and valsartan (Chow et al., 1995; Scheidegger et al., 1996; Tea et al., 2000). Such findings apparently apply to various substrains of SHR (Li et al., 2006). In one study with 10 week old SHR being treated for 4 weeks, both azilsartan and candesartan similarly lowered BP during the daytime resting phase of the animals and the nighttime active phase, but in the first two hours of the active phase, i.e. immediately after daily dosing, azilsartan caused greater BP reduction than candesartan (Isegawa et al., 2015). However, interpretation of these data is challenging as the chosen of azilsartan was more effective on overall BP than that of candesartan.

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While most of these chronic treatment studies have started at ages of 10-14 weeks, some started at much later time points e.g. at 8 months (Choisy et al., 2015; Koike et al., 2001; Li et al., 2006), 12 months (Mandarim-de-Lacerda & Pereira, 2004) or even 20 months (Jones et al., 2004; Jones et al., 2012); although it can be assumed that at such late time points structural alterations of the cardiovascular system including hypertrophy and fibrosis already contribute to the BP maintenance, ARBs were nevertheless successful in lowering it. In contrast AT2R antagonists did not lower BP in SHR (Tea et al., 2000) and also did not attenuate BP reduction by an ARB (Jones et al., 2004; Jones et al., 2012). Taken together these findings demonstrate that ARBs as a class are effective in BP lowering in SHR with established hypertension. On a pathophysiological level these studies demonstrate that ANG contributes to the maintenance of elevated BP in established hypertension, at least in the SHR model. 2.2.2. Effect on hypertension development

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Conceptually different from the question of an ANG contribution to the maintenance of elevated BP is whether ANG similarly contributes to hypertension development. As development of hypertension in SHR is a fairly predictable process, multiple studies have explored whether chronic administration of an ARB, either before onset of hypertension or in early phases thereof, can prevent BP elevation (Figure 1). Indeed preventive effects have been demonstrated for candesartan (Müller-Fielitz et al., 2012; Pollock & Morsing, 1999; Rizzoni et al., 1998), irbesartan (Intengan et al., 1999; Vacher et al., 1995; Zalba et al., 2001), losartan (Kawano et al., 2000a; Morton et al., 1992; Oddie et al., 1992), telmisartan (Miesel et al., 2012; Müller-Fielitz et al., 2012; Yoo et al., 2015; Zou et al., 2015) and valsartan (Scheidegger et al., 1996). In some cases even a very late start of ARB administration, i.e. after one year, still could attenuate further BP elevations (Mandarim-de-Lacerda & Pereira, 2004). Valsartan and enalapril also prevented hypertension development in New Zealand genetically hypertensive rats (Ledingham et al., 2000). Of note, depending on when treatment was started, such preventive ARB administration not only attenuated the genetically determined increase in BP but, at least in some cases, completely prevented it. While ARBs presently are only indicated for the treatment of hypertension, these data in SHR raise the possibility that this drug class may also have preventive effects on hereditary BP elevation; however, this has not been tested clinically. <> 2.2.3. Persistence of BP lowering after end of treatment

The chronic infusion of ANG can cause hypertension, and such hypertension can persist for a considerable time (at least weeks to months) after the ANG administration has stopped. This 14

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raises the possibility that ARB administration may also have BP lowering effects that extend well beyond the presence of the drug in the organism. An early observation in this regard comes from a study in which losartan was given to 3-week old SHR for 4 or 10 weeks (Morton et al., 1992). Both treatments prevented BP elevations and their effects at least partly persisted up to an age of 30 weeks after treatment had been stopped; in this regard the 10week treatment was more effective than the shorter one, which may be related to a stronger effect on vascular hypertrophy. Similar findings were reported from an independent study (Oddie et al., 1992), but those investigators found that the persistent effects of a 6-week treatment with losartan starting at the age of 14 weeks were only transient, and 3 months after the end of ARB administration BP had reapproached untreated SHR levels (Oddie et al., 1993). Similar findings were obtained with candesartan given for four weeks to 20-week old SHR (Paull et al., 2001; Paull & Widdop, 2001); in those studies the gradual return of BP towards values of vehicle treated rats was not necessarily accompanied by similar changes for regional vascular conductance (Paull et al., 2001). Irbesartan attenuated hypertension development when given to 4-week old SHR for 16 weeks; within 8 weeks after discontinuation of treatment, BP remained significantly reduced but thereafter gradually approached levels of untreated SHR (Vacher et al., 1995) (Figure 1). In another study, SHR weighing 300 g (age not specified) were treated with telmisartan for 5 days; BP remained significantly lowered for 4 days after the end of telmisartan treatment and numerically below values from vehicle-treated rats during the entire 14 day observation period (Wienen & Schierok, 2001). Similarly, the reduction of diastolic (but not systolic) BP achieved by treatment with valsartan was also largely retained for at least 4 weeks after treatment discontinuation in transgenic mice harboring human angiotensinogen and renin genes (Inaba et al., 2011).

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The withdrawal response to different drug classes has been directly compared. In one study 20 week old SHR were initially treated for 4 weeks with either candesartan, perindopril or hydralazine (Paull & Widdop, 2001). While the chosen doses of the three drugs produced comparable BP lowering during active treatment, mean arterial pressure returned to levels observed in vehicle-treated rats within 4 and 15 days after withdrawal of hydralazine or candesartan, respectively, but remained consistently reduced to some degree for the 8-week follow-up after withdrawal of perindopril treatment. 2.2.4. Stroke-prone SHR and otherwise aggravated hypertension in SHR

Hypertension in SHR can be aggravated either in a hereditary form, such as in the substrain of stroke-prone SHR, or by a range of other interventions including high-salt diet, partial nephrectomy or superimposed diabetes. Such aggravated models may be representative for some types of patients. Moreover, they can be associated with a more rapid development of the sequelae of hypertension, e.g. stroke, and hence be models for the prevention of such complications. Various ARBs were found to lower BP in stroke-prone SHR including candesartan (Kishi et al., 2012; Nakamura et al., 1994b; Takai et al., 2011; Xie et al., 1999), losartan (Wagner et al., 1998), olmesartan (Tomita et al., 2009), telmisartan (Kishi et al., 2012; Takai et al., 2007; Thoene-Reineke et al., 2011; Wagner et al., 1998) and valsartan (Pu et al., 2008; Takai et al., 2007; Webb et al., 1998a). Of note the beneficial effects of an ARB in stroke-prone SHR were shown not only in treatment but also in preventive study designs (Thoene-Reineke et al., 2011).

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A high-salt diet can also aggravate hypertension in SHR but with few exceptions (Yoshida et al., 2011) apparently ARBs have little effect under those conditions. E.g. an 8-week treatment with telmisartan did not lower BP in SHR on a high-salt diet (Susic et al., 2013). When a high-salt diet was given to stroke-prone SHR, neither telmisartan nor valsartan remained an effective antihypertensive drug, although they had lowered BP in stroke-prone SHR in the absence of a high-salt diet (Takai et al., 2007). Valsartan also failed to lower BP in strokeprone SHR on a high-salt diet and furthermore did not significantly enhance the BP lowering effect of amlodipine under these conditions (Dong et al., 2011). Similarly, eprosartan only produced rather limited BP lowering when given to stroke-prone SHR on a high-salt diet (Abrahamsen et al., 2002) or those on a mixed high-salt, high-fat diet (Barone et al., 2001; Behr et al., 2004). Finally, candesartan or telmisartan did not lower BP in salt-loaded strokeprone SHR continuously infused with ANG (Kishi et al., 2014). This lack of effect apparently is related to the suppression of RAS activity upon chronic administration of a high-salt diet. However, a major BP lowering to levels below those in SHR on a normal diet was seen in rats which had been on a high-salt diet for 8 weeks and then switched to a normal diet + telmisartan (Susic & Frohlich, 2014); under these conditions, 8/9 rats treated with telmisartan survived the normal diet phase, whereas only 1/9 rats survived when only switched to a normal diet. In salt-loaded stroke-prone SHR continuously infused with ANG candesartan and, to an even greater extent, telmisartan reduced mortality despite a lack of BP lowering (Kishi et al., 2014).

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Impaired renal function can also cause hypertension, and the 5/6 subtotal nephrectomy is a frequently used model of this condition (see section 2.3). When 5/6 nephrectomy was performed in SHR, candesartan continued to lower BP (Kohara et al., 1993), even when an additional high-salt diet was administered (Okada et al., 1995). The immunosuppressant cyclosporin A can also cause renal impairment, and hypertension is a frequent side effect of cyclosporin treatment; valsartan was shown to attenuate hypertension development in young SHR concomitantly treated with cyclosporin A and a high-salt diet (Lassila et al., 2000a).

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As hypertension and diabetes mellitus are frequent comorbidities (Michel et al., 2004), several groups have studied ARB effects in SHR with a superimposed STZ-induced diabetes; under such circumstances e.g. irbesartan (Bonnet et al., 2001; Cao et al., 2001), telmisartan (Wienen et al., 2001) and valsartan (Hulthen et al., 1996) remained effective BP-lowering drugs. When SHR were fed a cafeteria-style diet, a model for the association of hypertension and the metabolic syndrome, telmisartan also provided effective BP lowering (Müller-Fielitz et al., 2012). Taken together these data demonstrate that the antihypertensive effect of ARBs as a drug class in SHR is largely maintained under aggravating circumstances. The concomitant presence of a high-salt diet in SHR appears to be an exception to this rule; this is not surprising as a high-salt intake provides strong suppression of the RAS and hence leaves little room for a RAS inhibitor to work (but see section 2.2.5 for BP lowering by combination treatment). 2.2.5. Combination treatments

Many hypertensive patients require treatment with more than one anti-hypertensive drug to reach target BP values. Accordingly, several studies have addressed potential combinations of ARBs with representatives of other drug classes. In this regard most studies have focused on combinations with diuretics, with Ca2+ entry blockers, or more recently with neutral endopeptidase inhibitors; some of these combinations were expected to exhibit synergies, for 16

ACCEPTED MANUSCRIPT instance due to direct RAS activation by diuretics or indirect inactivation due to baroreflex activation by Ca2+ entry blockers.

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Studies on a combination with diuretics have largely involved hydrochlorothiazide. Importantly, it should be noted that the BP-lowering effect of thiazide diuretics in patients typically develops much more slowly than its diuretic effect and may take days to weeks. While this is in contrast to the antihypertensive effect of ARBs (see section 2.2.1), it implies that testing of ARB/diuretic combinations should be based on treatment of at least several days. A 5-day treatment with telmisartan lowered BP in SHR; while hydrochlorothiazide alone did not significantly lower BP, it significantly enhanced the effects of telmisartan starting on the first day and reaching maximum reductions on the last day of treatment (Wienen & Schierok, 2001). Additive to synergistic BP lowering were also observed with a combination of valsartan and hydrochlorothiazide during a 2-week treatment (Webb et al., 1998b). In a 2-week study with candesartan the addition of hydrochlorothiazide also exhibited synergistic BP reductions, which exceeded the effects of combinations with prazosin, atenolol or manidipine (Wada et al., 1996b). Enhanced BP lowering by a combination of candesartan and hydrochlorothiazide were also reported in stroke-prone SHR (Takai et al., 2011). While neither telmisartan nor hydrochlorothiazide significantly lowered BP during an 8-week treatment in SHR on a high-salt diet, their combination was effective (Susic et al., 2013); this is in line with the view that high-salt intake suppresses the RAS and that the presence of a diuretic resensitizes the animal to an ARB.

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Ca2+ entry blockers, particularly those of the dihydropyridine family, have also been tested in combination with ARBs in SHR. Most of these studies have used amlodipine, but studies with manidipine (Wada et al., 1996b), lercanidipine (Lee et al., 2010) or cilnidipine (Takai et al., 2013a) have also been reported. In acute interaction studies, in which multiple doses of either agent were tested, telmisartan and amlodipine showed synergistic BP lowering with the strongest synergism being observed when the two were given in a 6:1 ratio (Liu et al., 2011). Within the same study a 4-months combination treatment had produced greater BP lowering than either monotherapy, and also reduced BP variability. In SHR on a high-salt diet, amlodipine lowered BP during an 8-week treatment, whereas telmisartan alone was ineffective (see above); however, the combination of both agents lowered BP to an at least numerically greater degree than amlodipine alone (Susic et al., 2013). Greater blood pressure lowering effects of combinations with amlodipine have also been reported for candesartan (Takai et al., 2011), irbesartan (Shang et al., 2011), losartan (Choi et al., 2009) or valsartan (Takai et al., 2013a). However, a combination of low doses of candesartan and amlodipine produced lesser BP reductions in stroke-prone SHR than candesartan monotherapy (Kim et al., 2000). A combination of low dose valsartan with amlodipine also did not yield enhanced BP lowering in salt-loaded stroke-prone SHR (Dong et al., 2011). A third interesting option treatment was explored for the combination of an ARB with a neutral aminopeptidase inhibitor. Using valsartan and CGS 25354 in combination produced slightly greater BP lowering than valsartan alone during a 10 week treatment of stroke-prone SHR (Pu et al., 2008). Similar data were obtained when valsartan was combined with the endopeptidase inhibitor candoxatril (Hegde et al., 2011). Of note, these combination studies have evaluated not only BP reductions but also endpoints such as endothelium-dependent vasodilatation or cardiac fibrosis, and were found to be beneficial on these endpoints as well (see below). 2.2.6. Effects on mortality, particularly in stroke-prone SHR 17

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Insufficiently treated hypertension can lead to premature death, possibly the hardest of all possible study endpoints. On pragmatic grounds animal studies on potential survival benefits of ARBs have largely focused on models with a clearly increased mortality such as the saltloaded stroke-prone SHR. Several ARBs have been shown to enhance survival in such animals when treatment was started at an age of 10-14 weeks, e.g. candesartan (Bennai et al., 1999), eprosartan (Barone et al., 2001), olmesartan (Takai et al., 2009) or valsartan (Takai et al., 2009; Webb et al., 1998a). Interestingly, valsartan also improved survival in stroke-prone SHR on a high-salt diet, despite not lowering BP under these conditions; the reduction in mortality was similar to that achieved by amlodipine, and the valsartan/amlodipine combination completely prevented mortality in this model (Dong et al., 2011). A summary of mortality studies with ARBs in stroke-prone SHR and various other disease models is provided in Table 2. Interestingly, AR1R knock-out has been shown to increase longevity as compared to healthy wild-type mice (Benigni et al., 2009); based on comparable effects of such knock-out and of ARB treatment on presumed underlying physiological mechanisms, it has been proposed that ARBs may share this effect (Cassis et al., 2010).

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The kidney contributes to BP control in multiple ways. Firstly, it affects circulating blood volume by regulating the excretion of salt and fluid. Second, in reaction to the amount of sodium reaching the distal tubules, it mediates renin release which increases formation and release of ANG and aldosterone. Thirdly, pressure natriuresis is a powerful mechanism with infinite gain in BP control, a mechanism which in itself involves the RAS (Romero & Knox, 1988). Hence, reductions of renal perfusion or of nephron mass or function can activate the RAS and lead to renovascular and renal mass-associated hypertension, respectively. Therefore, ARBs typically cause strong BP reductions in animals with renovascular hypertension or advanced nephropathy.

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2.3.1. Renovascular hypertension

Renovascular hypertension can be induced experimental animals in multiple ways. The classic Goldblatt model uses animals which retain both kidneys and have a unilateral reduction of renal blood flow (RBF) due to a clip on the renal artery of one kidney. However, a one-kidney-one-clip model or acute single-sided renal artery ligation models have also been used. Most animal models of renovascular hypertension involve rats, but mice (Salguero et al., 2008), hamsters (Jin et al., 1998) and dogs (Brooks et al., 1992a; Ito et al., 1995; Koike et al., 2001; Kusumoto et al., 2011) have also been used to study ARB effects in renovascular hypertension. Renovascular hypertension models generally are characterized by strong RAS activation, which on theoretical grounds makes them very sensitive to RAS inhibition. Accordingly, each clinically used ARB has been shown to be effective in at least one model of renovascular hypertension. This includes studies with azilsartan (Kusumoto et al., 2011), candesartan (Inada et al., 1994; Ito et al., 1995; Li & Widdop, 1995; Wada et al., 1996a; Yu et al., 1993), eprosartan (Brooks et al., 1992b), irbesartan (Lacour et al., 1994), losartan (Brooks et al., 1992b; Hashimoto et al., 1997; Sigmon & Beierwaltes, 1993b; Wong et al., 1990a; Wong et al., 1990c; Wong et al., 1990e; Zhang et al., 1993b), olmesartan (Koike et al., 2001; Mire et al., 2005), telmisartan (Chaykovska et al., 2013; Kawai et al., 2009; Ries et al., 1993; 18

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These data indicate that ARBs as a class effectively lower BP in renovascular hypertension across different models and species. This may involve direct effects on the kidney and those on the extra-renal vasculature. For example in 2-kidney-1-clip renal hypertensive rats losartan lowered BP along with an increase of RBF of the clipped kidney but no change in the unclipped kidney (Sigmon & Beierwaltes, 1993b). On the other hand, similar to findings from the SHR, the BP lowering by candesartan in that model involved vasodilatation in multiple vascular beds (Li & Widdop, 1995). While most studies have administered an ARB early after induction of renal artery stenosis, treatment has also been started as late as 32 weeks after inducing renal artery stenosis and still provided similar BP lowering as with early treatment as reported with candesartan (Jin et al., 1998). Moreover, similar to the SHR, a combination of telmisartan and amlodipine produced synergistic BP lowering in two-kidney-one-clip rats (Liu et al., 2011).

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Hypertension can also be a consequence of renal parenchymal disease, which experimentally often is mimicked by surgically induced renal mass reduction. Renal mass reduction, with few exceptions (Toba et al., 2012), is accompanied by an increase in BP. This BP elevation was be prevented by treatment with candesartan (Hamaguchi et al., 1996; Li et al., 1999b; Mackenzie et al., 1994; Noda et al., 1997; Noda et al., 1999; Podjarny et al., 2007; Sugimoto et al., 1999), eprosartan (Gandhi et al., 1999; Wong et al., 2000), irbesartan (Cao et al., 2012; Ziai et al., 1996), losartan (Kanagy & Fink, 1993; Lafayette et al., 1992; Mayer et al., 1993; Pollock et al., 1993a; Toba et al., 2013), telmisartan (Kadhare et al., 2015; Toba et al., 2013; Tsunenari et al., 2005; Zou et al., 2014) or valsartan (Wu et al., 1997). In a rat renduced renal mass model, losartan also prevented the additional BP increase caused by a high-salt diet (Kanagy & Fink, 1993).

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Injection of an anti-Thy-1 monoclonal antibody is being used as a rat model of glomerulonephritis; in this model of renal hypertension candesartan also lowers BP (Nakamura et al., 1997). Such reductions were even found when anti-Thy-1 injection and uninephrectomy were combined (Nakamura et al., 1999). In PCK rats, an orthologous model of human autosomal recessive polycystic kidney disease, telmisartan lowered BP to a level lower than that in control rats (Yoshihara et al., 2013). Losartan lowered BP in rats with immune-mediated kidney disease (passive Heyman nephritis) (Hutchinson & Webster, 1992).

2.4.

Dahl salt-sensitive rats

The Dahl salt-sensitive rat represents an inbred strain which is genetically susceptible to BP elevation in response to a high-salt diet, typically applied as a 4-8% NaCl part of their overall chow, sometimes with additional NaCl in the drinking water; in contrast, there is a Dahl saltresistant inbred strain which does not exhibit major BP elevation upon exposure to a high-salt diet (Rapp, 1982). Whether and to which degree ARBs lower BP in Dahl salt-sensitive rats on a high-salt diet has remained controversial. For instance studies with lack of effect were reported for e.g. candesartan (Otsuka et al., 1998), losartan (von Lutterotti et al., 1992), olmesartan (Kiya et al., 2010) and telmisartan (Takenaka et al., 2006). Studies with moderate effects, as compared to the results in SHR or renal hypertensive rats, were reported for candesartan (Ideishi et al., 1994; Sugimoto et al., 1996), irbesartan (Leenen & Yuan, 2001), 19

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losartan (Liang & Leenen, 2008; von Lutterotti et al., 1992), telmisartan (Liang & Leenen, 2008) or valsartan (Aritomi et al., 2010; Biala et al., 2011; Nagasawa et al., 2015). However, a small number of studies reported considerable antihypertensive effects in this model with BP being reduced almost to the level of Dahl rats not exposed to a high-salt diet, e.g. with azilsartan (Jin et al., 2014), irbesartan (Takai et al., 2010), losartan (Liang & Leenen, 2008) or telmisartan (Liang & Leenen, 2008; Satoh et al., 2010a). It is difficult to determine whether these equivocal results represent differences between ARBs or between specific aspects of the model or assessment techniques being used. However, it appears that ARBs as a class are less effective BP lowering agents in the Dahl S model on a high-salt diet than in SHR or renal hypertensive rats. Given that a high salt intake suppresses the RAS activity, this finding is not entirely surprising. However, several investigators have turned this apparent disadvantage into an advantage by studying ARB effects on end-organ damage in the Dahl S rat, based upon the implication that such effects may argue in favor of BP-independent organ-protective ARB effects.

Mineralocorticoid-induced hypertension

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An interesting aspect of studies in this model is a potentially enhanced role for a local RAS in the brain. Thus, it was reported that an 11-12 day intracerebroventricular (i.c.v.) candesartan infusion lowered BP in Dahl salt-sensitive rats on a high-salt diet, an effect not seen in Dahl salt-resistant rats (Teruya et al., 1995). A partial inhibition of hypertension development was also reported with chronic intra-cerebroventricular irbesartan in Dahl salt-sensitive rats; as the applied dose did not affect pressor responses to peripherally administered ANG, it can be considered to have acted only within the brain (Leenen & Yuan, 2001).

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Another hypertension model with low RAS activity is based on the chronic administration of the mineralocorticoid deoxycorticosterone acetate (DOCA), mostly applied in combination with a high-salt diet. Similar to most studies in Dahl salt-sensitive rats on a high-salt diet, ARBs typically caused small if any BP lowering in DOCA-based rat models as shown with candesartan (Brown et al., 1999; Fujita et al., 1997; Inada et al., 1994; Kim et al., 1994b; Wada et al., 1995), irbesartan (Lacour et al., 1994), losartan (Inada et al., 1994; Wong et al., 1990a), olmesartan (Koike et al., 2001), telmisartan (Sharma & Singh, 2012) or valsartan (Yamamoto et al., 1997b). These findings are similar to those seen in Dahl salt-sensitive rats on a high-salt diet and not surprising given that mineralocorticoid treatment also suppresses the RAS. Also in analogy to the Dahl findings and in contrast to those with systemic ARB administration, i.c.v. candesartan was found to lower BP in DOCA rats (Nishimura et al., 1998a). Thus, DOCA rats have also been used as a model to study potential BP-independent ARB effects on end-organ damage (see corresponding sections).

2.6.

Dietary, obesity and diabetes models of hypertension

Human hypertension often is associated with high salt intake, obesity and/or diabetes mellitus, and all of these can be mimicked in animal models. Thus, a high-salt (3% of chow) diet given for 5 weeks to otherwise healthy rats elevated systolic BP by about 35 mm Hg, and this elevation was completely prevented by concomitant telmisartan treatment (Kamari et al., 2010); however, given the typical lack of BP lowering in SHR on a high-salt diet (see section 2.2.4) this finding is difficult to understand. A high-fat diet given for 12 weeks moderately increased BP in otherwise healthy rats, albeit to a lesser degree than the above high-salt diet, and concomitant telmisartan treatment normalized BP in such animals (Wang et al., 2012c). Even a moderately high-fat diet can induce obesity in rats, which is accompanied by an elevated BP; treatment with losartan initiated after induction of obesity also reduced BP in 20

ACCEPTED MANUSCRIPT such rats (Boustany et al., 2005). Hypertension induced by a high-fat diet was also improved by telmisartan in mice (Penna-de-Carvalho et al., 2014).

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Type 2 diabetes mellitus also is often associated with some degree of hypertension, possibly secondary to an impaired renal function. Fructose-fed rats are a frequently used model of type 2 diabetes. Such rats often but not always (Kobayashi et al., 1993) exhibit moderate hypertension, and in this model e.g. candesartan (Chen et al., 1996a; Chen et al., 1996b; Higashiura et al., 2000; Iimura et al., 1995), and telmisartan (Kamari et al., 2008; Rabie et al., 2015) have been shown to lower BP. In one of these studies telmisartan and aliskiren yielded similar BP reduction (Rabie et al., 2015).

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Telmisartan lowered BP in SHRSP.Z-Leprfa/IzmDmcr rats in some (Kagota et al., 2013; Kagota et al., 2014) but not all studies (Sugiyama et al., 2013). Another model of non-insulindependent diabetes is the Cohen-Rosenthal diabetic hypertensive rat, in which telmisartan and valsartan lowered BP (Younis et al., 2010); in this model telmisartan was also reported to prevent hypertension development when given early (Younis et al., 2012). Koletsky (fak/fak) rats carry a missense mutation in the leptin receptor on an SHR background, and azilsartan lowered BP in such rats (Zhao et al., 2011); similarly, SHRcp rats also are an SHR-derived strain with metabolic syndrome due to a spontaneous nonsense mutation in the leptin receptor gene, and telmisartan and valsartan lowered BP during a 5-week study, but telmisartan yielded a greater reduction in BP variability and in 24-h urinary noradrenaline excretion (Sueta et al., 2014). Olmesartan (Harada et al., 1999) and telmisartan (Zhao et al., 2013) were found to lower BP in Otsuka Long-Evans Tokushima fatty rats, and azilsartan (Hye Khan et al., 2014b) and irbesartan (O'Donnell et al., 1997) in obese Zucker rats. In db/db mice BP reduction was reported with losartan (Toyama et al., 2011), olmesartan (Shigahara et al., 2014), telmisartan (Shiota et al., 2012b; Toyama et al., 2011) and valsartan (Dong et al., 2010), although the numerical reduction did not reach statistical significance in all studies (Shiota et al., 2012b); interestingly, the ARB effect on BP was not mimicked by aliskiren in this model (Dong et al., 2010). In KK-Ay mice BP lowering was reported with azilsartan and candesartan (Matsumoto et al., 2014), but candesartan did not lower BP in another study (Iwai et al., 2007); losartan and telmisartan were both reported not to lower BP in this model (Iwanami et al., 2010). Administration of STZ is the most frequently used model of type 1 diabetes mellitus, and this diabetes model typically is not associated with hypertension. Nevertheless, and in contrast to most findings in normotensive rats (see section 2.1), ARBs have been reported to lower BP in the rat STZ model, e.g. candesartan (Kato et al., 1999), telmisartan (Salum et al., 2014; Schäfer et al., 2007; Tojo et al., 2015) or valsartan (Cao et al., 1998). Why STZ-treated rats are more sensitive to ARBs with regard to BP lowering than other nomotensive rats has not been explored.

2.7.

Transgenic and knock-out hypertension models

Rat strains such as the SHR and the Dahl salt-sensitive rat have been generated by generations of phenotype-driven inbreeding for many years. More recently, rodent hypertension models have become available which are based on molecular transgenic or knock-out technologies. While some of these models were generated to produce specific cardiovascular phenotypes, others have exhibited such phenotypes as a byproduct. While transgenic and knock-out mice are technically easier to generate than e.g. transgenic rats, phenotypic studies typically are technically easier in rats than in mice. Moreover, there is a greater body of physiological 21

ACCEPTED MANUSCRIPT background knowledge for rats, whereas there is a greater set of molecular tools for studies in mice. Therefore, both approaches have been applied and should be seen as complementary.

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The first transgenic model in the field was a rat harbouring the mouse Ren-2 renin gene, commonly referred to as the (mRen-2)27 rat (Mullins et al., 1990). These animals exhibit a hypertensive phenotype, with reduced renal renin expression but increased plasma levels of active renin, pro-renin and ANG; ANG tissue levels and plasma aldosterone are also elevated in this model (Campbell et al., 1995). In this model BP normalization was reported with eprosartan (Rothermund et al., 2001), irbesartan (Andreis et al., 2000; Rossi et al., 2000), olmesartan (DeMarco et al., 2011), telmisartan (Böhm et al., 1995; DeMarco et al., 2011) and valsartan (Kelly et al., 2000). Interestingly, ARBs also lowered BP in this model when STZinduced diabetes was concomitantly present (Kelly et al., 2000). Valsartan was also shown to lower BP in a model derived from a back-cross of (mRen-2)27 and Lewis rats (Moniwa et al., 2012) and in double transgenic rats harboring a human renin and an angiotensin gene (Mervaala et al., 1999).

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A model has been created by mating transgenic mice harboring a human renin or angiotensinogen gene. While either single transgenic remained normotensive, the double transgenic animals exhibited hypertension; a direct renin inhibitor lowered BP only in the double transgenic rats, whereas both captopril and losartan lowered BP in the double transgenic and in the normotensive single transgenic mice (Fukamizu et al., 1993), reflecting that human renin interacts poorly with the murine substrate. In another study in such double transgenic mice, BP was lowered by azilsartan or olmesartan, with the former being more effective at the chosen dose levels (Iwanami et al., 2014). Valsartan also lowered BP in this double transgenic model (Inaba et al., 2011). In the latter study, cessation of valsartan treatment after 4 weeks led to a rapid increase of systolic BP to levels of untreated mice, whereas diastolic BP increased to a much smaller extent.

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Among other genetically engineered models, a mouse with transgenic C-reactive protein expression exhibited an exaggerated BP response to ANG but the BP lowering effect of telmisartan was maintained (Vongpatanasin et al., 2007). Elevated BP can also occur in various knockout mouse models, and some studies have reported ARB effects in such mice. For instance telmisartan lowered BP in a knock-out mouse for endothelial NO synthase with superimposed STZ-induced diabetes (Alter et al., 2012), and valsartan abolished hypertension in collecting duct endothelin-1 knock-out mice (Ge et al., 2008). Knock-out mice lacking hypoxia-inducible factor-1α exhibt an exaggerated BP response in response to ANG infusion and an elevated resting BP which was lowered upon telmisartan treatment (Huang et al., 2013).

2.8.

Other hypertension models

A wide range of other hypertension models exists. Most of them are based on the administration of ANG, on physiological or surgical interventions or exhibit an elevated BP as a side effect of treatment with other drugs. However, hereditary hypertension in inbred rat strains is not limited to the SHR or Dahl salt-sensitive rats but has also been reported in several other strains. While the specific pathophysiology of other hypertensive rat strains has not been elucidated in as much detail as in the SHR (see section 2.10), it is noteworthy that ARBs typically were effective anti-hypertensive drugs in these other strains as well. This includes valsartan in New Zealand genetically hypertensive rats (Ledingham et al., 2000) or irbesartan in the SHHF/Mcc-facp rat, a model of spontaneous hypertension and heart failure (Carraway et al., 1999). However, a follow-up study in the latter model found that irbesartan 22

ACCEPTED MANUSCRIPT reduced BP in lean SHHF/Mcc-facp rats of either gender and in male obese but not in female obese animals (Sharkey et al., 2001); the reason for this gender difference, which has not been observed in most other hypertension models remains to be elucidated.

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Chronic infusion of ANG, i.e. for days to weeks, can cause hypertension which persists for a long time after discontinuation of ANG administration. Not surprisingly, this form of ANGinduced hypertension is prevented or at least attenuated by treatment with ARBs such as azilsartan (Carroll et al., 2015; Lastra et al., 2013), candesartan (Inscho et al., 1999), temisartan (Ren et al., 2012) or valsartan (Cao et al., 1999; Sakai et al., 2008). A physiologically more interesting model is based on the observation that exposure of rats to cold temperature (5°C) for 1-3 weeks raises BP, possibly by the cold-induced activation of the sympathetic nervous system; this cold-induced BP elevation was prevented by losartan (Fregly et al., 1993).

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Hypertension can also be induced by surgical intervention, e.g. aortic banding which causes cardiac pressure overload. In this model ARBs such as azilsartan (Quinn Baumann et al., 2013), telmisartan (Aswar et al., 2013; Liu et al., 2010; Zhang et al., 2014a) or valsartan (Obayashi et al., 1999) lowered BP and reduced systemic vascular resistance. A different surgical model causing hypertension is sino-aortic denervation, and e.g. telmisartan lowered BP in rats exposed to this intervention (Wang et al., 2006). Telmisartan lowered BP in rats when administered after induction of myocardial infarction, and such BP lowering was not affected by concomitant administration of GW9662 (Nagashima et al., 2012). Candesartan was reported to lower BP in a rat kidney allograft transplantation model (Mackenzie et al., 1997).

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Endothelium-derived NO is an important contributor to vascular tone, and its release can be reduced by NO synthase inhibitors such as L-nitro-arginine methyl ester (L-NAME). Accordingly, chronic treatment with L-NAME causes hypertension, and ARBs such as candesartan (Casellas et al., 1999; Fortepiani et al., 1999; Liu et al., 1998; Okamura et al., 1994), irbesartan (Luvara et al., 1998), losartan (Jover et al., 1993; Pollock et al., 1993b; Ribeiro et al., 1992; Sigmon & Beierwaltes, 1993a) or olmesartan (Moningka et al., 2012; Takemoto et al., 1997) lower BP in this model. Interestingly, the BP lowering effect in the LNAME rat model was observed on a low and a high sodium diet (Jover et al., 1993; Okamura et al., 1994). On the other hand, candesartan lowered L-NAME-induced BP in rats upon i.v. but not i.c.v. administration (Liu et al., 1998), indicating a peripheral site of action. Some clinically used drugs can have hypertension as a side effect and/or can be used experimentally to increase BP. For example the immunosuppressant cyclosporin A elevates BP, possibly due to its renal effects, and e.g. telmisartan (Al-Amran et al., 2011) and valsartan (Lassila et al., 2000b) lowered BP in a rat model thereof. The dual vascular epidermal growth factor/platelet-derived growth factor inhibitor linifanib, which is under clinical development for the treatment of certain types of cancer, also can cause hypertension as a side effect in rats; telmisartan prevented this BP elevation, but RAS inhibition did not impair its effect on tumor growth in mice (Franklin et al., 2009). Telmisartan was reported to attenuate the hypertension developing after a doxorubicin injuection (Rashikh et al., 2014). Experimentally, chronic infusion of isoprenaline can also cause hypertension, probably via elevation of cardiac output and stimulation of renin release, and that also was attenuated by telmisartan (Goyal & Mehta, 2012). The moderate BP elevation induced by the monoamine uptake inhibitor tesofensine was not mitigated by concomitant treatment with telmisartan (Bentzen et al., 2013). 23

ACCEPTED MANUSCRIPT 2.9.

Hypotensive states

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A limited number of studies has explored whether ARBs affect BP in already hypotensive conditions. Such work was primarily done in animals with acutely lowered BP. Thus, following hemorrhage the vasoconstriction response to ANG was attenuated in rats, and under such conditions the BP lowering response to telmisartan was also reduced (Pieber et al., 1999). However, it remains unclear whether this in part also reflects the lower baseline BP in such animals.While not affecting resting BP, ARBs such as losartan and telmisartan, at least in high doses, can affect the BP response to tilting in rats, an animal model of orthostatic hypotension (Bedette et al., 2008); a study with eprosartan did not observe orthostatic hypotension (Ohlstein et al., 1992). Microinjection of renin into the rat nucleus tractus solitarii causes a depressor response, which can be attenuated by concomitant application of ARBs such as losartan or valsartan (Cheng et al., 2012)

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Another experimental condition where basal blood pressure is low is the pithed rat, which eliminates the central nervous contribution to BP control. In such animals candesartan, eprosartan, irbesartan, losartan, EXP3174 or valsartan had no effect on basal BP despite blocking pressor effects of endogenously administered ANG (Balt et al., 2001c; Christophe et al., 1995; Criscione et al., 1993) although some early studies reported losartan-induced BP lowering in this model (Wong et al., 1990b), specifically when given on top of propranolol (Zhang et al., 1993a).

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The above parts of section 2 demonstrate that ARBs as a class consistently lower BP in almost all hypertension models ever studied, with models with a markedly suppressed RAS being the notable exception. Many of the above studies have also included information which helps to clarify the underlying mechanisms of the BP-lowering effects of ARBs. As expected based on the known effects of ANG, the following overall actions of ARBs have have been identified (Figure 2): - Effects on vascular smooth muscle cells (VSMC; see section 3.1) - Effects on cardiovascular remodeling (see sections 3 and 4) including those on angiogenesis (Carbajo-Lozoya et al., 2012; Chaudagar & Mehta, 2014; NelissenVrancken et al., 1993; Nenicu et al., 2014; Soliman et al., 2014; Verhoest et al., 2014) - Modulation of mineralo- and glucocorticoid release from the adrenals (Andreis et al., 2000; Criscione et al., 1993; Lymperopoulos et al., 2014; Miura et al., 2014; Wong et al., 1990c) - Effects on renovascular and tubular function (see section 7) - Alterations of baroreflex function - Non-renal effects on circulating volume - Modulation of release of vasoactive neurotransmitters from sympathetic nerve endings - Effects on the central nervous system (see section 2.10.1.) <<>> The main underlying tissue mechanism of acute BP reduction by ARBs is vasodilatation by inhibiting the vasoconstrictive effect of endogenous ANG mostly mediated by the blockade of AT1R on VSMC. However, there is a range of other effects which may also contribute to BP lowering by ARBs, but their relative contribution remains to be clarified and may differ between experimental models; moreover, some of them are more relevant for chronic than acute BP lowering. The mechanistic complexity of ARB-induced BP lowering is illustrated by 24

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pharmacodynamic interaction studies. Thus, the anti-hypertensive ARB effects in SHR may be attenuated by NO synthase inhibition (Cachofeiro et al., 1992) or concomitant treatment with capsaicin (Harada et al., 2009) but not by a kinin antagonist (Cachofeiro et al., 1992) or by concomitant ibuprofen treatment (Pollock & Morsing, 1999); however, the specific mechanisms in many of those interactions have not been explored in detail and it remains unclear whether they indeed reflect pharmacodynamic interactions or just interference with independent pressure-relevant mechanisms. Therefore, we will focus on the above eight mechanisms. Some will be covered in the indicated subsequent sections of the manuscript, whereas the others will be discussed in this section, with a dedicated subsection for the central nervous effects. In most cases these effects are assumed to primarily occur by inhibition of endogenous ANG. However, a role for inverse agonism, i.e. inhibition of receptor function in the absence of endogenous agonist, cannot be excluded. Inverse agonism has been demonstrated e.g. for azilsartan (Miura et al., 2013; Ojima et al., 2011), the losartan metabolite EXP3174 (Feng et al., 2005; Miura et al., 2003; Noda et al., 1996), olmesartan (Kiya et al., 2010; Miura et al., 2006), telmisartan (Bhuiyan et al., 2009) and valsartan (Bhuiyan et al., 2009; Miura et al., 2008); whether the parent compound losartan (Bhuiyan et al., 2009; Feng et al., 2005; Miura et al., 2003) or candesartan (Feng et al., 2005; Miura et al., 2013) exhibit inverse agonism, has remained controversial. While inverse agonism is a clearly defined in vitro property of some ARBs (Michel et al., 2013) and the structural basis of inverse agonism at AT1R is emerging (Takezako et al., 2015), it remains to be determined whether it contributes in a relevant manner to their in vivo effects.

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The baroreflex is primarily important for moment-to-moment BP control, but by changing its sensitivity it can also become important in longer term regulation (Hainsworth, 1991). In an early study in conscious SHR, both losartan and lisinopril given i.v. significantly increased the maximum gain of the baroreceptor reflex control of nerve activity whereas the AT2R antagonist CGP 42112A did not (Kumagai et al., 1993). In another study, SHR with a myocardial infarction exhibited a lowered baroreflex gain, but chronic treatment with candesartan not only restored this but raised it to levels above those of non-infarcted control rats (Nishizawa et al., 1997). An improved baroreflex sensitivy was also shown in SHR upon treatment with azilsartan or candesartan (Isegawa et al., 2015; Sueta et al., 2013). Losartan also improved baroreflex sensitivity in a rat heart failure model (DiBona et al., 1998). While microinjection of candesartan into the nucleus tractus solitarius did not change BP in normotensive rats or SHR, it increased the baroreflex sensitivity in both strains (Matsumara et al., 1998). However, i.c.v. administration of candesartan did not affect the baroreflex in DahlIwai salt-resistant rats (Teruya et al., 1995). Losartan also increased the baroreflex sensitivity in conscious rabbits (Wong et al., 1993). The baroreflex sensitization by systemically given ARBs may be independent of BP lowering, as in one study in SHR chronic treatment with irbesartan produced less if any BP reduction as compared to atenolol, nifedipine or hydrochlorothiazide but nevertheless yielded a quantitatively similar baroreflex sensitization; based on these findings it was speculated that baroreflex sensitization along with a reduction of BP variability may contribute to end-organ protection by ARBs (Xie et al., 2006). Thus, ARBs as a class appear to increase the sensitivity of the baroreflex and thereby stabilize moment-to-moment BP control. The circulating volume reflects a balance between the intake of fluid salt and renal excretion as well as the osmotically active substances in plasma and tissues. It contributes to BP control, and changes thereof may be part of the anti-hypertensive mechanism of diuretics. The most important long-term effect of ARBs on circulating volume may result from their direct effects on tubular sodium reabsorption; this will be discussed in section 7.1. Additionally, it has been found that atrial natriuretic peptide can promote a fluid shift from the intravascular to an 25

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interstitial compartment; this was antagonized by acute administration of losartan or enaprilat (Valentin et al., 1993), indicating a role for endogenous ANG in keeping intravascular volume stable. In isolated beating rat atria losartan and, to a much greater extent, telmisartan promoted release of atrial natriuretic peptide; addition of the PPAR-γ antagonist GW9662 reduced the telmisartan response to that seen with losartan (Gao et al., 2014b). Acute infusion of telmisartan increased plasma levels of atrial natriuretic peptide by approximately 80% (Gao et al., 2014b). Another important role in the control of circulating volume is played by the hormone vasopressin. Central nervous administration of ANG causes a pressor response, which is partly mediated by peripheral V1 vasopressin receptors, indicating that it may involve modulation of vasopressin release from the pituitary; this vasopressin-dependent part of the pressor response was inhibited by central administration of losartan (Toney & Porter, 1993). More direct evidence comes from studies in which centrally applied ANG stimulated vasopressin release into the circulation, and ARBs including irbesartan, losartan and telmisartan given i.v. or orally inhibited this response (Culman et al., 1999; Gohlke et al., 2001). However, the RAS can also affect circulating volume from the input side as centrally administered ANG will increase drinking behavior and appetite for salt; peripherally administed ARBs inhibit this drinking behavior (Culman et al., 1999; Gohlke et al., 2001). Thus, ARBs acting on multiple levels can prevent ANG-induced increases in circulating volume, and this may contribute to their effects on BP.

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Another important player in BP control is the sympathetic nervous system, which affects vascular tone directly but also indirectly e.g. by promoting renin release or by reducing diuresis. Post-ganglionic sympathetic neurons express AT1R which couple to facilitation of the release of noradrenaline and its cotransmitters (Nap et al., 2003). Thus, various ARBs were reported to reduce the release of sympathetic transmitter including noradrenaline and ATP as evoked by electrical field stimulation in various in vitro preparations e.g. in rat mesenteric artery (Balt et al., 2001a), atrium (Shetty & DelGrande, 2000) and ventricle (Guimaraes et al., 2011), rabbit thoracic aorta (Nap et al., 2002) and mesenteric artery (Balt et al., 2002), guinea pig mesenteric artery (Onaka et al., 1997) or atrium (Brasch et al., 1993) or canine pulmonary artery (Guimaraes et al., 2011). In vivo locally administered irbesartan inhibited ANG-stimulated catecholamine secretion from canine adrenals (Martineau et al., 1995). Most in vivo studies on prejunctional AT1R were performed in pithed rats, where ANG enhances electrical stimulation-induced noradrenaline release. In such studies various ARBs including candesartan, eprosartan, irbesartan, losartan, telmisartan and valsartan inhibited the ANG effect (Balt et al., 2001b; Balt et al., 2001c; Criscione et al., 1993; Dendorfer et al., 2002). One study has suggested that eprosartan but not irbesartan, losartan or valsartan inhibited the stimulating effect of ANG on sympathetic transmitter release (Ohlstein et al., 1997), but the reason for this finding inconsistent with a range of other studies remain unclear. More limited data indicates that ARBs can block an inhibitory effect of ANG on acetylcholine release from parasympathetic nerve endings (Rechtman & Majewski, 1993), a finding which is conceptually difficult to understand and in need of confirmation. 2.10.1. Central nervous contributions to blood pressure control

The brain expresses AT1R (Speth & Karamyan, 2008) and administration of ANG directly into the brain by microinjection into defined nuclei or i.c.v. administration can elevate systemic BP; i.c.v. administration of losartan prevented such elevations (Culman et al., 1999; DePasquale et al., 1992; Gohlke et al., 2001; Toney & Porter, 1993). This centrally evoked BP elevation apparently has one component related to activation of the sympathetic nervous system and one related to pituitary vasopressin release (Toney & Porter, 1993). While this raises the possibility that ARBs may lower BP in hypertension via a central nervous 26

ACCEPTED MANUSCRIPT mechanism of action, the two emerging questions are whether centrally administered ARBs will lower BP in hypertensive animals in the absence of exogenous ANG administration and whether peripherally given ARBs, i.e. in a therapeutic setting, would mimic that.

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It has been reported that i.c.v. injection of ARBs such as candesartan and losartan can lower BP in SHR (Bunting & Widdop, 1995). However, in that study BP lowering was observed only in high doses which also inhibited responses to peripherally administered ANG, i.e. a spill-over to the periphery cannot be excluded. In another study i.c.v. losartan did not lower BP in SHR whereas oral losartan did (DePasquale et al., 1992). Injection of losartan into the anterior hypothalmus of salt sensitive SHRs reduced BP, but injection into the posterior hypothalmus had no effect (Yang et al., 1992b). In a follow-up study from the same investigators both losartan and EXP3174 injected into the anterior hypothalmic area reduced BP in SHR on a low or high-salt diet (Yang et al., 1992a). Microinjection of candesartan into the nucleus tractus solitarius did not reduce BP in SHR, but interestingly nevertheless sensitized the baroreflex in the injected rats (Matsumara et al., 1998). I.c.v. candesartan also did not lower BP in the L-NAME model whereas i.v. administration did (Liu et al., 1998). Taken together these data do not yield a conclusive picture, and the possibility exists that ARBs must reach very specific nuclei within the brain in sufficient concentrations to exhibit antihypertensive effects.

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Even if it is considered that ARB-induced BP lowering can involve a central nervous system, mechanism, this will only be relevant to the clinical situation with peripheral drug administration if the ARB penetrates into the central nervous system; however, the evidence in favor of this possibility remains controversial. Moreover, central effects of peripherally administered ARBs may at least partly result from mediators released in response to their peripheral vasodilating effects. While prescribing information for all clinically used ARBs states poor to absent brain penetration (Michel et al., 2013), several experimental studies have suggested otherwise. Such studies mostly are based on assessment of brain functions following peripheral ARB administration. Notably, negative studies in this regard have largely used single administration of relative low ARB doses and/or have been based on whole body autoradiography upon systemic administration of radioactive labeled ARBs which has limited sensitivity to detect brain penetration (Song et al., 1991; Wienen et al., 2000). More recent PET studies have demonstrated that peripherally injected [11C]-telmisartan can reach therapeutically relevant concentrations in the brain of monkeys and humans (Noda et al., 2012; Shimizu et al., 2012). Of note telmisartan appears to be the most lipophilic ARB (Michel et al., 2013), which means that brain penetration by other ARBs may be less. On the other hand, positive studies have typically used relatively high ARB doses (Gohlke et al., 2002; Muders et al., 2001) and/or extended treatment durations (Groth et al., 2003; Hosomi et al., 2005; Leenen & Yuan, 2001; Muders et al., 2001). The BP lowering effect of i.v. candesartan in SHR was accompanied by an increased release of the inhibitory neurotransmitters glycine and GABA in rostro-ventro-lateral medulla, and blockade of glycine or GABA receptors attenuates BP lowering effect of candesartan; this apparently did not occur secondary to peripherally mediated BP lowering as administration of nitroglycerin did not change glycine or GABA release (Yamada et al., 1995). Chronic systemic treatment with candesartan was found to prevent the BP-increasing effect of ANG microinjection into the rostro-ventro-lateral medulla in SHR (Tsuchihashi et al., 1999). Upon chronic dosing telmisartan also was directly detected in the cerebrospinal fluid of rats (Gohlke et al., 2001). Accordingly, chronic telmisartan treatment reduced oxidative stress in SHR brain (Huang et al., 2012). 27

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While these data do not provide unequivocal evidence for central AT1R-mediated antihypertensive effects of peripherally administered ARBs, it needs to be considered that under some conditions the blood-brain-barrier may become leaky, e.g. with ageing (Farrall & Wardlaw, 2009), in hypertension (Pelisch et al., 2011) or in diabetes (Min et al., 2012) which may lead to CNS involvement in the effects of ARBs which otherwise have only poor brain access.

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2.11. Hypertension conclusions

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In conclusion, ARBs as a class are effective antihypertensive drugs in all animal models of hypertension except those with a markedly suppressed RAS activity (Table 1). They lower BP in established hypertension and can prevent the development of hypertension, at least to some degree, even after discontinuation of treatment. This BP lowering contributes to beneficial effects on end-organ damage (see sections 3, 4 and 7) and, at least to some degree, translates into a reduced mortality. All ARBs appear to have a similar potential for BP reduction. However, based on their pharmacological properties the duration of BP lowering of a given dose and the doses required to reach a certain degree of BP lowering differ considerably between ARBs (Michel et al., 2013). While acute BP lowering apparently is predominantly mediated by direct effects on vascular smooth muscle, the clinically more relevant chronic antihypertensive effects probably involve additional indirect effects including those on renal function and cardiovascular remodeling. Even animal models which are unresponsive to ARBs due to a suppressed RAS in most cases exhibit BP lowering when ARBs are administered in conjunction with other classes of antihypertensive drugs such as diuretics or Ca2+ entry blockers. ARB doses too low to reduce BP and animal models of hypertension with a suppressed RAS have been important to dissect BP-dependent and –independent components of ARB effects in end-organ protection (see section 3, 4 and 7).

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Of note, ARB doses required for effective BP lowering in rats and some other species are several fold higher than in humans. This may reflect various factors: Firstly, there is limited evidence to suggest intrinsic species differences in sensitivity for BP-lowering effects of ARBs (Sato et al., 1997). Second, inter-species comparisons of dose-response curves are complicated by differences in bioavailability; inter-species difference in bioavailability have been reported for several ARBs (Michel et al., 2013). Third, animal studies typically have been performed with pure compound whereas clinical studies have been performed with formulated drug product. In many cases drug product formulation has been optimized to yield desired pharmacokinetic profiles and, accordingly, often yields different drug exposure and exposure over time as compared to administration of pure compound. Therefore, doses required for BP lowering in one species such as rat cannot be directly extrapolated to another species such as human. However, the observation that some ARB effects, particularly on endorgan damage, can be observed at doses yielding little BP lowering within a model and species demonstrates that some ARB effects occur largely independent of BP lowering. As discussed based on specific examples in subsequent sections, this conclusion is further supported by the finding that anti-hypertensive drugs not acting via modulation of the RAS do not mimic ARB effects when given in doses causing comparable BP lowering. Therefore, we consider it very likely that this concept also applies to humans, although possibly with different specific doses as compared to those in animal studies.

3. Vascular function and atherosclerosis Most blood vessels acutely react to ANG with vasoconstriction. However, ANG may also be involved in vascular remodeling with hypertrophy of the vessel wall as well as 28

ACCEPTED MANUSCRIPT atherosclerosis. As all of these interact to some degree, this section discusses ARB effects on blood vessel function in general, and the subdivision into multiple sections serves to clarify the data, not to imply that the various effects occur independent of each other.

Normal vascular function

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AT1R are abundantly expressed in the vasculature. While most studies have focused on those in VSMCs, AT1R are also found in the endothelium (Watanabe et al., 2005). The expression of VSMC AT1R at the protein level has been demonstrated by radioligand binding studies (Criscione et al., 1993; Dickinson et al., 1994; Jaiswal et al., 1993; Kubo et al., 1996; Leung et al., 1992; Liang & Leenen, 2008; Lyall et al., 1992; Sachinidis et al., 1993; Virone-Oddos et al., 1997; Viswanathan et al., 1992; Wilson et al., 1989). Detection of AT1R at the protein level by receptor antibodies appears less reliable because most available AT1R antibodies have been found to lack target-specificity (Adams et al., 2008; Benicky et al., 2012; Herrera et al., 2013) and resulting data can not necessarily be interpreted at face value.

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The primary pathway of AT1R signal transduction is the activation of phospholipase C to generate inositol phosphates accompanied by an elevation of intracellular Ca2+ concentrations. ANG-induced inositol phosphate formation in rat VSMC is blocked e.g. by losartan and EXP3174 (Dickinson et al., 1994; Ko et al., 1992; Lyall et al., 1992; Sachinidis et al., 1993) and Ca2+ elevations in rat, porcine or human VSMC e.g. by candesartan (Iversen & Arendshorst, 1999; Jaiswal et al., 1993; Koh et al., 1994), eprosartan (Edwards et al., 1992a), irbesartan (Fortuno et al., 1999; Herbert et al., 1994) or losartan and EXP3174 (Herbert et al., 1994; Ko et al., 1992; Leung et al., 1992; Sachinidis et al., 1993). Interestingly, intracellular microinjection of ANG can also increase free Ca2+ concentrations in VSMCs, and this can also be blocked by candesartan (Haller et al., 1999); while the physiological relevance of this observation remains unclear, recent evidence suggests that G-protein-coupled receptors may exist in a functional state in intracellular compartments after internalization (Irannejad et al., 2013). Candesartan and telmisartan were also reported to inhibit Ca2+ elevations in human umbilical artery endothelial cells (Ko et al., 1997) and murine endothelium-derived bEnd.3 cells (Mulders et al., 2006). In those cells ANG promoted endothelin-1 release, blocked by candesartan, which may enhance the overall vasoconstriction response; however, in vivo ANG infusion did not raise plasma endothelin-1 levels in hypertensive patients (Ferri et al., 1999). Secondary signal transduction events in the vasculature sensitive to ARBs include the release of prostaglandin I2 (prostacyclin), e.g. in pigs (Jaiswal et al., 1993; Leung et al., 1992), the stimulation of a Na/H-antiporter, e.g. in rat (Ko et al., 1992), the activation of the mitogenactivated protein kinases extracellular signal-regulated kinase (ERK) and p38 in rat VSMCs (Touyz et al., 2001a) or the cellular acidification rate as assessed in a Cytosensor microphysiometer (Dickinson et al., 1994). Secondary signal transduction in the endothelium, at least in some vessels, includes stimulation of NO release, which apparently is mediated by activation of sphingosine kinase (Mulders et al., 2006). In human coronary artery endothelial cells in culture an ARB was reported to increase endothelial NO synthase expression (Kurokawa et al., 2015). While such signal transduction primarily causes acute changes of vascular tone, it can also lead to ARB-sensitive enhanced proto-oncogene expression (Lyall et al., 1992; Sachinidis et al., 1993; Touyz et al., 2001a) and DNA synthesis and cell proliferation in rat and human VSMC (Flesch et al., 1995; Herbert et al., 1994; Koh et al., 1994; Sachinidis et al., 1993; Sung et al., 1994), which may be involved in the development of vascular hypertrophy and athersclerosis (see section 3.3.1). It may also lead to increased expression of pro-angiogenic factors (Kurokawa et al., 2015). However, it should be noted that at least in some preparations such as rat portal vein it has been questioned whether ANG 29

ACCEPTED MANUSCRIPT effects on prostaglandin or NO release contribute to its overall losartan-sensitive effect on vascular tone (Zhang et al., 1993c).

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All ARBs have been tested for their quantitative ability to inhibit acute ANG-induced vasoconstriction in vitro. This work has mostly been performed in isolated rabbit aorta (Bernhart et al., 1993; Buhlmayer et al., 1991; Cazaubon et al., 1993; Criscione et al., 1993; Dickinson et al., 1994; Edwards et al., 1992a; Inada et al., 1999; Jin et al., 1997; Keiser et al., 1995; Lin et al., 1992; Liu et al., 1992b; Liu, 1993; Morsing et al., 1999; Noda et al., 1993; Ojima et al., 1997; Ojima et al., 2011; Schambye et al., 1994; Shibouta et al., 1993; Tamura et al., 1997a; Wienen et al., 1992; Wienen et al., 1993; Wong et al., 1990c; Wong et al., 1990e). However, antagonism of ANG-induced contraction of isolated blood vessels was also shown in many other preparations including rabbit mesenteric artery (Balt et al., 2002), rabbit renal artery (Li et al., 2001; Zhang et al., 1994), rat aorta (Fortuno et al., 1999; Inada et al., 1994), rat coronaries (Tschudi & Lüscher, 1995), rat carotid artery (Inoue et al., 1999), rat portal vein (Morsing et al., 1999; Zhang et al., 1993c), guinea pig aorta (Hashimoto et al., 1997; Leung et al., 1993; Mizuno et al., 1995), hamster aorta (Inoue et al., 1999; Nakamura et al., 1994a), dog pulmonary artery (Guimaraes et al., 2011), pig and human coronary artery (Maassen van den Brink et al., 1999) and human gastroepiploic artery (Jin et al., 1997) and human subcutaneous microvessels (Garcha et al., 1999). Similar inhibition was also observed for vasoconstriction induced by angiotensin I (Brooks et al., 1992b; Inoue et al., 1999) or angiotensin(1-7) (Osei et al., 1993a). Of note, ANG effects on the endothelium may at least partly counteract those on the VSMC (Li et al., 2001; Mulders et al., 2006).

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Accordingly, ARBs consistently inhibited acute ANG-induced BP elevations in vivo. In contrast to the above studies with isolated blood vessels, in vivo assessment of AT1R antagonism in the vasculature has largely been done in rats (see below), but some studies have also been performed in mice (Bivalacqua et al., 1999), hamsters (Jin et al., 1997; Trippodo et al., 1995), dogs (Brooks et al., 1992b; Cazaubon et al., 1993; Christ et al., 1994; Hashimoto et al., 1997; Hayashi et al., 1997b; Ito et al., 1995; Kubo et al., 1993), cats (Champion et al., 1999a; Champion & Kadowitz, 1997; Lambert et al., 1998; Osei et al., 1993b), pigs (New et al., 2000) or monkeys (Cazaubon et al., 1993; Criscione et al., 1993; Roccon et al., 1994) (for human studies see below). This involved studies under a variety of experimental conditions, including conscious, anesthetized or pithed animals and with oral or i.v. ARB administration. Such inhibition has been reported for azilsartan (Kohara et al., 1996; Kusumoto et al., 2011), candesartan (Champion et al., 1998; Dendorfer et al., 2002; Kohara et al., 1996; Koike et al., 2001; Kubo et al., 1993; Maillard et al., 2002; Nakano et al., 1997; Shibouta et al., 1993; Wada et al., 1994; Wada et al., 1996a), eprosartan (Brooks et al., 1992b; Dendorfer et al., 2002; Edwards et al., 1992a; Wang & Brooks, 1992), irbesartan (Cazaubon et al., 1993; Christophe et al., 1995; Culman et al., 1999; Dendorfer et al., 2002; Lacour et al., 1994; Maillard et al., 2002; Schuijt et al., 1999), losartan (Almansa et al., 1997; Brooks et al., 1992b; Christophe et al., 1995; Culman et al., 1999; Dendorfer et al., 2002; Gorbea-Oppliger et al., 1994; Kohara et al., 1996; Koike et al., 2001; Mizuno et al., 1995; Moreau et al., 1993; Shibouta et al., 1993; Tamura et al., 1997a; Wong et al., 1990b; Wong et al., 1990d; Wong et al., 1990e; Wong et al., 1992), olmesartan (Koike et al., 2001; Kusumoto et al., 2011; Mizuno et al., 1995; Yanagisawa et al., 1996), telmisartan (Maillard et al., 2002; Vincent et al., 2005; Wienen et al., 1993) and valsartan (Criscione et al., 1993; New et al., 2000). Such in vivo antagonism studies were performed not only in healthy normotensive rats, but e.g. also with chronic oral eprosartan treatment in conscious 5/6 nephrectomy rats (Gandhi et al., 1999), with losartan in SHR (Wong et al., 1990d) or with eprosartan as inhibitor of vasoconstriction induced by a 60 min treatment with lipopolysaccharide in an in situ hamster cheek pouch preparation (Gao et al., 1995). In some cases they also were not limited to the inhibition of the 30

ACCEPTED MANUSCRIPT systemic BP response to ANG but e.g. with valsartan have also explored antagonism for specific vascular beds (see below).

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A large number of studies have also explored direct vascular ARB effects based on their ability to inhibit ANG-induced BP elevations in human subjects, in most cases healthy volunteers. For instance telmisartan increased basal flow and blocked ANG-induced vasoconstriction in the human forearm in situ (Schinzari et al., 2011). However, most studies in humans in vivo have looked at BP to represent overall vasoconstriction. Such studies were reported with most ARBs for instance with candesartan (Belz et al., 1997; Belz et al., 2000; Delacretaz et al., 1995; Fuchs et al., 2000; Gleiter et al., 2004; Malerczyk et al., 1998; Ogihara et al., 1995), irbesartan (Belz et al., 1999; Belz et al., 2000; Maillard et al., 1999; Mazzolai et al., 1999), losartan (Belz et al., 1997; Belz et al., 1999; Belz et al., 2000; Christen et al., 1991; Fuchs et al., 2000; Gleiter et al., 2004; Maillard et al., 1999; Mazzolai et al., 1999; Munafo et al., 1992), telmisartan (Belz et al., 2000; Stangier et al., 2001), and valsartan (Belz et al., 1999; Belz et al., 2000; Maillard et al., 1999; Mazzolai et al., 1999; Morgan et al., 1997; Müller et al., 1994). Most of these studies were based on single or repeated oral ARB administration and have assessed the degree of antagonism at multiple time points. Some of these studies have assessed inhibition of vasoconstriction in parallel with AT1R occupancy as assessed by ex vivo radioreceptor assays. This approach allows determining whether potential quantitative differences between drugs related to chosen drug doses being on different parts of the respective dose-response curve or to other factors. Indeed evidence for the latter possibility was found in a study in whch candesartan yielded a stronger inhibition of ANGinduced BP elevation than losartan for a given level of receptor occupancy, indicating greater affinity and/or slower off-rate from receptor (Belz et al., 1997; Fuchs et al., 2000).

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However, the vasoconstricting effects of ANG do not occur uniformly in all vascular beds. Thus, reductions in blood flow upon systemic administration of ANG have been observed in mesenteric artery (Heinemann et al., 1998), gastric mucosa (Heinemann et al., 1999), hindquarters (Champion et al., 1999b; Osei et al., 1993b; Widdop et al., 1993), kidney (Ruan et al., 1999; Widdop et al., 1993), lung (McMahon et al., 1992), isolated eye preparation (Meyer et al., 1995) or cerebral vessels in an open-skull in situ preparation (Vincent et al., 2005) in rats, mice, cats and/or pigs. On the other hand, ANG only minimally affected perfusion in femoral artery where blood flow even increased with ANG administration (Heinemann et al., 1998). Earlier studies with antisera, ACE inhibitors or peptidic AT1R antagonists had confirmed this concept of non-uniform ANG effects on the vasculature. Correspondingly, ARBs including candesartan, losartan, telmisartan and valsartan increased blood flow when given alone and/or reversed the effects of ANG in gastric mucosa (Heinemann et al., 1999), hindquarters (Osei et al., 1993b; Widdop et al., 1993), kidney (Kanagawa et al., 1997; Ruan et al., 1999; Widdop et al., 1993), pulmonary circulation (McMahon et al., 1992) or isolated eye (Meyer et al., 1995) but had less consistent effects on splanchnic blood flow (Kanagawa et al., 1997) and lacked effects on blood flow to the brain, heart, adrenals, skin or skeletal muscle (Kanagawa et al., 1997). Of note biphasic pressor responses to ANG have been observed with an early losartan-sensitive increase followed by a later AT2R-mediated decrease (Scheuer & Perrone, 1993). Losartan also enhanced adenosine receptor-mediated vasodilatation in some vascular beds (Smits et al., 1993). All of these studies, however, exhibit some bias as they only reported on the vascular beds on which probes had been placed. A less biased approach has been applied in a study based on deposition of radioactive microspheres (Schuijt et al., 1999). This found that the ANGinduced decrease in systemic vascular conductance was caused by reduced regional conductances in the gastrointestinal tract (52%), kidney (63%), skeletal muscle (39%), skin 31

ACCEPTED MANUSCRIPT (63%), mesentery and pancreas (32%), adrenal (27%) and spleen (57%); irbesartan increased baseline vascular conductance in adrenal, brain and kidney, and reversed all ANG responses.

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Some ARBs, notably irbesartan, telmisartan and, in high concentrations, losartan can also inhibit vasoconstriction mediated by thromboxane A2 receptors, an effect not shared by other ARBs such as candesartan and apparently being mediated by a direct molecular interaction with TXA2 receptors (Asano et al., 2006; Fukuhara et al., 2001; Li et al., 2000; Monton et al., 2000). The degree to which this contributes to their effects on BP remains to be assessed. Similarly, irbesartan and losartan but not candesartan or valsartan also inhibited thromboxane A2-induced aggregation of human platelets (Li et al., 2000).

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Some ARBs are partial agonists at PPAR-γ (Michel et al., 2013), and PPAR-γ full agonists such as pioglitazone and rosiglitazone can cause vasodilatation in several vascular preparations (Salomone & Drago, 2012) including human pulmonary artery (Kozlowska et al., 2013). These effects may at least partly occur at the level of the endothelium (Balakumar & Kathuria, 2012). Therefore, it has been studied whether PPAR-γ-activating ARBs may at least in part cause vasodilatation via this mechanism. In mouse mesenteric vessels telmisartan inhibited vasoconstriction by RAS-independent agonists, and this inhibition was apparently NO-mediated; such inhibition was blocked by GW9662 and absent in PPAR-γ knock-out mice (Yuen et al., 2011). In rat femoral arteries telmisartan but not losartan reduced phenylephrine vasoconstriction and enhanced acetylcholine induced vasodilatation, and the telmisartan effects were blocked by GW9662 (Siarkos et al., 2011). However, it remains unclear whether these examples are representative for the overall vasculature as GW9662 did not attenuate the BP lowering effect by telmisartan in rats (Nagashima et al., 2012). Moreover, telmisartan affected mitochondrial function in a manner not shared by eprosartan; however, pioglitazone had opposite effects and the effects were maintained in VSMC of PPAR-γ knock-out mice (Takeuchi et al., 2013).

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The vascular effects of ANG in VSMCs and endothelium can be modulated by those on noradrenaline release from sympathetic nerve endings mediated by AT1R, as shown e.g. in rat mesenteric artery (Balt et al., 2001a), rabbit thoracic aorta (Nap et al., 2002) and mesenteric artery (Balt et al., 2002), guinea pig mesenteric artery (Onaka et al., 1997) or canine pulmonary artery (Guimaraes et al., 2011). Depending on the vascular bed this may result in α1-adrenoceptor-mediated vasoconstriction or β-adrenoceptor-mediated vasodilatation. Vasoconstriction can be inhibited by the presence of perivascular adipose tissue, and such inhibition diminishes under hypoxic conditions. Co-incubation of isolated segments of rat mesenteric artery with either telmisartan or captopril prevented the effects of hypoxia (Rosei et al., 2015). In conclusion, ANG can acutely affect vascular tone by acting on AT1Rs in VSMCs, endothelium and sympathetic nerve endings and AT2R in VSMCs. In most vascular beds under most conditions the AT1R VSMCs effect dominates, yielding vasoconstriction as the net response. However, stimulation of AT2R in VSMCs and AT1Rs in endothelium can attenuate the vasoconstriction response to some degree but in most cases contribute to the total vascular response in a minor way only. While ARBs can inhibit the ANG effects on VSMC and endothelium, their net effect almost always is vasodilatation as demonstrated in a large variety of isolated blood vessel preparations and vascular beds in vivo. Such inhibition was demonstrated in many species including humans and, in experimental animals, under a variety of experimental conditions. ARBs also can prevent agonist-induced desensitization of vascular responses (Robertson et al., 1994). While these data demonstrate the ability of ARBs 32

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to inhibit acute ANG-induced vasoconstriction, this does not necessarily mean that ARBs will cause vasodilatation in every blood vessel under all circumstances. While ARBs as a class inhibit ANG-induced vasoconstriction and cause vasodilatations, some ARBs can additionally cause vasodilatation by inhibiting thromboxane A2 and/or stimulating PPAR-γ receptors. While it remains to be investigated whether and to which degree relative to AT1R antagonism this contributes to BP lowering, the overall similarity in BP lowering potential between ARBs makes a major contribution of these additional mechanisms to overall BP lowering unlikely. Functional or morphological damage to the endothelium may shift the balance between vasoconstricting AT1R on VSMCs and vasodilating AT1R on endothelium and lead to a greater vasoconstriction in response to ANG (see below).

Altered endothelial function

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The endothelium is a physiological modulator of VSMC function. By release of NO and other factors it can cause vasodilatation, but it can also release mediators which cause VSMC contraction. While the release of vasodilating factors dominates in healthy endothelium in most cases, endothelial dysfunction can develop in pathological settings leading to a more pronounced vasoconstriction response. Moreover, endothelial dysfunction can be a step towards development of atherosclerosis. Endothelial function is typically assessed in vitro by determining the sensitivity to a muscarinic receptor agonist such as acetylcholine to induce relaxation of vascular preparations in an organ bath. Endothelial dysfunction can be observed in multiple models of hypertension and various other pathophysiological states. For example, acetylcholine-induced vasodilatation was markedly reduced in aortic rings from 30 week old SHR, but this was largely restored in SHR which had been treated with irbesartan from week 14-30 (Figure 3) (Zalba et al., 2001). Similar restoration of endothelial function was observed e.g. in small mesenteric arteries from SHR upon treatment with candesartan (Rizzoni et al., 1998), from obese SHR with azilsartan (Hye Khan et al., 2014a) or from obese SHRSP.ZLeprfa/IzmDmcr rats with telmisartan (Kagota et al., 2014). The endothelial dysfunction in SHR can be further worsened by ovariectomy, and irbesartan treatment restored it to a similar degree as estrogen replacement (Wassmann et al., 2001). The endothelial dysfunction in obese Zucker rats was fully restored by treatment with azilsartan (Hye Khan et al., 2014b) and at least partially restored upon treatment with azilsartan or candesartan in KK-Ay mice (Matsumoto et al., 2014). Endothelial dysfunction can also be induced by chronic arsenite exposure in rats and was also improved by telmisartan treatment (Nirwane et al., 2015). <<>> Two lines of evidence support the idea that ARB-induced normalization of endothelial function is not primarily mediated by BP reduction. Firstly, when given at doses causing similar BP reduction in obese SHR, telmisartan but not amlodipine or moxonidine restored acetylcholine-induced vasodilatation (Kagota et al., 2007). In Dahl S rats on a high salt diet a 2-week treatment with telmisartan or hydralazine lowered BP to a similar extent, but only telmisartan restored acetylcholine-induced relaxation of aortic rings (Satoh et al., 2010a). In another model with a suppressed RAS, DOCA-treated rats, telmisartan also improved endothelial function in aortic rings (Sharma & Singh, 2012). Thus, ARBs improved endothelial dysfunction in several hypertensive settings where effects were not explained by those on BP. Secondly, telmisartan and ramipril similarly improved endothelial function in ApoE knock-out mice on a high cholesterol diet, which are not hypertensive, (Schlimmer et al., 2011). Telmisartan also improved endothelial function in a normotensive, STZ-based rat model of type 1 diabetes, along with a reduction of reactive oxygen species (Wenzel et al., 2008); this improvement occurred similarly upon treatment with a telmisartan dose, which did 33

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not lower BP, and with a telmisartan-amlodipine combination treatment, which lowered BP (Mollnau et al., 2013). Improved endothelial function upon telmisartan but not losartan treatment was also reported in ApoE knock-out mice (Nakagami et al., 2008); however, another study reported that telmisartan and ramipril were similarly effective in restoring endothelial function in this model (Schlimmer et al., 2011). In Watanabe hyperlipidemic rabbits, another hereditary atherosclerosis model, telmisartan increased acetylcholine-induced NO release, a surrogate marker of endothelial function; telmisartan plus GW9662 or candesartan were less effective than telmisartan, suggesting a possible involvement of both AT1Rs and PPAR-γ (Ikejima et al., 2008). Some improvement of this surrogate marker was also observed with valsartan, and this was further enhanced by combination with pitavastatin (Imanishi et al., 2008a) or aliskiren (Imanishi et al., 2008b). In the same model irbesartan also improved endothelial function (Oelze et al., 2000). Thus, ARBs also improved endothelial dysfunction in non-hypertensive models. A beneficial effect of ARBs as a class on endothelial function is also supported by various clinical studies, in which ARBs and ACE inhibitors improved endothelial function to a greater extent not only as compared to placebo but also as compared to calcium channel blockers or β-adrenoceptor antagonists (Benndorf et al., 2007; Shahin et al., 2011), highlighting the role of ANG in endothelial pathophysiology and RAS inhibition in its treatment.

3.3.

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Endothelial dysfunction can be induced experimentally by treatment with nitroglycerin for several days; in this model telmisartan treatment prevented endothelial dysfunction and partially improved nitrate tolerance (Knorr et al., 2011). In a normotensive rat model with subtotal nephrectomy it has been found that telmisartan improves endothelial function in aortic rings, an effect blunted by GW9662 and only partly mimicked by losartan; this has led the investigators conclude that the improvement of endothelial function may be mediated by PPAR-γ (Toba et al., 2012). On the other hand, telmisartan also improved acetylcholineinduced dilatation of mesenteric vessels in SHRSP.Z Leprfa/IzmDmcr rats, a model of metabolic syndrome, but this was not mimicked by the PPAR-γ full agonist pioglitazone (Kagota et al., 2009; Kagota et al., 2011). Moreover, improved of endothelial function in several other models by e.g. candesartan or irbesartan (see above) also argues against a major role of PPAR-γ in this effect.

Vascular remodeling and atherosclerosis

During aging and in the context of various pathologies, most importantly arterial hypertension, the vasculature undergoes functional and morphological changes. These mainly involve vascular hypertrophy, which represents a relative or even absolute increase in the thickness of the arterial media leading to an increased media/lumen ratio, also referred to as inward remodeling. Long-standing hypertension as well as metabolic disease may also induce atherosclerosis. This occurs mainly in large and medium arteries; it leads to multiple types of cardiovascular disease and ultimately premature death. Following early phases of endothelial dysfunction, fatty streaks with macrophage and T-lymphocyte invasion develop, leading to intermediate to advanced lesions, narrowing of the vascular lumen and thrombosis (Ross, 1999). The development of atherosclerotic lesions, often accompanied by hypertrophy of vascular smooth muscle, involves a range of cell types; accordingly, several cell types in culture have been used as in vitro models of athersclerosis development. In vivo a large variety of models, largely related to hypertension and metabolic disease, have been used to study vascular remodelling and athersclerosis and its possible treatment. Interpretation of studies on vascular remodeling and atherosclerosis is hampered by the fact that investigators have used different pharmacological probes. Differing effects are 34

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preferentially interpreted to reflect different mechanisms induced by different mechanisms inherent to the molecules used. This makes comparisons between studies risky as they have used different experimental models, experimental conditions and/or probes with different pharmacokinetic profiles, particularly in a very complex setting such as atherosclerosis. This limitation should be kept in mind in the interpretation of the subsequent data.

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Although the development atherosclerosis is a complex process involving the interaction of many cell types in the circulation and the vessel wall, various in vitro models, largely based on culturing of one specific cell type, have been used to allow a better molecular understanding of effects in specific cell types. These include endothelial cells, VSMCs and inflammatory cells such as macrophages, monocytes and other white blood cells. ARB effects on adipocytes, which can also contribute to atherosclerosis via inflammatory mediators, will be covered in section 6.1. 3.3.1.1. Endothelial cells

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The endothelium not only plays an important role in the acute regulation of vascular tone (see section 3.1) but also is involved in the migration of inflammatory cells to the vascular wall (Ross, 1999) and in vascular development (Coultas et al., 2005); endothelial cell proliferation is a relevant step in the development of intima hyperplasia. Alterations of endothelial function may affect the permeability of large molecules from the lumen to the vascular wall. ANG can play an important role in these processes, but the effects of some ARBs on the endothelium may additionally involve PPAR-γ and are in line with those of selective PPAR-γ agonists (Cheang et al., 2015). Human umbilical vein endothelial cells (HUVECs) are a frequently used model of human endothelium.

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Atherosclerotic plaques primarily develop in arterial regions exposed to non-uniform shear stress, and are initiated by leukocyte-endothelial interactions (Ross, 1999). In isolated rabbit jugular vein segments, deformation induced expression of pre-pro-endothelin-1 and endothelin B receptor, which was inhibited by candesartan and irbesartan (Lauth et al., 2001). In the same study, such expression was also inhibited by irbesartan or ramiprilat in cultured porcine aortic endothelium. In HUVECs, exposure to non-uniform shear stress combined with TNF-α induced monocyte adherence, which was concentration-dependently inhibited by telmisartan (Cicha et al., 2011); this inhibition involved a reduced expression of the adhesion molecule VCAM-1 and was blocked by GW9662. Other studies in HUVECs, based on TNF-α stimulation alone, confirmed the inhibitory effect of telmisartan on vascular cell adhesion molecule (VCAM) 1 and also NF-κB expression; while this was not mimicked by losartan, EXP3174 or EXP3179, it also was not blocked by GW9662 (Cianchetti et al., 2008; Nakano et al., 2009). However, as part of these studies both losartan and telmisartan inhibited ANGinduced cell death (Cianchetti et al., 2008). Telmisartan also inhibited the tumor necrosis factor (TNF) α-induced expression of a secretory phospholipase A2 in HUVECs (Sonoki et al., 2012); while ANG did not induce this phospholipase, the PPAR-γ inhibitor GW3335 did not block it, indicating that the effect may be independent of both AT1Rs and PPAR-γ. Attenuation of the homocysteine-induced increased monocyte chemoattractant protein (MCP) 1 and VCAM-1 expression in HUVECs was attenuated by telmisartan; a PPAR-δ antagonist attenuated this and a PPAR-δ agonist mimicked the telmisartan response (Xu et al., 2014). In bovine retinal capillary cells asymmetric dimethylarginine, a NO synthase inhibitor, increased intercellular adhesion molecule-1 expression at the mRNA and protein level; at the mRNA level this was inhibited by both telmisartan and benazepril, indicating mediation via AT1Rs, 35

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whereas at the protein level only telmisartan yielded significant inhibition (Chen et al., 2011). While all of these studies agree that telmisartan can inhibit intercellular adhesion molecule-1 expression, they are equivocal with regard to the involvement of AT1Rs, PPAR-γ and, yet to be defined, other mechanisms. In cultured HUVECs telmisartan was also reported to downregulate zona occludens-1 mRNA and protein levels, disrupt its continuous pericellular distribution and increased their permeability; this effect was not mimicked by valsartan but blocked by GW9662 (Bian et al., 2009). Telmisartan also concentration-dependently prevented HUVEC apoptosis induced by the glucose metabolite methylglyoxal (Baden et al., 2008) and ANG-stimulated expression of matrix metalloproteinase-2 in HUVECs (Sato et al., 2014c). Telmisartan but not irbesartan or valsartan increased phosphorylation of endothelial NO synthase in HUVECs (Myojo et al., 2014). As part of the same study it was shown that in vivo treatment of mice with telmisartan resulted in increased phosphorylation of endothelial NO synthase in the aorta. Accordingly, endothelial dysfunction induced by ANG in isolated rabbit aortic rings was prevented by telmisartan (Hu et al., 2014).

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While NO production was stimulated by ANG in some types of endothelial cells (Mulders et al., 2006), in cultured bovine aortic endothelial cells valsartan stimulated NO formation (Su et al., 2009). The latter was related to an increased phosphorylation but not expression of endothelial NO synthase. Both telmisartan and benazepril attenuated endothelial cell proliferation induced by asymmetric dimethylarginine (Chen et al., 2011). In bovine aortic endothelial cells, both azilsartan and valsartan concentration-dependently inhibited proliferation in the absence of exogenous ANG but did not activate PPAR-γ (Kajiya et al., 2011). Losartan reduced intimal hypertrophy in an organ culture model of human saphenous vein (Varty et al., 1994). Detailed molecular studies on the mechanisms involved in endothelial cell proliferation, sprouting and neo-angiogenesis were also performed in primary bovine retinal and immortalized rat microvascular endothelial cells as well as in a mouse model of oxygen-induced proliferative retinopathy, where telmisartan and the AT2R antagonist PD 123319 had opposite effects which involved both Gi and G12/13 G-proteins and rho kinase (Carbajo-Lozoya et al., 2012). On the other hand, telmisartan, but neither losartan, valsartan nor the Ca2+-entry blocker amlodipine, also inhibited HUVEC proliferation and prevented their entry into the S-phase of the cell cycle (Siragusa & Sessa, 2013). This was linked to a specific change in gene expression as detected by microarray analysis, reduced phosphorylation of Akt and glycogen synthase-3β leading to p53 accumulation in the nucleus and rapid turnover of cyclin D1; on the other hand, telmisartan down-regulated pro-apoptotic genes and protected HUVECs from serum starvation-induced apoptosis. While not necessarily directly linked to atherosclerosis development, advanced glycosylation end products (AGE) and their receptor RAGE play a central role in the development of diabetic vascular complications. In human microvascular endothelial cells telmisartan inhibited RAGE expression, and in a clinical study also reduced circulating RAGE in human plasma (Nakamura et al., 2005). Inhibition of RAGE expression in human endothelial cells was confirmed in a later study and shown to be blocked by GW9662 (Yamagishi et al., 2008). Endothelial progenitor cells are a circulating pool of cells, which contribute to endothelial replenishment. They express both AT1R and AT2R (Endtmann et al., 2011), and have been used as a model system to study ARB effects. Telmisartan stimulated human endothelial progenitor cells proliferation in vitro, and inhibited TNF-α-induced apoptosis of such cells in a GW9662-sensitive manner (Cao et al., 2014; Honda et al., 2009). This was confirmed by other investigators, who also demonstrated that telmisartan also increased the number of such cells in bone marrow and peripheral blood in mice in vivo (Steinmetz et al., 2010). An in vivo increase in endothelial progenitor cells in circulating blood has also been reported with 36

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telmisartan in SHR (Yoo et al., 2015). In human endothelial progenitor cells ANG induced expression of NADPH oxidase and promoted senescence, and telmisartan inhibited such ANG effects (Li et al., 2011). In another study ANG induced apoptosis of human endothelial progenitor cells through a pathway involving c-Jun N-terminal kinase and p38 mitogenactivated protein kinase, decreased Bcl-2 and increased Bax, and activation of caspase 3; irbesartan inhibited those effects (Endtmann et al., 2011). Within the same study ANG infusion reduced the number of endothelial progenitor cells in wild-type but not AT1aR knock-out mice, whereas telmisartan treatment of patients with stable coronary artery disease increased circulating progenitor cell number. However, using rat bone marrow-derived endothelial progenitor cells, ANG was reported to inhibit apoptosis and to enhance NO release and adhesion potential, an effect being blocked by valsartan (Yin et al., 2008). Circulating endothelial progenitor cell numbers have been reported to be markedly increased in mice with renovascular hypertension, a phenomenon that could be mimicked by ANG administration; treatment with telmisartan, quinapril and hydralazine similarly reduced these circulating progenitor cells (Salguero et al., 2008), indicating an effect secondary to BP lowering.

3.3.1.2. VSMC

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Culturing for many passages can induce cellular senescence in endothelial cells. This was concentration-dependently reversed by telmisartan, an effect blocked by GW9662 or PPAR-γ siRNA, but not affected by co-incubation with ANG and not mimicked by eprosartan (Scalera et al., 2008). Taken together these data show a beneficial effect of AT1R inhibition on endothelium proliferation and endothelial progenitor cell number; telmisartan may have additional, PPAR-γ-mediated effects on these cells but the relative roles of AT1R-mediated and AT1R-independent effects remain unclear. Accordingly, a recent meta-analysis has indicated that ARBs as a class improve endothelial function in patients as assessed by flowmediated vasodilatation (Li et al., 2014b).

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While isolated and cultured VSMCs have frequently been used as in vitro models of vascular hypertrophy and atherosclerosis, it is important to realize that AT1R expression can decline to non-detectable levels after several passages in culture (Ko et al., 1997), a phenomenon that also applies to receptors for other pro-contractile mediators in other types of smooth muscle (Arrighi et al., 2013) and apparently represents a re-differentiation of smooth muscle cells in culture from a contractile to a synthetic phenotype (Hendriks-Balk et al., 2008). Such AT1R down-regulation after several passages in culture implies that the relative roles of AT1Rindependent ARB effects may be overestimated in late passages. With this caveat in mind inhibition of ANG-induced hypertrophy and/or proliferation has been shown in cultured VSMCs from multiple species including rats (Flesch et al., 1995; Fukuda et al., 1999; Graf et al., 1997; Itazaki et al., 1995; Ko et al., 1992; Koh et al., 1994; Kubo et al., 1996; Millet et al., 1992; Muniz et al., 2001; Sachinidis et al., 1992; Sachinidis et al., 1993; Sachinidis et al., 1996a; Sachinidis et al., 1996b; Sung et al., 1994; Touyz et al., 2001b; Virone-Oddos et al., 1997) and humans (Herbert et al., 1994; Mueck et al., 1999; Wang et al., 2012a). Its antagonism has been demonstrated with multiple ARBs including candesartan and its prodrug (Flesch et al., 1995; Fukuda et al., 1999; Itazaki et al., 1995; Koh et al., 1994; Kubo et al., 1996; Sachinidis et al., 1996a; Sachinidis et al., 1996b), eprosartan (Sung et al., 1994), irbesartan (Graf et al., 1997; Herbert et al., 1994; Muniz et al., 2001; Touyz et al., 2001b; Xi et al., 1999), losartan and its active metabolite EXP3174 (Herbert et al., 1994; Itazaki et al., 1995; Ko et al., 1992; Millet et al., 1992; Sachinidis et al., 1992; Sachinidis et al., 1993; Sung et al., 1994; Virone-Oddos et al., 1997), telmisartan (Wang et al., 2012a) and valsartan (Mueck et al., 1999; Wang et al., 2012a). However, a study in isolated rat aorta found that 37

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ANG stimulated protein but not DNA synthesis in a losartan-sensitive manner (Holycross et al., 1993), questioning whether the proliferation-enhancing effects observed with cultured VSMC also occur in situ. Moreover, it should be noted that the overall in vitro effects of ANG on vascular hypertrophy are rather limited, potentially due to lack of exposure to co-factors present in vivo (see next paragraph). However, direct peri-vascular ANG application to normotensive rat carotid arteries for 14 days induced dose-dependent adventitia thickening with increased DNA synthesis, neovascularization and collagen disposition; proliferation and collagen disposition was blocked by valsartan, whereas the observed neovascularization may be AT2R-mediated (Scheidegger & Wood, 1997). Perhaps independent of proliferation, ARBs may also affect the inflammatory part of atherosclerosis. Thus, in rat aortic VSMCs, telmisartan concentration-dependently reduced TNF-α-induced interleukin (IL) 6 expression; this was prevented by GW9662 and not mimicked by valsartan (Tian et al., 2009), suggesting PPARγ involvement.

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Of note, ANG can not only promote VSMC hypertrophy and proliferation when given alone, but may have synergistic effects when administered in concert with other mediators such as prostaglandins (Sachinidis et al., 1996b) or platelet-derived growth actor or epidermal growth factor (Sachinidis et al., 1996a); these synergistic effects were also sensitive to ARB treatment. On the other hand, it has remained controversial whether ARBs will affect serumstimulated VSMC proliferation, as this was inhibited by telmisartan (apparently not PPAR-γmediated) (Wang et al., 2012a), whereas inhibition by candesartan was found in one study (Kubo et al., 1996) but not in two others (Mueck et al., 1999; Wang et al., 2012a). The basal VSMC proliferation, i.e. in the absence of serum, was reported to be sensitive to candesartan in cells isolated from SHR but not those from WKY (Fukuda et al., 1999; Kubo et al., 1996). This may relate to the observation that SHR VSMCs express more angiotensinogen and ACE than those from WKY and exhibit a greater ANG release (Fukuda et al., 1999). In another study with human VSMCs, basal proliferation was inhibited by telmisartan but not by eprosartan, irbesartan or valsartan (Yamamoto et al., 2009b). Within the same study telmisartan, but not valsartan, similarly inhibited basal proliferation of VSMCs obtained from wild-type and PPAR-γ knock-out mice. Similarly, telmisartan but not candesartan, eprosartan or irbesartan concentration-dependently inhibited proliferation of rat VSMCs in the presence or absence of platelet-derived growth factor/insulin, but nonetheless this apparently was not PPAR-γ-related (Benson et al., 2008). Taken together these data indicate that ARBs can inhibit VSMC proliferation involving both AT1R and, for some ARBs, AT1R-unrelated but not PPAR-γ-mediated effects. ANG also stimulates adhesion of VSMC to collagen fibronectin in irbesartan-sensitive manner; while this β1-integrin dependent, ANG itself did not change integrin expression (Kappert et al., 2000). Mechanistically, the anti-proliferative effect of ARBs appears to involve an inhibition of ANG-induced elevations of intracellular Ca2+-concentrations (Herbert et al., 1994; Ko et al., 1997; Koh et al., 1994; Sakurada et al., 2013), activation of ERK (Graf et al., 1997; Kajiya et al., 2011; Sung et al., 1994; Touyz et al., 2001a; Touyz et al., 2001b; Xi et al., 1999; Zhong et al., 2011) and other members of the mitogen-activated protein kinase family (Touyz et al., 2001a; Touyz et al., 2001b; Zhong et al., 2011), tyrosine phosphorylation of focal adhesion molecules such as paxilin (Okuda et al., 1995), increased expression of c-fos and c-myc (Hirata et al., 1994; Lyall et al., 1992; Millet et al., 1992; Xi et al., 1999), reduction of NO synthase (Nakayama et al., 1994) and endothelin-1 expression (Sung et al., 1994). The ANGstimulated migration of VSMCs was also inhibited (Graf et al., 1997; Xi et al., 1999). Such findings have not only been obtained in VSMC from the general circulation but also from specialized vessels, e.g. from the porcine ciliary artery (Dubey et al., 1998); in this preparation ANG had only a low potency in stimulating proliferation; the potency of ANG to 38

ACCEPTED MANUSCRIPT promote proliferation was increased in the presence of insulin, whereas its potency in stimulating VSMC migration was independent of insulin. Moreover, as reported from murine aortic VSMCs, telmisartan may inhibit autophagy, possibly via a PPAR-γ pathway (Li et al., 2015).

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3.3.1.3. Macrophages, monocytes and other white blood cells

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The invasion of the vascular wall by inflammatory cells including macrophages and monocytes is an important step in the development of atherosclerotic lesions (Ross, 1999). Therefore, ARB effects have also been tested in isolated white blood cells. Circulating human monocytes typically express little AT1R mRNA or protein, and ARBs do not modify cytokine release from such cells; this expression pattern is not affected by exposure to ANG or lipopolysaccharide (Larrayoz et al., 2009; Pang et al., 2012). Nevertheless, candesartan and telmisartan were reported to inhibit the lipopolysaccharide-induced expression and secretion of cytokines such as TNFα, IL-1β and IL-6, whereas losartan had a smaller effect (Larrayoz et al., 2009; Pang et al., 2012). Inhibition of lipopolysaccharide-induced expression of TNFα and MCP-1 by telmisartan but not losartan, olmesartan or valsartan were also reported by others, and in that study the telmisartan effects were abrogated by small interfering RNA (siRNA) against PPAR-γ (Matsumura et al., 2011). Telmisartan also inhibited lipopolysaccharide-induced IL-6 and MCP-1 expression in the monocyte-derived THP1 cell line (Konda et al., 2014). Additionally, telmisartan was shown to concentration-dependently inhibit lipopolysaccharide-induced ERK and p38 phosphorylation, expression of PPAR-γ target genes NF-κb, CD36 and ABCG1, expression of cyclooxygenase-2 expression and prostaglandin E2 release, release of reactive oxygenase species, reduction of IκB expression, and monocyte migration (Pang et al., 2012). PPAR-γ silencing by corresponding siRNA abolished the effects of telmisartan on CD36 and ABCG1 expression as well as the inhibitory effects on IκB and TNF-α expression. The effect of telmisartan, and to a lesser extent of irbesartan, on CD36 expression in human monocytes has also been shown by other investigators (Kappert et al., 2009). Given the lack of AT1R expression in human monocytes (Larrayoz et al., 2009; Pang et al., 2012), the direct effects of candesartan, irbesartan and telmisartan on PPAR-γ (Erbe et al., 2006; Storka et al., 2008), and the attenuation of telmisartan effects by PPAR-γ gene silencing (Pang et al., 2012), it appears that the antiinflammatory effects of these ARBs in human monocytes are not mediated by AT1R but rather by PPAR-γ. In line with these in vitro effects, treatment of hypertensive patients with telmisartan was reported to lower the enhanced adhesiveness of monocytes obtained from such patients to human endothelial cells, which occurred largely independent of chemokine receptor expression (Syrbe et al., 2007). Monocytes and the human monocyte cell line THP1 can differentiate into macrophages or macrophage-like cells, respectively, and in contrast to monocytes, these cells may become responsive to ANG. Thus, ANG stimulated peroxide formation in differentiated THP1 cells and this was inhibited by candesartan (Yanagitani et al., 1999). Similarly, other investigators reported that ANG caused down-regulation of ABCA1 but not and ABCG1 (both considered as PPAR-γ target genes) in differentiated THP1 cells; these ANG effects were abolished by by telmisartan and valsartan, whereas an AT2R antagonist or GW9662 remained without effect, indicating mediation by AT1R (Chen et al., 2012a). These transporters are relevant to atherosclerosis because they mediate cholesterol efflux from macrophages. Telmisartan also increased the expression of ABCA1, ABCG1 and SR-B1 in differentiated THP1 cells in the absence of exogenous ANG, an effect that was suppressed by PPAR-γ knock-down (Nakaya et al., 2007). On the other hand, in murine peritoneal macrophages telmisartan increased PPAR-γ activity, whereas losartan, olmesartan or valsartan did not (Matsumura et al., 2011). 39

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In that study telmisartan induced CD36 and ABCA1 and ABCG1 expression and suppressed macrophage proliferation induced by oxidized LDL, effects which were abrogated by PPAR-γ gene silencing. Whether these differences regarding a role of PPAR-γ in ARB effects on the regulation of macrophage function represent those between species and/or those between primary cells and cell lines remains to be explored. Raw264.7 cells are another macrophagederived cell line; telmisartan reduced the inflammatory response to lipopolysaccharide administration (Balaji & Ramanathan, 2014).

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In isolated human lymphocytes telmisartan concentration-dependently inhibited expression of the β2-integrin MAC-1, irrespective of the presence of ANG, indicating an AT1Rindependent effect; in a placebo-controlled in vivo study in patients with hypertension and coronary artery disease, telmisartan also lowered MAC-1 expression, whereas that of Creactive protein, soluble intercellular adhesion molecule-1 and IL-6 were not affected (Link et al., 2006). Migration of CD4+ lymphocytes into the vessel wall is also an early step in the development of atherosclerosis. Migration of such lymphocytes is stimulated by stromalderived factor, and this was concentration-dependently inhibited by telmisartan (Walcher et al., 2008). Such inhibition was mimicked by several PPAR-γ activators but not by eprosartan and blocked by small interfering RNA against PPAR-γ. The molecular basis of the AT1Rindependent inhibition by telmisartan was an inhibition of phosphatidylinositol 3-kinase activity.

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3.3.2. In vivo models

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Vascular remodeling including arterial wall hypertrophy and increased media/lumen ratio and atherosclerosis are related phenomena. While remodeling is most prominent with chronically elevated BP, atherosclerosis occurs largely under hyperlipidemic conditions, as a consequence of diabetes and upon mechanical damage to the endothelium such as in the balloon injury models. Endothelial dysfunction can be an early step in the development of atherosclerosis (see section 3.2.). The importance of studying ARB effects on atherosclerosis not only in cultured cells but also in vivo is illustrated by a report that in an isolated artery ANG promoted protein synthesis but, in contrast to cultured VSMCs, not DNA synthesis (Holycross et al., 1993). The potential of ARBs in vascular remodeling is indicated by studies in which chronic administration of ANG induces aortic thickening in mice (Sakurada et al., 2013) or rats (Cao et al., 1999). 3.3.2.1. Hypertension models

Effects of hypertension on vascular remodeling have largely been studied in SHR and their stroke-prone substrain (Bennai et al., 1999; Kim et al., 1995c), but some studies have also been performed in New Zealand genetically hypertensive rats (Ledingham et al., 2000), Dahl salt-sensitive rats (Liang & Leenen, 2008) or in (mRen-2)27 transgenically hypertensive rats (Rossi et al., 2000); it has also been observed in pharmacologically induced hypertension, e.g. by NO synthase inhibitor treatment (Luvara et al., 1998; Takemoto et al., 1997) or in the surgically induced hypertension model of aortic banding (Obayashi et al., 1999). Vascular remodeling with media hypertrophy and hence increased media/lumen ratio is a consistent feature of hypertension. It can also be induced by chronic ANG treatment in normotensive rats, and this can be blocked by ARBs such as valsartan (Cao et al., 1999), reflecting the VSMC growth-promoting properties of AT1R. Multiple studies have addressed the more relevant question, i.e. whether endogenous ANG and AT1R contribute to vascular remodeling in animal models of hypertension. Accordingly, treatment of SHR starting at an 40

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early age when BP is still normal or only modestly elevated prevents vascular hypertrophy development as shown with candesartan (Rizzoni et al., 1998), irbesartan (Intengan et al., 1999; Vacher et al., 1995), losartan (Morton et al., 1992) and telmisartan (Yoo et al., 2015). Perhaps more relevant for the clinical situation, treatment with an ARB starting when hypertension has already developed can reverse the vascular hypertrophy as shown with candesartan (Kim et al., 1995c), losartan (Dai et al., 2007; Oddie et al., 1993; Soltis, 1993) (Figure 4) and telmisartan (Dai et al., 2007; Dupuis et al., 2005; Foulquier et al., 2014; Zhong et al., 2011). Similar observations were made in New Zealand genetically hypertensive rats treated with valsartan or enalapril but not with felodipine (Ledingham et al., 2000). A prevention or reversal of vascular hypertrophy by ARBs has been shown in multiple vascular beds including aorta (Kim et al., 1995c; Soltis, 1993; Vacher et al., 1995; Zhong et al., 2011), coronaries (Takemoto et al., 1997), mesenteric vessels (Intengan et al., 1999; Kim et al., 1995c; Ledingham et al., 2000; Morton et al., 1992; Oddie et al., 1993; Rizzoni et al., 1998), tail artery (Soltis, 1993), renal vessels (Bennai et al., 1999) or the cerebral vasculature (Bennai et al., 1999; Dupuis et al., 2005; Foulquier et al., 2014). It also has been observed in the stroke-prone substrain of SHR (Bennai et al., 1999; Kim et al., 1995c) or in diabetic SHR (Hulthen et al., 1996). On the other hand, it should be noted that such anti-hypertrophic effects of ARBs may not materialize in studies with short duration (Morton et al., 1992). Moreover, some vascular beds with strong auto-regulation may not exhibit beneficial effects on remodelling as reported e.g. with candesartan in intrarenal arcuate and interlobular arteries (Anderson et al., 1997). Similarly, irbesartan was reported ineffective in reducing mesenteric artery hypertrophy in (mRen-2) 27 rats despite lowering BP (Rossi et al., 2000). In an aortic banding rat model of cardiac pressure overload, only high doses of candesartan reduced vascular hypertrophy and arterial wave reflection, whereas both high and low doses reduced cardiac hypertrophy in the same study (Obayashi et al., 1999). A high dose of valsartan also reduced thoracic aorta hypertrophy in this model along with normalization of expression of RGS proteins 2, 4 and 5; abnormal contractile responses or aortic rings, however, were not normalized (Wang et al., 2008). <<>>

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Of note, the increase in peripheral resistance in hypertensive animals not only involves a narrowed lumen of specific arteries and aterioles but also an overall reduction of the number of microvessels, a phenomenon termed vessel rarefication (Struyker-Boudier et al., 1990). While such rarefication has been reported to be even worsened by benazeprilat, valsartan was neutral in SHR (Scheidegger et al., 1996). Neither valsartan nor benazeprilat affect the neovascularization of skeletal muscle following partial ligation of the iliac artery in normotensive rats (Scheidegger et al., 1997). On the other hand, candesartan even increased capillary density in the subendocardium of stroke-prone SHR (Xie et al., 1999). Several studies have compared the ability of ARBs and ACE inhibitors to prevent or reverse vascular hypertrophy in SHR. Except for one study in which the chosen dose of irbesartan was more effective than that of fosinopril (Intengan et al., 1999), the two approaches were generally found to be similarly effective with regard to vascular remodeling, e.g. with candesartan vs. enalapril (Rizzoni et al., 1998), candesartan vs. perindopril (Paull & Widdop, 2001), losartan vs. captopril (Morton et al., 1992), telmisartan vs. ramipril (Dupuis et al., 2005) or, in diabetic SHR, with valsartan vs. ramipril (Hulthen et al., 1996) or, in NO synthase inhibitor-treated rats, with olmesartan vs. temocapril (Takemoto et al., 1997). In the candesartan vs. perindopril study, beneficial effects on vascular hypertrophy were no longer visible after 8 weeks of treatment withdrawal (Paull & Widdop, 2001). In New Zealand genetically hypertensive rats valsartan and enalapril had similar effects on the media/lumen 41

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ratio, but the former caused hypertrophic and the latter eutrophic outward remodeling (Ledingham et al., 2000). Conversely, the beneficial effects of the ACE inhibitor temocapril were reported to be insensitive to concomitant administration of the bradykinin receptor antagonist icatibant (Takemoto et al., 1997), further supporting the central role of AT1R in vascular hypertrophy.

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Direct comparative studies of e.g. losartan vs. hydralazine in SHR (Soltis, 1993), valsartan vs. lacidipine in diabetic SHR (Hulthen et al., 1996) or valsartan vs. felodipine in New Zealand genetically hypertensive rats (Ledingham et al., 2000) indicate that despite similar levels of BP reduction, ARBs perform more favorably than to be expected based on their antihypertensive effect. Although supported by only few direct comparative studies, this specific role of RAS inhibiting vs. other anti-hypertensive agents is pathophysiologically plausible based on the direct ANG-induced and AT1R-mediated effects on VSMC growth (see section 3.3.1.2). In a mouse model with transgenic overexpression of human renin and human angiotensinogen, valsartan treatment prevented vascular remodeling and produced prolonged BP reduction, whereas a dose of hydralazine causing similar initial BP-lowering had less if any effects (Inaba et al., 2011). In endothelial NO synthase knock-out mice valsartan and the direct renin inhibitor aliskiren reduced coronary remodeling, an effect not shared by hydralazine; valsartan and aliskiren also prevented cuff injury-induced arterial intimal thickening (Yamamoto et al., 2009a).

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To explore mechanistic links between the in vitro studies on VSMC growth and the in vivo situation, various studies have determined ARB treatment effects on the expression of factors potentially contributing to vascular remodeling. Thus, beneficial effects of e.g. candesartan (Jones et al., 2004; Jones et al., 2012; Kim et al., 1995c), irbesartan (Luvara et al., 1998; Vacher et al., 1995), losartan (Dai et al., 2007; Liang & Leenen, 2008) or telmisartan (Dai et al., 2007; Jin et al., 2012; Liang & Leenen, 2008; Zhong et al., 2011) were reported on the mRNA and/or protein expression of ACE-2, profilin-1 and PPAR-γ (Jin et al., 2012; Zhong et al., 2011), endothelial NO synthase (Jin et al., 2012), inducible NO synthase, NADPH diaphorase, intercellular adhesion molecule and VCAM-1 (Luvara et al., 1998), MCP-1 and its its receptor CCR2 (Dai et al., 2007), several integrins (Intengan et al., 1999), transforming growth factor-β (TGF-β), and the extracellular matrix molecules fibronectin, laminin and collagen I, III and IV (Kim et al., 1995c) in the vascular wall; the latter was mirrored by a beneficial effect on aortic collagen content and collagen/elastin ratio (Intengan et al., 1999; Vacher et al., 1995) and on overall vascular fibrosis (Jones et al., 2004; Jones et al., 2012; Liang & Leenen, 2008; Takemoto et al., 1997), which should structurally affect the ability for vasodilatation. Of note, such ARB effects have been observed even if treatment started as late as 20 months of age (Jones et al., 2004; Jones et al., 2012). Mesenteric microvessels from SHR exhibit increased expression of transient receptor potential canonical channels TRPC1, TRPC3 and TRPC5 and enhanced vasomotion in response to norepinephrine in vitro, and the latter was blocked by TRPC1 or TRPC3 antibodies; a 16-week treatment with candesartan or telmisartan normalized the TRPC expression and vasomotion in response to noradrenaline whereas amlodipine at a dose producing similar BP reduction did not (Chen et al., 2010). The telmisartan-induced BP reduction in SHR was accompanied by restoration of heightened TRPC3 expression in aorta, and this was not mimicked by an amlodipine dose yielding similar BP lowering (Liu et al., 2009a). SHR also exhibit monocyte infiltration of the vascular wall; this was inhibited by treatment with losartan or telmisartan, even in doses not affecting BP (Dai et al., 2007). Although long-standing hypertension is a major risk factor for atherosclerosis, only few studies have used hypertension models to study atherosclerosis endpoints other than vascular 42

ACCEPTED MANUSCRIPT hypertrophy (Bennai et al., 1999). More direct approaches to ARB effects in atherosclerosis were studied in other models as described in the subsequent sections. 3.3.2.2. ApoE1 knock-out mice

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Disturbed lipid metabolism is an important risk factor for the development of atherosclerosis, and apolipoprotein E1 (ApoE) plays an important part in such metabolism. Accordingly, ApoE knock-out mice, which spontaneously develop hyperlipidemia and atherosclerosis, have become an important animal model for studying the pathophysiology, prevention and treatment of atherosclerosis. Interferon-γ (IFNγ), MCP-1, or the MCP-1 receptor CCR-2 are considered to be important pathophysiological factors for the development of atherosclerosis, specifically in the ApoE knock-out mouse.

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Multiple ARBs were effective in preventing the development of atherosclerosis in ApoE knock-out mice including irbesartan (Dol et al., 2001), losartan (Keidar et al., 1997; Nakagami et al., 2008), olmesartan (Tsuda et al., 2005), telmisartan (Blessing et al., 2008; Fukuda et al., 2010; Grothusen et al., 2005; Iwai et al., 2008; Matsumura et al., 2011; Schlimmer et al., 2011; Takaya et al., 2006) and valsartan (Li et al., 2004). ACE inhibitors such as captopril (Keidar, 1998), ramipril (Blessing et al., 2008; Matsumura et al., 2011; Schlimmer et al., 2011) or temocapril (Tsuda et al., 2005) were also effective in this model, pointing to a general role of ANG acting on AT1R. This general role is also supported by the finding that atherosclerotic lesion area and MCP-1 expression were lower in ApoE/AT1R double knock-out mice (Tomono et al., 2008). Moreover, transplantation of bone marrow from AT1aR knock-out mice into ApoE knock-out mice also improved atherosclerotic lesion development (Endtmann et al., 2011).

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The beneficial ARB effects in the ApoE knock-out model do not involve a reduction in circulating lipoprotein levels (Dol et al., 2001; Grothusen et al., 2005; Kikuchi et al., 2007; Nagy et al., 2010; Nakagami et al., 2008) or in ApoE/AT1R double knock-out mice (Fukuda et al., 2010). However, an increased HDL cholesterol was reported in one study (Fukuda et al., 2010), telmisartan was reported to increase total cholesterol, LDL, VLDL and triglycerides without altering HDL in the absence or presence of concomitant atorvastatin treatment (Grothusen et al., 2005), and irbesartan was reported to increase total cholesterol and triglyceride levels in such knock-out mice (Dol et al., 2001). Rather they may involve various levels of the pathway to athersclerotic lesions including reduced infiltration by macrophages (Dol et al., 2001), reduced oxidative stress (Schlimmer et al., 2011; Takaya et al., 2006; Tsuda et al., 2005) and reduced disposition of biglycans (Nagy et al., 2010) and lipids in the vascular wall and enhanced collagen and fibrous cap thickness (French et al., 2011; Fukuda et al., 2010; Grothusen et al., 2005). At the molecular level a reduced expression of MCP-1, its receptor CCR2, macrophage inflammatory protein-1α and -2 was observed whereas the cytokine receptor CXCR2 was up-regulated (Dol et al., 2001; Matsumura et al., 2011). Moreover, ARBs and ACE inhibitors reduced MCP-1 concentration in monocytes, number of macrophages and metalloproteinase-9 content (Grothusen et al., 2005). Importantly, the above effects are not necessarily mimicked by other BP-lowering medications (Nagy et al., 2010), indicating that they are at least to some degree specific for RAS inhibition. While similarly inhibiting the development of lipid-rich plaques and IL-6 expression, telmisartan but not losartan increased expression of hepatocyte growth factor (Nakagami et al., 2008). An additional knock-out of AT1aR in ApoE knock-out mice mimicked some of the effects of telmisartan, but treatment of such double-knock-out with telmisartan yielded further 43

ACCEPTED MANUSCRIPT improvement (Fukuda et al., 2010). Another study confirmed beneficial effects of a combined AT1aR and ApoE knock-out; in the absence of AT1R telmisartan did not cause additional improvement, and ramipril even worsened atherosclerotic lesion development, the latter effect apparently due to withdrawal of a protective AT2R-mediated effect (Tiyerili et al., 2012).

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Moreover, ARBs may not only prevent the development of atherosclerotic plaques but may also stabilize or even reduce them as shown e.g. with azilsartan (French et al., 2011), telmisartan (Blessing et al., 2008; Fukuda et al., 2010) (Figure 5) and valsartan (Aono et al., 2012). This finding is corroborated by studies with ApoE/AT1R double knock-out mice which exhibit less plaque rupture (Aono et al., 2012). Plaque stabilization has also been reported with valsartan in a model of atherosclerosis induced by a combination of acquired diabetes combined with a high-fat diet in Yorkshire swines (Chatzizisis et al., 2009).

3.3.2.3. Other hyperlipidemic animal models

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The Watanabe heritable hyperlipidemic rabbit is a frequently used model, in which telmisartan reduced atherosclerotic plaques; the telmisartan plus GW9662 combination or candesartan were less effective, indicating that both AT1R and PPAR-γ contributed to the protective effect (Ikejima et al., 2008). Some reduction of atherosclerotic plaque area was also reported with olmesartan (Koike et al., 2001) or valsartan in this model; the latter was further enhanced by concomitant treatment with pitavastatin (Imanishi et al., 2008a) or aliskiren (Imanishi et al., 2008b). In contrast, irbesartan reduced atherosclerotic plaques in this model only when administered in doses exceeding those required to block most of the pressor effect of exogenously administered ANG (Hope et al., 1999).

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Atherosclerosis can also be induced in rabbits not genetically prone for this condition by feeding them a high-cholesterol diet. In this model valsartan reduced triglycerides but not cholesterol and produced some reduction in aortic atherosclerotic plaque size, intima/media ratio and macrophage infiltration; however, these effects were less pronounced than those of benazepril, which also lowered cholesterol (Li et al., 1999a). In similar studies, valsartan did not lower BP or plasma cholesterol but reduced atherosclerotic lesions and vascular hypertrophy (de las Heras et al., 1999). A later study extended these findings with valsartan to the rabbit pulmonary artery (Li et al., 2010b). In monkeys fed a high cholesterol diet, olmesartan reduced atherosclerotic lesion size in the aorta (Koike et al., 2001). Taken together, ARBs as a class have some beneficial effect on heritable or diet-induced atherosclerosis in rabbits and probably other species; telmisartan may cause a greater effect due to additional stimulation of PPAR-γ. 3.3.2.4. Diabetes mellitus models

Although frequently associated with hypertension in clinical practice, diabetes mellitus per se does not increase BP. However, it is an important risk factor in the development of atherosclerosis. While most studies on atherosclerosis-related endpoints were based on the STZ-induced model of type 1 diabetes, some have also explored models of obesity and type 2 diabetes (Abdelsaid et al., 2014; Toyama et al., 2011). In an early study in rats with STZ-induced diabetes, valsartan, ramipril in two doses with and without concomitant icatibant, lacidipine or mibefradil were administered for 24 weeks (Cao et al., 1998). While none of the treatments affected the metabolic parameters, all except 44

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icatibant monotherapy reduced the media/lumen ratio of the mesenteric artery. Of note, this was true for BP-lowering treatments (valsartan and ramipril high dose with and without icatibant) as well as the BP-neutral treatments (valsartan and ramipril low dose, lacidipine or mibefradil). Others reported that the rat STZ-model of diabetes was associated with a reduced internal diameter, a narrower media, fewer elastic and more collagen fibers in the thoracic aorta; all of these changes were attenuated by telmisartan treatment (Salum et al., 2014). These data suggested that AT1R blockade can improve diabetes-induced vascular remodeling, probably independent of BP lowering.

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In Goto-Kakizaki rats, a model spontaneously developing type 2 diabetes in the absence of obesity, azilsartan thickening of the wall of cerebral vessels and reversed the loss of myogenic tone (Abdelsaid et al., 2014). However, these data data are difficult to interpret mechanistically as azilsartan also markedly lowered blood glucose in this model.

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A later study compared 8-week treatments with losartan, telmisartan and telmisartan plus GW9662 in db/db mice, a genetic model of diabetes (Toyama et al., 2011). Losartan and telmisartan, irrespective of concomitant GW9662 treatment, similarly lowered BP in these mice. None of the treatments affected fasting glucose but telmisartan lowered the insulin resistance index; the latter was blocked by GW9662 and not mimicked by losartan. The impaired endothelium-dependent aortic vasodilatation by acetylcholine was improved to a greater extent by telmisartan than by losartan, with the effect of the former being attenuated by GW9662; similar differences between treatments were observed for phosphorylation of aortic Akt and endothelial NO synthase, PPAR-γ expression and aortic and plasma TNFα levels; PPAR-α expression was not affected by any treatment. Thus, in contrast to the above rat study, these findings indicate a possible additional contribution of PPAR-γ activation to beneficial ARB effects in diabetes-associated atherosclerosis.

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Other studies shed important light on these apparently contradictory studies with regard to the roles of AT1R and PPAR-γ in diabetes-associated vascular remodeling and atherosclerosis. They were based on ApoE knock-out mice, in which diabetes was induced by STZ treatment. One study has explored the effects of treatment with olmesartan or telmisartan on aorta and has also involved ApoE knock-out mice (Ihara et al., 2007). While this study found that only telmisartan lowered LDL cholesterol, both ARBs prevented activation of the RAGE pathway, which in turn preceded AT1R up-regulation. Both treatments and and also an ApoE/AT1R double knock-out prevented aortic atherosclerosis development. Within that study the ANGinduced RAGE expression in cultured VSMCs was also AT1R-mediated. In another study using the same model and also looking at aorta, treatment with the PPAR-γ antagonist GW9662 resulted in the highest elevation of fasting glucose levels in diabetic ApoE and ApoE/AT1R knock-out mice, whereas telmisartan treatment and AT1R deficiency in ApoE knock-out mice showed the lowest fasting glucose levels (Tiyerili et al., 2013). Diabetic ApoE knock-out mice had severe impairment of endothelial function, enhanced oxidative stress and increased atherosclerotic lesion formation; all of these effects were significantly less pronounced in the ApoE/AT1R double knock-out or in telmisartan-treated ApoE knockout mice. Cotreatment of ApoE or ApoE/AT1R knock-out mice with GW9662 omitted the atheroprotective effects of AT1R deficiency or telmisartan. Taken together these data show a clear role for AT1Rs and hence ARBs in atherosclerosis development in diabetic animals. An additional role for PPAR-γ may exist, but the presently available data are insufficient to quantify the relative contributions of the AT1R and PPAR-γ, particularly as this may depend not only on the ARB being used but also on specific aspects of the experimental model being used. Thus, ARBs consistently reduce atheroscleoris in multiple models of type 1 and type 2 45

ACCEPTED MANUSCRIPT diabetes, but some ARBs may have additional AT1R-independent effects including those related to PPAR-γ stimulation. 3.3.2.5. Balloon injury model

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The balloon injury model, induced by temporarily inflating a balloon inside a blood vessel, is frequently used to study atherosclerosis and its treatment in experimental animals as it provides an intervention which is clearly identified with regard to time and location. Balloon injury causes local destruction of the endothelium followed by neointima formation and VSMC migration and proliferation. This model has mostly been used in rat carotid arteries, but some studies have also been performed in rat aorta (Moroi et al., 1997; Viswanathan et al., 1992), rabbit carotid artery (Azuma et al., 1992; Janiak et al., 1994) and dog carotid and femoral artery (Miyazaki et al., 1999a; Miyazaki et al., 1999b; Tea et al., 2000). ARB effects were generally comparable across these models and species.

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In such studies the ARB or comparator drug was administered prior to, concomitantly with or shortly after the balloon injury and then continued for several days to weeks. In one study, a single application at the site of injury was as effective as repeated systemic administration (Taguchi et al., 1993). The primary phenotype being studied in this model is the neointima formation at the site of injury, and that was reduced by a variety of ARBs including candesartan (Kawamura et al., 1993; Kino et al., 1994; Kino et al., 1995; Miyazaki et al., 1999b; Taguchi et al., 1994; Tea et al., 2000), losartan and EXP3174 (Farhy et al., 1992; Farhy et al., 1993; Janiak et al., 1994; Osterrieder et al., 1991; Prescott et al., 1991; Taguchi et al., 1993), valsartan (Miyazaki et al., 1999a) or experimental ARBs (Virone-Oddos et al., 1997) but also by ACE inhibitors such as benazeprilat (Prescott et al., 1991), captopril (Virone-Oddos et al., 1997), cilazapril (Osterrieder et al., 1991), enalapril (Farhy et al., 1992; Miyazaki et al., 1999b), lisinopril (Kino et al., 1994; Kino et al., 1995) or ramipril (Farhy et al., 1992; Farhy et al., 1993; Janiak et al., 1994; Taguchi et al., 1993). In contrast, neither AT2R antagonists (Janiak et al., 1994) nor RAS-independent antihypertensive drugs such as hydralazine reduced neointima formation (Tazawa et al., 1999), indicating that this is a specific effect of AT1R inhibition rather than of BP lowering. Moreover, such ARB effects have not only been shown in normotensive rats but also in SHR; while the extent of neointima formation was greater in SHR than in normotensive rats, the beneficial effect of an ARB or ACE inhibitor was maintained (Kino et al., 1995). However, some controversy exists about the comparison of neointima formation upon ARB and ACE inhibitor treatment. Most investigators found that both drug classes have a quantitatively similar effect (Janiak et al., 1994; Kino et al., 1994; Kino et al., 1995; Osterrieder et al., 1991; Prescott et al., 1991; Virone-Oddos et al., 1997), but some have reported ACE inhibitors to be more effective than ARBs (Farhy et al., 1992; Farhy et al., 1993; Taguchi et al., 1993). In their studies the greater effect of ACE inhibitors was abolished by the bradykinin receptor antagonist icatibant and/or an NO synthase inhibitor. Of note the studies reporting greater ACE inhibitor than ARB efficacy have all been based on the comparison of losartan with ramipril and may be explained by specific properties of one of these two drugs, but not even all losartan/ramipril comparisons supported a difference between the two drug classes (Janiak et al., 1994). On the other hand, a study in dog carotid and femoral artery found candesartan and enalapril to be equally effective in the femoral artery whereas, in contrast to candesartan, enalapril was ineffective in carotid artery (Miyazaki et al., 1999b). Thus, in the balance of all studies ARBs and ACE inhibitors apparently have comparable effects on neointima formation, but additional roles for kinins and NO cannot be excluded. 46

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The above studies have also explored the molecular, cellular and tissue mechanisms underlying neointima formation in this model and how they are affected by ARBs. Balloon injury affects the expression and functional status of various signaling molecules at the site of neointima formation. This includes a reduced expression of platelet-derived growth factor receptor α and β, which is not easy to understand as the phosphorylation status of both receptor subtypes was enhanced; the increased phosphorylation was apparently specific as it did not apply to epidermal growth factor receptors or insulin receptor substrate-1 (Abe et al., 1997). On the other hand, the expression of immediate-early genes including c-fos, c-jun, jun B, jun D, and Egr-1 as well as of ornithine decarboxylase, TGF-β1, fibronectin and collagen was found to be increased (Kim et al., 1995b; Kim & Iwao, 1997; Tazawa et al., 1999). All of these changes in expression and phosphorylation were ameliorated by candesartan treatment.

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It was also found that at the site of neointima formation the expression of ACE and the alternative ANG-generating enzyme chymase were up-regulated (Miyazaki et al., 1999a; Miyazaki et al., 1999b). Moreover, the expression of AT1R was also up-regulated, and this was prevented by candesartan but not hydralazine treatment (Tazawa et al., 1999; Viswanathan et al., 1992), indicating that it was not linked to BP lowering but rather specifically to AT1R blockade. On the other hand, it was reported that ANG reduces levels of the vasodilatory second messenger cGMP in rat aorta neointima after balloon injury by stimulating AT2R; in contrast, AT1R was not involved based on lack of inhibition by candesartan, and no reduction of cGMP levels by ANG was observed in normal aorta (Moroi et al., 1997).

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ARBs such as losartan (Azuma et al., 1992; Prescott et al., 1991; Taguchi et al., 1994) or candesartan (Kawamura et al., 1993; Taguchi et al., 1994) were also reported to inhibit migration of VSMCs to the site of injury and their proliferation in the media; the ACE inhibitor benzaprilat mimicked these effects but was quantitatively less effective (Prescott et al., 1991).

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A key feature of the balloon injury model is an imbalance in contraction/relaxation responses of the affected vessel. In a rabbit carotid artery model contractile responses of aortic strips in vitro to noradrenaline or ANG were significantly increased six weeks after the injury whereas those to endothelin-1 remained unaffected; losartan treatment, starting one week prior to the injury, did not affect the increased response to noradrenaline but similarly reduced that to ANG in both control and injured vessels (Azuma et al., 1992). In the same study balloon injury markedly reduced the vasodilation response to acetylcholine; while losartan treatment did not affect this response in control animals, it almost completely restored the response in the injured vessels. Both the impairment after balloon injury and the restoration upon losartan treatment apparently did not happen at the VSMC level, as vasodilatation responses to sodium nitroprusside were similar in all four study groups. Interestingly, the number of endothelial cells was greater in the injured than the control vessels six weeks after the injury, whereas losartan did not affect their number in either condition. In a rat carotid artery model balloon injury similarly caused intima thickening as detected 14 days after the injury, and in this case the thickening was attenuated either by candesartan or the ACE inhibitor cilazapril, started 6 days prior to the injury (Kawamura et al., 1993). Similar to the rabbit model, vasodilatation of isolated carotid artery in response to acetylcholine was almost completely abolished upon injury despite the intimal thickening but scanning electron microscopy revealed that the endothelial cells at the site of injury were morphologically abnormal; candesartan dosedependently restored acetylcholine-induced vasodilatation and normalized the morphological abnormalities, whereas the vasodilatation response to sodium nitroprusside was similar in all 47

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four treatment groups. Taken together these findings indicated a shift of vascular contractility away from endothelium-dependent vasdodilatation at the site of neointima formation, which is most likely explained by a functional and morphological impairment at the level of the endothelium. ARBs, at least when treatment is started prior to or concomitnant with the injury, can prevent such changes. These ARB effects apparently are not BP-related as they are not mimicked by other anti-hypertensive drugs and are not necessarily accompanied by BP lowering.

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A related model is the placement of a cuff on the femoral artery of mice. In this model, neointima area was greater in double transgenic mice harboring the human angiotensinogen and renin genes as compared to wild-type mice (Inaba et al., 2011). This neointima formation was prevented by valsartan but not by hydralazine.

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A similar phenotype as in the ballon model can be induced by placing a cuff on the femoral artery. When this was done in wild-type or mas knock-out mice, azilsartan reduced neointimal area and cell proliferation in the intima and media, which was accompanied by a reduced expression of inflammation markers including MCP-1, TNF-α and IL-1β (Ohshima et al., 2014).

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In a normotensive rat model of subtotal nephrectomy, endothelial dysfunction as assessed by carbachol-induced relaxation of aortic rings was normalized by telmisartan treatment (Toba et al., 2012). This was partly inhibited by GW9662 and only partly mimicked by losartan; the telmisartan + GW9662 combination caused quantitatively very similar improvement as losartan treatment. A similar pattern of telmisartan, telmisartan plus GW9662 and losartan was observed for vascular macrophage invasion, osteopontin-positive cells, and osteopontin, VCAM-1 and NADPH oxidase expression, indicating concomitant involvement of AT1R and PPAR-γ in all of these responses. The same group of investigators largely confirmed such findings in a study using 5/6 rather than subtotal nephrectomy (Toba et al., 2013).

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In a mouse model of heterotopic heart transplantation between MHC-antigen compatible strains, intima growth develops in the graft coronary arteries; in this model treatment with either candesartan or captopril reduced intima growth (Furukawa et al., 1996). Candesartan and enalapril also reduced intima proliferation of cardiac vessels in a rat heterotopic heart transplantation model treated with cyclosporin A, interestingly more so in small than in large ones (Richter et al., 1999). These findings indicate that RAS blockade may be beneficial to reduce coronary atherosclerosis in heart transplant recipients.

3.4.

Other effects on vascular function

Isolated studies on ARB effects on vascular function unrelated to hypertension or atherosclerosis have also been reported. For example, one study has explored effects of candesartan in isolated hearts obtained from WKY, SHR and SHR chronically treated with an NO synthase inhibitor (Fujita et al., 1995). In that study coronary flow was reduced in SHR and even more so in SHR with NO synthase inhibition. Candesartan restored coronary flow in SHR but not in those treated with the inhibitor. In another study, uric acid induced senescence and apoptosis in cultured HUVECs, and this was attenuated by telmisartan, enalaprilat or probenicid, the latter probably acting independent of the RAS (Yu et al., 2010); the relationship of this finding to gout remains to be explored. In a third approach, aneurysm was induced by perfusion of isolated abdominal aortic segments with elastase followed by a 14 48

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day treatment with telmisartan or hydralazine (Kaschina et al., 2008). Telmisartan, but not hydralazine treatment, reduced aortic diameter and aortic expression of matrix metallopeptidase 3, cathepsin D, NF-κB, TNF-α, TGF-1β as well as caspase 3, p53 and Fas ligand proteins; serum MCP-1 was also reduced. Chronic infusion of ANG can induce aortic aneurysm in mice; this was abolished by a low telmisartan dose, not lowering BP, in regulator of G-protein signaling-2 knock-out but not wild-type mice (Matsumoto et al., 2010). Concomitantly, telmisartan increased survival in knock-out but not wild-type mice. Using similar models, effects of irbesartan and telmisartan were explored in mouse models of abdominal aortic aneurysm induced by either chronic ANG infusion in ApoE knock-out mice or by transient intraaortic administration of porcine pancreatic elastase in C57BL/6 mice (Iida et al., 2012). In the ApoE knock-out model both telmisartan and irbesartan limited aneurysm enlargement, medial elastolysis, smooth muscle attenuation, macrophage infiltration, adventitial neocapillary formation, and the expression of proteinases and proinflammatory mediators, whereas doxycycline, fluvastatin or bosentan did not affect aneurysm progression. In this model, aortic aneurysms developed in 67% of ANG-infused mice and 40% died of rupture; strikingly no telmisartan-treated mouse developed an aneurysm, and mortality with irbesartan also was below 10%. Irbesartan and telmisartan also reduced the expression of many inflammatory genes, an effect not shared by the other treatments. Telmisartan was similarly effective in the elastase model (irbesartan not investigated).

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4. Heart function

Normal heart function

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AT1R in the heart have been directly demonstrated at the mRNA level in many studies, e.g. by in situ hybridization in rat heart (Gasc et al., 1994) or by Northern blots in the human heart (Mauzy et al., 1992). Due to the problems with AT1R antibodies (Adams et al., 2008; Benicky et al., 2012; Premer et al., 2013; Xue et al., 2011), reliable detection at the protein level has largely been based on radioligand binding studies (Booz & Baker, 1996; Feolde et al., 1993a; Liang & Leenen, 2008; Nakamura et al., 1994a; Sechi et al., 1992b). Of note, cardiac expression of AT1R at the mRNA and protein level is not limited to myocytes but also found in fibroblasts (Villareal et al., 1993).

4.1.1. Signal transduction and cellular function

ARB effects on cardiac function at the cellular level have primarily been studied in isolated cardiomyocytes from rats, but findings from rabbits (Habuchi et al., 1995) and hamsters (Nakamura et al., 1994a) have also been reported. Thus, losartan-sensitive activation of cardiac AT1R has directly been shown to involve G-protein activation (Sechi et al., 1992b). Accordingly, ARBs inhibit prototypical signal transduction pathways of AT1R in cardiomyocytes including inhibition of phospholipase C activation by losartan (Feolde et al., 1993b; Ishihata & Endoh, 1993) (Pönicke et al., 1997), and of Ca2+ elevations by candesartan (Kinugawa et al., 1993), irbesartan (Delisee et al., 1993) and losartan (Delisee et al., 1993; Feolde et al., 1993b). Alternatively, the latter signal transduction pathway has been assessed as Ca2+ efflux from isolated cardiomyocytes, which was stimulated by ANG and inhibited by candesartan and losartan (Fukuta et al., 1996). Ca2+ elevations can involve mobilization from various types of intracellular stores and from influx from the extracellular space, the latter mostly occurring via specific Ca2+ channels (Krebs et al., 2015). Candesartan blocked the ANG-induced L-type Ca2+ current in isolated rabbit sino-atrial node cells (Habuchi et al., 1995). A more down-stream signaling event activated via AT1R is the Na+/H+-exchanger, which may be relevant for cardiac responses to ischemia (Xiao & Allen, 2003). ARBs were 49

ACCEPTED MANUSCRIPT also reported to prevent ANG-induced down-regulation of adiponectin receptors in the H9C2 cardiomyocyte cell line (Wu & Guo, 2015).

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4.1.2. Heart rate and conduction system

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Of note, cardiac ANG effects do not necessarily occur at the cardiomyocyte level but may also be mediated by AT1R on fibroblasts (Schorb et al., 1995). In this regard it has been proposed that AT1R on cardiomyocytes and fibroblasts, using distinct signal transduction pathways, drive hypertrophy and proliferation, respectively (Yamazaki & Yazaki, 1999). Effects on fibroblasts may underly the beneficial ARB effects on cardiac fibrosis (see section 4.2.). Moreover, they may also occur by AT1R located in sympathetic nerve endings promoting noradrenaline release (Brasch et al., 1993; Guimaraes et al., 2011; Shetty & DelGrande, 2000) and parasympathetic nerve endings promoting acetylcholine release (Rechtman & Majewski, 1993), in the cardiac endothelium (Chua et al., 1993), or in coronary arteries (see section 4.1.4.). Effects of ARBs on hypertrophy and proliferation of cardiomyocytes and cardiac fibroblasts derived from healthy animals will be discussed in section 4.2.1.

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Autoradiographic studies, e.g. in rats, have shown that the cardiac conduction system expresses angiotensin receptors at the protein level, which based on their sensitivity to losartan appear to belong exclusively to the AT1R subtype (Saavedra et al., 1993). AT1R may also be involved in the direct communication between cardiomyocytes as ANG reduced junctional conductance between pairs of rat ventricular cells in culture in a losartan-sensitive manner; interestingly, enalapril had an opposite effect in this study (De Mello & Altieri, 1992). There may also be chronic effects of ANG on conductivity between cardiomyocytes. Thus, ANG released from fibroblasts was reported to suppress connexin-43 expression in rat cardiomyocytes in a losartan-sensitive manner (Salameh et al., 2013); this fibroblastdependent effect at least partly buffered the direct stimulation of connexin-43 expression by isoprenaline.

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Nonetheless, ARBs had only small effects if any on heart rate in in vivo studies in multiple animal species. Most of this work was done in rats, mostly SHR and other hypertension models, but some studies have reported similar findings in dogs (MacFadyen et al., 1992; Yamamoto et al., 1997b) and monkeys (DeGraaf et al., 1993; Lacour et al., 1993). Specifically, small if any effects on heart rate have been reported for candesartan (Fujii et al., 1998; Kito et al., 1996; Takishita et al., 1994), eprosartan (Behr et al., 2004; MukaddamDaher et al., 2009), irbesartan (Lacour et al., 1993; Lacour et al., 1994), losartan (DeGraaf et al., 1993; Lacour et al., 1993; Lacour et al., 1994; MacFadyen et al., 1992; Wong et al., 1990d), olmesartan (Tomita et al., 2009), telmisartan (Wang et al., 2006), and valsartan (Criscione et al., 1993; Kometani et al., 1997; Yamamoto et al., 1997b). This general lack of ARBs on heart rate when used in animal models of hypertension or heart disease does not necessarily contradict opposite findings in specific experimental conditions. For instance, candesartan attenuated the reflex tachycardia in response to a high dose of the Ca-channel blocker manidipine (Sato et al., 1996). Telmisartan was reported to prevent the heart rate increase in rats with unilateral nephrectomy and additional ethylene glycol injection (Kadhare et al., 2015). Moreover, ARBs including losartan may inhibit positive chronotropic effects of ANG in pithed rats (Knape & van Zwieten, 1988; Zhang et al., 1993a), most likely by acting at the prejunctional level.

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In line with these findings on signal transduction, candesartan was shown to inhibit the ANGinduced shortening (Kinugawa et al., 1993) and the spontaneous beating frequency (Yonemochi et al., 1997) of rat neonatal cardiomyocytes. Similarly, losartan blocked the ANG-induced positive inotropic effect in rabbit (Ishihata & Endoh, 1993) or guinea pig ventricular strips, whereas an AT2R inhibitor was ineffective (Feolde et al., 1993a); inhibition of positive inotropic ANG effects by losartan was also reported from isolated human atrium (Zerkowski et al., 1993) whereas studies in failing human ventricle reported enhanced calcineurin activity but only weak and inconsistent inotropic effects (Li et al., 2005). In conscious, salt-depleted dogs infusion of ANG caused a small reduction in cardiac output, which was blocked by losartan (MacFadyen et al., 1993). In the same study, losartan alone induced a small rise in cardiac output in sodium-depleted dogs, which was attributed to a rise in heart rate and stroke volume. However, in most studies in the absence of exogenous ANG, ARBs lacked major effects on cardiac function. For instance, candesartan even in high concentrations lacked effects on cardiac function in isolated perfused guinea pig heart, the spontaneous beating rate of the right atria or the contractile force of electrically-paced left atria (Kito et al., 1996). Moreover, candesartan did not affect the increase in left ventricular pressure induced by elevating perfusion pressure in a Langendorff preparation of Syrian hamster heart (Nakamura et al., 1994a). In isolated guinea pig perfused heart, candesartan had no effect on cardiac function and in isolated guinea-pig atria it had no effect on the spontaneous beating rate of the right atria or the contractile force of electrically-paced left atria (Kito et al., 1996). In vivo, candesartan had no effect on left ventricular systolic pressure, its dp/dtmax, or cardiac output in anesthetized dogs; moreover, even at high doses, candesartan had little effect on cardiac output, right atrial pressure, pulmonary artery pressure, pulmonary capillary wedge pressure or respiration in conscious dogs (Kito et al., 1996). Similarly, EXP3174 or enalprilat had little effect on cardiac function in anesthetized dogs unless the RAS had been activated by furosemide pre-treatment (Richard et al., 1993). ACE inhibitors were reported to cause up-regulation of cardiac β-adrenoceptors in neonatal rat cardiomyocytes but this was not mimicked by candesartan (Yonemochi et al., 1997) and rather mediated by bradykinin receptors (Yonemochi et al., 1998). In contrast to the data in healthy rats, treatment with telmisartan attenuated the reduction of dP/dtmax in those with STZ-induced diabetes (Salum et al., 2014). 4.1.4. Coronary blood flow

Immunohistochemical studies have demonstrated the presence of AT1R in human coronary arteries obtained as autopsy specimens (Gross et al., 2002). While the validity of the antibodies used in these studies has been questioned (see above), staining in nonatherosclerotic sections was confined to smooth muscle cells in the vascular media. However, with the advent of atherosclerosis, AT1R receptor expression was also present in atherosclerotic plaques and involved other cell types including inflammatory cells and myofibroblasts. These data are in line with those from animal models demonstrating an upregulation of AT1R in atherosclerotic lesions in the general circulation (see section 3.3.2.). Exogenously applied ANG causes contraction of isolated coronary arteries, and that was antagonized by valsartan in rats (Tschudi & Lüscher, 1995) and by irbesartan in pigs and humans (Maassen van den Brink et al., 1999). In vivo, losartan did not change cardiac perfusion or coronary resistance in anesthetized rats (Sigmon & Beierwaltes, 1993a) but increased coronary blood flow in anesthetized dogs (Sudhir et al., 1993). In the latter study, this effect was not affected by indomethacin or propranolol but partially inhibited by L51

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Heart hypertrophy and fibrosis

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NAME, indicating that it may have in part occurred via NO release. In another dog study, EXP3174 or enalaprilat decreased coronary resistance but did not affect myocardial perfusion (Richard et al., 1993). In vivo pre-treatment with candesartan at least partly normalized the elevated minimal coronary resistance of SHR as measured in a Langendorff preparation (Takeda et al., 1994).

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The heart can develop hypertrophy and fibrosis in the context of many diseases. While hypertrophy initially can support maintaining cardiac output, it eventually turns into a risk factor of its own. 4.2.1. In vitro models

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Cardiac overexpression of AT1R causes heart hypertrophy (Hein et al., 1997). This can be mimicked in vitro by prolonged exposure to ANG which also promotes the development of cardiomyocyte hypertrophy (Hunyady & Turu, 2004; Lijnen & Petrov, 1999). At the cellular level this has primarily been studied in cultured rat neonatal cardiomyocytes. In this model ANG induces a hypertrophy response, often assessed as increased protein synthesis, which involves intermediary formation and release of endothelin (Pönicke et al., 1997; Xia & Karmazyn, 2004). It also involves an increased expression of several proto-oncogenes, atrial natriuretic peptide (Hirata et al., 1994; Wang et al., 2012b), endothelin-1 (Hirata et al., 1994), brain natriuretic peptide and visfatin (Chang et al., 2012). DNA synthesis is also enhanced (Saris et al., 2002). At least some of these responses are intracellularly mediated by ERK activation (Aoki et al., 2000; Nyui et al., 1997) and/or microRNA-19b (Gao et al., 2014a). They are inhibited by various ARBs including candesartan (Aikawa et al., 1999; Hirata et al., 1994; Nyui et al., 1997), eprosartan (Saris et al., 2002), losartan (Booz & Baker, 1996; Ito et al., 1993) and telmisartan (Chang et al., 2012; Gao et al., 2014a). Moreover, ANG can not only promote hypertrophy but also apoptosis in rat neonatal cardiomyocytes, a process involving inhibition of STAT3 phosphorylation and promotion of DNA fragmentation; such apoptosis induction was also blocked by candesartan (Tone et al., 1998). However, not all ANG responses in rat cardiomyocytes may be AT1R-mediated. Thus, ANG caused an early (30 min) and a late (120 min) phosphorylation of members of the JAK and STAT family of transcription factors; candesartan inhibited the early but not the late phase (Kodama et al., 1998). ANG also increased expression of T-type Ca2+-channels in neonatal rat cardiomyocytes, and this was inhibited by telmisartan; valsartan did not mimic this telmisartan effect but inhibited it (Morishima et al., 2009). Thus, some ANG effects on cardiomyocytes may occur via non-AT1R sites. Cardiac hypertrophy has also been observed in mice with cardiomyocyte-specific transgenic expression of angiotensinogen (Mazzolai et al., 2000); this hypertrophy was attenuated by treatment with losartan or ramipril and was not accompanied by fibrosis or systemic BP elevation. The hypertrophy response in cultured cardiomyocytes can not only be elicited by addition of exogenous ANG but also by stretch. Mechanical stretch activates a range of protein kinase cascades in cardiomyocytes including protein kinase C, raf-1, S6 protein kinase and ERK; this in vitro response is often considered to be a model system for the development of heart hypertrophy and is associated with increased c-fos expression and protein synthesis. While neither candesartan nor irbesartan affected stretch-induced hypertrophy of rabbit cardiomyocytes (Blaauw et al., 2010), candesartan inhibited stretch responses in isolated mouse (Nyui et al., 1997) or rat cardiomyocytes (Kojima et al., 1994; Komuro et al., 1995; Yamazaki et al., 1995). In addition to the AT1R component, stretch-induced hypertrophy also 52

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has an endothelin A receptor component; ARBs inhibit only the former, and a combination of ARB and endothelin receptor antagonist is required for full inhibition of stretch effects (Yamazaki et al., 1996a; Yamazaki et al., 1998), apparently reflecting the finding that AT1R activation promotes endothelin-1 release from cardiomyocytes but is not the only factor to do so (Hirata et al., 1994; Ito et al., 1993). Conditioned medium from stretched myocytes also caused the hypertrophy response, and candesartan may provide stronger inhibition against conditioned medium than against stretch itself (Yamazaki et al., 1996b). Stretch also induced angiotensinogen mRNA expression in neonatal rat cardiomyocytes via a candesartan-sensitive mechanism, potentially creating a positive feed-back loop (Tamura et al., 1997b). The candesartan inhibition of stretch-induced ERK activation was absent in angiotensinogen knock-out mice (Nyui et al., 1997). Interestingly, the stretch-induced hypertrophy was even greater in angiotensinogen knock-out than wild-type mice, but candesartan inhibited the response only in the latter (Nyui et al., 1997). These studies established that ANG and AT1R are involved in the stretch-induced cardiomyocyte hypertrophy, but are not the sole mediators. Moreover, these findings indicate that a non-AT1R component of the RAS may exhibit an anti-hypertrophic response in the heart. The idea that this may be the AT2R (Diez, 2004) is supported by the finding that L-NAME treatment-induced cardiac hypertrophy is greater in AT2R knock-out than wild-type mice (Gross et al., 2004). ANG stimulates endothelin-1 release in cardiac myocytes (Ito et al., 1993); while endothelin may in part mediate the ANGinduced cardiac hypertrophy, the opposite is untrue, i.e. candesartan did not inhibit the hypertrophy response directly induced by exogenous endothelin (Yamazaki et al., 1996a). These data demonstrate that AT1R are involved in the development of cardiomyocyte hypertrophy in response not only to exogenous ANG but also to stretch, an in vitro surrogate of cardiac volume overload. However, they are not the only mediator of the stretch hypertrophy response and may not protect from hypertrophy e.g. due to increased exposure to endothelin. If indeed AT2R mediate an opposite response, this could mean that ARBs may provide more effective protection from hypertrophy development than ACE inhibitors. On the other hand, cardiomyocyte-specific transgenic expression of angiotensinogen causes stretch in the absence of systemic BP elevation, indicating that ANG can induce cardiomyocyte hypertrophy independent of stretch; this was blocked by losartan or ramipril, establishing an AT1R-mediated effect (Mazzolai et al., 2000). AT1R may not only play a role in cardiomyocytes but also in cardiac fibroblasts of multiple species including humans (Kawano et al., 2000b). AT1R have been detected on cardiac fibroblasts in radioligand binding studies (Schorb et al., 1993). Actually, fibroblasts may express a greater density of AT1R than myocytes (Kim et al., 1995a). They couple to elevation of intracellular Ca2+ concentrations (Brilla et al., 1997; Schorb et al., 1993) and ERK activation (Schorb et al., 1995). In the absence of exogenously added ANG, candesartan, eprosartan or irbesartan did not alter the spontaneous proliferation of rat cardiac fibroblast in culture, whereas telmisartan caused inhibition, apparently in an AT1R-independent manner (Benson et al., 2008). However, the presence of ANG resulted in increased collagen mRNA expression and protein synthesis (Brilla et al., 1997; Lijnen et al., 2001; Lijnen et al., 2000; Schorb et al., 1993) as well as enhanced expression of integrins, osteopontin and actininin (Kawano et al., 2000a). Telmisartan reduced ANG but not TGF-β1-induced collagen synthesis in rat cardiofibroblasts (Zhang et al., 2014b). While ANG alone did not affect secretion of matrix metalloproteinase 9 from rat cardiac fibroblasts, it enhanced IL-1-induced secretion thereof (Okada et al., 2010b). In isolated rat cardiac fibroblasts telmisartan and pioglitazone prevented the TNF-induced activity increase of matrix metalloproteinase-2; this was blocked by GW9662 and not mimicked by amlodipine (Nagashima et al., 2012), raising the possibility that some ARBs may have AT1R-independent effects on fibrosis. The above studies were performed with rat cells, but a study in human cardiac fibroblasts confirmed that 53

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ANG increased ERK activation, DNA synthesis, and expression of TGF-β1, laminin and fibronectin; moreover, it enhanced plasminogen activator inhibitor-1 expression, which inhibits metalloproteinases that degrade the extracellular matrix (Kawano et al., 2000b). Such effects were inhibited by candesartan (Brilla et al., 1997), irbesartan (Kawano et al., 2000a; Kawano et al., 2000b), losartan (Schorb et al., 1993; Schorb et al., 1995) or telmisartan (Brilla et al., 1997; Okada et al., 2010b) (Lijnen et al., 2001; Lijnen et al., 2000). In isolated rat heart and in isolated atrial myocytes and fibroblasts, ANG stimulated expression of connective tissue growth factor, an important stimulus for collagen synthesis; these effects were blocked by losartan and telmisartan (Ko et al., 2011). Upon culturing on hydrated collagen gels, rat cardiac fibroblasts exhibited spontaneous and ANG-induced contraction; stimulated, but not basal contraction was inhibited by telmisartan and an AT2R antagonist, P-186 (Lijnen et al., 2001; Lijnen et al., 2000).

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These ANG and AT1R effects on cardiac fibroblasts are of potential importance for cardiac fibrosis but may also be relevant for cardiac hypertrophy as evidenced by studies using coculturing of cardiac myocytes and fibroblasts. Thus, early studies demonstrated that ANG had little effect on protein synthesis of cardiomyocytes, but conditioned medium from ANGtreated fibroblasts enhanced protein synthesis; this was prevented by addition of losartan to the conditioned medium (Kim et al., 1995a). In a similar vein, other investigators reported that the ANG-induced protein synthesis in cardiomyocytes largely disappeared when a purified preparation was used but was restored when non-myocytes were also present (Pönicke et al., 1997). Similarly, stretch-induced cardiomyocyte expression of atrial and brain natriuretic peptides was only evident upon co-culture with non-myocytes and inhibited by candesartan or an endothelin A receptor antagonist (Hara et al., 1997). Stretch-induced neonatal rat cardiomyocyte hypertrophy was more pronounced in co-culture; based on the surrogate marker of production of atrial natriuretic peptide and brain natriuretic peptide, hypertrophy responses were greater in the presence of non-myocytes and blocked by candesartan (Harada et al., 1998b). The interaction of cardiac fibroblasts and cardiomyocytes in promoting cardiac hypertrophy and fibrosis is schematically depicted in Figure 6.

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4.2.2. In vivo models of hypertension- or resistance-associated cardiovascular hypertrophy

Chronic arterial hypertension inevitably leads to cardiac hypertrophy as the heart has to pump against an elevated peripheral resistance. As cardiac hypertrophy is an independent risk factor for morbidity and mortality in hypertensive patients, ARB effects on cardiac hypertrophy have been studied in almost as many animal models of hypertension as their antihypertensive effects. Of note, the hypertension-induced cardiac hypertrophy often is associated with cardiac fibrosis; as this may impair diastolic filling, it has an additional adverse effect on cardiac function. In an early study, a 7 or 14 day ANG infusion increased left ventricular/body weight ratio by 18.6% and 17.3%, respectively; concomitant treatment with losartan prevented this hypertrophy development, whereas hydralazine in doses yielding similar BP lowering or enalapril did not (Dostal & Baker, 1992), providing initial evidence that AT1R blockade may provide anti-hypertrophic effects beyond BP lowering. In a similar study, ANG infusion for up to 10 days induced hypertrophy and fibrosis along with increased expression of fibronectin, collagen type I and IV and atrial natriuretic factor (Crawford et al., 1994). The hypertrophy was abolished by concomitant losartan treatment, whereas hydralazine or prazosin, causing similar or greater BP lowering, did not; in this model of exogenous ANG 54

ACCEPTED MANUSCRIPT administration, trandolapril neither lowered BP nor reduced cardiac hypertrophy. Similar findings were also reported with azilsartan (Carroll et al., 2015). Acute pressure overload induced by a 2 h vasopressin infusion increased atrial and ventricular brain natriuretic peptide expression in SHR; these were blocked by bosentan but not by losartan (Magga et al., 1997).

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4.2.2.1. SHR

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As with the antihypertensive effects, those on cardiac hypertrophy were most often studied in SHR, a model in which ACE inhibitors are well established to reduced cardiac hypertrophy (Huang et al., 2014). Most of these studies assessed cardiac hypertrophy as total heart or left ventricular weight in absolute terms (Inada et al., 1997; Jones et al., 2004; Jones et al., 2012; Kawano et al., 2000a; Mizuno et al., 1994; Paull & Widdop, 2001; Takeda et al., 1994; Vacher et al., 1995; Xie et al., 1999) or relative to body weight (Choisy et al., 2015; Dai et al., 2007; Inada et al., 1997; Ishimitsu et al., 2010; Kagoshima et al., 1994; Oddie et al., 1993; Zhang et al., 1997) but some more recent studies have also used echocardiography, sometimes even in longitudinal assessments (Barone et al., 2001; Behr et al., 2004; Hye Khan et al., 2014a; Kawano et al., 2000a; Kojima et al., 1994; Mukaddam-Daher et al., 2009; Zou et al., 2015). Studies were performed in a preventive approach with ARB administration starting prior to onset of hypertension (Kawano et al., 2000a; Kojima et al., 1994; Mizuno et al., 1992c; Oddie et al., 1992; Vacher et al., 1995) and also in a treatment design with young adult but already hypertensive SHR (Barone et al., 2001; Ishimitsu et al., 2010; Kohya et al., 1995; Oddie et al., 1993; Zou et al., 2015) and older SHR with established hypertension (Choisy et al., 2015; Dai et al., 2007; Hye Khan et al., 2014a; Inada et al., 1997; Kagoshima et al., 1994; Kim et al., 1995c; Koike et al., 2001; Mandarim-de-Lacerda & Pereira, 2004; Mizuno et al., 1994; Mukaddam-Daher et al., 2009; Takeda et al., 1994; Xie et al., 1999; Zhang et al., 1997). Notably, some of these studies reported regression of cardiac hypertrophy even when treatment was started at an age of 20 months (Jones et al., 2004; Jones et al., 2012; Paull & Widdop, 2001). Telmisartan also reduced cardiac hypertrophy in SHR when hypertension had been aggravated by a high-salt diet (Susic & Frohlich, 2014). Moreover, studies were also performed in spontaneously hypertensive obese rats (Hye Khan et al., 2014a), the strokeprone substrain of SHR exhibiting aggravated hypertension (Barone et al., 2001; Behr et al., 2004; Inada et al., 1997; Kim et al., 1995c; Mukaddam-Daher et al., 2009; Xie et al., 1999) or in SHR in which hypertension and hypertrophy had been aggravated by STZ-induced diabetes (Wienen et al., 2001) and in salt-loaded stroke-prone SHR continuously infused with ANG (Kishi et al., 2014). Similar to the BP lowering, the antihypertrophy effects lasted considerably longer than the presence of the ARB can be expected based on pharmacokinetic properties (Oddie et al., 1992). With few exceptions in candesartan studies (Mori et al., 1995), such studies have uniformly reported reduction or prevention of cardiac hypertrophy. Such studies were reported e.g. with azilsartan (Hye Khan et al., 2014a), candesartan (Choisy et al., 2015; Inada et al., 1997; Ishimitsu et al., 2010; Jones et al., 2004; Jones et al., 2012; Kagoshima et al., 1994; Kim et al., 1995c; Kohya et al., 1995; Kojima et al., 1994; Mizuno et al., 1994; Paull & Widdop, 2001; Takeda et al., 1994; Xie et al., 1999; Zhang et al., 1997), eprosartan (Barone et al., 2001; Behr et al., 2004; Mukaddam-Daher et al., 2009), irbesartan (Vacher et al., 1995), losartan (Dai et al., 2007; Kawano et al., 2000a; Mizuno et al., 1992c; Oddie et al., 1992; Oddie et al., 1993), olmesartan (Koike et al., 2001) and telmisartan (Dai et al., 2007; Mandarim-de-Lacerda & Pereira, 2004; Wagner et al., 1998; Wienen et al., 2001; Zou et al., 2015). However, some data indicate that regression of cardiac hypertrophy with losartan may be limited to BP-lowering doses (Dai et al., 2007). On the other hand, a reduction of cardiac hypertrophy by candesartan in 20 months old SHR was not mimicked by treatment with hydralazine (Jones et al., 2012) or only partially mimicked (Paull & Widdop, 2001). Interestingly, when treatment was withdrawn for 8 weeks, a reduction of left 55

ACCEPTED MANUSCRIPT ventricle/body weight ratio was no longer observed in SHR previously treated with hydralazine but remained detectable in those previously treated with candesartan or perindopril (Paull & Widdop, 2001)

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Some of these studies also revealed an additional potential layer of complexity: treatment with candesartan produced similar BP lowering in the absence and presence of concomitant administration of the AT2R antagonist PD123319, but only candesartan monotherapy reduced cardiac hypertrophy (Jones et al., 2004; Jones et al., 2012). On the other hand, such apparent interaction between AT1R and AT2R blockade was not observed in 20 week old SHR (Jones et al., 2012). Treatment with valsartan or enalapril but not with felodipine also reduced cardiac hypertrophy in New Zealand genetically hypertensive rats (Ledingham et al., 2000), supporting the view that anti-hypertrophic effect are specific for RAS inhibition and not simply a reflection of BP lowering.

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Similar protection was also provided against cardiac fibrosis in many studies in SHR (Ishimitsu et al., 2010; Kagoshima et al., 1994; Kojima et al., 1994; Mandarim-de-Lacerda & Pereira, 2004), including that aggravated by concomitant L-NAME treatment (Akashiba et al., 2008). Moreover, biochemical indicators of fibrosis such as expression of collagen, fibronectin, integrins, osteopontin or α-actinin, which are elevated in SHR, were also normalized by ARBs (Kawano et al., 2000a; Kim et al., 1995c; Zou et al., 2015), including a study in which a dose of amlodipine providing similar BP reduction did not prevent cardiac collagen disposition (Zou et al., 2015). However, one study with a 14-week treatment with candesartan starting at an age of 32 weeks did not detect regression of fibrosis in the atrium (Choisy et al., 2015).

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Several studies were performed to explore the relative roles of RAS inhibition vs. BP reduction or of AT1R antagonism vs. ACE inhibition for reduction or prevention of cardiac hypertrophy and fibrosis (Table 3). In such studies ARBs performed consistently better than hydralazine (Kagoshima et al., 1994; Kawano et al., 2000a; Kohya et al., 1995; Kojima et al., 1994; Zhang et al., 1997) or hydrochlorothiazide (Zhang et al., 1997) when studied at doses yielding comparable BP reduction, supporting the specific role of the RAS in cardiac hypertrophy development; although in some of these studies hydralazine was not only less effective for hypertrophy reversal but also for BP reduction (Choisy et al., 2015). In one study the anti-hypertrophic effect of telmisartan was not mimicked by an amlodipine dose yielding comparable BP lowering (Zou et al., 2015). While in one study candesartan was less effective than lisinopril (Mori et al., 1995) or telmisartan more effective than captopril (Wagner et al., 1998), the overall picture supports that ARBs and ACEIs have similar effects on cardiac hypertrophy, e.g. in studies comparing candesartan with captopril (Kohya et al., 1995), derapril (Zhang et al., 1997) or enalapril (Inada et al., 1997; Kagoshima et al., 1994) in SHR or telmisartan with lisinopril in diabetic SHR (Wienen et al., 2001). One study reported greater anti-hypertrophy effects of telmisartan as compared to losartan in SHR (Wagner et al., 1998). As ARB treatment lowered cardiac but increased plasma ANG levels it was proposed that part of their anti-hypertrophic action may relate to the tissue rather than systemic RAS (Mizuno et al., 1992c; Mizuno et al., 1994). Taken together these studies demonstrate that ARBs as a class prevent and reduce cardiac hypertrophy more than antihypertensive drugs not acting on the RAS and to a comparable degree as ACE inhibitors. Findings consistent with this conclusion were also obtained in other animal models of hereditary hypertension, e.g. irbesartan in SHHF/Mcc-facp rats (Carraway et al., 1999) or valsartan in New Zealand genetically hypertensive rats (Ledingham et al., 2000). <<>> 56

ACCEPTED MANUSCRIPT 4.2.2.2. Renal hypertension

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Iin Goldblatt renal hypertensive rats, treatment with candesartan or perindopril similarly reversed cardiac hypertrophy and fibrosis as well as atrial natriuretic peptide expression (Nagai et al., 2004). In a follow-up study, treatment with an anti-hypertensive and a low BPneutral telmisartan dose equally reduced cardiac hypertrophy and fibrosis (Kawai et al., 2009), as confirmed by other investigators in the same model (Chaykovska et al., 2013; Ye et al., 2010). In a 2-kidney-2-clip rat model, valsartan improved cardiac function and reduced cardiac hypertrophy and fibrosis as well as mRNA levels of several matrix metalloproteinases (Fang et al., 2010). In rats with 5/6 nephrectomy, eprosartan attenuated cardiac hypertrophy development but did not significantly alter the modestly increased mRNA levels of TGF-β or fibronectin (Wong et al., 2000). Telmisartan was also reported to prevent cardiac hypertrophy in rats with uninephrectomy additionally receiving an ethylene glycol injection (Kadhare et al., 2015). Taken together these data extend the findings on beneficial ARB effects on cardiac hypertrophy and fibrosis from SHR to models of renal hypertension.

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4.2.2.3. Low renin models

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Low renin models of hypertension are interesting for studies on cardiac hypertrophy as they exhibit limited ARB effects on BP (see section 2.4. and 2.5.) and accordingly offer another possibility to study BP-independent effects of AT1R inhibition. In Dahl salt-sensitive rats on a high sodium diet, candesartan had little effect on cardiac hypertrophy in one study (Sugimoto et al., 1996), whereas in another study using this model both candesartan and captopril reduced cardiac hypertrophy to a similar extent (Ideishi et al., 1994). Azilsartan reduced cardiac hypertrophy in Dahl salt-sensitive rats on a high-salt, high-fat diet (Jin et al., 2014). Others reported that both losartan and telmisartan prevented cardiac hypertrophy and fibrosis in Dahl salt-sensitive rats on a high-salt diet (Liang & Leenen, 2008). The antihypertrophic and antifibrotic effects of telmisartan as well as those on expression of endothelial NO synthase were reported to be attenuated upon cotreatment with GW9662 (Kobayashi et al., 2008), indicating an additional role for PPAR-γ activation. Valsartan also reduced cardiac hypertrophy in Dahl salt-sensitive rats on a high-salt diet (Nagasawa et al., 2015). While addition of the two Ca2+ entry blockers had no major effects on overall cardiac hypertrophy, their addition yielded greater reduction in cardiomyocyte cross-sectional area, interstitial fibrosis, collagen content and macrophage infiltration; in this regard, addition of cilnidipine had greater effects on fibrosis, collagen content and macrophage infiltration than amlodipine. The more pronounced effects of cilnidipine were attributed to a greater inhibition of cardiac ACE, AT1R and mineralocorticoid receptor expression as well as urinary dopamine and adrenaline excretion. In rats with hypertension induced by treatment with DOCA, candesartan did not affect BP but prevented the development of cardiac hypertrophy (Fujita et al., 1997). Both candesartan and captopril reversed cardiac fibrosis, collagen and fibronectin expression and diastolic stiffness with treatment starting two weeks after that of mineralocorticoid administration (Brown et al., 1999). Taken together these findings further corroborate the concept that antihypertrophic and antifibrotic effects of ARBs in the heart occur at least partly independent of those on BP. 4.2.2.4. Surgically induced models

Cardiac hypertrophy can be induced surgically by procedures causing pressure or volume overload of the heart. The most frequently used surgical model of pressure overload is based 57

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on banding of the ascending (Weinberg et al., 1997), transverse (Li et al., 2012a; Li et al., 2010a; Müller et al., 2013; Quinn Baumann et al., 2013), or abdominal aorta (Aswar et al., 2013; Guan et al., 2011; Linz et al., 1991; Liu et al., 2010; Mukawa et al., 1997; Obayashi et al., 1997; Wang et al., 2008; Zhang et al., 2014a). In most cases the start of RAS inhibitor treatment was before or right after induction of pressure overload (Li et al., 2010a; Linz et al., 1991; Mukawa et al., 1997; Müller et al., 2013; Obayashi et al., 1997; Obayashi et al., 1999; Quinn Baumann et al., 2013), but in some cases it has also been applied one to six weeks after the surgical procedure (Aswar et al., 2013; Guan et al., 2011; Linz et al., 1991; Liu et al., 2010; Weinberg et al., 1997; Zhang et al., 2014a). Most all ARB pressure overload studies have used rats, but in some cases similar models were also applied to mice (Li et al., 2012a; Li et al., 2010a; Müller et al., 2013; Quinn Baumann et al., 2013).

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In one study the chosen losartan dose caused greater BP reduction than ramipril, but ramipril exhibited a greater beneficial effect on cardiac hypertrophy than losartan, both in a preventive and in a treatment design (Linz et al., 1991). In a later study, however, candesartan and quinapril had similar effects on left ventricular hypertrophy in a preventive design but only when either was used in a BP lowering dose; across all study groups, left ventricular weight and BP at end of treatment were well correlated, indicating a major role for BP lowering in the anti-hypertrophic effects in this model (Mukawa et al., 1997). Other studies confirmed that BP-lowering doses of candesartan reduced left ventricular hypertrophy and fibrosis in a preventive design aortic banding model (Obayashi et al., 1997; Obayashi et al., 1999). Using in vivo pressure measurements as well as ex vivo cardiomyocyte morphometry, they found that ARB treatment reduced not only the width but also the length of myocytes, leading to a reduction of midwall radius and hence wall stress, which in turn may contribute to a preservation of cardiac output. An antihypertensive dose of telmisartan, started one week after aortic banding, reduced not only left ventricular hypertrophy but also reversed the changed cardiac expression patterns in a proteomics analysis (Liu et al., 2010). In other studies in this model telmisartan also reduced cardiac hypertrophy and fibrosis; additionally, a reduced cardiomyocyte apoptosis and endplasmic reticulum stress markers were observed (Guan et al., 2011; Zhang et al., 2014a). Irbesartan or amlodipine started six weeks after surgery and maintained for another 15 weeks at doses not lowering BP did not reduce cardiac hypertrophy but improved left ventricular end-diastolic pressure (Weinberg et al., 1997). An interesting mouse study has used transverse aorta constriction in wild-type and AT1R knock-out animals, and compared effects of five ARBs (candesartan, losartan, olmesartan, telmisartan and valsartan) in both strains (Li et al., 2010a). Aortic banding increased heart/body weight and cross-sectional area of individual cardiomyocytes and altered cardiac gene expression to a similar extent in wild-type and knock-out mice, raising the possibility that endogenous ANG may not be a pathogenic factor in this mouse model. All five ARBs reduced cardiac hypertrophy to levels close to those in sham-operated mice, whereas crosssectional cardiomyocyte area was only partly normalized. While effects of candesartan, losartan and olmesartan were fully maintained in the knock-out mice, those of telmisartan and valsartan were attenuated. These differential effects of ARBs, particularly in the knock-out mice, are difficult to understand when considering their pharmacological profile with regard to AT1R-independent effects (Michel et al., 2013) and may more likely reflect specific experimental circumstances including relative differences in doses than true biological differences. In similar mouse models, but restricted to wild-type animals, a BP-lowering doses of azilsartan (Quinn Baumann et al., 2013) and telmisartan (Li et al., 2012a) reduced overall cardiac hypertrophy and fibrosis; the abstract of the latter report also claims a significant reduction in mortality, but corresponding data were missing in the manuscript. A third study in this model reported that telmisartan and ramipril both prevented development of cardiac 58

ACCEPTED MANUSCRIPT hypertrophy and fibrosis; while telmisartan effects were slightly greater, it also exhibited somewhat greater BP reductions (Müller et al., 2013).

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Animal models of cardiac volume overload have largely been based on aorto-caval shunts. In one study a BP-lowering olmesartan dose prevented the development of cardiac hypertrophy and concomitantly the up-regulation of atrial natriuretic peptide and collagen III and the down-regulation of Ca2+-ATPase; the chosen dose of temocapril as comparator had smaller effects on most of these parameters but also produced less BP lowering, which makes it difficult to exclude that the differences were due to relative underdosing of the ACEI (Kim et al., 1997). Antihypertrophic effects of olmesartan in this model were confirmed in a later study (Koike et al., 2001). In a similar model eprosartan also reduced heart hypertrophy to a greater extent than enalapril, but BP data were not provided to place this into perspective (Brodsky et al., 1998). A BP-lowering dose of candesartan also partly prevented cardiac hypertrophy in an aorto-caval shunt model of volume overload; it also normalized expression of several genes, including several involved in cellular Ca2+ handlng (Hashida et al., 1999).

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In conclusion, ARBs similar to ACEIs prevent or reduce cardiac hypertrophy and fibrosis induced by surgically induced pressure or volume overload. As these effects were seen only with BP-lowering doses in most studies, they may largely be due to their BP lowering effects; this contrasts the findings in the SHR and renal hypertensive models. However, even in the absence of beneficial effects on BP or hypertrophy, they may improve cardiac function.

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4.2.2.5. Transgenic and knock-out models

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Several studies have explored ARB effects in (mRen-2)27 transgenic rats. Of note, RAS activation is no longer controlled by the physiological negative feed-back loops. Thus, the degree of RAS activation in such animals by far exceeds what is observed in normal animals or under any pathophysiological condition, necessating caution in extrapolating findings from these transgenic rats to other conditions. In a study with telmisartan, even a dose not affecting BP reduced cardiac hypertrophy in this model (Böhm et al., 1995). Similarly, eprosartan also reduced cardiac hypertrophy and normalized sarcoplasmic Ca2+ handling in this model in doses not lowering BP; however, reduction of fibrosis was only observed at the higher, antihypertensive eprosartan dose (Rothermund et al., 2001). In another study, olmesartan and telmisartan, when given in equally BP-lowering doses had similar effects against heart hypertrophy and fibrosis and improved diastolic function (DeMarco et al., 2011). In double transgenic mice harbouring the human renin and angiotensinogen genes, azilsartan and olmesartan reduced cardiac hypertrophy (Iwanami et al., 2014). In mice with hypertension due to knock-out of the endothelial NO synthase, valsartan and the direct renin inhibitor aliskiren markedly and similarly suppressed cardiac hypertrophy, inflammation and fibrosis, as well as coronary remodeling; these effects were not shared by hydralazine, indicating that they are not explained by the anti-hypertensive effects of the two RAS inhibitors (Yamamoto et al., 2009a). 4.2.2.6. Other hypertension and hypotension models

ARB effects on cardiac hypertrophy and fibrosis have also been explored in several other hypertensive models. For instance, in hypertensive, obese and diabetic SHRSP.ZLeprfa/IzmDmcr rats telmisartan lowered BP and restored reduced cardiac expression of phospholamban (Kagota et al., 2013). A 1-3 week cold exposure raised BP and caused cardiac hypertrophy in rats, and losartan treatment blocked this (Fregly et al., 1993). Prolonged infusion of the β-adrenoceptor agonist isoprenaline also caused hypertension and cardiac 59

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hypertrophy and fibrosis in rats, and this was attenuated by candesartan (Omura et al., 1994) or telmisartan (Goyal & Mehta, 2012). In a similar study but with a slightly different design, neither captopril nor telmisartan prevented isoprenaline-induced fibrosis in rat heart, and captopril even worsened it; the latter was linked to an augmentation of increased expression of matrix metalloproteinase 9 by captopril (Okada et al., 2010a). Injection of very high isoprenaline doses on two consecutive days caused reductions of BP and maximal positive left ventricular pressure; a 14 day pre-treatment with valsartan prevented those (Goyal et al., 2011c). Chronic treatment with the NO synthase inhibitor L-NAME also caused hypertension and cardiac hypertrophy and fibrosis, and this was attenuated by treatment with irbesartan (Luvara et al., 1998) or olmesartan (Takemoto et al., 1997). The former also attenuated increased expression of inducible NO synthase or VCAM-1 and ICAM-1 expression. Studies on cardiac hypertrophy and fibrosis occurring after a myocardial infarction will be discussed in section 4.2.3.

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Interestingly, cardiac hypertrophy can not only result from high BP but also as a response to chronic treatment with a strong vasodilator, minoxidil (Ruzicka & Leenen, 1993), possibly due to volume overload. In minoxidil-treated rats, losartan did not decrease left ventricular end-diastolic pressure but prevented cardiac remodeling; in contrast, enalapril improved left ventricular end-diastolic pressure but did not affect cardiac hypertrophy.

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In line with the above experimental data in various animal models of hypertension, clinical data also support that the use of ARBs in management of hypertensive patients offers target organ protection over and above their antihypertensive activity (Silverstein et al., 2004; Thürmann et al., 1998).

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Cardiac hypertrophy and fibrosis often develop after a survived myocardial infarction in order to compensate for the loss of contractile function from the infarcted area. Effects of ARBs on post-infarction hypertrophy and fibrosis have mostly been explored in rats, but some studies were also done in mice (Bonda et al., 2012; Nakamura et al., 2013), rabbits (Makino et al., 2003; Miki et al., 2000) and pigs (van Kats et al., 2000). In most of these studies ARB administration started within one week after the infarction but duration of treatment and follow-up varied between one week and 7.5 months. A prevention of post-infarction cardiac hypertrophy development was observed in most studies, including with azilsartan (Nakamura et al., 2013), candesartan (Hanatani et al., 1995; Nishizawa et al., 1997; Yamagishi et al., 1993), eprosartan (van Kats et al., 2000), irbesartan (Ambrose et al., 1999; Gervais et al., 2000; Richer et al., 1999), losartan (Smits et al., 1992), telmisartan (Nagashima et al., 2012), and valsartan (Makino et al., 2003; Miki et al., 2000; Song et al., 2014) across all species that have been investigated (Figure 7). However, in a dog model based not on coronary artery ligation but on microembolization, valsartan did not affect cardiac hypertrophy development (Tanimura et al., 1999). SHR and other hypertensive animals exhibit baseline cardiac hypertrophy which can be prevented or reduced by ARBs (see section 4.2.2.1.), but ARBs also prevented the additional hypertrophy induced by myocardial infarction in SHR (Nishikimi et al., 1995b; Nishizawa et al., 1997). Importantly, during follow-up for 7.5 months, irbesartan treatment also reduced mortality (Richer et al., 1999). The anti-hypertrophic effects of telmisartan were not attenuated by concomitant treatment with GW9662 (Nagashima et al., 2012). Although prevention of hypertrophy has been reported in several studies with only few weeks of treatment, one study with irbesartan found that this was detectable after 6 months but not after 6 weeks of treatment (Gervais et al., 60

ACCEPTED MANUSCRIPT 2000). Mechanistic studies indicated that the prevention of post-infarction cardiac hypertrophy by ARBs involve normalized expression of atrial natriuretic peptide (Ambrose et al., 1999; Hanatani et al., 1995), TGF-β1 (Hanatani et al., 1995; Yoshiyama et al., 1999), βmyosin heavy chain and α-skeletal actin (Yoshiyama et al., 1999).

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However, ARBs may differentially affect post-infarction cardiac hypertrophy and fibrosis. Thus, valsartan treatment for 6 weeks did not prevent fibrosis despite reducing hypertrophy in rats (Gervais et al., 1999) and neither did a 3-months treatment in a dog microembolization model (Tanimura et al., 1999). Similarly, irbesartan treatment for 7.5 months had little effect on subendocardial fibrosis despite reducing hypertrophy (Richer et al., 1999). Correspondingly, candesartan or cilazapril only inconsistently inhibited the increased expression of collagen I and III after myocardial infarction (Hanatani et al., 1995; Yoshiyama et al., 1999). On the other hand, losartan fully prevented fibrosis induced by myocardial infarction (Smits et al., 1992) and a minor but dose-dependent reduction of fibrosis was observed with azilsartan treatment in a mouse infarction model (Nakamura et al., 2013). Moreover, a 14 day treatment with a BP-lowering dose of telmisartan reduced heart/body weight ratio, cardiomyocyte cross-sectional area, number of matrix metalloproteinase-2 positive cells and fibrosis; while amlodipine or telmisartan in combination with GW9662 yielded similar BP lowering and hypertrophy reduction, these two treatments did not significantly reduce matrix metalloproteinase-2 positive cells or attenuate fibrosis (Nagashima et al., 2012). To mechanistically explain the observed fibrosis prevention with telmisartan, one study in mice demonstrated that during a 12-week follow-up telmisartan prevented the infarction-induced up-regulation of cysteine-rich angiogenic inducer 61 and connective tissue growth factor at the protein level (Bonda et al., 2012), which both are implicated in fibrosis development. Additional experiments in isolated rat cardiac fibroblasts showed that telmisartan, similar to pioglitazone, suppressed TNF-α-induced matrix metalloproteinase-2 activity (Nagashima et al., 2012); this inhibition was abolished by GW9662, indicating that it may relate to the PPAR-γ agonism by telmisartan. This could explain why post-infarction cardiac fibrosis was prevented by ARBs with PPAR-γ agonism including losartan and telmisartan but not other ARBs. Several studies have compared ARB and ACEI effects on post-infarction hypertrophy development. A quantitatively similar effect on hypertrophy was found with candesartan and delapril (Nishikimi et al., 1995b; Yamagishi et al., 1993) or candesartan and cilazapril in rats (Yoshiyama et al., 1999) or with eprosartan and captopril in pigs (van Kats et al., 2000), indicating that this may be a general feature of RAS inhibition. To explore whether such effects are specific for RAS inhibitors, one study compared telmisartan with amlodipine. In doses yielding similar BP reduction, both also had similar effects on myocyte cross-sectional area and heart/body weight ratio, although this did not reach significance for amlodipine effects on the former (Nagashima et al., 2012). An alternative approach is the comparison of ARB doses with and without BP-lowering effects. In one such study, post-infarction hypertrophy was prevented by a BP lowering but not a BP neutral valsartan dose (Gervais et al., 1999). In another study, a low and a high dose of either candesartan or cilazepril both lowered BP, although to a somewhat different extent, but had quantitatively similar effects on left ventricular weight (Yoshiyama et al., 1999). In conclusions RAS inhibitors as a class appear effective in preventing post-infarction cardiac hypertrophy. While the evidence is not fully conclusive, this can most likely be explained by BP lowering. In contrast to other pro-hypertrophic models, prevention of fibrosis has not 61

ACCEPTED MANUSCRIPT consistently been observed and, in the positive cases, may be AT1R-independent. These alterations along with the lack of contractility of the scar tissue affect cardiac function; such functional effects are discussed in section 4.3.

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While cardiac hypertrophy in most cases is a result of increased hemodynamic demand to the heart, fibrosis is a uniform response of the organism to a variety of causative stimuli. Invariably, this involves chronic inflammation and a complex signaling network, in which TGF as well as some interleukins play crucial roles, leading to activation of fibroblasts and their transition to myofibroblasts and resulting in increased extracellular matrix deposition (Denton et al., 2006; Wynn & Ramalingam, 2012). Accordingly, ARB effects on cardiac fibrosis have been tested in different non-hypertensive models. In a rat STZ model of type 1 diabetes, for instance, irbesartan (Tang et al., 2013) and telmisartan (Goyal et al., 2008) attenuated cardiac hypertrophy and fibrosis. In this model telmisartan also normalized elevated time to peak tension and time to half-maximal relaxation in isolated hearts (Emre et al., 2010). When administered for 8 weeks, starting 5 weeks after STZ injection, valsartan and captopril treatment similarly improved cardiac ultrastructure as well as circulating levels of marker enzymes creatine kinase MB and lactate dehydrogenase (Zhang et al., 2008b). Valsartan treatment, starting directly after the STZ injection, also improved cardiac fibrosis (Wang et al., 2009).

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In a rat type 2 diabetes model, induced by a low dose of STZ on top of a high-fat, high-sugar diet, telmisartan prevented cardiac hypertrophy and improved cardiac function; concomitantly the decreased cardiac expression of adiponectin and its receptor was restored (Guo et al., 2011). Administration of STZ to neonatal rats also can be used to mimic type 2 diabetes; in this model, telmisartan reduced cardiac hypertrophy and fibrosis (Goyal et al., 2011b). In other type 2 diabetes rat models, valsartan partly reversed cardiac hypertrophy, fibrosis and apoptotic index (Yang & Peng, 2010), irbesartan reduced hypertrophy and fibrosis (Tang et al., 2013). Telmisartan also reduced cardiac hypertrophy in a type 2 diabetes model; moreover, although it had no effect on atrial weight, it at least partly partly prevented the enhancement of negative inotropic effects of adenosine in isolated atria while not improving the depressed positive inotropic effect of phenylephrine (Hussein et al., 2011). Telmisartan (Sukumaran et al., 2012) and valsartan (Liu et al., 2009b) also improved cardiac hypertrophy, fibrosis and inflammation in a rat model of auto-immune myocarditis, induced by immunization against cardiac myosin. Similarly, candesartan improved cardiac hypertrophy and inflammation in mice which had been inoculated with encephalomyocarditis virus without affecting viral replication; importantly, in this model the ARB at least numerically improved survival (Tanaka et al., 1994). The cardiac hypertrophy and fibrosis induced by a high dose of L-thyroxin was alleviated by imidapril and valsartan (Zhang et al., 2007). Candesartan and captopril similarly reduced fibrosis in a mouse model of heterotopic heart transplantation between MHC-antigen compatible strains (Furukawa et al., 1996). Losartan reduced cardiac hypertrophy and fibrosis in transgenic mice overexpressing calsequestrin (Günther et al., 2010). Given the crucial role of TGF-β in the development of fibrosis (Denton et al., 2006; Wynn & Ramalingam, 2012), it is not surprising that mice with transgenic overexpression of TGF-β develop cardiac hypertrophy and fibrosis. In such mice telmisartan reduced both hypertrophy and fibrosis by normalizing the ratio between matrix metalloproteinases and their endogenous inhibitors, TIMP-1and TIMP-4; in contrast, metoprolol or a TGF antibody had antihypertrophic but little antifibrotic effects in this model (Seeland et al., 2009). In a follow62

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up study, however, metoprolol appeared more effective than telmisartan (Huntgeburth et al., 2011). Moreover, effects by additional β-adrenoceptor stimulation with isoprenaline were attenuated by metoprolol but not by telmisartan. Caveolin-1 knock-out mice spontaneously develop cardiac hypertrophy and fibrosis; telmisartan treatment reduced the hypertrophy, collagen expression and associated perivascular fibrosis of intramyocardial vessels (Krieger et al., 2010).

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While not directly related to cardiomyocyte function, myxomatous degeneration of the mitral valve is a pathological hallmark of mitral valve prolapse; in cultured human valvular interstitial cells, TGF-β produced extracellular matrix production, which was abolished by candesartan, losartan and telmisartan (Geirsson et al., 2012). Similarly, pericardial thickening and fibrosis can be a problem in patients having undergone cardiac surgery; the pericardial fibrosis induced by percardiectomy was attenuated by valsartan in a pig model (Loging et al., 1999).

Myocardial infarction

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Taken together these data demonstrate that ARBs can prevent and reduce cardiac hypertrophy and fibrosis in a broad range of experimental models. In many cases these effects can already be observed in doses not lowering BP, and the effects of BP-lowering doses typically considerably exceed those observed with other antihypertensive drugs. This leads to improved cardiac function, particularly diastolic function. Possible exceptions are models of cardiac pressure or volume overload, where the beneficial ARB effects are largely BP-dependent.

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ARB effects on myocardial infarction have been studied in several settings. One group of studies has tested whether pre-treatment with an ARB affects damage and recovery after myocardial infarction; this may relate to the clinical question whether patients on ARB treatment will incur less damage if a heart attack occurs (see section 4.3.1). A second group of studies has applied ARBs immediately (within few hours to one day) after myocardial infarction, mimicking a situation where a patient receives acute treatment in an intensive care unit (see section 4.3.2.). Findings of an up-regulation of both AT1R and AT2R in the infarcted and the non-infarcted parts of the heart (Nio et al., 1995) or of increased ANG in the scar (but not the overall heart) (Yamagishi et al., 1993), provide additional rationale for such studies. A third group of studies has explored how ARB treatment started several days or later after an infarction may affect chronic consequences, which could be interpreted as models of secondary prevention (see section 4.3.3.). Some of the studies from all three groups have investigated ARB effects on long-term consequences of myocardial infarction; while the functional consequences are discussed in this section, those on cardiac hypertrophy and fibrosis were discussed in section 4.2.3. Effects on the prevention of heart failure are discussed in section 4.4. Finally, some studies have explored whether ARBs interfere with ischemic preconditioning (see section 4.3.4.). Most studies in this field have used rat models, but some were based on mice (Chen et al., 2014; Higashikuni et al., 2012; Xu et al., 2009; Ye et al., 2011; Zeng et al., 2013b), rabbits (Hartman et al., 1993; Zhao et al., 2009), pigs (Schmermund et al., 2000) or dogs (Dörge et al., 1999; Jalowy et al., 1998; Nakai et al., 1999; Preckel et al., 2000). Most studies were based coronary ligation, but some have also applied other models such as microembolization (Schmermund et al., 2000; Tanimura et al., 1999), exposure to hypoxia (Maejima et al., 2011; Zeng et al., 2013b) or to a high dose of isoprenaline (Bayir et al., 2012; Goyal et al., 2011c). While most studies were performed in in vivo settings, including open-chest preparations (Preckel et al., 2000), some have also used in vitro models including isolated cardiomyocytes 63

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(Maejima et al., 2011; Matoba et al., 1999) or isolated heart preparations (Hartman et al., 1993; Kim et al., 2012a; Shimizu et al., 1999; Yoshiyama et al., 1994). Of note, such in vitro models mostly have used hypoxia to mimic myocardial infarction, but hypoxia mimics only the oxygen supply side of the event and not the lack of removal of metabolic breakdown products. Moreover, some studies have used only short periods of myocardial ischemia to induce stunning rather than full-fledge myocardial infarction (Dörge et al., 1999; Nakai et al., 1999). Finally, some studies have explored ARB effects in models with underlying other disease such as type 1 (Goyal et al., 2010; Goyal et al., 2011d) or type 2 diabetes (Rinaldi et al., 2012; Ye et al., 2011). 4.3.1. Preventive ARB administration

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A study acutely exposed rat neonatal cardiomyocytes to candesartan, cilazepril or bradykinin, followed by 5.5 h of hypoxia and 1 h of reoxygenation (Matoba et al., 1999). While cilazepril and bradykinin reduced hypoxia-induced release of creatine kinase, candesartan did not; moreover, icatibant prevented the cilazepril effects, suggesting that protective ACEI effects in this in vitro model are due to an increased bradykinin exposure and not to RAS inhibition. Other in vitro studies have used rat isolated heart in a Langendorff preparation. In one study, rats were treated for one week with candesartan or delapril; thereafter, ischemia was induced in isolated hearts (Yoshiyama et al., 1994). Both treatments similarly reduced release of creatine kinase and improved post-ischemia cardiac function; injection of ANG prior to ischemia worsened cardiac damage, and such worsening was attenuated only by the ARB. Other in vitro studies did not rely on in vivo pre-treatment but rather directly exposed the isolated heart to ARBs. Under such conditions, high concentrations of neither valsartan (100 µM) nor enalapril (20 µM) given immediately prior to ischemia induction affected the impaired cardiac function during the reperfusion period in a rat Langendorff preparation (Hayashi et al., 1997a). On the other hand, 100 nM candesartan improved the double-product in ischemia and reduced the no-flow area; 1 nM candesartan, however, was ineffective (Shimizu et al., 1999). Similarly, acute exposure to losartan reduced infarct size in an isolated rat heart preparation, and also attenuated the infarct-increasing effect of icatibant (Sato et al., 2000). In vivo treatment with candesartan for 2 weeks reduced infarct size due to ischemia in isolated mouse heart, whereas ramipril treatment did not (Lange et al., 2007). Similarly, a 2-week pretreatment with valsartan reduced infarct size in an in vivo ischemia/reperfusion setting (Yang et al., 2009). Pre-treatment with telmisartan reduced infarct size in Zucker diabetic fatty rats and improved systolic function as assessed 2 h after the ischemia period (Di Filippo et al., 2014). However, one study found that telmisartan (but not losartan) in concentrations of 30 µM and more actually induced infarction; this was apparently due to calcium overload caused by inactivation of voltage-gated Na+ channel (Kim et al., 2012a). Based on a telmisartan affinity of 5 nM for the AT1R (Michel et al., 2013), this finding based on 60,000 fold therapeutic concentration may not be applicable to a therapeutic setting. The cardioplegic solution applied during open heart surgery can also be considered a model of cardiac ischemia. When this was applied to rabbit heart in the absence and presence of RAS inhibition, losartan exhibited much smaller cardioprotective effects than captopril (Hoyer et al., 2014). In another rabbit study, not reporting on infarct size, pre-treatment with candesartan or telmisartan reduced myocardial neutrophil infiltration and increased cardiac microvessel cross-sectional area as observed after a 60 minutes ischemia period (Zeng et al., 2013a).

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Short periods of ischemia often do not cause myocardial infarction but a reversible state of impaired heart function, stunning. In a dog study, both telmisartan and enalapril enhanced fractional shortening during the reperfusion period following a 10 min coronary artery ligation; while icatibant prevented the protection by enalapril, it did not affect that by telmisartan (Nakai et al., 1999). Whether this reflected inconsistent effects of RAS inhibition or a non-AT1R effect of telmisartan, has not been explored. In another dog study, balloon pumps were used to maintain stable systemic hemodynamics and regional myocardial blood flow during a short period of ischemia; under these conditions candesartan improved left ventricular function (Dörge et al., 1999).

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The key questions regarding preventive ARB administration and myocardial infarction are whether it reduces the incidence of heart attacks and whether the severity (infarct size) of the remaining ones is reduced. While addressing the first one is difficult in animal models, the second one has frequently been explored, mostly based on coronary artery occlusion models; however, the answer to this question has been equivocal. Thus, in a mouse model neither AT1R knock-out nor pre-treatment with candesartan affected infarct size (Harada et al., 1998a). Similarly, treatment with irbesartan starting 24 h before ligation and maintained for 6 weeks did not reduce the size of transmural infarction in rats as determined histologically at study end (Ambrose et al., 1999). In a dog study using occlusion of the left anterior descending artery in an open-chest preparation, acute irbesartan pre-treatment increased collateral blood flow to ischemia area but the observed reduction in infarct size did not reach significance (Preckel et al., 2000). On the other hand, a 2-week pre-treatment with two doses of valsartan reduced infarct size in rats by about half (Yang et al., 2009). This was accompanied by inhibition of release of creatine kinase and lactate dehydrogenase, TNF-α and IL-6 levels and of TLR4 and NF-κB expression. Similarly, in a pig model, acute pretreatment with candesartan also reduced infarct size by about half (Jalowy et al., 1998). In that study, the AT2R antagonist PD123319, the bradykinin B2 receptor antagonist icatibant or the cyclooxygenase inhibitor indomethacin had no effects on infarct size when given alone but abolished the reduction by candesartan. In diabetic db/db mice on a Western diet, valsartan, aliskiren and their combination reduced infarct size in a dose-dependent manner; however, this reduction did not reach statistical significance for the lower valsartan dose, and all treatments were less effective than in non-diabetic mice (Ye et al., 2011). In Zucker diabetic fatty rats, pre-treatment with telmisartan also dose-dependently reduced infarct size (Rinaldi et al., 2012). This was accompanied by lowered levels of plasma and cardiac adiponectin and a reduced expression of inflammation markers NF-κB, TLR2, TLR4 and TNF-α; the increased expression of inducible NO synthase was abolished by telmisartan. The inconclusive picture emerging from these studies is not easily explained by difference in species, ischemia protocol or specific aspects of the ARB under investigation. Therefore, it remains unresolved whether pre-treatment with an ARB reduces infarct size. However, even in studies where infarct size was not reduced by pre-treatment with an ARB, myocardial function was improved. For instance, irbesartan normalized the increased expression of atrial natriuretic peptide and reduced cardiac hypertrophy in a rat model despite a lack of reduction of infarct size (Ambrose et al., 1999). As an alternative to coronary artery ligation, myocardial ischemia can also be induced by microembolization. When such a model was applied to pigs, irbesartan administered immediately before the microspheres increased the ratio of regional intramyocardial blood volume and perfusion (assessed by electron-beam computed tomography) (Schmermund et al., 2000). When irbesartan treatment was continued for 4 weeks, this ratio was also moderately increased, along with an increase in ejection fraction; of note, this is a model in which ischemia did not lead to cardiac hypertrophy. While not involving ischemia, chronic 65

ACCEPTED MANUSCRIPT intermittent hypoxia caused oxidative stress in mouse heart, and this was attenuated by telmisartan (Zeng et al., 2013b). Moreover, telmisartan also reduced excessive NO formation, IL-6 release and apoptosis in the heart of rats exposed to intermittent hypoxia (Yuan et al., 2015).

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An alternative to coronary artery occlusion or microembolization is the induction of myocardial infarction by injection or infusion of a high dose of the β-adrenoceptor agonist isoprenaline. One group has reported a number of studies using this model in rats. In an early study, a 13 day pre-treatment with telmisartan counter-acted the isoprenaline-induced alterations of inotropy, lusitropy and left ventricular end-diastolic pressure, levels of damage markers creatine kinase and lactate dehydrogenase and the oxidative stress markers superoxide dismutase and malondialdehyde (Goyal et al., 2009). In another study of similar design, valsartan pre-treatment for 15 days prior to isoprenaline administration improved myocardial function and levels of creatine kinase, lactate dehydrogenase and anti-oxidant enzymes superoxide dismutase and catalase (Goyal et al., 2011c). To determine whether such protection was specific for ARBs, a general feature of RAS inhibition or a consequence of BP lowering, effects of valsartan, ramipril and lacidipine were compared with isoprenaline administration after 1-2 or after 29-30 days of treatment (Keles et al., 2009). Short and long treatment with all three drugs prevented the isoprenaline-induced reduction of NO, the increase in creatine kinase and lactate dehydrogenase and the DNA damage measured in circulating leukocytes. A later study of similar design but only using a 30 day pre-treatment period supported this conclusion: all three drugs similarly attenuated the isoprenaline-induced increase in superoxide dismutase, catalase and malondialdehyde (Bayir et al., 2012). While these studies indicated the possibility that the protective effects on an ARB may occur secondary to BP lowering, somewhat different conclusions can be derived from using the same model in rats with STZ-induced diabetes. In such studies, telmisartan improved redox status and functional recovery, inhibited Bax expression, and reduced TUNEL-positive cells, necrosis and edema; all of these telmisartan effects were attenuated by GW9662 (Goyal et al., 2010). In a follow-up study, telmisartan normalized BP, left ventricular contractility (assessed as dP/dt) and end-diastolic pressure and reduced infarct size; telmisartan also reduced endogenous antioxidants, creatine kinase and lactate dehydrogenase release, prevented the increase in TNF-α and malondialdehyde, and also reduced Bax expression and TUNELpositive cells, inflammation and myocardiac necrosis; GW9662 when given alone had opposite effects on most of these parameters, and when administered in combination attenuated the effects of telmisartan (Goyal et al., 2011d). These studies do not support the concept that ARB effects in the isoprenaline model of myocardial infarction occur secondary to BP lowering and rather raise the possibility that at least those of telmisartan may occur independent of BP and involve PPAR-γ agonism. Whether the conflicting findings of the valsartan/ramipril/lacidipine and the telmisartan/GW9662 studies can be explained by the presence of diabetes remains to be determined. 4.3.2. Early post-infarction ARB administration

While it remains controversial whether animals receiving ARB treatment exhibit a reduced infarct size (see section 4.3.1.), with exception of one azilsartan study (Nakamura et al., 2013) it has consistently been reported that early post-infarction treatment with an ARB (up to 24 h after the occlusion) does not affect infarct size. For instance, such findings were reported for candesartan (Hanatani et al., 1995; Nio et al., 1995; Yamagishi et al., 1993), elmisartan (Maejima et al., 2011) and valsartan in rats (Hayashi et al., 1997a) and for losartan (Hartman et al., 1993) and valsartan in rabbits (Makino et al., 2003; Miki et al., 2000). Interestingly, one of these studies reported a reduced infarct size by ramipril and a ramipril plus ANG 66

ACCEPTED MANUSCRIPT combination (Hartman et al., 1993), raising the possibility of an AT1R-independent effect of the ACE inhibitor.

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Based on a somewhat unusual study design, it has been reported that valsartan or enalapril when given immediately after coronary ligation in anesthetized dogs lowered BP and heart rate and prevented the rise in left ventricular end-diastolic pressure and the decrease in cardiac output (Yamamoto et al., 1997a). However, most studies have started ARB treatment one day after the infarction. One early study reported that losartan did not restore cardiac output in a rat model, in which captopril had done so in previous experiments by these investigators (Smits et al., 1992). Cardiac output was also unaffected in rats upon either valsartan or enalapril treatment (Hayashi et al., 1997a) or in rabbits upon valsartan or temocapril treatment (Makino et al., 2003), questioning whether this indeed reflects a difference between the two drug classes. However, when other parameters of cardiac function were explored, administration of an ARB early after myocardial infarction typically had beneficial functional effects despite a lack of effect on infarct size (for effects on hypertrophy and fibrosis see section 4.2.3.). Thus, candesartan (Nishikimi et al., 1995b; Yamagishi et al., 1993) and valsartan (Gervais et al., 1999; Hayashi et al., 1997a) reduced left ventricular end-diastolic pressure; candesartan (Yoshiyama et al., 1999), telmisartan (Maejima et al., 2011) and valsartan (Makino et al., 2003) improved left ventricular dilatation, systolic and diastolic function and ejection fraction; candesartan improved fractional shortening (Hanatani et al., 1998); candesartan lowered cardiac expression of atrial natriuretic peptide (Hanatani et al., 1998), and telmisartan lowered plasma levels of brain natriuretic peptide (Maejima et al., 2011). Valsartan treatment reduced elevated plasma noradrenaline and expression of βadrenoceptor kinase and Giα G-proteins and restored lowered β-adrenoceptor density, depressed adenylyl cyclase activity (Makino et al., 2003). Improvement of heart function upon ARB treatment after a myocardial infarction was similarly seen in both normotensive rats and SHR (Nishikimi et al., 1995b). However, one study in a mouse model of myocardial infarction did not observe major telmisartan effects on systolic or diastolic function nor on cardiac expression of atrial natriuretic peptide, but prevented up-regulation of CNN gene expression (Bonda et al., 2015).

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One study explored ARB effects on atrial fibrillation by treating rabbits with valsartan starting immediately after the infarction for a period of 12 weeks (Zhao et al., 2009). In this study, atrial fibrillation was observed in 4 rabbits of the infarction group as compared to 2 each in the control and the valsartan-treated infarction group. Moreover, the mean duration of atrial fibrillation episodes was 39 s in the infarction vs. 9 s in the other two groups. A numerical reduction in the density of IKACh current density in left atrium upon valsartan treatment did not reach statistical significance. Given the overall small numbers, however, these findings are difficult to interpret. In another study, ventricular fibrillation was induced, followed by closed chest resuscitation and defibrillation; telmisartan, administered 10 min after the resuscitation, increased myocardial contractility without affecting cardiac index, systemic or pulmonary vascular resistance, or myocardial perfusion (Strohmenger et al., 1997). In a rat study on coronary artery function after myocardial infarction, a low valsartan dose not affecting BP slightly improved basal left and right ventricular coronary flow and resistance values, but decreased left and right coronary vascular reserve (Gervais et al., 1999). A high valsartan dose decreased BP and slightly improved basal left ventricular flow and resistance values but only the right ventricular coronary vascular reserve was increased. Several studies have explored factors which may explain the observed functional improvements upon ARB administration in the absence of infarct size reduction. For instance, candesartan prevented the up-regulation of cardiac AT1R (Nio et al., 1995) and mitigated the 67

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increased expression of β-myosin heavy chain and α-skeletal actin (Yoshiyama et al., 1999). Telmisartan in doses not lowering BP reduced macrophage infiltration, TUNEL staining, activation of matrix metalloproteinase-2 and -9 and expression of TGF-β1, connective tissue growth factor and osteopontin expression and increased expression of the endogenous matrix metalloproteinase inhibitor TIMP-1 as compared to rats treated with vehicle, losartan or telmisartan plus GW9662 (Maejima et al., 2011). In additional experiments with 24 h in vitro hypoxia, telmisartan increased expression of TIMP-1 and reduced expression of TGF-β1 in neonatal rat cardiomyocytes, whereas it reduced activity of metalloproteinase-2 and -9 and expression connective tissue growth factor and osteopontin in cardiac fibroblasts. As these telmisartan effects were abolished by concomitant GW9662 and not mimicked by losartan, they may not represent class effects but rather specific properties of telmisartan. However, this remains to be confirmed in additional studies.

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Finally, some studies have compared the functional effects of early administration of ARBs with those of other RAS inhibitors or RAS-independent anti-hypertensive drugs. In such studies ARBs and ACEIs had similar effects, for instance candesartan and cilazapril on systolic and diastolic function, left ventricular dilatation and expression of β-myosin heavy chain, α-skeletal actin and atrial natriuretic peptides in rats (Yoshiyama et al., 1999). Candesartan and delapril also had similar effects on left ventricular end-diastolic pressure in both normotensive rats and in SHR (Nishikimi et al., 1995b; Yamagishi et al., 1993). In C57BL/6 mice, valsartan and aliskiren had similar beneficial effects on hemodynamics as assessed by echocardiography, and their combination was even more effective; however, hydralazine did not mimic these effects (Higashikuni et al., 2012), indicating that they may be specific for RAS inhibitors. In other studies valsartan and enalapril in rats (Hayashi et al., 1997a) and valsartan and temocapril in rabbits (Makino et al., 2003) also yielded similar improvement of cardiac function after myocardial infarction. Taken together these data show that early post-infarction administration of ARBs improves functional outcomes in experimental animals despite not affecting infarct size. The relevance of this is demonstrated by a study in which olmesartan increased rupture-free survival in a mouse model, apparently at least in part by reducing post-infarction inflammation and cardiomyocyte apoptosis (Chen et al., 2014). 4.3.3. Late post-infarction ARB administration

It has also been explored how ARBs affect outcomes if treatment was started 1 week or later after the heart attack; however, start of treatment was very heterogeneous in such studies, ranging from 7-21 days after the infarction, which makes comparison of the results difficult. Similar to treatment starting early after myocardial infarction (see section 4.3.2.), ARB administration starting 1 week or later after the event also did not affect infarct size (Nishizawa et al., 1997; Richer et al., 1999; Xu et al., 2009). Nonetheless it typically improved cardiac function and morphology in most studies. One of the few exceptions was use of losartan in rats during days 21-35 after infarction, which did not restore cardiac output, while captopril had done so in a previous study using the same protocol (Smits et al., 1992). For instance, in both normotensive rats and SHR candesartan starting 1 week after ligation and maintained for 3 weeks almost normalized left ventricular end-diastolic pressure as well as right and left ventricular hypertrophy (Nishizawa et al., 1997). Irbesartan treatment in rats starting 1 week after the infarction and maintained for 7.5 months dose-dependently decreased left ventricular end-diastolic pressure whereas cardiac hypertrophy was improved but not normalized with a BP-lowering and a lower, BP neutral dose; moreover, the elevated urinary cGMP excretion was dose-dependently reduced (Richer et al., 1999). In another study irbesartan starting after 8 days was maintained for 6 weeks or 6 months (Gervais et al., 2000); 68

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long-, but not short-term treatment prevented endothelial function decline, opposed right ventricular coronary dilatation reserves (dipyridamole testing) impairment, prevented pericoronary fibrosis development, and improved systemic hemodynamics. In another study, valsartan or ramipril were administered starting 3 weeks after myocardial infarction and maintained for 5 weeks to wild-type and bradykinin B1 receptor knock-out mice (Xu et al., 2009); both drugs reduced left ventricular hypertrophy and myocyte cross-sectional area, collagen deposition and left ventricular diastolic dimension and improved ejection fraction, but all to a smaller extent in the B1 receptor knock-out mice. In dog model where myocardial ischemia had been induced by microembolization, a 3-months treatment with valsartan restored ejection fraction and left ventricular end-systolic and end-diastolic volume (Tanimura et al., 1999).

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Secondary hyperaldosteronism is a frequent consequence of myocardial infarction and may contribute to cardiovascular remodeling. The ANG-induced adrenal aldosterone release can be blocked by various ARBs including azilsartan, irbesartan, losartan, olmesartan or telmisartan whereas AT2R antagonists are ineffective (Miura et al., 2014); however, ANGstimulated aldosterone release is not mediated by canonical AT1R signaling but rather by βarrestin 1 (Lymperopoulos et al., 2009). Based on adrenal-targeted, adenovirus-mediated gene transfer, β-arrestin 1 also appears to mediate hyperaldosteronism in a post-infarction setting (Lymperopoulos et al., 2011). In light of biased agonism exhibited by ARBs (Tilley, 2011; Wilson et al., 2013) and the role of β-arrestin 1 in aldosterone release, it is interesting that in a post-infarction setting in mice candesartan and valsartan but not by irbesartan or losartan reduced hyper-aldosteronism (Lymperopoulos et al., 2011; Lymperopoulos et al., 2014); similarly, candesartan and valsartan had stronger beneficial effects on ejection fraction and isoprenaline-induced inotropy (increase in dP/dtmax). Other than differential tissue penetration and PPAR-γ activation (Michel et al., 2013), this represents an additional mechanism how ARBs can have differential effects.

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Taken together these data demonstrate that ARBs as a class improve cardiac morphology and function in experimental animals even when treatments start a week or later after the event. These beneficial effects are similar to those of other RAS inhibitors. Most importantly, ARBs including irbesartan (Richer et al., 1999) and olmesartan (Koike et al., 2001) improved longterm survival after myocardial infarction; this may require a BP-lowering dose but apparently is unrelated to infarct size. 4.3.4. Ischemic preconditioning

Ischemic preconditioning is an endogenous protective mechanism in which short periods of ischemia protect against tissue damage induced by later, more pronounced ischemia (Ferdinandy et al., 2014). Experimental conditioning studies typically use infarct size due to the later ischemia episode as the primary outcome parameter. Ischemic preconditioning can be mimicked by activation of various receptors, including those for adenosine or bradykinin, coupling to protein kinase C activation with subsequent opening of the mitochondrial ATPsensitive K+ channels (Miki et al., 2000). Members of the mitogen-activated protein kinase family also appear to be important mediators of such preconditioning (Michel et al., 2001). As AT1R stimulation can activate protein kinase C and mitogen-activated protein kinases (Michel et al., 2013), ANG-induced preconditioning has been explored. Indeed, exogenously administered ANG induced some degree of preconditioning in isolated rabbit heart (Liu et al., 1995; Miki et al., 2000; Sharma & Singh, 1999); this was prevented by ARBs including candesartan and losartan, but not by an AT2R antagonist, indicating mediation via AT1R. Moreover, the ANG-induced preconditioning was no longer detected in rabbit hearts with 69

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hypertrophy due to a coronary artery ligation two weeks earlier (Miki et al., 2000). Endogenous ANG apparently is also capable of cardiac preconditioning, as the degree of preconditioning observed in transgenic (mREN-2)-27 rats was greater than in normotensive Sprague-Dawley rats (Randall et al., 1997). Accordingly, candesartan attenuated ANGinduced preconditioning in isolated rat heart; this was similarly observed whether infarct size or ischemia-induced release of lactate dehydrogenase or creatine kinase was assessed (Sharma & Singh, 1999). However, if ANG is given on top of ischemic preconditioning, it may abolish the protective effect of preconditioning, an effect sensitive to losartan (Xiao & Allen, 2003).

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Based on reports of ANG-induced preconditioning, it may be assumed that ARBs could have the opposite effect. Indeed it was reported from a rabbit in vivo model that candesartan, given 20 min prior to the initial ischemia period, attenuated preconditioning by a 3 or 30 min ischemia episode (Nakano et al., 1997). However, in isolated rat heart, acute exposure to candesartan (Sharma & Singh, 1999) or losartan (Xiao & Allen, 2003) had no effect on ischemic preconditioning. Similarly, a 2-week pretreatment with candesartan in an in vivo mouse model did not enhance the protection offered by ischemic preconditioning; however, these data are not easy to interpret as candesartan alone reduced infarct size to a similar degree as ischemic preconditioning alone (Lange et al., 2007). On the other hand, a 1-week in vivo pre-treatment with losartan enhanced the effect of cardioprotective effects of ischemic preconditioning as assessed in isolated heart (Butler et al., 1999). This study had included an additional group of rats with high-salt diet-induced cardiac hypertrophy; in these animals the effect of preconditioning was greater than in control rats, but similarly enhanced by losartan (Butler et al., 1999). Finally, a study in rabbits has used a rather complex design (Miki et al., 2000). The animals underwent a sham procedure, coronary artery ligation or ligation with subsequent valsartan treatment. Two weeks later, hearts were isolated, mounted in a Langendorff set-up and exposed to ischemic preconditioning; at this time, ligated but not ligated rats with valsartan treatment had developed cardiac hypertrophy. In vitro ischemic preconditioning reduced infarct size in control rabbits but not in those with cardiac hypertrophy; in valsartan-treated rats, effects of preconditioning were observed but to a smaller extent than in control animals. Taken together, ischemic preconditioning can be mimicked by exogenous ANG acting via AT1R. However, in most studies ARBs did not attenuate effects of ischemic preconditioning; in some cases with chronic pre-treatment ARBs even enhanced cardiac protection by preconditioning.

Heart failure

Congestive heart failure (CHF) often has a poor prognosis, which is worse than that of many malignancies. It is a condition with many possible causes including chronic arterial hypertension, acutely survived myocardial infarction and viral infections; moreover, in many cases a specific cause cannot be identified, which are referred to as idiopathic. Based on previous knowledge of beneficial effects of ACE inhibitors in patients with CHF and animal models thereof, numerous studies have been performed to explore ARB effects in animal models of CHF; many of them were specifically designed to compare ARB and ACE inhibitor effects. In line with the many causes of CHF, a wide range of models were used in such research (Hongo et al., 1997); while most of them were done in rats, mice (Chen et al., 2014; Tarikuz Zaman et al., 2013) including genetically modified ones (Günther et al., 2010), hamsters (Nakamura et al., 1994a; Taniyama et al., 2000; Trippodo et al., 1995), rabbits (Makino et al., 2003), dogs (Spinale et al., 1997b; Suzuki et al., 2001; Tanimura et al., 1999; Yamamoto et al., 1997a), pigs (Clair et al., 2000; Krombach et al., 1998; Spinale et al., 1997c) and sheep (Fitzpatrick et al., 1992) were also used. ARB effects on cardiac hypertrophy and fibrosis as well as long-term functional outcomes after a myocardial infarction have been 70

ACCEPTED MANUSCRIPT discussed in sections 4.2 and 4.3; this section will focus on in vivo studies in animal models of CHF.

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The Syrian hamster is a species genetically prone to develop CHF; specifically several inbred strains including BIO14.6, BIO53.58, BIO TO2 or UM-X 7.1 are often used as an animal model of CHF and its treatment. They are also suitable for ARB studies as they exhibit elevated plasma ANG (Nakamura et al., 1994a). Confirming earlier findings with an ACE inhibitor and a neutral endopeptidase inhibitor, acute administration of irbesartan or an endopeptidas inhibitor did not improve left ventricular end-diastolic pressure, whereas their combination did (Trippodo et al., 1995). An 8-week treatment with candesartan did not affect heart function in control hamsters as assessed in a Langendorff preparation but improved inotropic function in those with CHF (Nakamura et al., 1994a). However, candesartan did not improve the lowered body weight or cardiac hypertrophy in this study. In another study, cardiomyopathic hamsters were treated for 8 weeks with olmesartan, temocapril, hydralazine or vehicle (Taniyama et al., 2000). While both RAS inhibitors restored lowered cardiac expression of hepatocyte growth factor and reduced elevated collagen expression and fibrotic area, hydralazine did not share these effects at a dose producing at least as much BP lowering as olmesartan or temocapril, indicating that these beneficial effects were not a consequence of hemodynamic alterations. Valsartan and the renin inhibitor aliskiren similarly improved cardiac function but their combination did not yield additional improvement (Crespo et al., 2012), indicating that this BP-independent effect may be shared by RAS inhibitors in general. Transgenic mice with overexpression of calsequestrin also spontaneously develop CHF; in such mice losartan treatment reduced cardiac hypertrophy and fibrosis, improved cardiac function and, most importantly, reduced mortality resulting in an almost doubling of lifespan (Günther et al., 2010).

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Continued (mostly 3-5 weeks) and substantial elevation of heart rate (more than 200 beats per minute) based on electrical pacing also is a frequently applied CHF model, most often used in large animals such as dogs and pigs. The model is characterized by an increased left ventricular pre- and after-load, low cardiac output and left ventricular dilatation (Suzuki et al., 2001). It has been used in preventive designs, i.e. with ARB administration concomitant with pacing, or in a treatment design, i.e. with acute or chronic ARB treatment starting after CHF had developed. In a preventive setting, concomitant irbesartan treatment for 3 weeks reduced left ventricular end-diastolic dimension and peak wall stress (Clair et al., 2000). Within that study a vasopressin V1a receptor antagonist had similar effects; however, only combination treatment increased left ventricular fractional shortening. In a dog pacing model valsartan dose-dependently prevented the increase in left ventricular end-diastolic pressure and, at least in the higher dose, the decline of inotropy (assessed as dp/dt) (Yamamoto et al., 1997a). Spinale and colleagues reported effects of valsartan in a pig pacing model in five articles, two of which apparently reporting at least partly the same data. In the first study, they compared effects of valsartan, benazepril and their combination with vehicle; the doses of the two RAS inhibitors had been chosen to yield a 50% reduction in pressor responses to angiotensin I and ANG without lowering of mean arterial pressure (Spinale et al., 1997c; Spinale et al., 1998). While the ACE inhibitor and the ARB similarly normalized myocyte action potential duration, only benazepril or the combination treatment prevented the reduction in left ventricular fractional shortening and myocyte shortening velocity; however, combination treatment was superior to each monotherapy for all parameters. In a follow-up study using a similar design but considerably higher doses of both RAS inhibitors, valsartan and benazepril alone attenuated the fall in cardiac output, but only the combination fully prevented it, both at rest and during exercise (Krombach et al., 1998). Both monotherapies reduced systemic and pulmonary resistance, but only combination treatment normalized it. All three treatments 71

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attenuated the elevation of plasma noradrenaline and endothelin levels, but only combination treatment restored left ventricular myocardial blood flow. In a third study, the high valsartan dose normalized left ventricular fractional shortening and end-diastolic dimension (Clair et al., 1998a). However, valsartan did not appear to favorably affect the degree of left ventricular dilation and myocardial collagen structure in this study. A fourth study compared the effects of the low valsartan dose, bosentan or their combination (New et al., 2000). Neither monotherapy improved left ventricular stroke volume or plasma noradrenaline, but the combination did. Only bosentan and the combination reduced left ventricular wall stress and restored left ventricular myocyte shortening velocity. Taken together these studies indicate that ARBs can prevent or at least attenuate some but not all parameters of pacing-induced CHF; however, greater effects are observed when either a high dose is used or the ARB is combined with a vasopressin or an endothelin receptor antagonist.

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Other studies using pacing models have explored whether ARBs can improve cardiac function when given in a treatment setting, i.e. when CHF has already developed. In an early study in an ovine pacing model, acute administration of losartan lowered plasma aldosterone and atrial natriuretic peptide and mean arterial and left atrial pressure; glomerular filtration rate (GFR) and urinary sodium excretion were maintained despite a fall in renal perfusion pressure; all of these responses were similar to those seen with captopril (Fitzpatrick et al., 1992). Other investigators, using a similar design, reported that single doses of losartan, its active metabolite EXP3174 (dogs do not generate this metabolite) and enalapril similarly reduced pulmonary artery pressure, pulmonary capillary wedge pressure, peripheral resistance and mean arterial pressure and increased stroke volume; left ventricular pressure (assessed as dp/dt) was not affected (Suzuki et al., 2001). In similar studies, except for using a pig pacing model, valsartan acutely increased cardiac output and reduced systemic vascular resistance (Clair et al., 1998b). While such acute dosing provides pathophysiological insight, it reflects the clinical situation in a limited way only. More relevant in this regard are chronic treatment studies. In one such study CHF was induced by pacing in dogs, and irbesartan was administered for 4 weeks thereafter (Spinale et al., 1997b). This improved cardiac hypertrophy, ejection fraction and myocyte shortening velocity to control levels but did not restore left-ventricular dilatation (Spinale et al., 1997b). In a parallel report on apparently the same dogs but with an additional fosinopril group, fosinopril improved myocyte geometry and function; both treatments increasd β-adrenoceptor density, but only fosinopril also normalized cAMP response (Spinale et al., 1997a), indicating that the ARB only partly mimicked the ACE inhibitor effects. Other investigators have applied a hybrid between a preventive and a treatment design by starting two doses of candesartan treatment after 8 days of pacing in dogs and then continued pacing and candesartan treatment for another 14 days (Maeda et al., 1997). This resulted in a partial recovery of right atrial pressure and cardiac output and an enhanced renal plasma flow, GFR, urinary flow rate and sodium excretion; elevations of plasma aldosterone were abolished, those of noradrenaline attenuated, but those of atrial natriuretic peptide not altered as compared to vehicle-treated dogs. Aorta-caval shunts are used to generate models of cardiac volume overload. An aorta-caval shunt increased left ventricular end-diastolic pressure, right atrial pressure, right ventricular systolic pressure, left and right ventricular weight, and left ventricular end-diastolic volume index as compared to sham-operated rats (Nishikimi et al., 1995a). In contrast, in rats treated with candesartan or delapril none of these parameters differed significantly from sham controls. Losartan and captopril treatment started 3 weeks after shunt surgery in rats similarly decreased mean arterial pressure and normalized left ventricular end-diastolic pressure and regressed cardiac hypertrophy; right atrial pressure and cardiac contractility (assessed as dp/dt) were not affected by either treatment (Qing & Garcia, 1992). Others reported that 30 72

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days after shunt surgery, CHF rats exhibited a blunted atrial natriuretic peptide response to acute volume expansion; acute treatment with valsartan or ramipril normalized this (Willenbrock et al., 1999). In a rat aorta-caval shunt model, in which treatment started 3 days prior to surgery, losartan and enalapril had similar effects on cardiac and systemic hemodynamics, including a major reduction in left ventricular end-diastolic pressure (Ruzicka et al., 1993). However, losartan reduced left ventricular dilation and left and right ventricular hypertrophy, whereas enalapril caused only modest dilation and did not affect hypertrophy.

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One group of investigators has explored telmisartan effects in a rat model of autoimmune myocarditis based on immunization of Lewis rats with cardiac myosin. In their initial study telmisartan treatment was started 4 weeks after immunization and maintained for another 4 weeks (Sukumaran et al., 2010). Telmisartan at least partly restored cardiac function as measured by echocardiography, most importantly ejection fraction. It also reduced cardiac fibrosis and reversed cardiac hypertrophy. It also at least partly reversed elevated cardiac expression of the inflammatory cytokines interleukin-1β and -6 and MCP-1. Most importantly, telmisartan treatment markedly improved survival (60% vs. 90% vs. 100% in vehicle vs. telmisartan vs. healthy control rats, respectively). In follow-up studies, telmisartan treatment started immediately after immunization and was maintained for 3 weeks (Sukumaran et al., 2011a; Sukumaran et al., 2011b). In both studies, and similar to the initial study, telmisartan at least partly restored left ventricular end-diastolic pressure and cardiac contractility (assessed as dp/dt) and prevented cardiac hypertrophy and fibrosis development. Telmisartan treatment also at least partly normalized increased expression of cytokines interleukins 1β and 6, tumor necrosis factor-α and interferon-γ, NADPH oxidase subunits p47phox, Nox-4, and gp91phox, tumor necrosis factor receptor-associated factor-2, C/EBP homologous protein and glucose-regulated protein 78. Moreover, telmisartan treatment decreased the protein expression levels of total and phosphorylated mitogen-activated protein kinases ERK, JNK and p38, and of myocardial apoptotic markers. In contrast myocardial protein levels of ACE-2 and the ANG 1-7 mas receptor were upregulated.

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Aortic banding induces pressure overload leading to cardiac hypertrophy; 4 weeks after the banding procedure, mice were put on a high-salt diet for another 4 weeks, causing additional hypertrophy, cardiac dysfunction and sympathetic hyperactivity (Ito et al., 2009). In such mice a 28-day i.c.v. administration of telmisartan reduced sympathetic activation and cardiac hypertrophy and increased fractional shortening. When aortic banding was performed in mice on a high-fat diet, treatment with azilsartan reduced cardiac hypertrophy and fibrosis and improved cardiac output (Tarikuz Zaman et al., 2013). CHF can also be induced in Dahl S rats by feeding a high-salt diet; in this model, a 5-week telmisartan treatment of rats with CHF moderately reduced heart weight, normalized fractional shortening and lung weight and, most importantly, reduced mortality (Takenaka et al., 2006). It can also be induced in rats by chronic alcohol intake; in this model valsartan treatment improved left ventricular enddiastolic pressure, ejection fraction and fractional shortening (Sang et al., 2011). Others have explored ARB effects in rat models of CHF caused by cancer chemotherapeutics including 5-fluorouracil, adriamycin or doxorubicin. Pre-treatment with telmisartan was reported to prevent the 5-fluorouracil-induced increase on serum levels of cardiac troponin T, aspartate aminotransferase and alanine aminotransferase (Khudhair & Numan, 2014). Losartan and telmisartan attenuated the adriamycin-induced growth retardation and at least partly reversed the reduced left ventricular ejection fraction; expression of angiotensin(1-7) and AT1R but not AT2R or mas was also restored (Zong et al., 2011). Treatment with telmisartan for 1 week at least partly restored left ventricular end-diastolic pressure and ejection fraction in doxorubicin-induced cardiotoxicity (Hadi et al., 2012). Based on 73

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expression levels of various cytokines, the authors primarily attributed this to reduced inflammation. In a preventive design, others treated rats for 12 days with telmisartan or vehicle and injected doxorubicin on day 7 (Abd El Samad & Raafat, 2012). In this setting telmisartan prevented doxorubicin-induced fibrosis development, assessed as collagen area percentage, reduction of cardiomyocyte diameter.

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Injection of monocrotaline produces a model of pulmonary hypertension but can also be used as a (right ventricular) heart failure model (Campian et al., 2006). The cardiac hypertrophy and fibrosis observed in this model was attenuated by telmisartan (Guo et al., 2014; Li et al., 2014a; Okada et al., 2009) or valsartan (Campian et al., 2009). Telmisartan also reduced the pulmonary hypertension and pulmonary vascular inflammation in monocrotaline-treated rats (Guo et al., 2014) and improved endothelial function in the pulmonary artery (Li et al., 2014a). Similar to other CHF models, monocrotaline injection can lead to skeletal muscle myopathy. To address this, two weeks after monocrotaline injection rats were assigned to irbesartan or nifedipine treatment for another 2 weeks (dalla Libera et al., 2001). Irbesartan did not improve the lowered left/right ventricular mass ratio or the right ventricular cavity dilatation but decreased skeletal muscle apoptosis and atrophy as well as circulating tumor necrosis factor-α levels; nifedipine behaved similarly for the lack of cardiac improvement but did not mimic the beneficial effects on skeletal muscle.

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In conclusion, ARBs improve cardiac function in a wide range of CHF models. A limited number of studies indicate that they do so beyond what can be expected based on their BP effects. Whether ARBs or ACE inhibitors do better, has remained controversial as evidence in both directions has been reported. This equivocal evidence may represent scatter around overall similar effects or differences between specific models.

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5. Embolism and stroke

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Arterial hypertension is a major cause of stroke; accordingly, some degree of stroke prevention can be expected from ARBs based on their anti-hypertensive effects. This section will look at ARB effects on platelet function and coagulation, cerebral blood flow, stroke incidence and stroke outcomes. High concentrations (> 1 µM) of some ARBs, specifically irbesartan (Fukuhara et al., 2001; Li et al., 2000; Monton et al., 2000), losartan (Li et al., 2000; Liu et al., 1992a; Monton et al., 2000), telmisartan (Asano et al., 2006; Monton et al., 2000) and valsartan (Monton et al., 2000) also have AT1R-independent, direct antagonistic effects on thromboxane A2 receptors. Direct thromboxane A2 receptor antagonism by some ARBs results in platelet inhibition (Champion et al., 1999a; Lambert et al., 1998; Li et al., 2000; Liu et al., 1992a; Monton et al., 2000). Based on these studies, the ratio between concentrations required for inhibition of AT1R and of thromboxane A2 receptors appears lowest for losartan, as it has relatively low AT1R and relatively high thromboxane A2 receptor affinity. In vivo, telmisartan treatment of type 1 diabetic rats normalized the elevated activation state of platelets as assessed by fibrinogen binding (Schäfer et al., 2007). In patients with concomitant hypertension and chronic-stage ischemic stroke, a 4-week treatment with losartan (but not telmisartan) reduced platelet activation as assessed by CD62P expression; however, neither losartan nor telmisartan altered spontaneous platelet aggregation in such patients (Yamada et al., 2007). Losartan also failed to affect pulmonary vascular resistance in a lamb model of acute pulmonary embolism induced by silicon microsphere injections (Dias-Junior et al., 2012). In contrast to the abovementioned ARBs, candesartan consistently failed to inhibit thromboxane A2 receptors (Fukuhara et al., 2001; Li et al., 2000; Monton et al., 2000). Accordingly, candesartan did not 74

ACCEPTED MANUSCRIPT inhibit thrombus formation or aortic expression of plasminogen activator inhibitor-1 protein in an SHR model of thrombosis; improvement by imidapril in this model were attributed to AT2R activation (Mitsui et al., 1999). Thus, the overall importance of direct thromboxane A2 receptor antagonism by some ARBs remains unclear.

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Auto-regulation of cerebral blood flow is an important mechanism to maintain constant perfusion of the brain in the presence of fluctuating BP. To achieve this, cerebral resistance vessels dilate if perfusion pressure drops and constrict if it increases. The auto-regulation of cerebral blood flow can be altered in SHR, where cerebral blood flow stays constant up to higher levels of BP as compared to WKY (Nishimura et al., 1998b). Candesartan did not affect baseline cerebral blood flow but lowered the threshold for autoregulation in WKY and SHR, i.e. the BP range over which cerebral blood flow stayed constant was shifted to lower levels (Vraamark et al., 1995). It also at least partially normalized cerebral blood flow autoregulation in SHR in other studies (Nishimura et al., 1998b). Improvement of cerebral blood flow auto-regulation was also reported in SHR e.g. with telmisartan even in low doses not lowering BP (Kumai et al., 2008). However, other investigators found that low telmisartan or ramipril doses that were insufficient to improve cerebral blood flow as monotherapy did so in when administered in combination (Dupuis et al., 2010).

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ARBs have been shown to reduce the incidence of stroke in rat models, e.g. losartan in Dahl S rats on a high-salt diet (von Lutterotti et al., 1992) or candesartan (Inada et al., 1997) or telmisartan in stroke-prone SHR (Thoene-Reineke et al., 2011). These effects were shared by enalapril and ramipril, respectively, pointing to general role of RAS inhibition rather than a specific role of AT1R antagonism; moreover, they may be BP-independent as a low candesartan dose, not lowering BP, similarly reduced stroke incidence.

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Many more studies have explored whether ARBs, given in a preventive or early treatment setting will improve outcomes, particularly of ischemic stroke. Such research was stimulated by reports on effects of ANG, ARBs and ACE inhibitors on brain plasticity, which have recently been reviewed elsewhere (Horiuchi & Mogi, 2011). Most studies were based on pretreatment with an ARB for 1 to 4 weeks, followed by occlusion of the middle cerebral artery in rats or mice. For instance, a 7-day pre-treatment of normotensive rats with telmisartan reduced infarct volume, prevented histopathological neuronal changes in the peri-infarct corticle regions, and improved sensomotoric function in forelimb and hindlimb placing tests (Kobayashi et al., 2009). A reduction in infarct size was also demonstrated in normal mice upon pre-treatment with telmisartan (Haraguchi et al., 2009) or valsartan (Li et al., 2008a), by losartan and telmisartan in KK-Ay diabetic mice (Iwanami et al., 2010), by telmisartan in ApoE knock-out mice (Iwai et al., 2008), or by valsartan in chimeric mice harboring transgenes for both human renin and human angiotensinogen (Inaba et al., 2009); where investigated, this was accompanied in improved neurological function. Reductions in neurological damage were also reported in prevention settings in models of transient cerebral ischemia, including with valsartan in rats with transient forebrain ischemia induced by bilateral common carotid artery ligation (Wakai et al., 2011) or with irbesartan (Dai et al., 1999), telmisartan (Thoene-Reineke et al., 2011) or valsartan in rats (Groth et al., 2003) or mice (Miyamoto et al., 2008) with transient occlusion of the middle cerebral artery. The neuroprotection associated with ARB pre-treatment appears to involve reduced inflammation (Haraguchi et al., 2009; Thoene-Reineke et al., 2011) and superoxide anion formation (Inaba et al., 2009), increased capillary density (Li et al., 2008a), and up-regulation of NO synthase and NO production (Li et al., 2008a; Thoene-Reineke et al., 2011); whether alterations of MCP-1 or TNF-α are involved has remained controversial (Li et al., 2008a; Thoene-Reineke et al., 2011). Interestingly, the protective effect of valsartan in mice were at least partly 75

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retained if preventive treatment was discontinued 2 weeks prior to ischemia; this was attributed to an increased capillary density upon valsartan treatment (Li et al., 2008a). On the other hand, a preventive effect of telmisartan in ApoE knock-out mice was partly attributed to a reduced atherosclerosis (Iwai et al., 2008). These data suggest that ARBs as a class can have preventive effects on the size of the ischemic region in rodent models of stroke. According to some studies (Haraguchi et al., 2009), even a 2-hour pre-treatment may already exert preventive effects. As telmisartan and ramipril were similarly effective in a transient brain ischemia rat model (Thoene-Reineke et al., 2011), it appears that these effects may extend to RAS inhibitors in general. On the other hand, data with permanent ischemia in KK-Ay diabetic mice indicate that losartan only partly mimicked the telmisartan effects, whereas GW9662 partly blocked them (Iwanami et al., 2010). Partial inhibition of telmisartan pretreatment-associated infarct size reduction by GW9662 was also reported in non-diabetic mice (Haraguchi et al., 2009), indicating the possibility that a PPAR-γ-mediated component may exist on top of that of RAS inhibition. In one of the few studies in which an ARB was administered after the ischemia period, candesartan was reported to promote angiogenesis in a rat model of transient middle cerebral artery occlusion (Soliman et al., 2014). However, in the interpretation of all ARB data in stroke prevention and treatment, it needs to be considered that animal models of prevention of stroke incidence or stroke size reduction have not shown consistent predictive value with regard to reduction of stroke incidence in patients (Tymiianski, 2015).

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In a large series of manuscripts one group has extensively evaluated the effect of a low BPneutral and a higher BP-lowering dose of telmisartan administered for up to 15 months after a 90 minute ligation of the middle cerebral artery; it appears that all of these reports are based on the same or at least strongly overlapping set of animals but most of the articles do not explicitly say so. This model apparently was used not only to study ARB effects on postcerebral ischemia recovery but also used it as a model of dementia development. In this model telmisartan treatment increased the number of PPAR-γ-positive neurons (Deguchi et al., 2014; Omote et al., 2015) and reduced that of AT1R- or insulin receptor-positive neurons (Deguchi et al., 2014; Omote et al., 2015), oxidative brain damage as assessed as presence of AGE, 4hydroxynonenal and phosphorylated α-synuclein (Fukui et al., 2014; Sato et al., 2014b), brain inflammation as assessed by presence of MCP-1 and TNF-α (Sato et al., 2014a), damage to the neurovascular unit (Kono et al., 2015; Liu et al., 2015b), ApoE and LDL receptor expression (Yamashita et al., 2014) and amyloid-β-, phosphorylated τ- and TNF-α-positive neurons and senile plaques (Kurata et al., 2014a; Kurata et al., 2014b). All of these effects were already seen with the low telmisartan dose but became more prominent with the higher dose. While the above data largely relate to ischemic stroke, hemorrhagic stroke and subarachnoidal hemorrhage can also occur. When given at doses with similar BP effects, telmisartan but not candesartan improved internal diameter of pial blood vessels at baseline and during hemorrhage-induced hypotension; this difference was attributed to PPARγ activation, as the effects of candesartan in combination with pioglitazone were similar to those of telmisartan (Foulquier et al., 2012). A single dose of telmisartan, administerd 2 hours after intracerebrovascular hemorrhage, reduced hemorrhage volume, brain edema, and inflammatory or apoptotic cells in the perihematomal area and, most importantly, neurological deficits (Jung et al., 2007). In a rat model of subarachnoidal hemorrhage, acute pre-treatment with peripherally or intracisternally administered irbesartan aggravated the hemorrhageinduced decrease in cerebral perfusion pressure; central, but not peripheral, irbesartan administration increased intra-cranial pressure in this model (Fassot et al., 2001). 76

ACCEPTED MANUSCRIPT 6. Metabolic effects

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AT1R are found at the mRNA and protein level in multiple cell types and tissues relevant for carbohydrate and lipid metabolism. In rat gut enterocytes, AT1R were found in the brush border membrane; exposure of such cells to ANG inhibited SGLT1-mediated glucose transport in a losartan-sensitive manner (Wong et al., 2007). The pancreas expresses multiple components of the RAS including angiotensinogen, ACE and AT1R (Hasegawa et al., 2009). Accordingly, telmisartan decreased the accumulation of palmitate-induced reactive oxygen species in isolated mouse pancreatic β cells and in the β cell-derived cell line MIN6 (Saitoh et al., 2009). In in vivo studies ARBs such as telmisartan or valsartan or ACE inhibitors such as perindopril were reported to improve pancreatic islet function in models of hereditary and genetic diabetes including STZ-treated rats (Li et al., 2009; Li et al., 2012b; Yuan et al., 2010), db/db mice (Cheng et al., 2008) and diabetic Torii rats (Hasegawa et al., 2009). The production of RAS components by adipocytes is exacerbated during obesity; among those, angiotensinogen has been detected more consistently than renin or ACE (Frigolet et al., 2013). As reviewed elsewhere, at least part of the beneficial effects of RAS inhibition may be mediated by an interaction with the sympathetic nervous regulation of metabolic function (Ernsberger & Koletsky, 2006).

6.1.

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ARB effects on glucose and lipid metabolism have been explored in numerous animal models. While realizing the relatedness of glucose and lipid metabolism, we have separated our discussion of corresponding in vivo data into five main sections, one each on body weight and food intake, on glucose metabolism, on lipid metabolism, on adipose tissue mass and function and on liver morphology and function. As the data may differ between species and animal models under investigation, some of these sections have been divided into subsections on lean euglycemic animals, lean animals with diabetes, and obese animals with and without diabetes. The latter two groups include models in which diabetes and/or obesity were induced, for instance by specific toxins or diets, and those with hereditary causes including those induced by molecular genetic techniques. In sections 6.2-6.5 data are analyzed first for mouse and then for rat studies, and within each group studies on induced models are discussed prior to those on hereditary models.

In vitro studies

Many in vitro studies on ARB effects in adipocytes were performed using the mouse preadipocyte cell line 3T3-L1. In undifferentiated 3T3-L1 cells a high concentration of telmisartan (10 μM) was reported to reduce lipid accumulation (Konda et al., 2014). However, 3T3-L1 cells can be differentiated into mature adipocytes using a cocktail of insulin, a glucocorticoid and a phosphodiesterase inhibitor. Therefore, it has repeatedly been explored how ARBs affect this differentiation process of 3T3-L1 cells. The typically used differentiation protocols led to an up-regulation of renin, angiotensinogen and ACE, leading to an increased ANG formation; concomitantly, AT1R mRNA was reduced, whereas AT2R mRNA was increased (Hung et al., 2011). In the same study, differentiation increased mRNA expression of apelin, adiponectin, RBP4, resistin and visfatin; losartan, PD123,319, captopril or perindopril further enhanced apelin mRNA and protein expression, but inhibited mRNA expression of the other four typical adipocyte molecules. Moreover, differentiation markedly increased lipid accumulation and reactive oxygen species generation, which both were also inhibited by all four RAS inhibitors. The up-regulation of adiponectin mRNA and protein during 3T3-L1 cell differentiation was confirmed by others, who additionally showed an increased expression and release of IL-6 (Hasan et al., 2014). During 3T3-L1 differentiation an increased expression of lipoprotein lipase mRNA and protein level expression as well as 77

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functional activity was also observed, which was further enhanced by valsartan (Saiki et al., 2008). In a somewhat different approach, 3T3-L1 cells were exposed to the differentiation cocktail for 24 hours only, followed by azilsartan or valsartan treatment for 7 days (Kajiya et al., 2011). At the single tested concentration of 10 μM each, the two ARBs differentially affected the expression profile of 3T3-L1 cells: both stimulated lipid accumulation, although azilsartan more than valsartan; azilsartan induced mRNA expression for PPAR-α, PPAR-δ, adipsin and adiponectin, whereas valsartan reduced mRNA for PPAR-γ. Telmisartan but not valsartan promoted differentiation of 3T3-L1 cells as assessed by lipid accumulation (Fujimoto et al., 2004). When human pre-adipocytes were isolated and differentiated in vitro presence of irbesartan, losartan or telmisartan increased lipid content as marker of adipogenesis, whereas eprosartan did not; however, the bell-shaped concentration-response curve for telmisartan (minor stimulation at 0.1 μM, strong at 1 μM and none at 10 μM) is difficult to interpret mechanistically (Janke et al., 2006). Within that study irbesartan, losartan, telmisartan and the PPAR-γ agonist pioglitazone but not eprosartan enhanced gene expression of PPAR-γ target genes adiponectin and lipoprotein lipase; GW9662 abolished the effects of irbesartan and pioglitazone, inhibition of effects of other ARBs was not reported. However, a therapeutic dose of irbesartan had no effect on the expression of either PPAR-γ target gene abdominal subcutaneous adipose tissue of patients. Taken together these findings demonstrate that 3T3-L1 cells express a local RAS, which is regulated during their differentiation into mature adipocytes. ARBs and ACE inhibitors can interfere with this differentiation, indicating autocrine feedback loops in this model.

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Other studies exposed 3T3-L1 cells to ARBs after differentiation into adipocytes had been completed. Valsartan was reported to reduce the elevated expression and the enhanced release of IL-6 (Hasan et al., 2014) but had limited effect on the expression of several metabolically relevant proteins and their phosphorylation status such as insulin receptor β, insulin receptor substrate 1 and the glucose transporter GLUT4; moreover, it had no effect on the regulation of these proteins by TNF-α (Iwashita et al., 2012). Exposure to uric acid for 2 days up-regulated the local RAS in 3T3-L1 cells and increased generation of reactive oxygen species; apparently, the up-regulation of the local RAS had stronger effects on reactive oxygen species than the direct anti-oxidant effect of uric acid (Zhang et al., 2015). Losartan or captopril inhibited the increase in reactive oxygen speciesA reduction of reactive oxygen species in the absence of uric acid was reported with telmisartan (Rios et al., 2012). In that study mRNA expression of endothelial and inducible NO synthase was reduced, but the phosphorylation pattern of endothelial NO synthase suggested an increased enzymatic activity; unfortunately, no functional data were included in that report. Various studies have reported that some ARBs can stimulate adiponectin expression in 3T3-L1 cells; this was consistently reported with telmisartan (Brody et al., 2009; Fujimoto et al., 2004; Inomata-Kurashiki et al., 2010; Yamada et al., 2008), an effect mimicked by losartan (Brody et al., 2009) but not by candesartan (Yamada et al., 2008). As this pattern matches that for PPAR-γ activation, AT1R-dependent and independent ARB effects may exist in these cells. Thus, telmisartan and pioglitazone but neither candesartan nor ANG stimulated expression of a reporter gene under control of the adiponectin promotor (Moriuchi et al., 2007). Similarly, the telmisartan-induced up-regulation of adiponectin mRNA was mimicked by pioglitazone but not by candesartan; however, the relevance of these mRNA changes did not become fully clear, as the corresponding protein data were equivocal (Yamada et al., 2008). However, in the same study telmisartan caused greater increases in plasma adiponectin than candesartan in hypertensive diabetic patients during a 3 month treatment. A telmisartan-induced increase in adiponectin mRNA and protein, along with increased Sirt1 expression, was also reported by others; the role of PPARγ in this regulation is difficult to determine as GW9662 had differential effects at the mRNA and protein level (Shiota et al., 2012a). On the other hand, ANG was also reported to enhance 78

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adiponectin expression in 3T3-L1 cells (Clasen et al., 2005). The apparent puzzle that ARBs can have the same effect was resolved by the observation that the ANG effect was inhibited by an AT2R antagonist, whereas irbesartan enhanced the ANG effect on adiponectin expression (Clasen et al., 2005). Finally, telmisartan but not valsartan was reported to increase protein expression of fatty acid binding protein-4, insulin receptor β, and glucose transporters GLUT1 and GLUT4 and to enhance glucose uptake; these findings were mimicked by troglitazone but not by candesartan and were reduced by the PPAR-γ antagonist T0070907 (Fujimoto et al., 2004; Furukawa et al., 2011). In human subcutaneous adipocytes, telmisartan induced a pattern of gene expression changes compatible with a differentiation towards brown fat (Konda et al., 2014). Taken together these studies suggest that ARBs can regulate the expression of many genes important for adipocyte function at the mRNA and protein level. Most of the ARB effects on adipocytes are anabolic, i.e. favor glucose uptake and, thereby, are likely to improve glucose metabolism in metabolic syndrome and diabetes. Telmisartan may have additional effects on some parameters via PPAR-γ.

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Hepatocytes express several members of the RAS including renin, angiotensinogen, ACE and AT1R; some of these are up-regulated upon TGF-β treatment (Sakamoto et al., 2009). In guinea pig hepatocytes irbesartan and losartan inhibited ANG-induced inositol phosphate formation and, as a down-stream consequence, phosphorylase stimulation (Garcia-Sainz et al., 1997). Inhibition of inositol phosphate formation was also demonstrated for irbesartan and losartan in a rat hepatic epithelium-derived cell line (Hines et al., 1999). In the human hepatic stellate cell line, LX2, losartan and telmisartan suppressed TGF-β-induced expression of fibrosis markers procollagen, metallomatrixproteinase 13 and α-smooth muscle actin and also that of AT1R but the effects of telmisartan were more pronounced and/or occurred at earlier time points than those of losartan; as telmisartan but not losartan prevented TGF-β-induced down-regulation of PPAR-γ, it was concluded that an AT1R-mediated and an AT1Rindependent, possibly PPAR-γ-mediated effect may be involved (Sakamoto et al., 2009). Telmisartan in the absence of exogenous ANG stimulated expression of PPAR-α target genes in the human hepatoma cell line, HepG2, and in murine hepatic AML12 cells, and such expression was blocked by PPAR-α siRNA (Clemenz et al., 2008; Yin et al., 2014). Telmisartan also increased the expression of lipoprotein lipase in HepG2 cells, apparently via a pathway involving PPAR-α (Yin et al., 2014). In another human hepatoma cell line, Hep3b, AGE induced generation of reactive oxygen species and subsequent expression of C-reactive mRNA and protein (Yoshida et al., 2006). Telmisartan inhibited this AGE effect; such inhibition was prevented by GW9662 and was not mimicked by candesartan, indicating that they occur via PPAR-γ stimulation. Telmisartan also concentration-dependently downregulated RAGE mRNA and basal and AGE-induced RAGE protein expression (Yoshida et al., 2006). In a follow-up study in Hep3b cells, AGE markedly increased phosphorylation of serine-307 of the insulin receptor substrate-1 and reduced its association with phosphatidylinositol-3-kinase as well as insulin-stimulated glycogen synthesis (Yoshida et al., 2008). Telmisartan inhibited these AGE effects; this was again blocked by GW9662 and was not mimicked by candesartan, confirming a role for PPAR-γ in the telmisartan effects in Hep3b cells. Thus, at the cellular level ANG induces pro-fibrotic gene expression, ARBs as a class can inhibit hepatocyte AT1R signal transduction, and some ARBs may have additional AT1R-independent, PPAR-γ-mediated effects. A smaller number of studies have explored ARB effects on skeletal muscle cells and cell lines derived from them. In cultured murine myotubes telmisartan up-regulated levels of PPAR-δ and phosphorylated adenosine monophosphate-activated protein kinase α (Feng et al., 2011). As these effects were absent in myotubes derived from PPAR-δ knock-out mice, PPAR-δ appears to be involved, although not necessarily as a direct molecular target of the ARB. 79

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In vivo studies on body weight and food intake

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Follow-up work from the same group explored telmisartan effects in palmitate-induced insulin-resistant C2C12 myotubes (Li et al., 2013). In this model the ARB enhanced insulinstimulated Akt and Akt substrate of 160 kDa phosphorylation as well as translocation of the glucose transporter GLUT4 to the plasma membrane. Consistent with the previous knock-out mouse data, these effects were inhibited by antagonists of PPAR-δ and phosphatidylinositol-3 kinase, but not by those of PPAR-α and PPAR-γ.

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6.2.1. Studies in lean euglycemic animals

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In contrast to humans, rodents exhibit a physiological body weight gain during most of their adult life span without necessarily developing obesity. Several studies have explored whether ARBs can interfere with this physiological body weight gain but have yielded equivocal results. In wild-type mice a lack of effect has been shown with telmisartan (Araki et al., 2006; Iwanami et al., 2010; Zidek et al., 2013) and valsartan (Cole et al., 2010). However, others reported inhibition of physiological weight gain with ageing in mice with telmisartan (Feng et al., 2011; He et al., 2010; Penna-de-Carvalho et al., 2014); interestingly, this inhibition was absent in concomitantly studied PPAR-δ KO mice (Feng et al., 2011; He et al., 2010). However, addition of telmisartan did not induce weight loss (He et al., 2010).

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Similarly equivocal data on ARB effects on physiological body weight gain have been reported in rats. Thus, a lack of effect has been found with azilsartan in normal rats (Abdelsaid et al., 2014; Sueta et al., 2013) and during chronic ANG infusion (Lastra et al., 2013) and with candesartan (Kobayashi et al., 1993) and valsartan in normal rats (Komers & Cooper, 1995). Some studies with telmisartan confirmed such a lack of effect (Bentzen et al., 2013; Emre et al., 2010; Goyal et al., 2008; Kushwaha & Jena, 2013; Xu et al., 2011; Yoshihara et al., 2013), whereas others (partly from the same investigators) reported inhibition of physiological weight gain with ageing in healthy rats (Goyal et al., 2011a; Goyal et al., 2011b; Liang et al., 2011b). In normotensive rabbits neither candesartan nor telmisartan (alone or in combination with GW9662) affected body weight during a 2-week treatment (Zeng et al., 2013a). In SHR azilsartan (Sueta et al., 2013), candesartan (Anderson et al., 1997) or valsartan did not affect physiological body weight gain (Chow et al., 1995; Chow et al., 1997) or mitigated the slowing of body weight gain (Ishimitsu et al., 2010); candesartan failed to inhibit body weight gain in (m-Ren2)27 rats (Kelly et al., 2000). The same was observed with a low and medium dose of candesartan, whereas a high dose caused some inhibition (16 vs. 2 or 6 mg/kg/d) (Müller-Fielitz et al., 2011); similarly, in lean Zucker rats low doses of candesartan had no effect (Ecelbarger et al., 2010) while a high dose caused inhibition (Müller-Fielitz et al., 2011). In contrast, inhibition of body weight gain in SHR was repeatedly reported with telmisartan at low doses (He et al., 2010; Younis et al., 2010). Some of the rat studies also included a group treated with an ACE inhibitor. Except for one study with benazepril (Chow et al., 1995), the ACE inhibitors benazepril (Chow et al., 1997) and ramipril (Komers & Cooper, 1995) also failed to affect physiological body weight gain. The body weight reduction in salt-loaded stroke-prone SHR was attenuated by valsartan (Yoshida et al., 2011). Feeding SHR a high-salt diet resulted in a loss of body weight, and that was ameliorated by telmisartan treatment (Susic & Frohlich, 2014). Food intake was reported not be altered by telmisartan in normotensive mice (Penna-deCarvalho et al., 2014) or rats (Bentzen et al., 2013; Goyal et al., 2011a) or in Dahl salt80

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sensitive rats on a high-fat, high-salt diet (Jin et al., 2014) or by candesartan in SHR unless the latter was given in very high doses (Müller-Fielitz et al., 2011); a very high dose of candesartan also lowered food intake in lean Zucker rats (Müller-Fielitz et al., 2011). Telmisartan also did not affect the reduction in food intake in rats induced by treatment with the monoamine uptake inhibitor tesofensine (Bentzen et al., 2013). As body weight effects of telmisartan were proposed to occur via an effect on the central nervous system (Müller-Fielitz et al., 2011), differences between ARBs in this regard may be explained by their different quantitative ability to pass the blood-brain-barrier (Michel et al., 2013).

6.2.2. Studies in lean diabetic animals

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Rats with 5/6 nephrectomy loose weight; this was prevented by telmisartan or troglitazone treatment but not by telmisartan + GW9662 treatment, indicating a role of PPAR-γ in the protection from weight loss in this model (Zou et al., 2014). Telmisartan was also reported to prevent the weight loss in rats with uninephrectomy additionally receiving an ethylene glycol injection (Kadhare et al., 2015). Thus, ARBs and other RAS inhibitors did not exhibit consistent effects on physiological body weight gain in lean euglycemic animals.

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STZ treatment destroys pancreatic β-cells and is the most frequently used means to generate animal models of type 1 diabetes. Similar to the situation in most patients with type 1 diabetes, the STZ model is typically associated with weight loss. Neither telmisartan nor valsartan prevented the weight loss upon STZ treatment in wild-type mice (Kurihara et al., 2008), neither olmesartan nor telmisartan prevented body weight loss in STZ-treated ApoE knock-out mice (Ihara et al., 2007), and telmisartan did not prevent it in STZ-treated endothelial NO synthase knock-out mice (Komala et al., 2014). Similarly, in otherwise healthy rats neither olmesartan (Gandhi et al., 2012), nor telmisartan (whether given alone or in combination with amlodipine) (Emre et al., 2010; Goyal et al., 2008; Kushwaha & Jena, 2013; Mollnau et al., 2013; Salum et al., 2014; Tojo et al., 2015) or valsartan (Allen et al., 1997; Cao et al., 1998; Komers & Cooper, 1995; Remuzzi et al., 1998; Siragy & Huang, 2008) prevented the reduced body loss in STZ-treated animals. Some of these studies have also included other RAS inhibitors including aliskiren (Gandhi et al., 2012), benazepril (Remuzzi et al., 1998) or ramipril (Allen et al., 1997; Cao et al., 1998), which also did not prevent reduced body weight gain. Similarly, neither irbesartan nor captopril or their combination (Cao et al., 2001), neither telmisartan nor lisinopril (Wienen et al., 2001) and neither valsartan nor ramipril (Hulthen et al., 1996) preserved body weight in STZ-treated SHR. Valsartan also failed to do so in STZ-treated diabetic (mRen-2)27 rats (Kelly et al., 2000). The body weight reduction upon STZ treatment holds up even when animals are fed a high-fat diet; under such conditions neither valsartan nor perindopril prevented body weight loss in otherwise healthy rats (Li et al., 2009; Yuan et al., 2010) and irbesartan yielded similar results in STZ-treated SHR on a high-sucrose, high-cholesterol diet (Tang et al., 2013). Taken together, RAS inhibition does not prevent the body weight loss observed in STZ-induced models of type 1 diabetes in normotensive and hypertensive animals, even in the presence a high-fat diet, which in the absence of STZ treatment induces obesity. In Goto-Kakizaki rats, a lean model of type 2 diabetes, azilsartan did not affect body weight (Abdelsaid et al., 2014). 6.2.3. Studies in obese animals with and without diabetes

Obesity is a medical problem of globally growing prevalence (Scully, 2014). It can lead to type 2 diabetes and is a risk factor for many other conditions including various types of cardiovascular disease. Therefore, using a wide range of mouse and rat models including those with type 2 diabetes, it was explored whether ARBs can prevent or reverse obesity. 81

ACCEPTED MANUSCRIPT Some of these models have applied a preventive design, i.e. started treatment with an ARB prior to or concomitant with the obesity-inducing intervention.

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In otherwise healthy mice the body weight gain induced by a high-fat diet was inhibited by telmisartan (Foryst-Ludwig et al., 2010; Noma et al., 2011; Rong et al., 2010). Interestingly, such inhibition was maintained with telmisartan in AT1R knock-out mice (Noma et al., 2011; Rong et al., 2010).

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Similar preventive study designs have also been applied to rats with high-fat diet-induced obesity. In such animals losartan did not affect body weight gain (Boustany et al., 2005), and neither did azilsartan in Dahl salt-sensitive rats on a high-fat, high-salt diet (Jin et al., 2014). In contrast, inhibition was observed with telmisartan in normal rats on a high-fat diet (Schuchard et al., 2015), with candesartan (Müller-Fielitz et al., 2012; Wang et al., 2012d) and telmisartan (Liang et al., 2011b; Miesel et al., 2012; Müller-Fielitz et al., 2012; MüllerFielitz et al., 2015; Wang et al., 2012c; Zanchi et al., 2007). Telmisartan also reduced body weight gain in SHR on a high-fat diet (He et al., 2010) and in normal rats and Zucker rats on a high-fat diet with additional pioglitazone treatment (Zanchi et al., 2007). Of note, the attenuation of body weight gain in direct comparative studies was larger with telmisartan than ramipril (Miesel et al., 2012). Amlodipine in a dose providing similar BP lowering as telmisartan did not reduce body weight gain (Müller-Fielitz et al., 2014). Thus, such prevention if it is observed apparently does not occur secondary to BP lowering and may only partly be explained by RAS inhibition.

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Preventive designs have also been applied to other models of induced obesity. Thus, telmisartan inhibited body weight gain in mice induced by pioglitazone, regardless whether pioglitazone was given alone or on top of a high-fat diet (Aubert et al., 2010). Obesity induced by fructose feeding was not prevented by candesartan in a study in which the ACE inhibitor alacepril caused inhibition (Chen et al., 1996a; Chen et al., 1996b). It also was not prevented by telmisartan or aliskiren (Rabie et al., 2015). In contrast, telmisartan inhibited body weight gain in fructose-fed rats, in those with olanzapine treatment-induced obesity and in those with on a high-fructose diet combined with olanzapine treatment (Ramesh Petchi et al., 2012). While prevention of obesity or attenuation of obesity development by ARBs is an interesting concept, it probably is clinically more relevant whether ARBs will lower body weight when given to already obese animals, i.e. whether they are effective in a treatment design. In a treatment setting of high-fat diet-induced obesity in mice, irbesartan was without effect (Aubert et al., 2010), whereas candesartan (Fujisaka et al., 2011) and telmisartan lowered body weight (Araki et al., 2006; Aubert et al., 2010; Fujisaka et al., 2011; He et al., 2010; Li et al., 2013; Penna-de-Carvalho et al., 2014; Souza-Mello et al., 2010), although one of these telmisartan studies did not confirm this; moreover, one of the ‘positive’ studies found the effect of telmisartan to be maintained in AT1R knock-out mice. Telmisartan also lowered body weight under such conditions in rats (Goyal et al., 2011a; Müller-Fielitz et al., 2014) or guinea pigs (Xu et al., 2015) on a high-fat diet. In one of these studies, this effect was not observed with amlodipine or an amlodipine + telmisartan combination (Müller-Fielitz et al., 2014). Such body weight reduction was also observed for a telmisartan + metformin combination or, with even greater effects, a telmisartan + sitagliptin combination (SouzaMello et al., 2010; Souza-Mello et al., 2011). Some studies in a treatment design were performed in hereditary rat models of obesity. In Zucker diabetic fatty rats neither candesartan (Ecelbarger et al., 2010) nor irbesartan lowered 82

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body weight (Munoz et al., 2009). Similarly, olmesartan (Harada et al., 1999; Kikuchi et al., 2007) and telmisartan (Zhao et al., 2013) were ineffective in Otsuka Long-Evans Tokushima Fatty rats. Azilsartan treatment caused a minor reduction in body weight gain in spontaneously hypertensive obese rats (Hye Khan et al., 2014a) and a somewhat greater attenuation of body weight gain in Koletsky rats (Zhao et al., 2011). In SHRSP.ZLeprfa/IzmDmcr rats telmisartan did not affect body weight gain (Kagota et al., 2013; Kagota et al., 2014; Sugiyama et al., 2013). Thus, ARBs have yielded inconsistent effects on body weight in mouse and rat models of obesity, regardless whether given in a preventive or in a treatment setting. In the absence of studies to the contrary, this drug class may have a modest effect on body weight in obesity which may be of insufficient magnitude to allow consistent detection. Effects with telmisartan may be more consistent, which may relate to better penetration into the brain (Michel et al., 2013) and/or AT1R-independent effects (Aubert et al., 2010; Rong et al., 2010).

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Type 2 diabetes is most often found in association with obesity, both in animal models and patients. However, some animal models exist in which type 2 diabetes is observed in the absence of obesity such as non-obese Cohen diabetic or Cohen-Rosenthal non-obese diabetic hypertensive rats on a high-sugar, copper-free diet (Younis et al., 2010). Among the hereditary mouse models of obesity, neither losartan nor telmisartan affected body weight gain in KK-Ay mice (Iwanami et al., 2010), whereas azilsartan (Iwai et al., 2007) and telmisartan (Noma et al., 2011) attenuated it in such mice on a high-fat diet; interestingly, telmisartan even reduced body weight gain when pair-feeding was used to eliminate differences in food intake as a possible cause. In contrast, lack of prevention of body weight gain by ARBs was reported in KK-Ay mice with azilsartan (Matsumoto et al., 2014), candesartan (Iwai et al., 2007; Matsumoto et al., 2014), telmisartan or valsartan (Takasu et al., 2006; Tomono et al., 2008; Ushijima et al., 2013). Lack of inhibition of body weight gain was also reported in in KK/Ta mice with candesartan (Fan et al., 2004), in db/db mice with losartan (Nagata et al., 2013), telmisartan (Shigahara et al., 2014; Shiota et al., 2012b) or valsartan (Dong et al., 2010; Gao et al., 2010)(Ye et al., 2011), and in ob/ob mice with valsartan (Imran et al., 2010). However, one study reported a minor reduction of body weight gain in db/db mice upon olmesartan treatment (Shigahara et al., 2014). Among rat models of type 2 diabetes, a lack of inhibition of body weight gain was reported with telmisartan in rats which had received STZ in the neonatal phase (Goyal et al., 2011b). On the other hand, body weight gain during development was attenuated by telmisartan as compared to vehicle or, in a second series of experiments, by telmisartan as compared to valsartan or rosiglitazone in non-obese Cohen diabetic rats on a high-sugar, copper-free diet (Younis et al., 2010). As part of the same study, telmisartan did not affect body weight gain relative to vehicle in Cohen-Rosenthal non-obese diabetic hypertensive rats on a high-sugar, copper-free diet, but in a second series of experiments yielded smaller body weight increases than valsartan (Younis et al., 2010). Treatment designs have also been applied to other type 2 diabetes models. Under such conditions, telmisartan reduced body weight in rats which had been treated with a combination of nicotinamide and STZ, whereas a similarly glucose lowering combination of metformin + pioglitazone + glimepiride did not have this effect (Sharma et al., 2014). In the type 2 diabetes model of a high-fat diet combined with an STZ injection, a reduction of body weight upon telmisartan treatment was reported by one group (Hussein et al., 2011) whereas another even reported an increase in body weight relative to vehicle treatment (Guo et al., 2011); in a similar model, neither valsartan nor perindopril affected body weight (Li et al., 2009). Thus, conflicting data have been reported in animal models of type 2 diabetes; in the 83

ACCEPTED MANUSCRIPT absence of reports on enhanced body weight gain, ARBs may exert an effect which is too small for robust detection.

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Prevention of body weight gain or body weight reductions can theoretically result from increased energy expenditure and/or reduced eating. The latter has been assessed by measurement of food intake (grams of food pellets being consumed) or of energy intake (multiplying food intake times energy content of food). While the latter is more precise, for the present analysis results from both approaches are combined. In healthy wild-type mice and PPAR-δ knock-out mice telmisartan did not affect food intake (Feng et al., 2011). In db/db mice neither olmesartan nor telmisartan affected food intake (Shigahara et al., 2014). In wild-type mice on high-fat diet neither candesartan (Fujisaka et al., 2011) nor irbesartan (Aubert et al., 2010) affected food intake whereas mixed data were reported for telmisartan; thus, telmisartan was reported not to affect food intake (Araki et al., 2006; Foryst-Ludwig et al., 2010; Fujisaka et al., 2011; Li et al., 2013; Rong et al., 2010; Shiota et al., 2012a) or to reduce it (Aubert et al., 2010; Noma et al., 2011; Souza-Mello et al., 2010). Within one of these studies, the telmisartan-induced reduction of food intake was confirmed in a second group of mice in which it was not mimicked by irbesartan; within that study telmisartan also reduced food intake in wild-type mice with concomitant high-fat diet + pioglitazone treatment (Aubert et al., 2010). Within a study where telmisartan did not alter food intake, it also failed to do so in AT1R knock-out mice on a high-fat diet (Rong et al., 2010). On the other hand, within studies where telmisartan did lower food intake, this effect was maintained in AT1R knock-out mice (Aubert et al., 2010; Noma et al., 2011). Where a reduced food intake had been observed in wild-type mice on a high-fat diet, it was not due to taste aversion (Noma et al., 2011). In conclusion, data on food intake in mice are inconsistent; if anything, telmisartan may have an effect in this regard. However, a study in which prevention of body weight gain upon telmisartan treatment was maintained in mice with forced pair-feeding (Noma et al., 2011) would argue that an effect on food intake may not be necessary to prevent body weight gain. However, what else may explain it is unclear as in the same study telmisartan did not alter energy expenditure (Noma et al., 2011).

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In rats on a high-fat diet, candesartan did not affect food intake (Sugimoto et al., 2006), whereas losartan lowered food intake in obesity-prone and –resistant rats on a high-fat diet (Boustany et al., 2005). Similar to mice, equivocal food intake data were reported with telmisartan in rats on a high-fat diet. Thus, reduced food intake with telmisartan treatment was reported in some studies including one where candesartan was ineffective (Goyal et al., 2011a; Müller-Fielitz et al., 2015; Sugimoto et al., 2006); in SHR on a cafeteria diet both candesartan and telmisartan lowered food intake (Müller-Fielitz et al., 2012). On the other hand, in some studies telmisartan even increased food intake in SHR on a cafeteria diet relative to control or ramipril treatment (Miesel et al., 2012; Müller-Fielitz et al., 2014). Even a high dose of candesartan failed to reduce food intake in obese Zucker rats (Müller-Fielitz et al., 2011), whereas the candesartan analog azilsartan reduced food intake in obese Koletsky rats (Zhao et al., 2011). One study has reported that telmisartan but not valsartan improved caloric expenditure in rats on a high-fat diet (Sugimoto et al., 2006). However, comparison of the food intake data from various studies is complicated by the fact that investigators assessed this for different time points and/or intervals. Telmisartan did not alter food intake in guinea pigs on a high-fat diet (Xu et al., 2015). Similarly controversial data were reported in animal models of type 2 diabets. In such models telmisartan was reported to reduce food intake in neonatally STZ-treated rats (Goyal et al., 2011b) or in KK-Ay mice on a high-fat diet (Noma et al., 2011), whereas others did not find such effects with azilsartan or candesartan (Iwai et al., 2007), telmisartan or valsartan in KK84

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Ay mice (Takasu et al., 2006; Ushijima et al., 2013) or with losartan or telmisartan in db/db mice (Nagata et al., 2013; Toyama et al., 2011) or valsartan in such mice on a high-fat diet (Dong et al., 2010; Shiota et al., 2012b). Whether these divergent findings represent differences between animal models, study designs or ARBs or simply reflect data variability remains to be determined. However, with very few studies reporting increased food intake upon ARB treatment, the overall data if anything argue in favor of a possible reduction as a contributor to often reported attenuation of body weight gain or body weight reduction. As ARBs apparently have only small and inconsistent effects on food intake, more consistently observed reductions in body weight or body weight gain seem to be related to glucose and fat metabolism in general as discussed in the next sections.

In vivo studies on glucose homeostasis

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The most frequently studied parameters of glucose homeostasis are blood or plasma concentrations of glucose, measured under steady state conditions or after a fasting period, insulin and HbA1c. Insulin sensitivity has often been studied as changes of glucose and/or insulin after a glucose or insulin tolerance test, or based on the HOMA insulin-resistance index. 6.3.1. Studies in lean euglycemic animals

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Most data on ARB effects on glucose homeostasis in euglycemic animals come from the control groups of studies in obese and/or diabetic rodents. In such studies, neither telmisartan (Araki et al., 2006; Emre et al., 2010; Feng et al., 2011; Iwanami et al., 2010; Zidek et al., 2013) nor valsartan (Cole et al., 2010) affected plasma glucose or insulin in normal mice nor did telmisartan in ApoE knock-out mice (Feng et al., 2011) or losartan or telmisartan in such knock-out mice on a high-fat diet (Nakagami et al., 2008). In lean euglycemic rats ARBs typically did not affect plasma glucose or insulin for example with azilsartan in in normotensive rats (Abdelsaid et al., 2014), in chronically ANG-infused rats (Lastra et al., 2013) or in SHR (Kusumoto et al., 2011), candesartan in normotensive rats (Kobayashi et al., 1993) or SHR (Müller-Fielitz et al., 2011), olmesartan in SHR (Kusumoto et al., 2011), telmisartan in normotensive rats (Emre et al., 2010; Goyal et al., 2011a; Goyal et al., 2008; Kushwaha & Jena, 2013; Ola et al., 2013; Xu et al., 2011), losartan or telmisartan in rats with 5/6 nephrectomy (Toba et al., 2013; Zou et al., 2014), or valsartan in normotensive rats (Komers & Cooper, 1995) or in hypertensive (mRen-2)27 rats (Kelly et al., 2000). Telmisartan also did not affect HbA1c in normotensive rats (Emre et al., 2010). However, telmisartan was reported to lower glucose and insulin in SHR but not in the SHR/NIH substrain which harbors a deletion mutation in the fatty acid translocase CD36, which is a known target gene of PPAR-γ (Li et al., 2006). Nevertheless, ARBs may improve insulin sensitivity in lean euglycemic animals, but the data are equivocal. Thus, increased insulin sensitivity as assessed by glucose infusion rate in a hyperinsulinemic, euglycemic clamp experiment was reported with azilsartan and olmesartan in SHR (Kusumoto et al., 2011). Moreover, telmisartan reduced glucose excursions during an oral glucose tolerance test in mice (Penna-de-Carvalho et al., 2014). On the other hand, valsartan treatment did not change insulin sensitivity in one study in SHR, whereas benazepril did so (Chow et al., 1995); in a follow-up study from the same group the lack of effect of valsartan was confirmed, whereas benazepril now also was ineffective (Chow et al., 1997). 6.3.2. Studies in lean diabetic animals 85

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Only few studies have explored glucose homeostasis in lean diabetic mice. For instance, neither telmisartan nor valsartan affected the markedly elevated blood glucose upon STZ treatment in C57BL/6 mice (Kurihara et al., 2008). A similar finding was reportd with olmesartan and telmisartan in STZ-treated ApoE knock-out mice (Ihara et al., 2007) and for telmisartan in STZ-treated endothelial NO synthase knock-out mice (Komala et al., 2014). Within the former study AT1R knock-out on top of the ApoE knock-out also did not attenuate the glucose elevation upon STZ treatment.

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In rat models with STZ-induced diabetes, glucose levels typically were not affected by ARBs including candesartan (Kato et al., 1999), irbesartan (Tang et al., 2013), losartan (Senapathy et al., 2014), telmisartan (Alter et al., 2012; Bishnoi et al., 2012; Kosugi et al., 2010; Kushwaha & Jena, 2013; Lakshmanan et al., 2011; Mollnau et al., 2013; Mudagal et al., 2011; Mulay et al., 2010; Ola et al., 2013; Onozato et al., 2008; Satoh et al., 2010b; Senapathy et al., 2014; Singh et al., 2006; Wienen et al., 2001; Yao et al., 2007; Zhang et al., 2012) and valsartan (Cao et al., 1998; Hulthen et al., 1996; Kelly et al., 2000; Komers & Cooper, 1995; Li et al., 2009; Remuzzi et al., 1998; Senapathy et al., 2014; Siragy & Huang, 2008; Tang et al., 2012; Yuan et al., 2010; Zhang et al., 2008b) (Figure 8). Similarly, HbA1c was not improved by irbesartan (Bonnet et al., 2001; Cao et al., 2001; Tang et al., 2013), telmisartan (Tojo et al., 2015) or valsartan (Allen et al., 1997; Cao et al., 1998; Remuzzi et al., 1998)). Moreover, irbesartan also failed to improve the HOMA insulin-resistance index (Tang et al., 2013). Several of these studies also included an ACE inhibitor treatment group, in which plasma glucose (Cao et al., 1998; Kato et al., 1999; Komers & Cooper, 1995; Li et al., 2009; Yuan et al., 2010; Zhang et al., 2008b) or HbA1c (Allen et al., 1997; Cao et al., 1998) also was not altered. However, a few exceptions from this pattern exist. Thus, in contrast to several other studies, olmesartan or aliskiren lowered glucose and increased insulin in one study (Gandhi et al., 2012) and telmisartan or trandolapril lowered plasma glucose (Goyal et al., 2008; Hamed & Malek, 2007; Rao et al., 2011) or HbA1c in others (Emre et al., 2010) (Hamed & Malek, 2007). Moreover, in a study where neither valsartan nor perindopril had major effects on plasma glucose, both increased fasting blood insulin concentration and pancreatic islet function as assessed by insulin concentration per time (Li et al., 2009). In a follow-up study, the same group also reported that both drugs moderately improved glucose excursions during an i.v. glucose tolerance test (Yuan et al., 2010) (Figure 8). <<>>

ARB effects on glucose homeostasis have also been studied in STZ-treated hypertensive rat strains. Thus, in STZ-treated SHR neither telmisartan nor lisinopril (Wienen et al., 2001) and neither valsartan nor ramipril affected glucose levels (Hulthen et al., 1996). Similarly, in other studies neither irbesartan nor captopril nor their combination affected HbA1c levels in SHR (Bonnet et al., 2001; Cao et al., 2001). STZ-induced diabetes has also been studied in MWF rats, which also spontaneously develop hypertension but to a lesser degree than SHR; in this model, neither valsartan nor benazepril affected glucose levels which had been increased by STZ-treatment (Remuzzi et al., 1998). Finally, valsartan treatment also did not affect plasma glucose in transgenic (mRen-2)27 rats pre-treated with STZ (Kelly et al., 2000). Thus, RAS inhibitors as a class have little effect on glucose homeostasis in lean animal models of diabetes. Similarly, telmisartan had little effect on blood glucose or HbA1c levels in Akita (Ins2+/C96Y) mice, a non-obese insulin-dependent model of spontaneous type 1 diabetes (Fujita et al., 2012; Koshizaka et al., 2012). Similar to STZ, injection of alloxan destroys pancreatic β cells and, accordingly, can also be used as a model of type 1 diabetes. Patients with a genetic defect in catalase have a high 86

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incidence of diabetes, and alloxan-induced diabetes is aggravated in catalase deficient mice. Treatment with a low telmisartan dose (0.1 mg/kg per day) starting one day before alloxan injection and continued for another 7 days markedly reduced occurrence of diabetes upon alloxan injection from 36.4% to 0% in wild-type mice and from 65.2% to 20% in catalasedeficient mice (Kikumoto et al., 2010). Unfortunately, other ARBs have not been tested in this model to the best of our knowledge. In Goto-Kakizaki rats, azilsartan treatment starting an age of 12 or 18 weeks and continued until week 18 or 22, respectively, markedly lowered but did not normalize blood glucose levels (Abdelsaid et al., 2014). Of note, study designs starting ARB administration prior to or concomitant with the β cell toxin may affect the onset of pancreatic damage as they may mitigate oxidative damage to the β cells induced by the toxin. 6.3.3. Studies in obese animals with and without diabetes

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Dedicated studies on ARB effects on glucose homeostasis were performed in mouse and rat models of obesity with and without concomitant type 2 diabetes. When given to mice on a high-fat diet, valsartan improved glucose tolerance and reduced fasting blood glucose levels and serum insulin levels (Cole et al., 2010). Using a similar model, others reported that telmisartan, sitagliptin, metformin or dual combinations thereof reduced hyperinsulinemia and improved responses to an oral glucose tolerance or an intra-peritoneal insulin tolerance test (Souza-Mello et al., 2010). In a follow-up paper based on the same set of mice (Souza-Mello et al., 2011), a lowering of fasting plasma glucose, the insulin/glucose and the insulin/glucagon ratio were also reported upon telmisartan, sitagliptin or combination treatment. Using a similar model, other investigators reported that telmisartan normalized insulin levels and glucose excursions during intraperitoneal glucose and insulin tolerance testing (Li et al., 2013). Improved glucose excursions with telmisartan upon an intraperioneal or oral glucose tolerance test or an insulin tolerance test in mice on a high-fat diet were confirmed by others (Foryst-Ludwig et al., 2010; Penna-de-Carvalho et al., 2014). However, telmisartan did not alter glucose and insulin levels in mice on a high-fat diet in other studies (Aubert et al., 2010). While these data indicate an ARB class effect, telmisartan also normalized fasting plasma glucose and insulin as well as glucose concentrations after an insulin challenge in AT1R knock-out mice on a high-fat diet (Rong et al., 2010), indicating possible AT1R-independent effects. This idea is supported by the observation that a partial PPAR-γ agonist without AT1R antagonist properties also improved fasting glucose levels and glucose excursions during an oral glucose tolerance test in a model of type 2 diabetes, rats on a high-fat diet with additional STZ injection (Liu et al., 2015a). STZ injections in ApoE knock-out mice are a model of non-obese type 2 diabetes; in this model, olmesartan actually increased plasma glucose, whereas it was not affected by telmisartan or by olmesartan in ApoE/AT1R double-knock-out mice (Ihara et al., 2007). The db/db mouse is an animal model of obesity, diabetes and dyslipidemia that is caused by a point mutation in the gene encoding the leptin receptor. In db/db mice valsartan did not alter blood glucose with treatment duration of 4-8 weeks (Dong et al., 2010; Gao et al., 2010), but one of these studies reported a minor reduction after 12 weeks of treatment (Gao et al., 2010); moreover, another study in db/db mice found a reduced glucose excursion in an oral glucose tolerance test (Cheng et al., 2008). Similarly, inclusive results were reported with losartan which did not reduce plasma glucose or HbA1c after 8 weeks of treatment (Nagata et al., 2013; Toyama et al., 2011), but in one of these studies a reduction was observed after 4 weeks (Nagata et al., 2013). Similarly, both olmesartan and telmisartan did not affect glucose levels in db/db mice after 2 or 6 weeks of treatment but caused a minor reduction after 4 weeks (Shigahara et al., 2014). In another study, telmisartan also failed to affect fasting glucose 87

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levels after 8 weeks, but nevertheless reduced the HOMA insulin-resistance index; the latter effect was abolished by concomitant treatment with GW9662 and not mimicked by losartan (Toyama et al., 2011). In another study in db/db mice on a high-fat diet telmisartan also did not alter basal glucose or insulin levels but slightly improved glucose excursions in an insulin tolerance test (Shiota et al., 2012b). KK-Ay are a different inbred mouse strain spontaneously developing obesity and diabetes; in these mice azilsartan and candesartan (Iwai et al., 2007; Matsumoto et al., 2014), losartan (Iwanami et al., 2010) or telmisartan (Iwanami et al., 2010; Takasu et al., 2006) did not lower glucose levels, although telmisartan lowered basal insulin in one of these studies. However, azilsartan and candesartan lowered glucose during an oral glucose tolerance test (Iwai et al., 2007); similarly, azilsartan and candesartan (Iwai et al., 2007) or telmisartan and valsartan enhanced glucose lowering during an insulin-tolerance test in KK-Ay mice (Ushijima et al., 2013) with the latter two additionally reducing the HOMA insulin-resistance index. However, one study did not find improvements in glucose or insulin tolerance tests in KK-Ay mice upon treatment with azilsartan or candesartan (Matsumoto et al., 2014). Using the opposite approach, a 2-week infusion of ANG enhanced glucose excursions in an oral glucose tolerance test or an insulin tolerance test, providing a mechanistic basis why ARBs can improve glucose tolerance (Ohshima et al., 2012). This argument was further strengthened within the study, as these enhanced glucose excursions were attenuated by concomitant treatment with telmisartan but not with hydralazine, indicating that they did not occur secondary to BP lowering. KK/Ta mice spontaneously develop type 2 diabetes associated with fasting hyperglyacemia, glucose intolerance, hyperinsulinaemia, mild obesity and dyslipidaemia; in such mice candesartan treatment for up to 22 weeks did not affect fasting glucose or peak glucose levels during a glucose tolerance test (Fan et al., 2004).

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Other investigators have used otherwise healthy rats or SHR on a high-fat or cafeteria diet to explore ARB effects on glucose homeostasis but these experiments yielded conflicting results. Candesartan did not change HbA1c or plasma insulin levels or HOMA insulin-resistence index in normotensive rats or SHR on a high-fat diet in one study (Chung et al., 2010). In another study, candesartan did not alter fasting plasma glucose but lowered fasting plasma insulin in rats on a high-fat diet (Wang et al., 2012d). It also improved glucose excursions in an oral glucose tolerance test and glucose infusion rate during euglycemic-hyperinsulinemic clamp analysis. Irbesartan improved insulin sensitivity in rats when chronically administered concomitantly with a high-fat diet (de las Heras et al., 2009). In rats on a high-fat diet both valsartan and perindopril improved insulin sensitivity, an effect attributed to an increase in pancreatic microvessel density (Li et al., 2009). Studies with telmisartan have yielded conflicting findings. One group reported that telmisartan treatment attenuated the increase in fasting plasma glucose upon in rats a high-fat diet and improved the HOMA insulin-resistance index (Wang et al., 2012c). Another group found that telmisartan did not alter plasma glucose in rats on a normal diet but attenuated the glucose elevation in rat on a high-fat diet to a similar degree as low dose metformin (Goyal et al., 2011a). Others reported a lowering of glucose in rats on a high-fat diet; while losartan also lowered blood glucose, its effect was weaker at the chosen doses (Rosseli et al., 2009); in that study only telmisartan but not losartan lowered plasma insulin and the HOMA insulin-resistance index. Yet another group observed that telmisartan did not alter fasting plasma glucose or insulin or the HOMA insulinresistance index in rats on a normal diet but attenuated the elevation of both in rats on a highfat diet (Xu et al., 2011). The findings of a fourth group point in the opposite direction. In their hands, telmisartan and telmisartan + ramipril but not ramipril alone increased fasting plasma glucose without alterations of fasting insulin in SHR on a cafeteria diet (Miesel et al., 2012); glucagon levels were increased by all three treatments, but more so with telmisartan than with ramipril. In an oral glucose tolerance test, glucose excursions were not affected 88

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while insulin excursions were attenuated by telmisartan and telmisartan + ramipril. In another study from the same group, telmisartan but not candesartan increased fasting plasma glucose but both ARBs similarly prevented the high-fat diet-induced lowering of plasma glucagon and actually increased it to levels above those observed in SHR on a normal diet (Müller-Fielitz et al., 2012). In an additional follow-up study from that group telmisartan and telmisartan + amlodipine treatment but not amlodipine alone also increased fasting plasma glucose and insulin and the HOMA insulin-resistance index (Müller-Fielitz et al., 2014). Glucose or insulin excursions during an oral glucose tolerance test were not affected by telmisartan. In their most recent study they reported that telmisartan did not alter basal glucose or insulin levels; during an oral glucose tolerance test, however, glucose excursions remained unaffected whereas insulin excursions were reduced by about half (Schuchard et al., 2015). In rats on a high-fat, high-calorie diet neither valsartan nor perindopril affected plasma glucose or insulin concentrations (Li et al., 2009). In Dahl salt-sensitive rats on a high-fat, high-salt diet treatment witz azilsartan did not alter plasma glucose (Jin et al., 2014). Glucose excursions during an intra-peritoneal glucose tolerance test in telmisartan-treated rats on a high-fat diet were somewhat greater in unilaterally nephrectomized than in intact rats (Chin et al., 2015), but these findings are difficult to interpret as no direct comparison with values in the absence of telmisartan treatment was reported.

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A-ZIP/F1 mice are a transgenic mouse model lacking adipose tissue but having increased food intake and body weight with insulin-resistance. In this model telmisartan treatment did not affect body weight, food intake, or plasma glucose but lowered plasma insulin and the HOMA insulin-resistance index; it also increased insulin sensitivity in an orgal glucose tolerance test and an insulin tolerance test, whereas liver hypertrophy was not affected (Rong et al., 2009), indicating that at least for telmisartan beneficial effects on glucose homeostasis may occur independent of adipose tissue.

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Potential ARB effects on glucose homeostasis have also been explored in rat models of acquired and hereditary type 2 diabetes. One model of acquired type 2 diabetes is the fructose-fed rat, characterized by hyperinsulinemia, increased plasma ANG and mild hypertension. In early studies, candesartan was reported not to affect plasma glucose or insulin concentrations (Kobayashi et al., 1993). This was confirmed by other investigators who also reported a lack of alteration of elevated plasma glucose and insulin levels upon candesartan or alacepril treatment; they also reported a lack of effect on glucose or insulin excursions upon intra-peritoneal glucose loading (Chen et al., 1996a). However, in a later study the same group reported a reduced glucose and insulin excursion upon both candesartan and alacepril treatment in this model without providing an explanation for these apparently contradictory results (Chen et al., 1996b). Reduced steady-state plasma glucose and insulin upon candesartan treatment of fructose-fed rats was reported by others (Shimamoto et al., 1994). A minor lowering of plasma glucose upon candesartan or delapril treatment was also reported by others in fructose-fed rats (Iimura et al., 1995). In later studies, the latter group did not detect changes of fasting plasma glucose with either olmesartan or temocapril in fructose-fed rats; however, they noticed an improved insulin sensitivity as assessed during glucose infusion upon both treatments (Higashiura et al., 2000). A reduced plasma glucose was also reported with telmisartan treatment of fructose-fed rats, olanzapine-treated rats or fructose-fed/onlanzepine-treated rats (Ramesh Petchi et al., 2012); reduced insulin levels were also reported in this model with telmisartan (Kamari et al., 2008). Others also reported reduced glucose and insulin levels in fructose-fed rats with telmisartan and aliskiren (Rabie et al., 2015). Thus, ARB effects on glucose homeostasis in the fructose-fed rat model of type 2 diabetes remain equivocal, with apparently inconsistent results even reported within research groups; when ARBs were reported to improve glucose homeostasis in this model, this effect 89

ACCEPTED MANUSCRIPT typically was shared by an ACE inhibitor, indicating that it may be a general effect of RAS inhibition, albeit perhaps too small to allow robust detection.

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While injection of STZ is primarily used as a model of type 1 diabetes, lower doses of STZ, particularly when applied on top of other interventions, can also be used as a model of type 2 diabetes. Thus, multiple ARBs have been tested in rats, including SHR (Tang et al., 2013), which had received an STZ injection on top of a high-fat diet. In this model, valsartan started 4 weeks after diabetes induction did not improve insulin sensitivity (Yang & Peng, 2010). Others reported that both valsartan and perindopril did not alter fasting blood glucose concentration in this model but increased fasting blood insulin and insulin excursions during acute glucose loading (Li et al., 2009). These investigators confirmed their findings in a follow-up study which also showed reduced plasma glucose excursions after an acute glucose load in rats treated with valsartan or perindopril (Yuan et al., 2010). They also reported that telmisartan reduced glucose and increased insulin excursions after an acute glucose load in this model (Li et al., 2012b). Reduced plasma glucose and insulin concentrations in this type 2 diabetes model upon telmisartan treatment were also reported by other groups (Guo et al., 2011; Hussein et al., 2011). One of these studies found the effects of telmisartan on both parameters and also on the HOMA insulin resistance index to be weaker than those of rosiglitazone, but the combination of rosiglitazone + telmisartan was more effective than rosiglitazone alone (Hussein et al., 2011). In contrast, irbesartan was found not to alter HbA1c or the HOMA insulin-resistance index in this model (Tang et al., 2013). In a direct comparative study in rats on a high-fat diet combined with a STZ injection, telmisartan, lisinopril and aliskiren caused a similar reduction in serum glucose, indicating that this is a general feature of RAS inhibition in this model (Hassanin & Malek, 2014). STZ injection can also create a type 2 diabetes model when administered directly after injection of nicotinamide. In this model telmisartan reduced fasting blood glucose to a similar extent as metformin + pioglitazone + glimepiride combination but in contrast to that combination also markedly lowered body weight (Sharma et al., 2014). A type 2 diabetes model can also be generated by injecting STZ into neonatal rats; in this model telmisartan also lowered serum glucose and insulin concentrations (Goyal et al., 2011b). Taken together, ARBs and ACE inhibitors improved glucose homeostasis in most studies with type 2 diabetes rat models involving STZ injections. ARB effects have also been studied in inbred rat models spontaneously developing obesity and/or type 2 diabetes. In spontaneously hypertensive/NIH-corpulent rat (SHR/NDmcr-cp) valsartan up to very high doses failed to improve elevated glucose levels (Tominaga et al., 2009), and a similar lack of glucose alteration was reported with azilsartan in Koletsky obese hypertensive rats (Zhao et al., 2011); however, in the latter model plasma insulin and the HOMA insulin-resistance index were lowered by azilsartan treatment. In SHRSP.ZLeprfa/IzmDmcr rats telmisartan did not affect basal glucose or insulin levels or their excursion during an oral glucose tolerance test (Kagota et al., 2013; Kagota et al., 2014). In Otsuka Long-Evans Tokushima Fatty rats, olmesartan did not affect basal glucose or insulin levels or glucose infusion rate under euglycemic hyperinsulinemic clamp conditions (Harada et al., 1999); a later study confirmed the lack of effect of olmesartan on glucose levels in this model but reported for a high (but not low) dose of olmesartan a reduction in HbA1c (Kikuchi et al., 2007). Telmisartan and valsartan also did not affect basal glucose levels in these rats; however, telmisartan but not valsartan or amlodipine lowered insulin levels (Mori et al., 2007). Despite a lack of effect of telmisartan on fasting plasma glucose, telmisartan attenuated glucose and insulin excusions in an orgal glucose tolerance test (Zhao et al., 2013). Zucker diabetic fatty rats spontaneously develop obesity and diabetes due to a mutation in the leptin receptor, i.e. can be considered a rat homolog of the db/db mouse. In obese Zucker rats 90

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candesartan did not affect basal glucose or insulin levels (Müller-Fielitz et al., 2011) and actually increased glucose excursions during a glucose tolerance test (Ecelbarger et al., 2010); in contrast, azilsartan was reported to lower glucose AUC during an i.p. glucose tolerance test in this model (Hye Khan et al., 2014b). In this model olmesartan was reported not to affect serum glucose but to lower serum insulin levels (Munoz et al., 2009), whereas irbesartan was found to improve the HOMA insulin sensitivity index (Clasen et al., 2005). In CohenRosenthal non-obese diabetic hypertensive rats, neither rosiglitazone, nor telmisartan nor valsartan altered insulin levels (Younis et al., 2010). However, telmisartan but not valsartan lowered basal glucose levels and improved the HOMA insulin-resistance index. Spontaneously Diabetic Torii rats are a model of non-obese type 2 diabetes; in this model, treatment with telmisartan starting at 8 weeks of age suppressed subsequent diabetes development, apparently by protecting pancreatic β-cells (Hasegawa et al., 2009). Similarly, an increased pancreatic β-cell mass has been reported after 6 weeks of treatment with olmesartan or telmisartan in db/db mice (Shigahara et al., 2014). In spontaneously hypertensive obese rats azilsartan treatment did not alter fasting blood glucose but increased the AUC during a glucose tolerance test (Hye Khan et al., 2014a).

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Taken together, ARBs as a class can improve glucose homeostasis in many rodent models of obesity and type 2 diabetes but the results are inconsistent and observed effect sizes often are small; part of these inconsistencies may relate to differences between the experimental models. Beneficial ARB effects were more often detected by dynamic assessment of glucose homeostasis, i.e. glucose or insulin tolerance tests, than by static assessments, i.e. basal glucose or insulin concentrations. Thus, findings of improved insulin sensitivity as assessed by glucose or insulin tolerance tests or by the HOMA insulin-resistance index were often observed in the absence of improvements in basal glucose or insulin levels, indicating possible effects on glucose disposition. Studies reporting positive metabolic ARB effects in obesity and/or type 2 diabetes models typically found that this was a shared effect of ARBs and ACE inhibitors, indicating a general feature of RAS inhibition. In line with these nonclinical observations, ARBs and ACE inhibitors have improved glucose tolerance and insulin sensitivity in patients in controlled clinical trials (van der Zijl et al., 2011) and in large observational studies (Michel et al., 2004). Moreover, ARBs reduced the incidence of diabetes in large clinical outcome studies (Andraws & Brown, 2007; Mathur et al., 2007). In additional to such class effects, telmisartan exerted apparently AT1R-independent beneficial effects in some rodent studies. Accordingly, a systematic meta-analysis of clinical studies comparing telmisartan with other ARBs in patients with insulin resistance or diabetes confirmed that the former had greater beneficial effects on fasting glucose, fasting insulin and insulin sensitivity than other ARBs (Suksomboon et al., 2012).

6.4.

In vivo studies on lipid metabolism

Studies of potential ARB effects on lipid metabolism have largely been performed in the same animal models as those on glucose homeostasis, often within the same studies. The most frequently explored outcome parameters were serum and plasma cholesterol including its subfractions and triglycerides. 6.4.1. Studies in lean euglycemic animals

Telmisartan did not affect triglyceride levels in PPAR-δ knock-out mice (Feng et al., 2011). in ApoE knock-out mice (Blessing et al., 2008), nor did mice on a high-fat diet (Nagy et al., 2010; Nakagami et al., 2008) or in ApoE/AT1R double knock-out mice (Fukuda et al., 2010) but increased HDL in the latter. On the other hand, one study in ApoE knock-out mice 91

ACCEPTED MANUSCRIPT reported telmisartan (in the absence or presence of concomitant atorvastatin treatment) to increase total cholesterol, LDL, VLDL and triglycerides without altering HDL (Grothusen et al., 2005); similarly, irbesartan was reported to increase blood cholesterol and triglyceride levels in such knock-out mice (Dol et al., 2001).

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In stroke-resistant SHR, a 15 months treatment with telmisartan did not affect triglycerides or total or LDL cholesterol (Kurata et al., 2014a; Kurata et al., 2014b). The Imai rat is a hereditary model of hypertension and hyperlipidemia. In such rats olmesartan almost normalized plasma levels of total, LDL and VLDL cholesterol as well as triglycerides (Rodriguez-Iturbe et al., 2004). Chronic exposure of rats to arsenite can increase plasma cholesterol and triglycerides, and this was preventd by telmisartan (Nirwane et al., 2015). Thus, ARBs as a class apparently have little effect on plasma lipids in lean euglycemic animals.

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In ApoE knock-out mice with additional STZ treatment, olmesartan treatment or additional AT1R knock-out did not alter elevated total cholesterol or LDL and also did not affect triglycerides; telmisartan treatment reduced LDL within the same study (Ihara et al., 2007). In a rat model of non-obese type 2 diabetes, induced by neonatal STZ treatment, telmisartan treatment lowered total, LDL and VLDL cholesterol and triglycerides and increased HDL cholesterol (Goyal et al., 2011b). In rats first treated with a high-sucrose, high-cholesterol diet and then with STZ, subsequent irbesartan treatment did not alter total or LDL cholesterol or triglycerides (Tang et al., 2013).

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6.4.3. Studies in obese animals with and without diabetes

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In otherwise healthy mice on a high-fat diet telmisartan treatment was reported to lower triglyceride levels in serum, liver and skeletal muscle of mice on a high-fat diet (Araki et al., 2006; Clemenz et al., 2008). Other investigators treated mice on a high-fat diet with either telmisartan, sitagliptin or their combination (Souza-Mello et al., 2011). In that study triglycerides were not affected by either monotherapy but reduced by approximately 50% by combination treatment. Total cholesterol was reduced by telmisartan and combination treatment but not by sitagliptin monotherapy, whereas LDL cholesterol was lowered by all three treatments with the weakest effect for sitagliptin and the strongest for combination treatment. Neither treatment affected HDL cholesterol. Telmisartan was also reported to lower triglyceride levels in KK-Ay mice (Takasu et al., 2006) and A-ZIP/F-1 transgenic mice (Rong et al., 2009); in the latter animals triglyceride levels were also reduced in the liver. In diabetic db/db mice treatment with valsartan for 4, 8 and 12 weeks lowered total, LDL and HDL cholesterol and triglycerides (Gao et al., 2010). In rats on a high-fructose diet cholesterol and triglyceride levels were reduced by alecapril but not candesartan (Chen et al., 1996b); using a similar model, others reported a reduction of triglycerides (Kamari et al., 2008) or of total and LDL cholesterol and of triglycerides accompanied by increased HDL cholesterol upon telmisartan treatment (Ramesh Petchi et al., 2012). In rats on a high-fat diet candesartan lowered triglyceride levels by about 40% without reaching statistical significance (Guo et al., 2008), and irbesartan treatment reduced total cholesterol (de las Heras et al., 2009). Using a similar model, others reported that telmisartan lowered total, LDL and VLDL cholesterol as well as triglycerides and concomitantly increased HDL cholesterol (Goyal et al., 2011a; Rabie et al., 2015); in the latter studies both drugs also reduced glucose excursions during an oral glucose tolerance test. Another group 92

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confirmed these findings except for those on HDL cholesterol (Xu et al., 2011). Others confirmed the telmisartan effect on LDL cholesterol and triglycerides but did not observe alterations of total or HDL cholesterol (Wang et al., 2012c). Yet other investigators reported in a rat high-fat diet model that telmisartan decreased triglycerides and increased HDL cholesterol without alterations of total or LDL cholesterol (Müller-Fielitz et al., 2014; MüllerFielitz et al., 2015). In Dahl salt-sensitive rats on a high-fat, high-salt diet azilsartan treatment reduced total cholesterol with minor if any effects of triglycerides (Jin et al., 2014). In rats on a high-fat diet with additional STZ treatment, monotherapy with either telmisartan or rosiglitazone lowered total and LDL cholesterol and triglycerides and increased HDL cholesterol; combination treatment had even stronger effects on all four parameters (Hussein et al., 2011). Lowering of total cholesterol and triglycerides upon telmisartan treatment in a similar model was confirmed by others (Guo et al., 2011). In a direct comparative study in rats on a high-fat diet combined with a STZ injection, telmisartan, lisinopril and aliskiren caused a similar reduction in triglycerides and total and LDL cholesterol, indicating that this is a general feature of RAS inhibition in this model (Hassanin & Malek, 2014) (Figure 9). A reduction of total and LDL cholesterol and of triglycerides along with an increase in HDL cholesterol upon telmisartan treatment was also reported in a rat model of olanzapine-induced obesity (Ramesh Petchi et al., 2012). Among rat models of hereditary obesity, azilsartan did not alter triglycerides in Koletsky rats (Zhao et al., 2011) or cholesterol or triglycrides in obese Zucker rats (Hye Khan et al., 2014b) and telmisartan caused only minor reductions of total cholesterol or triglycerides in SHRSP.Z-Leprfa/IzmDmcr rats (Kagota et al., 2013; Kagota et al., 2014). However, azilsartan lowered serum cholesterol and triglycerides in spontaneously hypertensive obese rats (Hye Khan et al., 2014a), candesartan lowered triglycerides in obese Zucker rats (Ecelbarger et al., 2010), irbesartan lowered total cholesterol and triglycerides in obese Zucker rats (Munoz et al., 2009), and valsartan lowered total cholesterol and triglycerides in SHR/NDmcr-cp rats (Tominaga et al., 2009). In Otsuka Long-Evans Tokushima Fatty rats olmesartan lowered total cholesterol (but only in a high dose) but not triglycerides in (Kikuchi et al., 2007). In the latter strain, a 16-week treatment with telmisartan (but not equally anti-hypertensive doses of valsartan or amlodipine) reduced triglyceride excursions in an acute oral fat tolerance test (Mori et al., 2007). Telmisartan treatment also lowered LDL cholesterol in such rats, which at least for the higher of two doses was accompanied by an increase in HDL cholesterol (Zhao et al., 2013). <<< insert Figure 9 approximately here>>>

In guinea pigs on a high-fat diet, treatment with telmisartan slightly lowered plasma triglycerides whereas total, LDL or HDL cholesterol were not affected (Xu et al., 2015). Moreover, it did not enhances the cholesterol-lowering effect of pitavastatin in this model. In conclusion, in contrast to most studies in lean euglycemic animals, ARBs lowered total and LDL cholesterol as well as triglycerides and increased HDL cholesterol in many studies. While this was not observed consistently, in the absence of studies reporting opposite effects ARBs as a class appear to exert a beneficial effect on serum and plasma lipid profiles in obese animals with and without concomitant diabetes, albeit perhaps not robust enough to allow consistent detection.

6.5.

In vivo studies on adipose mass and function

AT1R, AT2R along with angiotensinogen, renin and ACE are also expressed in adipocytes, both in native tissue (Putnam et al., 2012) and in the 3T3-L1 mouse adipocyte cell line (Hung et al., 2011; Zhang et al., 2015), which has frequently been used in functional studies. Obesity 93

ACCEPTED MANUSCRIPT is characterized by an increase in total body fat, most often accompanied by an increase of individual adipocte size. Experimental studies with ARBs typically have not assessed total body fat mass but rather that of one or more anatomically defined regions such as visceral or subcutaneous fat depots.

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Our search did not identify any study reporting increased fat depots upon treatment with an ARB. In only a few studies a lack of effect of ARB treatment on the size of fat depots was reported; these included studies in healthy mice with telmisartan (Penna-de-Carvalho et al., 2014) or valsartan (Tomono et al., 2008), otherwise healthy PPAR-δ knock-out mice with telmisartan (Feng et al., 2011; He et al., 2010), mice on a high-fat diet with candesartan (Fujisaka et al., 2011), in KK-Ay mice with azilsartan or candesartan (Iwai et al., 2007; Matsumoto et al., 2014), in ob/ob mice with valsartan (Imran et al., 2010), in healthy rats with telmisartan (Goyal et al., 2011a), in rats on a high-fat diet with telmisartan (Zanchi et al., 2007) and in obese Koletsky rats with azilsartan (Zhao et al., 2011). However, most studies have reported that treatment with an ARB, particularly in rodent models of obesity, reduces fat mass and/or adipocyte size (Table 4). These included studies in normal healthy mice with telmisartan (Feng et al., 2011; He et al., 2010; Kudo et al., 2009; Zidek et al., 2013), in mice on a high fat diet with irbesartan (Aubert et al., 2010) and telmisartan (Aubert et al., 2010; Fujisaka et al., 2011; Penna-de-Carvalho et al., 2014; Souza-Mello et al., 2010), mice on a high-fat diet with additional pioglitazone treatment with telmisartan (Aubert et al., 2010), ApoE knock-out mice on a high-fat diet with losartan or telmisartan (Nakagami et al., 2008), AT1R knock-out mice on a high-fat diet with telmisartan (Rong et al., 2010), mice on a methionine- and choline-deficient high-fat diet with telmisartan (Kudo et al., 2009), and in KK-Ay mice with azilsartan (Iwai et al., 2007) or valsartan (Tomono et al., 2008). It also included several rat studies including those in otherwise healthy SHR with telmisartan (He et al., 2010), in rats on a high-fat diet with candesartan (Müller-Fielitz et al., 2012), irbesartan (de las Heras et al., 2009), losartan (Rosseli et al., 2009), telmisartan (Goyal et al., 2011a; Miesel et al., 2012; Müller-Fielitz et al., 2012; Müller-Fielitz et al., 2014; Rosseli et al., 2009; Sugimoto et al., 2006; Wang et al., 2012c) or valsartan (Guo et al., 2008; Sugimoto et al., 2006) including those in SHR with candesartan (Chung et al., 2010) or telmisartan (He et al., 2010), in rats on a high-fat diet and additional pioglitazone treatment with telmisartan(Zanchi et al., 2007), studies in fructose-fed rats with telmisartan (Rabie et al., 2015; Ramesh Petchi et al., 2012), in rats with olanzapine-induced obesity with telmisartan (Ramesh Petchi et al., 2012), or in Otsuka Long-Evans Tokushima Fatty rat with olmesartan (Harada et al., 1999), telmisartan (Mori et al., 2007; Zhao et al., 2013) or valsartan (Mori et al., 2007). Telmisartan also reduced the size of epididymal and retroperitoneal fat pads in guinea pigs on a high-fat diet; when those guinea pigs were treated with a telmisartan + pitavastatin combination, reductions in fat pad size were greater than to be expected based on the two monotherapies (Xu et al., 2015). <> Although the effects were not fully consistent across all time points (Fujisaka et al., 2011) or all anatomical locations (Müller-Fielitz et al., 2014) within some experimental series, the vast majority of studies suggest that ARBs as a class can reduce body fat mass in rodent models of obesity and probably even lean mice and rats. In rats on a high-fructose diet the reduction in adipocyte mass was similar with telmisartan and aliskiren (Rabie et al., 2015), arguing for a shared effect of all RAS inhibitors. On the other hand, experiments with ramipril mimicked the effects of telmisartan for a reduction of visceral but not that of subcutaneous fat depots (Miesel et al., 2012) and provided evidence that such reductions may not be a general feature of RAS inhibition. While losartan and telmisartan had similar effects in ApoE knock-out mice 94

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on a high-fat diet in direct comparative studies (Nakagami et al., 2008), irbesartan was less effective than telmisartan in wild-type mice on a high-fat diet (Aubert et al., 2010), valsartan less than telmisartan in rats on a high-fat diet (Sugimoto et al., 2006) or valsartan less than telmisartan in Otsuka Long-Evans Tokushima Fatty rat (Mori et al., 2007); epididymal and perirenal fat mass was lower in telmisartan- than losartan-treated polydactylus/Cub rats on a high-sucrose, high-fat diet (Sugimoto et al., 2008). Together with the observation that telmisartan also reduced body fat in AT1R knock-out mice on a high-fat diet (Rong et al., 2010), these data suggest that on top of the fat mass reducing effects of ARBs as a class, telmisartan may exert additional AT1R-independent effects. Whether the PPAR-δ-dependent component of adipose tissue reduction of telmisartan (Feng et al., 2011) represents the ARB class or the telmisartan-specific AT1R-independent part remains to be determined. From a physiological point of view, it remains to be understood why ARBs had only inconsistent effects on body weight and much more consistent ones on adipose tissue mass. We do not propose that ARBs increase the mass of other body compartments. Rather we feel that it is most likely that effects of adipocyte mass are of insufficient magnitude to have consistent effects on overall body weight.

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Adiponectin is a polypeptide secreted from adipose tissue which regulates glucose homeostasis and fatty acid oxidation. Adiponectin expression is stimulated by at least some ARBs in cultured adipocytes (Brody et al., 2009; Inomata-Kurashiki et al., 2010; Yamada et al., 2008). This is reflected by various in vivo studies. While some studies with telmisartan in ob/ob mice (Araki et al., 2006), rats on a high-fat diet (Müller-Fielitz et al., 2014) or fructosered rats (Kamari et al., 2008) have reported no change of adiponectin levels, and some found a lack of effect for both candesartan and telmisartan in SHR on a high-fat diet (Müller-Fielitz et al., 2012), most studies in the field reported that ARBs increase plasma adiponectin. These include studies with irbesartan in Zucker diabetic fatty rats (Clasen et al., 2005; Munoz et al., 2009), with olmesartan or aliskiren in the STZ model of type 1 diabetes (Gandhi et al., 2012) with high but not low dose olmesartan (Kikuchi et al., 2007) and with two doses of telmisartan (Zhao et al., 2013) in Otsuka Long-Evans Tokushima Fatty rats, and with telmisartan in SHR (Younis et al., 2010), mice (Araki et al., 2006; Souza-Mello et al., 2010; Zidek et al., 2013) or rats on a high-fat diet (Wang et al., 2012c), rats on such a diet with additional STZ injection (Hussein et al., 2011), mice on a methionine- and choline-deficient high-fat diet (Kudo et al., 2009), Cohen diabetic rats and Cohen-Rosenthal non-obese diabetic hypertensive rats (Younis et al., 2010). In a direct comparative study in rats on a high-fat diet combined with a STZ injection, telmisartan, lisinopril and aliskiren caused a similar normalization of adiponectin levels, indicating that this is a general feature of RAS inhibition in this model (Hassanin & Malek, 2014). Of note, the telmisartan-induced increase in serum adiponectin in mice on a high-fructose diet was absent in adipocyte-specific but not skeletal muscle-specific PPAR-γ knock-out mice (Zidek et al., 2013). Thus, ARBs as a class seem to increase adiponectin levels in most studies, thus reversing the reduced adiponectin levels typically observed in obese animal models and humans. Interestingly, increased plasma adiponectin levels upon ARB treatment have been observed not only in models of obesity and diabetes but also in rats on a high-salt diet; in such animals plasma adiponectin was reduced by telmisartan but not by clonidine or hydralazine (Kamari et al., 2010). The lack of hydralazine effect indicates that elevation of adiponectin levels does not occur secondary to improved blood supply to adipose tissue as a result of vasodilatation.

6.6.

Liver morphology and function

The liver is an important organ in glucose and fat metabolism and may at least in part mediate ARB effects on plasma levels of glucose, cholesterol and triglycerides. On the other hand, 95

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obesity and diabetes may alter morphology and function of the liver, i.e. induce hypertrophy, steatosis and fibrosis. Such alterations of liver morphology may at least partly result from activation of the RAS. Thus, AT1R are expressed in the liver of various mammalian species (de Gasparo & Whitebread, 1995; Garcia-Sainz et al., 1997; Hines et al., 1999). In primary human hepatic stellate cells, ANG incubation for 3 days induced protein expression of Arhgef1, rho kinase and α-smooth muscle actin, probably secondary to activation of Jak2, which are believed to play a role in induction of liver fibrosis; losartan inhibited these responses to a similar degree as the JAK inhibitor AG490 (Granzow et al., 2014); within the same study it was found that chronic infusion of ANG can lead to activation of such hepatic stellate cells and cause liver fibrosis in in wild-type but not AT1R knock-out mice (Granzow et al., 2014).

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Against this background several in vivo studies, mostly primarily designed to explore ARB effects on glucose and lipid metabolism, have reported on liver mass as an outcome parameter. None of them reported an increase in liver mass upon treatment with an ARB, and in few cases a lack of effect was reported; the latter included a study in normal mice with telmisartan (Kudo et al., 2009), one in mice on a high-fat diet with losartan (Nakagami et al., 2008), one in SHR (Li et al., 2006), two in rats on a high-fat diet with telmisartan (MüllerFielitz et al., 2014; Schuchard et al., 2015). However, in many studies a reduced liver mass was found upon treatment with an ARB. These included studies in wild-type mice on a highfat diet with telmisartan (Nakagami et al., 2008; Souza-Mello et al., 2010), in AT1R knockout mice on a high-fat diet with telmisartan (Rong et al., 2010), mice on a methionine- and choline-deficient high-fat diet with telmisartan (Kudo et al., 2009), rats on a high-fat diet with candesartan (Müller-Fielitz et al., 2012), losartan (Rosseli et al., 2009) or telmisartan (Miesel et al., 2012; Müller-Fielitz et al., 2012; Rosseli et al., 2009; Sugimoto et al., 2006), rats with neonatal STZ treatment with telmisartan (Klein et al., 2014), fructose-fed rats with telmisartan (Ramesh Petchi et al., 2012), and olanzapine-treated rats with telmisartan (Ramesh Petchi et al., 2012). Taken together these data demonstrate a reduction of liver mass by about 15% in both mice and rats likely to develop non-alcoholic liver steatohepatitis upon treatment with ARBs as a class (Figure 10). Telmisartan may have additional effects partly because it concentrates in liver (Clemenz et al., 2008) and partly AT1R-independently; in favor of the latter it was reported that its effects in rats on a high-fat diet was not mimicked by ramipril (Miesel et al., 2012) and that it reduced liver mass in AT1R knock-out mice on a high-fat diet (Rong et al., 2010). <<>> Indeed morphological evidence for amelioration of non-alcoholic steatohepatitis with ARBs was found in several studies. In mice on a high-fat diet telmisartan was reported to promote expression of PPAR-α-dependent gene expression (Clemenz et al., 2008). In mice on a highfat diet which had received a low dose of STZ 2 days after birth, treatment with telmisartan reduced hepatic inflammation and fibrosis (Cynis et al., 2013). In mice on a methionine- and choline-deficient high-fat diet telmisartan treatment attenuated liver steatosis with decreased hepatic triglycerides and fibrogenesis with decreased type I collagen and TGF-β1 mRNA expressions (Kudo et al., 2009); it also suppressed the infiltration of macrophages into the liver and decreased hepatic MCP-1 and its receptor mRNA expressions. In other studies, the fatty liver induced by a high-fat diet in rats was reverted by treatment with losartan or telmisartan (Rosseli et al., 2009). Similarly, telmisartan reduced liver fibrosis that had been induced in rats by a short- or long-term methionine- and choline-deficient diet (Nakagami et al., 2010). Telmisartan also prevented liver fibrosis in a rat bile duct ligation model (Yi et al., 2012), in high-fat diet-induced progression from simple steatosis to non-alcoholic 96

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7. Renal effects and nephroprotection

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steatohepatitis in a fish (Oryzias latipes) model (Kuwashiro et al., 2011), in PCK rats (Yoshihara et al., 2013) and in a rat model of Schistozoma mansoni infection (Attia et al., 2013). In rats on a choline- and L-amino acid-deficient diet, telmisartan not only prevented development of fibrosis but also the occurrence of hepatocellular carcinoma (0/10 vs. 6/11 animals) (Tamaki et al., 2013). In rats on a high-fructose diet, telmisartan and aliskiren fibrosis in the portal vein along with a reduction of congestion in the portal vein and of inflammatory cell infiltration in the portal area and between hepatocytes (Rabie et al., 2015). In a murine genetic model of liver fibrosis, Abcb4 knock-out mice), telmisartan as monotherapy did not prevent fibrosis development but exhibited a strong effect when administered in combination with propranolol (Mende et al., 2013). Losartan attenuated ischemia/reperfusion-induced liver damage in mice in the absence but not in the presence of GW9662, indicating a PPAR-γ-mediated effect (Koh et al., 2013).

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RAS inhibition is a clinically well established principle of nephroprotection, particularly in the context of diabetic nephropathy (Bernstein et al., 2013; Hughes & Britton, 2005; Strippoli et al., 2006). While BP lowering per se can have nephroprotective effects, the beneficial effects of RAS inhibitors appear to exceed those of other antihypertensive drug classes in patients (Griffin & Standridge, 2012). Moreover, PPARs, which can be activated by some ARBs (Michel et al., 2013), can also contribute to the regulation of renal function (Röszer & Ricote, 2010). Against this background, numerous studies have explored ARB effects on renal function and its alterations in disease.

Normal renal function

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The presence of renal AT1R at the protein level has e.g. been demonstrated as binding sites for radiolabeled ANG analogs or for [3H]-losartan in whole rat kidney (Wienen & Entzeroth, 1994), rat glomeruli (Chansel et al., 1993b; Gauquelin & Garcia, 1992; Wilson et al., 1989), rat mesangial cells (Ernsberger et al., 1992; Zhou et al., 1993), rat tubule fragments (Poggioli et al., 1992), rat renal epithelial cells (Hines et al., 1999) or rabbit whole kidney (Edwards et al., 1992b; Liang & Leenen, 2008). Autoradiographic studies have demonstrated that most ANG receptors in rat and human kidney are AT1R and mainly found in glomeruli and medullary vascular bundles (Correa et al., 1995; Sechi et al., 1992a). 7.1.1. Signal transduction and in vitro studies

The canonical signaling pathway of AT1R is coupling to PLC via a pertussis toxin-insensitive G-protein, Gq, but additional pathways exist which in concert lead to engagement of various down-stream signaling pathways (de Gasparo et al., 2000). Thus, ANG elicits a variety of signal transduction responses in the kidney and isolated renal cells, which are blocked by ARBs. Stimulation of inositol phosphate accumulation and inhibition by ARBs was observed in rat mesangial cells (Madhun et al., 1993) and in freshly isolated fragments from rat proximal tubules (Poggioli et al., 1992). The latter effect was confirmed in cultured rat proximal tubule cells, where it was observed only with administration from the basolateral but not from the apical side (Schelling et al., 1992). In line with the role of inositol phosphates in releasing Ca2+ from intracellular stores, ANG also caused an elevation of intracellular Ca2+ in rat mesangial cells (Madhun et al., 1993), isolated preglomerular arterioles (Iversen & Arendshorst, 1999) and proximal tubules (Poggioli et al., 1992; Zhang & Mayeux, 2012), which was blocked by candesartan, eprosartan or losartan. An AT1R-mediated elevation of intracellular Ca2+ has also been observed in a canine tubular cell line, MDCK cells (Touyz et 97

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al., 2001c). In high micromolar concentrations, losartan was reported to elevate intracellular Ca2+ in mesangial cells, but this was not accompanied by increased inositol phosphate formation and is likely to be an artefact attributable non-specific effects at very high concentrations (Chansel et al., 1993a). ARB-sensitive inhibition of forskolin-stimulated cAMP formation was reported in rat isolated glomeruli (Edwards & Stack, 1993), mesangial cells (Madhun et al., 1993; Zhou et al., 1993) and proximal tubules (Poggioli et al., 1992). Thus, renal AT1R couple to the well-known primary signaling pathways of PLC activation, intracellular Ca2+ elevation and inhibition of adenylyl cyclase.

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Several lines of evidence indicate that ANG can modulate the formation and release of NO in the kidney via AT1R, but such modulation appears to be complex. Direct evidence for ANGstimulated NO release has been obtained in perfused microdissected rat renal resistance arteries; these were inhibited by candesartan and losartan, which did not alter basal NO release in the absence of exogeneous ANG (Thorup et al., 1999). Indirect evidence comes from the observation that ANG-stimulated, eprosartan-sensitive cGMP formation in rat proximal tubules was blocked by an NO synthase inhibitor (Zhang & Mayeux, 2012). On the other hand, infusion of valsartan increased renal interstitial fluid levels of NO metabolites, cGMP and bradykinin in conscious rats on a low-sodium diet, whereas the AT2R or icatibant had opposite effects; a combination of valsartan with either PD 123319 or with icatibant abolished the elevations seen with valsartan (Siragy et al., 2000). The latter data support a concept where ANG does not but an ARB promotes NO release, apparently secondary to release of bradykinin. In line with the latter observations, acute pre-treatment with an NO synthase inhibitor did not affect the BP response to candesartan in anesthetized normotensive rats but blocked the candesartan-induced reduction of renal vascular resistance; concomitant administration of L-arginine restored the renovasodilatory effect of candesartan (Demeilliers et al., 1999). Finally, a study in cultured rat mesangial cells reported that ANG (via a candesartan-sensitive pathway) and TGF-β inhibited IL-1β-induced NO release; in this regard a reduced NO synthase mRNA expression appeared to play a role in TGF-β but not ANG effects (Ihara et al., 1999). Thus, both stimulatory and inhibitory effects of ANG and AT1R inhibition of NO release have been reported, apparently reflecting a mixture of direct and indirect effects which at least partly mitigate each other. Other AT1R-activated signaling pathways in the kidney which have been less well characterized include activation of the mitogen-activated protein kinase JNK via pertussis toxin-sensitive and –insensitive pathways independent of protein kinase C in rat mesangial cells (Huwiler et al., 1998), activation of a Na+/H+-antiporter in an isolated rat macula densa preparation (Bell & Peti-Peterdi, 1999), activation of a Na+/HCO3-cotransporter in rabbit renal tubules (Eiam-Ong et al., 2015), and a reduction of free intracellular Mg2+ concentrations, which depended on the concentration of extracellular Na+ but not intracellular free Ca2+ (Touyz et al., 2001c). A net effect of all of the above signaling events, at least in mesangial cells, is an ANG-stimulated, ARB-sensitive increase in protein synthesis; whether this also results in proliferation remains controversial (Anderson et al., 1993; Bakris & Re, 1993; Madhun et al., 1993). The kidney also has important endocrine functions by releasing renin and erythropoietin. Renin release is an important step in the homeostatic systems regulating ANG formation, and as part of the physiological feed-back loop ANG itself suppresses renin release (Hackenthal et al., 1990). This partly reflects a direct effect on the macula densa but may also partly occur indirectly due to alterations of BP lowering and sodium and fluid homeostasis. Moreover, the sympathetic nervous systems via β-adrenoceptor stimulation, in humans β1-adrenoceptor stimulation (Motomura et al., 1990), is an important stimulus of renin release, and activity of 98

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the RAS and the sympathetic nervous system can cross-regulate each other. Accordingly, all drugs inhibiting formation or action of ANG increase renin secretion, as reported in numerous studies across a wide range of models and species including humans. More recent evidence demonstrates that at the macula densa level this may involve the convergent activation of cAMP formation, probably through indirect augmentation via the activity of prostaglandins E2 and I2, β-adrenoceptors and NO (Kim et al., 2012b). Formation and release of the hematopoietic hormone erythropoietin is another important endocrine function of the kidney. While anemia and lowered oxygen saturation are key stimulants for erythropoietin release, ANG has consistently been found to stimulate erythropoietin expression in experimental models and in humans; experimentally this can be inhibited by ARBs (Kim et al., 2014) but this appears to reflect a minor modulatory role only as lowering of hemoglobin or hematocrit typically is not observed upon treatment with RAS inhibitors.

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Radioligand binding studies with isolated rat renal microvessels demonstrated that vascular ANG receptors belong predominantly if not exclusively to the AT1R subtype (De Leon & Garcia, 1992). Studies in several in vitro preparations have demonstrated that ANG causes vasoconstriction of renal vessels via an AT1R. This includes experiments with losartan in isolated rat (Hayashi et al., 1993) and rabbit renal artery (Zhang et al., 1994) and with telmisartan in isolated rat kidney (Wienen & Entzeroth, 1994). In rabbits ANG can cause vasoconstriction of afferent and efferent glomerular arterioles via AT1R; this effect was enhanced when AT2R were blocked by PD 123319, and in the presence of candesartan the vasoconstrictor response to ANG was turned into a vasodilator response of the efferent arteriole (Endo et al., 1997).

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Several studies have explored ARB effects on RBF in the presence or absence of exogenous ANG; of note some of these studies did not report RBF but rather renovascular resistance or renal plasma flow. Thus, the ANG-induced reduction of RBF was attenuated for instance by candesartan (Li & Widdop, 1996; Ruan et al., 1999). However, the situation may be more complex, as ANG was reported to reduce cortical but to increase papillary RBF in rats; while both effects were blocked by losartan, the increased papillary flow apparently was caused by locally released mediators including prostaglandins and the kallikrein system (Nobes et al., 1991). An ANG-induced reduction in cortical blood flow was confirmed by others and shown to be candesartan-sensitive (Cervenka et al., 1998). ANG also caused a reduction of RBF in mice, which was markedly attenuated in AT1R knock-out mice and by candesartan in wild type mice (Ruan et al., 1999). ARB effects on RBF in the absence of exogenous ANG administration may be speciesdependent. Thus, in the absence of exogenous ANG, candesartan increased RBF and decreased renovascular resistance in wild type mice (Traynor & Schnermann, 1999). Within that study a lowered renovascular resistance was also found with untreated AT1R knock-out mice, confirming a role of endogenous ANG in the regulation of renal perfusion. Moreover, similar to studies in rats, candesartan impaired autoregulation of RBF in wild type mice in that study. In guinea pigs, a losartan-induced RBF increase in the absence of exogenous ANG has also been shown and was shared by an ACE inhibitor and a human renin inhibitor also inhibiting guinea pig renin (El Amrani et al., 1993), suggesting that this RBF effect of losartan is a general feature of RAS inhibition. In healthy normotensive dogs ARBs consistently increased RBF and/or renal plasma flow in doses typically not affecting BP. This was shown for instance with candesartan (He et al., 1994; Ito et al., 1995; Kito et al., 1996), losartan (Chan et al., 1992; Keiser et al., 1992; Nishiyama et al., 1992) or valsartan (Hayashi 99

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et al., 1997b). While most of the dog studies had been performed in anesthetized animals, some were also reported from conscious dogs (Ito et al., 1995). A different situation may exist in rats. In isolated perfused rat kidney, telmisartan increased perfusate flow (Wienen & Entzeroth, 1994). Candesartan increased RBF in rats and attenuated the reduction caused by the NO synthase inhibitor L-NAME (Kawata et al., 1996). While one study reported an eprosartan-induced increase in RBF in rats in the absence of exogenous ANG (mostly due to an increase in cortical with smaller effects on medullary blood flow) (Brodsky et al., 1998), other investigators did not observe this with eprosartan in healthy rats (Wang & Brooks, 1992). Losartan and enalapril also lacked effect on basal RBF/renal plasma flow (Baylis et al., 1993; Sigmon & Beierwaltes, 1993a; Zhuo et al., 1992). In another study, three different candesartan doses were compared in the absence of exogenous ANG (Cervenka et al., 1998); the low dose slightly increased RBF, the medium dose had no effect and the high dose, which also considerably reduced BP, actually reduced it. Taken together these studies suggest that ARBs as a class as well other other types of RAS inhibitors increase RBF in healthy mice, guinea pigs and dogs but have smaller and inconsistent effects in rats. This suggests that the RAS appears to exert less tonic control over renovascular resistance in healthy rats than in other species and/or that major BP reductions more easily overrule direct RBF effects in this species. Endogenously released ANG may also play a role in the autoregulation of RBF, as this was inhibited by losartan (Cupples, 1993). Of note, the above studies are based on acute ARB administration and do not necessarily allow conclusions on chronic administration.

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The GFR is an important indicator of overall renal function; other than colloid osmotic pressure, the GFR is affected by the pressure and resistance of the afferent and efferent arteriole. Reported ARB effects on GFR in healthy animals have been inconsistent. In isolated rat kidney, telmisartan increased GFR (Wienen & Entzeroth, 1994). In vivo one study found an increased GFR upon acute losartan administration in rats (Zhuo et al., 1992), but a lack of effect on GFR was reported with eprosartan (Brodsky et al., 1998; Wang & Brooks, 1992), losartan or enalapril (Baylis et al., 1993). In one study comparing three different candesartan doses, the low dose not affecting BP increased GFR, the middle one with minor BP lowering was neutral on GFR, and the high dose lowering BP also reduced GFR (Cervenka et al., 1998). In guinea pigs, losartan and lisinopril increased GFR to a minor extent whereas a renin inhibitor did so to a greater extent (El Amrani et al., 1993). Dog studies have also yielded inconsistent results. While an increase in GFR was reported with losartan (Chan et al., 1992; Nishiyama et al., 1992), a lack of effect was found in another losartan study (Keiser et al., 1992) and with valsartan (Hayashi et al., 1997b); for candesartan both lack of effect (Ito et al., 1995) and a GFR increase were reported (He et al., 1994). In a rat study in which eprosartan alone did alter RBF or GFR, it attenuated the increase caused by glycine infusion-induced hyperfiltation (Wang & Brooks, 1992). Therefore, the above studies do not allow clear conclusions on ARB effects on GFR in healthy, normotensive animals. However, in each study ACE inhibitors or renin inhibitors exhibited similar effects (or lack thereof) as ARBs, suggesting class effects of RAS inhibition. Studies comparing multiple doses of a given ARB suggest that the absence or presence of BP lowering importantly contributes to observed GFR changes. 7.1.3. Tubular function and diuresis

Some studies have explored ARB effects on tubular function using isolated proximal tubule preparations. In isolated perfused rabbit proximal tubules eprosartan, irbesartan, losartan and valsartan inhibited para-amino-hippurate secretion (Edwards et al., 1999). Losartan was also a substrate of that transporter. In transfected cells all tested ARBs inhibited uptake of uric acid or estrone-3-sulfate by OAT1, OAT3 and OAT4 with olmesartan and valsartan exhibiting 100

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IC50 values comparable to their clinically observed unbound peak plasma concentrations; candesartan, losartan and telmisartan inhibited uric acid uptake by MRP4, but only losartan had an IC50 comparable to its estimated kidney concentration (Sato et al., 2008). In similar studies in transfected Xenopus laevis oocytes others reported inhibition of uric acid uptake by irbesartan, losartan, telmisartan and valsartan but not by candesartan (Nakamura et al., 2010). Such in vitro findings translated into alterations of uric acid excretion in rats (Li et al., 2008b). While they may be relevant for the use of some ARBs in patients with hyperuricemia/gout (Dang et al., 2006) or for some drug-drug-interactions, they have limited implications for overall kidney function.

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Several studies have explored ARB effects on diuresis, natriuresis and kaliuresis in healthy experimental animals, mostly in rats and dogs. In an early study in rats, candesartan was reported not to affect diuresis, natriuresis or kaliuresis in oral doses up to 300 mg/kg (Kito et al., 1996). In a later study, study an i.v. low candesartan dose (0.01 mg/kg) increased sodium excretion, while higher doses (0.1-1 mg/kg) along with the lowering of BP, RBF and GFR also reduced natriuresis (Cervenka et al., 1998). Azilsartan also did not change diuresis but inhibited the increase in urine production upon a 10-day ANG infusion (Carroll et al., 2015). Losartan, in a dose not affecting BP, was reported to increase diuresis and natriuresis whereas enalapril did not affect either parameter (Baylis et al., 1993). Losartan also increased diuresis and natriuresis (but not kaliuresis) in another rat study, in which fractional proximal fluid reabsorption decreased from 73% to 64% and fractional distal sodium reabsorption from 98% to 94% (Zhuo et al., 1992). Telmisartan increased urine flow in isolated perfused rat kidney, and in vivo increased diuresis and natriuresis (but not kaliuresis) in at least some doses in rats (Wienen & Entzeroth, 1994). In dogs acute treatment with ARBs consistently increased diuresis and natriuresis as reported with candesartan (He et al., 1994), losartan (Chan et al., 1992; Nishiyama et al., 1992) or telmisartan (Schierok et al., 2001). Taken together ARBs as a class, similar to ACE inhibitors, appear to increase diuresis and natriuresis but not kaliuresis in rats and dogs, but the effect was less consistently reported in rats. Part of the lack of consistency in findings may relate to the observation that high ARB doses may cause a degree of BP lowering which may overrule direct effects on tubular function.

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The micropuncture technique has been used to obtain a more mechanistic understanding of the observed diurectic ARB effects. Early micropuncture studies established that losartan and the AT2R antagonist PD123177 similarly inhibit bicarbonate, chloride, sodium and water absorption in the S1 proximal tubule segment, but their combination was not more effective than either alone (Cogan et al., 1991). Inhibition of proximal tubule fluid absorption by losartan was confirmed by others, and the extent of this effect was similar to that of enalapril (Zhuo et al., 1992). Similar findings were also obtained by investigators using candesartan, EXP3154 or losartan (Smart et al., 1999). Tubuloglomerular feedback is an important mechanism in the auto-regulation of renal function and markedly inhibited in rats by candesartan; moreover, candesartan inhibited the enhancements caused by the NO-synthase inhibitor L-NAME (Kawata et al., 1996). In mice tubuloglomerular feedback was abolished by treatment with candesartan or in those with AT1R or ACE knock-out (Traynor & Schnermann, 1999). In addition to their direct effects on renal function, ARBs might modify the response to pathophysiological interventions or to other drugs in healthy animals. In sodium-restricted rats losartan lowered BP but had only minor effects on cumulative urine volume, sodium excretion and water intake (Jover et al., 1991). Similarly, neither losartan nor captopril inhibited the anti-natriuretic response to acute volume depletion in uninephrectomized conscious dogs (Nelson & Osborn, 1993). On the other hand, candesartan, losartan and ACE 101

ACCEPTED MANUSCRIPT inhibitor derapil similarly inhibited furosemide-induced increase in renal prostaglandin E2 excretion in rats (Fujimura et al., 1994). Moreover, losartan and captopril blocked the inhibition of sodium excretion by erythropoietin in isolated rat kidney (Brier et al., 1993).

Development and progression of nephropathy

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Nephropathy can develop from kidney-specific conditions such as chronic glomerulonephritis but can also be the result of primarily non-renal conditions such as arterial hypertension, ureteral obstruction or diabetes. A range of animal models exist for these conditions; while some of them mimic extra-renal causes, e.g. the SHR or the STZ-induced diabetes, others are based on direct renal damage, e.g. the 5/6 nephrectomy model. The most frequently used functional and morphological outcome parameters from in vivo studies are albuminuria and fibrosis, respectively.

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Based on the large number of cell types contributing to renal function, multiple models have been used to study renal pathology and its therapeutic modulation at the cellular level. The most frequently used ones are mesangial and tubular cells. Cultured mesangial cells are frequently used to model glomerulonephritis and glomerulosclerosis. ANG stimulated protein synthesis in mesangial cells from rats (Madhun et al., 1993), mice (Anderson et al., 1993) and humans (Bakris & Re, 1993; Orth et al., 1995) in a losartan-sensitive manner. In microarray experiments with human mesangial cells, ANG induced expression of 27 and inhibited that of 7 genes, and this was inhibited by valsartan in all cases (Naito et al., 2009). The up-regulated genes included those encoding pro-inflammatory mediators such as MCP-1, LIF and cyclooxagenase-2; interestingly, this was observed similarly in the presence of normal and elevated glucose concentrations and also in the absence and presence of simvastatin. In rat mesangial cells ANG also affected the intracellular localization of proteins which shuffle cyclooxygenase-2 and plasminogen activator inhibitor-1 in a valsartan-sensitive manner; similar observations were made in acutely ANG-infused rats in vivo (Doller et al., 2009). In mouse mesangial cells ANG induced mRNA expression and release of IL-6, which was blocked by candesartan (Moriyama et al., 1995). Moreover, both ANG and IL-6 stimulated proliferation as assessed by [3H]-thymidine incorporation, and the stimulatory ANG effect was blocked by an IL-6 antibody. However, the overall role of ANG in mesangial cell proliferation has remained controversial as this has been observed by some (Orth et al., 1995) but not other investigators (Madhun et al., 1993). These experiments establish ANG, acting on AT1R, as a local inducer of inflammatory and perhaps proliferative responses in the glomerulum. In cultured mouse podocytes ANG or AGE induced DNA damage and cell detachment, both of which were prevented by telmisartan (Fukami et al., 2013). In the absence of exogenous ANG, the AGE-induced increase in superoxide generation, expression of RAGE and VCAM-1 and reduced expression of ACE2 were inhibited in mesangial cells by olmesartan but not azilsartan (Ishibashi et al., 2014). Exposure of rat mesangial cells and glomerular epithelial cells to a high glucose concentration caused oxidative stress and increased mRNA and protein expression of MCP-1, TGF-β and CTGF; all of these effects were prevented by valsartan (Huang et al., 2011; Jiao et al., 2011). In cultured human mesangial cells telmisartan reduced RAGE expression and AGE-induced superoxide generation and MCP-1 expression; these reductions were prevented by GW9662 and not mimicked by candesartan (Matsui et al., 2007).

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Glomerulosclerosis, similar to other fibrotic conditions, is characterized by deposition of extracellular matrix, and TGF-β is an important mediator in this process. In rat mesangial cells in the absence of exogenous ANG, telmisartan but not valsartan up-regulated PPAR-γ expression and inhibited the TGF-β-induced extracellular matrix accumulation; such inhibition was sensitive to GW9662 indicating PPAR-γ involvement (Yao et al., 2008). In rat mesangial cells TGF-β in the absence of exogenous ANG also activated protein kinase A and and stimulated CREB expression; both telmisartan and troglitazone inhibited the TGF-β effects and the expression of collagen and smooth muscle actin (Zou et al., 2010). In human mesangial cells telmisartan prevented TGF-β-induced collagen expression, apparently involving a PPAR-δ pathway (Mikami et al., 2014). Therefore, the overall evidence from cellular models supports a role of ARBs as a class in the prevention or treatment of glomerulonephritis and glomerulosclerosis, for instance in the context of diabetic nephropathy; this may partly occur by antagonizing locally formed ANG. Telmisartan may have an additional PPAR-γ-mediated effect on glomerular pathology.

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In proximal tubule cells, TNF-α caused release of lactate dehydrogenase and N-acetyl-βglucosaminidase, and this was inhibited by olmesartan and valsartan (Kagawa et al., 2008). Release of N-acetyl-β-glucosaminidase was also stimulated by exposure to albumin, and that was also inhibited by olmesartan and valsartan; such inhibition in the absence of exogenous ANG can be explained by the observation that albumin caused ANG release from tubule epithelial cells (Takao et al., 2009). In a human proximal tubule cell line, HK-2, ANG reduced the expression of bone morphogenetic protein-7, a renoprotective peptide, and such reduction was abolished by telmisartan (Bramlage et al., 2010). In the absence of exogenous ANG, TNF-α induced epithelial-to-mesenchymal transition in HK-2 cells; this was blocked by telmisartan in the absence but not presence of GW9662 (Chen et al., 2012b). In primary human proximal tubule cells, TNF-α induced the production of vascular endothelial growth factor at the mRNA and protein level, and this was inhibited by telmisartan; the TNF-α effect apparently involved phosphorylation of mitogen-activated protein kinase p38 and of heatshock protein 27, which also was inhibited by telmisartan (Kimura et al., 2014). The latter telmisartan effects apparently occurred independent of PPAR-γ and rather involved PPAR-δ. In Madin-Darby Canine Kidney cells, a distal tubule-derived cell line, ANG stimulated expression of the NF-κB-dependent transcription factor Ets-1 in a losartan-sensitive manner (Kumar et al., 2010). Thus, these data support the concept that ARBs as a class can protect tubule epithelial cells from pathological stimuli including TNF-α, and this may at least partly be due to blocking effects of ANG released by such stimuli; however, these protective ARB effects may also involve AT1R-independent mechanisms, suggesting that their extent may vary between ARBs. 7.2.2. Models of primary renal disease

The most frequently used in vivo model of primary renal disease is based on a surgical reduction of renal mass. This is most often performed as 5/6 nephrectomy in rats, but more severe models such as subtotal (17/18) nephrectomy (Toba et al., 2012) and less severe models such as unilateral nephrectomy (Matsuo et al., 2013; Ziai et al., 1996) have also been used. Nephrectomy in already hypertensive rats (Ziai et al., 1996) or in those receiving an additional ethylene glycol injection (Kadhare et al., 2015) has also been studied. Other than by surgical renal mass reduction, nephropathy can also be induced immunologically, for instance by injection of a monoclonal antibody against Thy-1 (Nakamura et al., 1997), which has also been applied in conjunction with unilateral nephrectomy (Song et al., 2012). In rat reduced renal mass models, ARBs as a class reduced albuminuria, the most frequently 103

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used functional parameter (Figure 11). This was shown for instance with candesartan (Hamaguchi et al., 1996; Hamar et al., 1999; Li et al., 1999b; Mackenzie et al., 1994; Noda et al., 1997; Noda et al., 1999; Podjarny et al., 2007; Shibouta et al., 1996; Sugimoto et al., 1999), eprosartan (Gandhi et al., 1999; Sasser et al., 2012; Wong et al., 2000), irbesartan (Cao et al., 2012; Tu et al., 2008; Ziai et al., 1996), losartan (Lafayette et al., 1992; Pollock et al., 1993a; Toba et al., 2012; Toba et al., 2013), telmisartan (Toba et al., 2012; Toba et al., 2013; Tsunenari et al., 2005; Zou et al., 2014), or valsartan (Wu et al., 1997; Zhang et al., 2008a). Interestingly, losartan and enalapril even lowered proteinuria in doses where they did not reverse the BP elevation caused by renal mass reduction (Pollock et al., 1993a). While most studies started ARB treatment immediately after renal mass reduction, studies with candesartan (Shibouta et al., 1996), losartan (Mayer et al., 1993) or telmisartan (Zou et al., 2014) reported improved proteinuria even when treatment started 4-11 weeks after the 5/6 ablation, suggesting that ARBs may not only be beneficial in a prevention but also in a treatment setting. Importantly, candesartan treatment has been reported to reduce mortality in rats with reduced renal mass (Podjarny et al., 2007).

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Some of these studies have directly compared improvement of albuminuria upon treatment with an ARB with that caused by an ACE inhibitor or a RAS-independent antihypertensive drug. In such studies ARBs and ACE inhibitors exhibited comparable improvements of albuminuria, for instance with candesartan vs. enalapril (Hamar et al., 1999; Noda et al., 1997; Noda et al., 1999; Shibouta et al., 1996; Sugimoto et al., 1999), candesartan vs. ramipril (Li et al., 1999b), irbesartan vs. enalapril (Ziai et al., 1996), irbesartan vs. perindopril (Cao et al., 2012), losartan vs. enalapril (Lafayette et al., 1992; Pollock et al., 1993a), telmisartan vs. enalapril (Tsunenari et al., 2005), or valsartan vs. ramipril (Wu et al., 1997) (Zhang et al., 2008a). The two classes of RAS inhibitor typically improved proteinuria to a similar extent, demonstrating that renoprotection in this model is a general feature of RAS inhibition. This conclusion is further supported by direct comparative studies with losartan and telmisartan, which also showed that the PPAR-γ antagonist GW9662 did not affect the telmisartaninduced reduction in proteinuria (Toba et al., 2012; Toba et al., 2013). In some studies, the effect of an ARB and ACE inhibitor combination was larger than that of either drug alone (Cao et al., 2012), but in others it was reported to be smaller than with each monotherapy (Lafayette et al., 1992); the former finding may be explained by submaximally effective doses of either agent, whereas the latter is somewhat difficult to explain based on the known pharmacology of both agents. Moreover, it was reported that losartan and enalapril lowered proteinuria also in doses where they did not reverse the BP elevation (Pollock et al., 1993a), pointing to a BP-independent effect. To explore this in more detail, some investigators compared effects of ARBs with those of RAS-unrelated BP-lowering medications. In one such study, the reduction of proteinuria was comparable but numerically greater with candesartan than with manidipine (Hamaguchi et al., 1996). In another study, three active treatments were compared which all caused similar BP lowering, losartan, enalapril and a reserpine/hydralazine/hydrochlorothiazide combination (Lafayette et al., 1992). While losartan and enalapril caused a similar improvement of albuminuria, the RAS-independent drug combination did not. Moreover, the β-adrenoceptor biased ligand nebivolol (Frazier et al., 2011), when given in a dose not lowering BP, did not improve proteinuria in a study where an ARB did (Sasser et al., 2012). On the other hand, clopidogrel improved proteinuria in 5/6 nephrectomy rats, and the clopidogrel + irbesartan combination was more effective in this regard than either drug alone (Tu et al., 2008) 104

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While proteinuria is the most frequently used indicator of damage in studies with renal mass reduction, other functional and also morphological parameters have also been used. Thus, renal mass reduction leads to glomerulosclerosis, and this has been reported to be reduced by treatment with candesartan (Mackenzie et al., 1994; Noda et al., 1997; Noda et al., 1999; Shibouta et al., 1996), eprosartan (Gandhi et al., 1999), irbesartan (Cao et al., 2012; Ziai et al., 1996), losartan (Lafayette et al., 1992), telmisartan (Tsunenari et al., 2005; Zou et al., 2014) or valsartan (Wu et al., 1997). In all cases where an ACE inhibitor was studied in comparison, it had similar effects as the ARB, for instance in the comparison of candesartan with enalapril (Noda et al., 1997; Sugimoto et al., 1999), losartan with enalapril (Lafayette et al., 1992), telmisartan with enalapril (Tsunenari et al., 2005) or valsartan with ramipril (Wu et al., 1997). However, when given in a later stage of the condition, candesartan had a greater effect than enalapril (Noda et al., 1997). On the other hand, the ARB effect on histologically detected glomerulosclerosis was also shared by RAS-independent agents such as manidipine (Hamaguchi et al., 1996), indicating that it may at least partly be a consequence of BP lowering. Interestingly, the inhibition of glomerulosclerosis upon ARB treatment was observed not only when the drug was given early, i.e. mimicking a preventive approach, but also when treatment started several months after the renal mass reduction, i.e. an treatment approach (Noda et al., 1999).

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ARBs including candesartan (Noda et al., 1997; Noda et al., 1999; Shibouta et al., 1996), eprosartan (Wong et al., 2000), telmisartan (Matsuo et al., 2013; Tsunenari et al., 2005; Zou et al., 2014) and valsartan (Wu et al., 1997) also reduced interstitial fibrosis and/or collagen expression in the remaining renal tissue, an effect being shared by ACE inhibitors such as enalapril (Noda et al., 1997; Tsunenari et al., 2005; Wu et al., 1997).

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ARBs also prevented renal infiltration by inflammatory cells, as shown for instance with candesartan (Hamar et al., 1999; Noda et al., 1997), telmisartan (Tsunenari et al., 2005) or valsartan (Wu et al., 1997). In one study the candesartan effect on macrophage infiltration was mimicked by the IL-2 synthesis inhibitor tacrolimus, and the combined administration of both drugs caused even greater reduction (Hamar et al., 1999). Reduced renal mass models typically exhibit an increased level of TGF-β1 expression in the remnant tissue. This increase was inhibited by various ARBS, including candesartan (Noda et al., 1997; Noda et al., 1999), eprosartan (Wong et al., 2000), irbesartan (Tu et al., 2008), telmisartan (Tsunenari et al., 2005) and valsartan (Wu et al., 1997). At the functional levels ARBs reduced the elevated glomerular capillary pressure typically observed after renal mass reduction, as reported e.g. with candesartan (Mackenzie et al., 1994), irbesartan (Ziai et al., 1996) and losartan (Lafayette et al., 1992; Mayer et al., 1993). Taken together these data demonstrate that ARBs as a class, similar to ACE inhibitors, are effective in the prevention and treatment of renal damage induced by renal mass reduction. This can be observed by morphological parameters including glomerulosclerosis and fibrosis but also functionally as shown by a reduced proteinuria. BP reduction may partly explain this effect, but parts of it apparently are independent of BP reduction. The most frequently used alternative to renal mass reduction for the generation of animal models of primary kidney disease is the immunological induction of renal damage. Early work on the latter was based on injection of an antibody directed against glomerular basement membrane in rats. This induced transient elevation of both plasma renin and angiotensinogen, followed by acute nephritis as indicated by proteinuria, hypoalbuminemia, hypercholesterolemia and an increase in serum creatinine; candesarten administed 2 h before an injection with the antibody prevented the nephritis, whereas successive administration of 105

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this drug for 1 week from 3 d after the injection of antibody did not (Yayama et al., 1995b), indicating that AT1R play a pathophysiological role in the development of this nephritis model but not in its maintenance. In this model glomerulonephritis is accompanied by an early increase in TGF-β1 mRNA in the renal cortex, which can be suppressed by repeated candesartan administration, apparently inhibiting locally generated ANG (Yayama et al., 1995a).

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For studies in a treatment design, more informative data come from injection of a monoclonal antibody against Thy-1, a 25 kDa protein expressed on the endothelium-facing surface of mesangial cells; this antibody is sometimes used in combination with unilateral nephrectomy as an animal model of mesangioproliferative glomerulonephritis (Cheng et al., 1995). In the anti-Thy-1 rat model, administration of irbesartan or losartan 24 h prior to antibody injection largely prevented the early up-regulation of MCP-1 at the mRNA and protein level (Wolf et al., 1998). A 10-week treatment with candesartan starting shortly after injection of the antibody prevented the increase in proteinuria and in renal expression of TGF-β1 and type I and III collagen; hydralazine administerd in equally BP-lowering doses dit not mimic the candesartan effect (Nakamura et al., 1997). A follow-up study from the same group compared effects of candesartan with those of cilazapril, cilazapril + icatibant, and hydralazine (Nakamura et al., 1999). This study confirmed the beneficial effects of candesartan and the lack of effect of hydralazine. Cilazapril mimicked the candesartan effect, and this was not attenuated by concomitant icatibant.

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From a treatment perspective possibly even more interesting are studies in which an ARB was administered several days or weeks after the anti-Thy-1 injection. In one study a low telmisartan dose, a high telmisartan dose and a hydrochlorothiazide + hydralazine combination were started 4 days after disease induction; while the low telmisartan dose did not lower BP, the high dose and the diuretic + hydralazine dose lowered BP to a similar extent (Villa et al., 2011). All three treatments reduced glomerular endothelial cell proliferation and glomerular and interstitial matrix deposition on day 14; however, only the high telmisartan dose reduced podocyte damage and tubular cell dedifferentiation on day 9 and mesangial cell activation on day 14. In a follow-up study from the same group, treatment with a low and a high telmisartan dose, atenolol or a hydrochlorothiazide + hydralazine combination was started 28 days after disease induction and maintained until day 131 (Villa et al., 2013). At study end, the high telmisartan dose but neither atenolol nor the hydrochlorothiazide + hydralazine combination prevented loss of renal function and reduced proteinuria compared to control rats; moreover, only the high telmisartan dose ameliorated glomerulosclerosis, tubulointerstitial damage, cortical matrix deposition, podocyte damage and macrophage infiltration and reduced cortical expression of platelet derived growth factor receptor-α and -β as well as TGF-β1. The low telmisartan dose exhibited minor but significant efficacy in the absence of antihypertensive effects. In another study, valsartan given from week 2 to 8 after anti-Thy-1 injection prevented glomerulosclerosis progression (Song et al., 2012). Taken together these data demonstrate that both ARBs and ACE inhibitors are renoprotective in the Thy-1 model of glomerulonephritis. Such protection is observed in preventive and treatment designs, even if treatment started several weeks after disease induction. BP lowering does not play a major role in renoprotection in this model as several classes of anti-hypertensive drugs had only little beneficial effect on renal function. In a rat model of IgA nephropathy induced by a combination of bovine serum albumin, lipopolysaccharide and CCl4, a 10 week treatment with losartan, telmisartan or pioglitazone markedly improved proteinuria, mesangial cell proliferation and inflammatory cell infiltration with telmisartan having the quantitatively greatest effect (Xing et al., 2010). In a mouse model 106

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anti-glomerular basement membrane nephritis, induced by injection of a rabbit anti-serum against mouse glomerular basement membrane, treatment with telmisartan but not with hydralazine suppressed glomerular damage (Hamano et al., 2011). Co-administration of GW9662 attenuated the renoprotective effect of telmisartan, and losartan only partly mimicked the telmisartan effect.

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In rats with passive Heyman nephritis, a high losartan dose and enalapril reduced proteinuria numerically to the same level, but this was difficult to interpret as control group for the losartan treated rats exhibited a very low proteinuria prior to treatment (Hutchinson & Webster, 1992). Within that study a lower losartan dose had the expected basal proteinuria, and against this baseline level a reduction with losartan was observed. In contrast to enalapril, both losartan doses increased GFR in that study. Taken together these data show a renoprotective ARB effect in animal models of primary renal disease which is independent of BP lowering; some ARBs may have an additional PPAR-γ-mediated component of renoprotection.

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Some vascular beds exhibit a greater sensitivity to ANG-induced vasoconstriction in SHR as compared to normotensive rats. Therefore, several investigators have explored ARB effects on RBF. Isolated pre-glomerular microvessels from SHR and WKY exhibited a similar density of ANG binding sites, which largely consisted of AT1R in both strains (Chatziantoniou & Arendshorst, 1993). Basal intracellular Ca2+ concentrations and candesartan-sensitive ANG-induced elevations thereof were similar in smooth muscle cells isolated from preglomerular vessels of WKY and SHR (Iversen & Arendshorst, 1999). In isolated afferent arterioles ANG induced a stronger concentration-dependent vasoconstriction in vessels from young, pre-hypertensive SHR as compared to age-matched WKY; however, this strain difference was attributed to a loss of AT2R-mediated vasodilatation in SHR and not to an increased sensitivity of AT1R (Endo et al., 1998). In line with these observations, ANG similarly reduced RBF in SHR and WKY in some studies (Chatziantoniou & Arendshorst, 1993). However, in other studies an exaggerated ANG-induced RBF reduction in SHR as compared to WKY have been reported (Li & Widdop, 1996; Ruan et al., 1999); this was observed either as a greater RBF reduction for a given ANG dose or as a greater ANG potency to cause a certain degree of RBF lowering. Irrespective of whether RBF reduction was enhanced, inhibition of ANG-induced reduction by ARBs was similar in both strains as shown for instance with candesartan (Li & Widdop, 1996; Ruan et al., 1999) or losartan (Chatziantoniou & Arendshorst, 1993). In the absence of exogenous ANG, candesartan (Inishi et al., 1997; Li & Widdop, 1996; Takishita et al., 1994), losartan (Kline & Liu, 1994; Li & Widdop, 1996), olmesartan (Koike et al., 2001) and valsartan (Hayashi et al., 1997b) increased RBF in SHR, which is in contrast to the equivocal RBF data in normotensive rats (see section 7.1.2). In direct comparative studies, the ARB-induced RBF elevation was only seen in the hypertensive rats but not normotensive rats (Li & Widdop, 1996). Within SHR, the ARB-induced increase in perfusion was specific for the kidney as it was not observed in several other tissues (Koike et al., 2001). Moreover, it occurred at BP-lowering ARB doses, whereas a similarly BP lowering dose of nicardipine reduced RBF (Takishita et al., 1994). In the absence of exogenous ANG, GFR was increased in SHR by candesartan (Inishi et al., 1997) but not by losartan (Kline & Liu, 1994). These data demonstrate that ARBs as a class increase RBF in SHR, and this does not occur as a consequence of but rather despite BP lowering.

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Other studies have explored ARB effects on tubular function in the SHR. The activity of the tubuloglomerular feedback mechanisms, as assessed by micropuncture and stop-flow studies, was increased in 7-week old SHR as compared to two different normotensive rat strains, and this exaggerated response was normalized by treatment with candesartan (Brännström & Arendshorst, 1999). Reports on ARB effects on diuresis and natriuresis are inconsistent. Thus, acute exposure to a BP-lowering dose of losartan did not affect urine flow in SHR (Wong et al., 1990d). At renal artery pressures between 130 and 175 mm Hg losartan also failed to alter urine flow or total or fractional sodium excretion, but sensitized the pressure-natriuresis mechanism (Kline & Liu, 1994). Treatment with candesartan for up to two weeks caused only minor and inconsistent enhancements of diuresis and natriureses in SHR (Wada et al., 1996b). Treatment with a BP-lowering dose of telmisartan for up to two weeks had only minor effects on diuresis and natriuresis in SHR, but tended to enhance the diuretic effect of hydrochlorothiazide (Wienen & Schierok, 2001). Azilsartan did not alter natriuresis relative to vehicle in normotensive rats or SHR but increased it in the hypertensive/NIH-corpulent SHR/NDmcr-cp strain (Sueta et al., 2013). Thus, ARBs as a class appear to have only minor effects on diuresis and natriuresis in SHR but may sensitize the pressure-natriuresis mechanism; moreover, they may enhance the effects of concomitantly administered diuretics.

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Based on nephroprotective findings in animal models of primary renal disease (see section 7.2.2), it has been explored whether ARBs also provide nephroprotection in SHR. Of note indicators of renal damage such as protein excretion only develop at an age of 6 months and older and typically are less pronounced than in the renal mass reduction models. In young SHR candesartan treatment starting before the development of over hypertension did not affect the number or volume of glomerula (Kett et al., 1996). In 15 week old SHR, a 12 months treatment with candesartan almost normalized urinary protein excretion and histologically assessed glomerulosclerosis (Ishimitsu et al., 2010). In 32 week old SHR, a 6 week treatment with olmesartan dose-dependently attenuated proteinuria, interestingly to a considerably greater extent than in DOCA-salt-treated rats (Koike et al., 2001). In 1 year old SHR, a 15-week treatment with telmisartan reduced glomerular volume whereas proteinuria and plasma and urine creatinine concentrations were not affected to a major extent (Mandarim-de-Lacerda & Pereira, 2004). A considerably larger reduction of renal albumin excretion was reported in another study in which 20 week old SHR were treated with telmisartan for 16 weeks; glomerular extracellular matrix was also reduced in that study (Zhu et al., 2012). Azilsartan was reported to fully prevent development of proteinuria in spontaneously hypertensive obese rats along with a reduction in a glomerular injury score (Hye Khan et al., 2014a). Of note the negative study reported a much smaller urinary protein excretion in the control group than the positive studies. As nephropathy develops more slowly in SHR than in the reduced renal mass models, several investigators have used SHR with aggravated hypertension; this includes the stroke-prone strain of SHR and SHR with additional reduction of renal mass, treatment with cyclosporine A or salt-loading (data on diabetes in SHR will be discussed in section 7.2.6). In one study stroke-prone SHR were treated with one of three candesartan doses or with enalapril from week 22 to 32 weeks and at that point were compared to age-matched WKY rats (Kim et al., 1994a; Kim et al., 1994c). The low candesartan dose did not reduce BP, the middle dose reduced it to a similar extent as enalapril, and the high dose normalized BP. Vehicle-treated stroke-prone SHR had a greater kidney weight than WKY rats, and all four treatments reduced renal weight, although not reaching WKY levels. Candesartan dose-dependently reduced renal protein excretion; the lowest dose not lowering BP already by about half, the middle dose similarly to enalapril, but even the highest dose did not reduce it to WKY levels. All four treatments prevented the rise in blood urea nitrogen and markedly reduced the serum 108

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creatinine elevation. Moreover, all four treatments markedly reduced the strongly increased mRNA expression of TGF-β1, fibronectin and collagens I, III and IV. In a quantitative analysis of histological examination, the low candesartan dose was without effect on all parameters, whereas the high dose reduced fibrinoid necrosis of the glomerulus, tubular atrophy, hypertrophy of the tubular basement membrane, and hypertrophy and fibrinoid necrosis of the afferent arteriole; however, none of the treatments affected interstitial fibrosis. A follow-up study from the same group using a similar design confirmed that even a low candesartan dose without BP effects markedly reduced proteinuria after 10 weeks of treatment; however, when assessed 4 weeks after start of treatment, a reduction of proteinuria was not longer detectable for the low candesartan dose whereas the two other doses and enalapril exhibited a similar degree of proteinuria improvement as after 10 weeks of treatment (Inada et al., 1997). Other investigators compared nephroprotection by candesartan with that by hydralazine, applied at dose yielding similar BP lowering; under these conditions candesartan exhibited a greater reduction of proteinuria and glomerulosclerosis index (Nakamura et al., 1994b). A reduction in urinary albumin excretion and TGF-β1 and fibronectin mRNA expression by candesartan but not by hydralazine was also reported in another study in stroke-prone SHR (Obata et al., 1997). Other investigators treated 4 months old stroke-prone SHR with losartan, two doses of telmisartan or captopril for 38 days (Wagner et al., 1998). All four treatments lowered BP, although observed to a much smaller exent with the lower telmisartan dose; nevertheless, all four treatments yielded a similar reduction in renal albumin excretion and glomerulosclerosis index. Taken together these findings consistently demonstrate that ARBs and ACE inhibitors as a class provide nephroprotection in stroke-prone SHR. These effects were also seen at ARB doses providing limited or no BP lowering and they were not mimicked by hydralazine, demonstrating that the nephroprotection in this animal model occurs largely independent of BP lowering. However, low ARB doses not lowering BP may require a longer treatment time to achieve nephroprotection.

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To achieve an even greater aggravation of hypertension, ARB effects have also been tested in stroke-prone SHR which had additionally received a high-salt diet or a high-salt, high-fat diet. In salt-loaded stroke-prone SHR, treatment with valsartan or benazepril starting at 10.5 weeks of age lowered BP, prevented proteinuria, decreased the severity of histopathological changes in the kidney and, most importantly, prolonged survival (Webb et al., 1998a). In another study 10 week old stroke-prone SHR received 2 weeks of a high-salt diet accompanied by valsartan, hydralazine or no drug (Yoshida et al., 2011). Compared to untreated WKY rats, stroke-prone SHR on a high-salt diet exhibited a markedly increased tubuloinsterstitial injury score, which was strongly reduced by valsartan but not by hydralazine. Other investigators put stroke-prone SHR on a high-salt/high-fat diet for 2 weeks and then added control or eprosartan treatment for up to 12 weeks (Barone et al., 2001). While hypertensive rats on a normal diet exhibited only a minor degree of proteinuria in this study, those on the high-salt/high-fat diet had a higher BP and a much greater renal protein excretion; eprosartan normalized this to levels observed in WKY. Importantly, rats receiving control treatment started dying as early as 4 weeks after initiation of treatment and almost all died after 9-12 weeks; in contrast, not a single rat died in the eprosartan group. The reduction of proteinuria by eprosartan in strokeprone rats on a high-salt/high-fat diet was confirmed in a study of similar design (Abrahamsen et al., 2002). Additionally, this study reported that eprosartan reduced body weight loss by the diet and the development of renal hypertrophy, largely restored histologically observed renal damage, and the markedly elevated mRNA expression of TGF-β1, fibronectin and collagen I and III.

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Candesartan has also been tested in SHR in which renal damage had been aggravated by renal mass reduction surgery. In one such study, 12 week old SHR underwent a 5/6 nephrectomy and 1 week later were randomized to receive candesartan, delapril, hydralazine or vehicle for 4 weeks (Kohara et al., 1993). The three active treatments similarly lowered BP but only the RAS inhibitors reduced proteinuria, serum creatinine and histologically assessed glomerulosclerosis. However, all three treatments improved blood urea nitrogen and reduced mortality. In another study, SHR underwent a 5/6 nephrectomy followed by a high-salt diet and treatment with candesartan or enalapril or no treatment for 8 weeks (Kanno et al., 1994; Okada et al., 1995). Both RAS inhibitors lowered BP, reduced proteinuria and improved histopathological changes. The immunosuppressant cyclosporine A elevates BP and causes renal damage (Marcen, 2009). SHR aged 8-9 weeks were placed on a combination of highsalt diet and cyclosporine A treatment and additionally received treatment with enalapril or two doses of valsartan or no treatment (Lassila et al., 2000b). All active treatments prevented the cyclosporine A-induced BP increase and similarly lowered urine volume, serum creatinine, proteinuria and histologically determined glomerular damage index. A separate set of animals received enalapril or an enalapril + icatibant combination, but addition of the bradykinin receptor antagonist affected neither BP lowering nor nephroprotection. In conclusion, RAS inhibition whether by ARBs or ACE inhibitors provides nephroprotection in SHR, including those in which hypertension and/or renal damage had been aggravated by various approaches. Such nephroprotection occurs apparently largely independent of BP lowering. Similar improvement of proteinuria has also been reported in another genetic model of hypertension with renal disease, the Imai rat, upon treatment with olmesartan (RodriguezIturbe et al., 2004). 7.2.4. Other hypertension models

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Against the background of nephroprotective effects of RAS inhibition in SHR, several other animal models of hypertension have also been used to explore renal effects of ARBs. Acute infusion of ANG in healthy rats had only minor effects on renal plasma flow, GFR or filtration fraction but dose-dependently increased renal protein excretion in an irbesartansensitive manner (Lapinski et al., 1996). Chronic ANG infusion was reported to deteriorate renal autoregulatory capability in a candesartan-sensitive manner (Inscho et al., 1999). Moreover, a chronic ANG infusion caused podocyte injury and markedly increased urinary albumin excretion in rats which were mitigated by concomitant telmisartan treatment (Ren et al., 2012). Chronic ANG infusion in mice induced expression of various pro-inflammatory molecules in the tubules in a telmisartan-sensitive manner (Kumar et al., 2010). Moreover, a 5-day ANG infusion also caused renal fibrosis, and this was attenuated by valsartan in a dose where it only weakly mitigated the ANG-induced BP elevation (Sakai et al., 2008). However, not only ANG but also chronic aldosterone administration caused nephropathy in rats, which was characterized by enhanced urinary albumin excretion, that was attenuated by telmisartan and eplerenone but not by amlodipine, despite the latter causing the greatest BP reduction (Liang et al., 2011a). Aldosterone administration also caused glomerulosclerosis and podocyte apoptosis which were inhibited by telmisartan and amlodipine, although to a smaller extent than by eplerenone. Studies in animal models of renovascular hypertension have largely focused on vascular, glomerular and tubular function in the 2-kidney-1-clip Goldblatt model in rats and dogs. Acute treatment with losartan reduced renal vascular resistance in such rats with renovascular hypertension; while the effect was shared by enalapril but not by furosemide (Wang et al., 1992). Others have acutely administered candesartan directly into the renal artery of the nonclipped kidney in a dose not affecting systemic BP but sufficient to suppress renal 110

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vasoconstricting responses to ANG (Cervenka et al., 1999). At this dose, in the absence of exogenous ANG, candesartan increased RBF, GFR and absolute and fractional sodium excretion to a similar extent as in sham-operated normotensive rats. A third study applied micropuncture to assess effects of a 5 day treatment with losartan or captopril on glycineinduced hyperfiltration (De Nicola et al., 1992). In comparison to normotensive control rats, renal hypertension was associated with a loss of renal functional reserve and a decrease in absolute proximal reabsorption during glycine administration. Losartan and captopril normalized systemic and glomerular capillary pressure and prevented the glycine-induced decrease in proximal reabsorption, but only captoptil increased single nephron GFR, suggesting involvement of AT1R-mediated and AT1R-independent effects contributing to the captopril effect. Looking at chronic effects, investigator have treated 2-kidney-1-clip rats with telmisartan, the dipeptidylpeptidase IV inhibitor linagliptin or their combination for 16 weeks (Chaykovska et al., 2013). Telmisartan alone or in combination normalized BP and prevented development of proteinuria in such animals, whereas linagliptin alone did not. Neither treatment affected histologically determined glomerulosclerosis in the clipped or the nonclipped kidney; telmisartan but not linagliptin or the combination increased fibrosis in the clipped kidney but none of the treatments did so in the non-clipped kidney. In 2-kidney-1-clip dogs an acute administration of candesartan or enalapril markedly lowered BP but did not alter effective renal plasma flow or GFR (Ito et al., 1995). A 14 day treatment of 2-kidney-1clip renal hypertensive dogs with olmesartan also lowered BP and moderately increased diuresis and natriuresis (Koike et al., 2001).

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Dahl salt-sensitive rats on a high-salt diet are an interesting model to study renal effects of ARBs as RAS activity is suppressed in these animals. Treatment of Dahl salt-sensitive rats on a high-salt, high-fat diet with azilsartan, chlorthalidone or their combination for 26 days (starting concomitantly with the diet) did not alter GFR, diuresis, natriuresis or kaliuresis but reduced proteinuria, renovascular and tubular injury, glomerulosclerosis and renal fibrosis (Jin et al., 2014). In most cases, however, ARB treatment was started several weeks after initiation of the high-salt diet. Under these conditions, a 4 week treatment with candesartan or captopril similarly reduced glomerulosclerosis score and urinary protein excretion in saltloaded Dahl salt-sensitive rats (Ideishi et al., 1994). In another study, candesartan and cilazapril similarly reduced proteinuria and improved histologically determined glomerulosclerosis despite little BP lowering (Otsuka et al., 1998). A 4 week treatment with irbesartan reduced glomerulosclerosis score, proteinuria and renal mRNA expression of TGFβ, MCP-1 and collagen I; none of these effects was mimicked by amlodipine, despite both drugs yielding similar BP lowering (Takai et al., 2010). A 10 week treatment with losartan had only little effect on BP but delayed the histologically determined occurrence of renal damage and concomitantly increased survival (von Lutterotti et al., 1992). A 5-week treatment with losartan or telmisartan similarly reduced tubular fibrosis (Liang & Leenen, 2008). In another study, telmisartan and hydralazine lowered BP to a similar extent, but telmisartan reduced glomerular damage and renal protein excretion considerably more than hydralazine (Satoh et al., 2010a). Olmesartan treatment also reduced renal protein excretion (Kiya et al., 2010). In a doses causing moderate BP lowering, valsartan but not amlodipine reduced renal protein excretion but the glomerulosclerosis score was not altered by either treatment (Aritomi et al., 2010). Taken together, ARBs as a class and ACE inhibitors similarly improved renal morphology and function in Dahl salt-sensitive rats on a high-salt diet. These effects were observed regardless of whether BP lowering was observed and, in cases where BP lowering was seen were not shared by RAS-independent anti-hypertensive drugs.

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A limited number of studies have also explored ARB effects in the rat DOCA-salt model of hypertension. Treatment with candesartan or enalapril did not affect BP but similarly reduced renal protein excretion and mRNA expression of TGF-β, fibronectin and several collagen isoforms; upon histological examination this was accompanied by decreased epithelial atrophy, basement membrane hypertrophy and glomerular fibrinoid necrosis (Kim et al., 1994b). A reduction of urinary protein excretion and an improvement of renal histology in the absence of BP changes were confirmed in another study with candesartan treatment, in which these effects were mimicked by enalapril (Wada et al., 1995). An attenuated proteinuria in the absence of BP lowering in DOCA-salt hypertensive rats was also reported with olmesartan (Koike et al., 2001). Taken together these data further support the concept of nephroprotection by ARBs as a class, which is shared with ACE inhibitors and occurs independent of BP lowering.

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In transgenic (mRen-2)27 rats acute treatment with candesartan or captopril reversed the rightward-shift of the pressure natriuresis curve typically observed in these animals (Gross et al., 1995). In this model a 9 week treatment with telmisartan dose-dependently reduced glomerulosclerosis and albuminuria (Figure 12); of note, the lowest telmisartan already reduced albumin excretion by more than half despite not lowering BP, whereas the highest dose had an even greater effect on albuminuria (Böhm et al., 1995). Other investigators confirmed a reduction of proteinuria with a BP-lowering dose of telmisartan and extended these findings to an amelioration of loss of adhesion, proximal tubule basolateral remodeling and tubulo-interstitial fibrosis (Whaley-Connell et al., 2011). A third group used heterozygote rats transgenically expressing the mouse Ren-2 gene, which resulted in a milder phenotype without major proteinuria; these were treated with valsartan, bosentan or vehicle (Kelly et al., 2000). Despite having a markedly elevated BP (reduced by both active treatments), these rats did not exhibit major albuminuria; valsartan did not change this but bosentan markedly increased urinary albumin excretion. However, vehicle-treated rats had an increased glomerulosclerosis index, which was reduced by both active treatments – albeit to a greater extent by valsartan. In congenic mRen-2.Lewis hypertensive rats monotherapy with valsartan or aliskiren for 2 weeks lowered BP to a similar extent; neither monotherapy had major effects on urinary protein excretion but combination treatment lowered it by half (Moniwa et al., 2013). However, the combination group exhibited extensive cortical areas of peritubular and periglomerular fibrosis, disorganized tubular proliferation, and tubular dilatation/atrophy; many congestive, enlarged glomeruli with aneurysmatic capillary dilatations as well as some nonperfused (sclerotic) glomeruli were also observed in this group. The authors interpreted this as a renal homeostatic stress response attributable to loss of tubuloglomerular feedback upon combined AT1R blockade and renin inhibition. In double-transgenic rats harboring both the human renin and angiotensinogen genes, valsartan, cilazapril or the human renin inhibitor, RO 65-7219, prevented the development of albuminuria (Mervaala et al., 1999). While only the ARB and the ACE inhibitor lowered BP, all three treatments similarly prevented monocyte/macrophage infiltration and the renal overexpression of adhesion molecules, plasminogen activator inhibitor-1, and fibronectin. Taken together these data confirm the role of ARBs and ACE inhibitors as nephroprotective agents and extend them to additional animal models; however, they also raise the possibility that dual RAS inhibition may have adverse effects, but it remains unclear whether this a specific feature of mRen-2.Lewis rats or also applies to other models. <<>> ARB effects on renal function have also been explored upon absence or inhibition of one or more isoforms of NO synthase. Endothelial NO synthase knock-out mice with additional cuff 112

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injury of the femoral artery developed prominent glomerulosclerosis and glomerular macrophage infiltration along with increased urinary albumin excretion as compared to wildtype mice (Yamamoto et al., 2009a). These parameters were not affected by treatment with hydralazine but similarly improved by treatment with valsartan or aliskiren and even more so by combination of the two RAS inhibitors. A more frequently applied model is the inhibition of all NO synthases by treatment with L-NAME, typically applied to rats. Acute administration of L-NAME markedly reduced RBF in anesthetized and conscious rats; concomitant losartan treatment abolished this RBF reduction in anesthetized but not in conscious animals (Sigmon & Beierwaltes, 1993a). Chronic administration of L-NAME leads to damage of pre-glomerular vessels, a reduced RBF and GFR, a right-shifted pressurenatriuresis curve and an increased urinary albumin excretion. Treatment with ARBs including candesartan, losartan and olmesartan reduced vascular damage (Casellas et al., 1999), RBF and GFR reduction (Fortepiani et al., 1999; Moningka et al., 2012; Ribeiro et al., 1992), a right-shifted pressure-natriuresis curve (Fortepiani et al., 1999), glomerular damage (Moningka et al., 2012; Okamura et al., 1994; Ribeiro et al., 1992), and attenuated albumin excretion (Casellas et al., 1999; Moningka et al., 2012; Okamura et al., 1994). In direct comparative studies, candesartan was more effective than hydralazine for protection of preglomerular vessels while both similarly lowered BP and albumin excretion (Casellas et al., 1999). While candesartan, captopril and the Ca2+ entry blocker verapamil similarly lowered BP and improved renal hemodynamics, neither normalized pressure-natriuresis (Fortepiani et al., 1999). Olmersartan, the atypical β-blocker nebivolol and their combination similarly improved GFR and normalized BP and urinary protein excretion (Moningka et al., 2012). These data demonstrate that in NO synthase inhibition models ARBs and other RAS inhibitors similarly improve renal function and morphology; these effects are partly but not fully shared by other BP lowering treatments. The beneficial renal effects of an ARB were maintained when a high-salt diet was given in addition to L-NAME treatment (Okamura et al., 1994). Taken together, the studies in this section consistently support a role of the RAS in hypertensive nephropathy. 7.2.5. Ureteral obstruction

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Obstruction of the ureter imposes retrograde pressure in kidney which may lead to renal damage, specifically fibrosis. As renal dysfunction secondary to ureteral obstruction is likely to have a very different pathophysiology than other models of renal disease, it is an interesting extension to studies of potential renoprotective effects of ARBs. Studies in this area are typically based on unilateral obstruction, which allows using the contra-lateral kidney as internal control. The damage imposed by such obstruction can be detected by proxy parameters as early as 12 hours after obstruction (Moriyama et al., 1997), but most studies have applied treatment phases of several days or weeks. Some studies have focused on the effects of ureteral obstruction on the intrarenal vasculature. To better observe vascular effects of RAS inhibition, an early study has pre-treated rats with either losartan or lisinopril for 5 days, surgically induced unilateral ureteral obstruction for 1 day and then measured renovascular function 3 hours after release of the obstruction (Pimentel et al., 1993). The obstructed as compared to the contra-lateral kidney exhibited marked reductions in GFR and renal plasma flow which were attenuated but not restored to control levels by either RAS inhibitor in a dose lowering systemic BP. Based on increased mRNA expression of renin and ACE in the renal vasculature of the obstructed kidney, these changes in renal hemodynamics were interpreted as reflecting at least partly inhibition of the local RAS. Both drugs also increased diuresis, natriuresis and kaliuresis from the obstructed kidney. An increase in renal levels of ACE and ANG was also reported in a study following 113

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obstructed animals for up to 5 weeks; in that model losartan also lowered the elevated BP (ElDahr et al., 1993). Another study explored acute effects of candesartan in comparison to manidipine two months after surgically induced hydronephrosis in stroke-prone SHR (Kimura et al., 1994). In this model both drugs reduced BP and dilated the afferent and efferent arterioles, thereby increasing glomerular blood flow.

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Other investigations focused on the renal fibrosis occurring secondary to ureteral obstruction. In mice, a 7-day treatment with telmisartan (Sugiyama et al., 2005) or a 28-day treatment with valsartan markedly reduced renal fibrosis in the obstructed kidney (Sakai et al., 2008). In rats, candesartan and lisinopril similarly ameliorated the fibrotic changes observed in the renal interstitium 7 days after the obstruction; this accompanied by reduced mRNA expression of type I collagen and the collagen-specific molecular chaperone HSP47 (Moriyama et al., 1997). In another study the effects of valsartan, aliskiren and their combination were tested 7 and 14 days after induction of the obstruction, using drug doses which yielded similar small reductions of BP (Wu et al., 2010). Both drugs similarly attenuated the increase in tubular dilatation, interstitial volume, α-smooth muscle actin expression and interstitial collagen deposition. For all of these parameters the combination was more effective than either drug alone, but still failed to inhibit more than half of the obstruction-induced increase. Of note, the renal damage caused by ureteral obstruction did not induce proteinuria. In contrast, proteinuria was observed in a mouse model of ureteral obstruction, and this was ameliorated by telmisartan treatment starting 5 days prior to ureteral ligation; renal fibrosis was also reduced in this model (Wong et al., 2014).

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Taken together, ureteral obstruction leads to activation of the renal tissue RAS. BP-lowering doses of RAS inhibitors as a class and perhaps also RAS-independent BP lowering drugs can attenuate the renovascular changes and fibrosis in the obstructed kidney but fail to restore normal renal function and morphology, even when applied prior to obstruction. In AT1R knock-out mice with hydronephrosis induced by unilateral ureteral obstruction telmisartan reduced renal fibrosis, glomerular and tubulointerstitial injury, and area staining positive for macrophages, myofibroblasts or TGF-β1 as observed 14 days after induction of the obstruction (Kusunoki et al., 2012). For all of these parameters losartan exhibited smaller effects or even a lack of effect; moreover, the effects of telmisartan were blocked by GW 9662. Presence of hepatocyte growth factor in the obstructed kidney and in serum was increased by telmisartan but not by losartan or by obstruction alone, an effect also prevented by GW 9662; this appears of interest as an antibody against hepatocyte growth factor similarly to GW 9662 prevented all beneficial effects of telmisartan in the obstructed kidney. These data support the idea that some ARBs may improve renal morphology and function in the ureteral obstruction model beyond the benefit derived from RAS inhibition. 7.2.6. Diabetes mellitus and obesity 7.2.6.1. Studies in lean diabetic animals

RAS inhibitors are established agents to prevent or ameliorate renal damage caused by longstanding diabetes mellitus. Correspondingly, many studies have assessed nephroprotective effects of ARBs, alone or in combination with other drugs, in variety of animal models of type 1 and type 2 diabetes. Among models of type 1 diabetes, rats and mice treated with STZ have been used most often to explore nephroprotective effects of ARBs. Of note, this is a model in which ARBs typically have little effect on blood glucose (see section 6.3.2), indicating that observed nephroprotective effects are not related to an anti-diabetic action per 114

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se. Thus, ARBs as a class consistently reduced proteinuria in the STZ model of type 1 diabetes as observed for instance with candesartan (Kato et al., 1999), irbesartan (Bonnet et al., 2001; Cao et al., 2001), telmisartan (Bishnoi et al., 2012; Mudagal et al., 2011; Rao et al., 2011; Satoh et al., 2010b; Singh et al., 2006; Tojo et al., 2015; Zhang et al., 2012), or valsartan (Allen et al., 1997; Chen et al., 2008; Kelly et al., 2000; Remuzzi et al., 1998; Tang et al., 2012). Of note, several of these studies have been performed under condition of aggravated renal damage, for instance with STZ application to hypertensive rat strains such as SHR (Bonnet et al., 2001; Cao et al., 2001; Wienen et al., 2001), MWF rats (Remuzzi et al., 1998) or transgenic (mRen-2)27 rats (Kelly et al., 2000) or following 5/6 nephrectomy (Chen et al., 2008). One study provided evidence that an ARB may also suppress renal gluconeogenesis in a rat STZ model (Tojo et al., 2015).

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In line with the clinical data, several studies have demonstrated a similar extent of nephroprotection by an ARB and an ACE inhibitor, for example candesartan vs. enalapril (Kato et al., 1999), telmisartan vs. benazepril (Singh et al., 2006), telmisartan vs. Lisinopril, valsartan vs. benazepril (Remuzzi et al., 1998) or valsartan vs. ramipril (Allen et al., 1997). However, in few studies the ARB has been reported to more effectively reduce proteinuria than the ACE inhibitor, for instance with irbesartan vs. captopril (Cao et al., 2001). Some studies have also explored the effect of combination treatments on proteinuria. In a study in which both telmisartan and trandolapril rduced proteinuria, their combination was even more effective (Rao et al., 2011). In contrast, in a study where captopril monotherapy lacked effect, the combination of irbesartan and captopril did not reduce albuminuria to a greater extent than irbesartan alone (Cao et al., 2001). Other studies in which combination treatment provided greater nephroprotection than ARB monotherapy included studies with telmisartan combined with fenofibrate (Bishnoi et al., 2012), with aspirin (Mulay et al., 2010), or with atorvastatin (Mudagal et al., 2011).

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While the above studies show a class effect of ARBs for reduction of proteinuria in rat models of STZ-induced diabetes, studies on other parameters of renal damage have yielded less consistent results. Thus, a reduction of serum concentrations of urea was reported in some (Bishnoi et al., 2012; Singh et al., 2006; Zhang et al., 2012) but not other studies (Mulay et al., 2010; Satoh et al., 2010b). Similarly, a reduced serum creatinine was found in some (Bishnoi et al., 2012; Mudagal et al., 2011; Tang et al., 2012; Zhang et al., 2012) but not other studies (Mudagal et al., 2011; Mulay et al., 2010; Satoh et al., 2010b; Wienen et al., 2001). Effects on GFR were also inconsistent across studies (Kelly et al., 2000; Onozato et al., 2008; Remuzzi et al., 1998; Satoh et al., 2010b; Tojo et al., 2015; Wienen et al., 2001; Zhang et al., 2012). While a lack of alteration in the diabetic as compared to the control group may explain a lack of ARB treatment in some instances, we have not identified a general reason for the lack of consistency in this regard. Differences in glucose levels in the STZ model between labs and between animals may have contributed to these inconsistent findings. ARBs were also reported to reduce histologically observed glomerular and tubular damage in the rat STZ model of type 1 diabetes, for instance with irbesartan (Cao et al., 2001), telmisartan (Mudagal et al., 2011; Satoh et al., 2010b; Singh et al., 2006) or valsartan (Allen et al., 1997; Kelly et al., 2000; Remuzzi et al., 1998; Tang et al., 2012). Other reported ARB effects in this model include a reduction of elevated renal lipid peroxides and increase of lowered superoxide dismutase and reduced glutathione (Bishnoi et al., 2012; Hamed & Malek, 2007) and a reduction of the endogenous inhibitor of NO synthase, asymmetric dimethyl-arginine, and concomitant increase in renal NO metabolite excretion (Onozato et al., 2008). In an array study 1541 genes were found to be differentially expressed upon telmisartan treatment, including those known to be associated with PPAR-γ pathway (Zhang 115

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et al., 2012). While the latter set of genes may be specifically regulated by ARBs with known PPAR-γ agonism, in line with the array studies several ARBs were found to normalize the expression of mRNA expression of HIF-1α, tissue inhibitor of metalloproteinase-1 and endothelin-1 (Tang et al., 2012) or the inflammation markers NFκB, TNFα, TGFβ and cyclooxygenase-2 (Cao et al., 2001; Chen et al., 2008; Huang et al., 2011; Kato et al., 1999; Kelly et al., 2000; Mulay et al., 2010; Rao et al., 2011) or the fibrosis marker fibronectin (Rao et al., 2011).

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The STZ model of type diabetes has also been applied to mice. In line with the findings in rats, telmisartan reduced proteinuria along with a normalization of the renal expression of protein kinase C-α, TGF-β1 and vascular endothelial growth factor (Yao et al., 2007). Others reported that telmisartan inhibited renal fibrosis along with normalization of the expression of TGF-β1 and collagen III (Lakshmanan et al., 2011). A reduction in proteinuria by telmisartan was confirmed by others in STZ-treated mice along with a reduction in serum creatinine, mesangial expansion and fibronectin and collagen III and IV deposition; all of these effects were similarly observed with enalapril treatment, indicating that they represent class effects of RAS inhibition (Kosugi et al., 2010). Comparable findings have been obtained within that study when the experiments were performed in endothelial NO synthase knock-out mice, and a reduction in proteinuria in such knock-out mice was confirmed in later studies along with a reduced glomerulosclerosis, tubular apoptosis and collagen disposition and parenchymal macrophage infiltration (Komala et al., 2014). Others reported in STZ-treated knock-out mice that the proteinuria was numerically reduced by about 40% by a low telmisartan dose not affecting BP; however, the reduction of proteinuria only reached statistical significance when a combination of telmisartan and the dipeptidylpeptidase inhibitor linagliptin was administed which attenuated proteinuria by about 60% (Alter et al., 2012; Ott et al., 2012).

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In the Akita mouse model of spontaneously occurring non-obese insulin-dependent diabetes, telmisartan lowered proteinuria and TGF-β and VEGF expression but did not affect glomerulosclerosis (Koshizaka et al., 2012). The reduction in proteinuria was confirmed by other investigators, but this was not mimicked by amlodipine treatment (Fujita et al., 2012). In that model telmisartan, but not amlodipine, reduced the elevated GFR but not a mesangial expansion score. Thus, ARB effects on proteinuria in rat and mouse models of type 1 diabetes may be shared features of RAS inhibitors but apparently do not occur secondary to BP lowering. 7.2.6.2. Studies in obese animals with and without diabetes

Effective lowering of blood glucose can at least partly prevent diabetic nephropathy, for instance with sulfonylureas or thiazolidinediones in db/db mice (Tong et al., 2015). In contrast, ARBs have only small and inconsistent effects on blood glucose or insulin levels in animal models of obesity and type 2 diabetes (see section 6.3.3.). Potential nephroprotective effects of ARBs have also been explored in rodent models of obesity with and without concomitant type 2 diabetes. In rats receiving a high-fat, high-carbohydrate diet in combination with an STZ injection, telmisartan reduced albuminuria and blood urea nitrogen and creatinine (Wu et al., 2013). In spontaneously hypertensive/NIH-corpulent (SHR/NDmcrcp) rats olmesartan, nifedipine and atenolol similarly normalized BP but only olmesartan reduced proteinuria (from 139.1 to 63.4 mg/d as compared to 8.6 mg/d in Wistar Kyoto rats) and a histology-based glomerular damage score (Izuhara et al., 2005) (Figure 13). In a later study from the same group seven doses of valsartan have been tested during an 8-week treatment (Tominaga et al., 2009). While a reduction in proteinuria was already detectable at 116

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the lowest dose of 4 mg/kg/d, the reductions became dose-dependently greater reaching a maximum at 160 mg/kg/d. The same was true for BP reductions, and there was tight correlation between systolic BP and urinary protein level across the eight treatment groups, except that low valsartan doses had proportionally greater effects on proteinuria than on systolic BP (Figure 14). A reduction of proteinuria was also shown for telmisartan but not hydralazine in SHRSP.Z-Leprfa/IzmDmcr rats, which was accompanied by reduced glomerulosclerosis and interstitial fibrosis (Sugiyama et al., 2013). Otsuka-Long-EvansTokushima-Fatty rats are another model of spontaneously developing type 2 diabetes. In such animals both telmisartan and the mineralocorticoid receptor antagonist eplerenone similarly reduced the proteinuria developing over a 52 week period, and their combination brought it to levels observed in a non-diabetic control strain (Nishiyama et al., 2010). As the combination did not lower BP more than either monotherapy, this nephroprotective effect was apparently BP-independent. The idea of a BP-independent effect was further supported by the observation that both monotherapies and their combination similarly reduced glomerulosclerosis, an effect not observed with hydralazine treatment. Several studies have used another model of obesity and type 2 diabetes, Zucker diabetic fatty rats. In these rats treatment with an ARB including azilsartan (Hye Khan et al., 2014b), candesartan (Ecelbarger et al., 2010), olmesartan (Koike et al., 2001; Pugsley, 2005) or telmisartan (Ohmura et al., 2012) also reduced proteinuria, but one study with irbesartan did not confirm this (O'Donnell et al., 1997). In some of the studies in this repeatedly used model beneficial ARB effects were also reported for other parameters of nephroprotection including serum creatinine (Ecelbarger et al., 2010), glomerulosclerosis (Hye Khan et al., 2014b; O'Donnell et al., 1997; Ohmura et al., 2012; Pugsley, 2005), tubular injury (Koike et al., 2001; O'Donnell et al., 1997; Ohmura et al., 2012; Pugsley, 2005), renal fibrosis (Ecelbarger et al., 2010; Ohmura et al., 2012), inflammatory cytokines such as IL-1, IL-2, IL-4 IL-6, IL-10, IL-18, TNF-α, TGF-β1 and MCP-1 (Ecelbarger et al., 2010). One of these studies also included multiple telmisartan doses and a single dose of enalapril; the two drug classes had comparable effects but the chosen enalapril dose for most parameters including proteinuria was weaker than than highest ARB dose (10 vs. 10 mg/kg/day) (Ohmura et al., 2012). In Imai hyperlipidemic rats losartan and enalapril similarly reduced proteinuria and glomerulosclerosis (SAkemi & Baba, 1993). <<>> <<>>

Similar investigators have been reported from mouse models of hereditary obesity and type 2 diabetes. In db/db mice valsartan and aliskiren similarly reduced proteinuria, but their combination had even greater effects; all three treatments also reduced mesangial matrix expression, macrophage infiltration, proliferation, collagen disposition and TGF-β1 expression (Dong et al., 2010). Others have confirmed the reduction of proteinuria in db/db mice upon valsartan treatment (Gao et al., 2010). In another study in db/db mice, treatment with the experimental ACE2 inhibitor MLN-4760 increased proteinuria, and such increase was prevented by concomitant telmisartan treatment; the effect of ARB monotherapy was not reported in this study (Ye et al., 2006). In a cross-over study, telmisartan and captopril similarly improved proteinuria in db/db mice (Wysocki et al., 2013). In KK/Ta mice candesartan similarly prevented albuminuria and lowered nitro-oxidative stress whether treatment was started at an age of 6 or 12 weeks (Fan et al., 2004). Among rodent models of acquired obesity a high-fat diet has been used repeatedly to explore ARB effects on nephropathy. In one study, normotensive rats or SHR on a high-fat diet did not exhibit major alterations of urinary protein excretion or creatinine clearance; candesartan treatment was reported to lower protein excretion in normotensive but not hypertensive rats 117

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and did not affect GFR (Chung et al., 2010). Others reported that a high-fat diet increased proteinuria in SHR, but that telmisartan and valsartan lowered protein by a similar margin irrespective of the rats being on a normal or high-fat diet (Khan & Imig, 2011). The combined treatment of rats with nicotinamide and STZ is another model of acquired type 2 diabetes. In this model treatment with telmisartan lowered fasting blood glucose to a comparable extent as either a metformin/pioglitazone/glimepiride combination or the dipeptidylpeptidase IV inhibitor vildagliptin, and the telmisartan vildagliptin combination exhibited the numerically largest glucose reduction; all four treatments lowered urinary albumin excretion, serum creatinine and blood urea nitrogen and improved glomerulosclerosis and interstitial fibrosis (Sharma et al., 2014). Others used the nicotinamide + STZ model with additional unilateral nephrectomy, followed by 2 weeks treatment with telmisartan or pioglitazone and then induction of ischemia/reperfusion injury in the remaining kidney and follow-up for another 2 weeks (Tawfik, 2012). In samples obtained 24 h after the ischemia/reperfusion injury both treatments markedly lowered glucose, creatinine and urea and ameliorated the histologically assessed injury.

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In conclusion ARBs as a class, probably to a similar extent as ACE inhibitors, prevent development or even reduce renal damage in a wide variety of rodent models of diabetic nephropathy. This occurs largely independent of anti-diabetic or BP lowering effects. Accordingly, the chance of reversal of albuminuria by an ARB was greater than by an RASunrelated antihypertensive agent in clinical studies (Viberti & Wheeldon, 2002).

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Potential nephroprotective effects of ARBs have also been explored in multiple other models of renal disease, but within each of these models only to a limited extent. Some of these models related to pathologies in the cardiovascular system. For instance, in a rat model of congestive heart failure with volume overload induced by creation of an aorto-caval fistula infusion of eprosartan reduced renovascular resistance and increased RBF and GFR (Brodsky et al., 1998). Several groups have explored ARB effects on ischemia/reperfusion injury in the kidney. A 7-day pre-treatment of rats with a low dose of telmisartan attenuated the ischemia/reperfusion injury-induced increases in blood urea nitrogen, serum creatinine, renal malondialdehyde, TNF-α, nitric oxide, caspase-3 activity, and serum and renal homocysteine levels (Fouad et al., 2010). Others reported that pre-treatment with fenofibrate prevented the ischemia/reperfusion injury-induced increase in serum creatinine and attenuated that of serum urea; addition of telmisartan to the pre-treatment did not further alter creatinine but led to normalization of serum urea and homocysteine (Bhalodia et al., 2010). Within that study, such experiments were also performed in rats on a high-cholesterol diet, and also under these conditions addition of telmisartan further improved the renoprotective effect of fenofibrate. Others have evaluated the effect of pre-treatment with telmisartan or pioglitazone on renal ischemia/reperfusion injury in a rat model of type 2 diabetes induced by a combination of nicotinamide + STZ, in which animals had undergone unilateral nephrectomy prior to any treatment (Tawfik, 2012). Both treatments similarly prevented the increase in serum creatinine and urea and renal TNF-α levels. In a model of chronic renal allograft failure bilaterally nephrectomized Lewis rats were transplanted with a single kidney from Fisher 334 rats (Mackenzie et al., 1997). In this model treatment with candesartan normalized glomerular capillary pressure and reduced proteinuria and allograft glomerulosclerosis. Another group of studies has explored protective effects of ARBs in models of renal damage induced by anti-cancer chemotherapeutics or other toxic agents. In rats injected with puromycin aminonucleoside oral losartan treatment for 15 days starting concomitant with the 118

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injection dose-dependently lowered proteinuria and at the highest dose prevented the increase in blood urea nitrogen (Yayama et al., 1993). Beneficial effects against puromycin injectioninduced renal damage in rats was also reported with irbesartan which exhibited a similar degree of nephroprotection as enalapril (Zhou et al., 2012). Similarly, in rats treated with daunorubicin a 6-week treatment with telmisartan attenuated proteinuria, scores of histologically observed glomerular congestion, tubular necrosis and renal fibrosis and the increase in serum creatinine and urea; furthermore, telmisartan treatment decreased the renal levels of ANG and AT1R but increased that of PPAR-γ (Arozal et al., 2011). Importantly, 6 out of 12 daunorubicin-treated rats died during the study, whereas no deaths were observed in the 12 rats receiving daunorubicin + telmisartan. In a rat model of adriamycin-induced nephropathy, telmisartan reduced proteinuria, glomerulosclerosis and podocyte apoptosis, and even more so when combined with oxacalcitriol (Jeong et al., 2015). In a study not involving an ARB, the PPAR-γ agonist pioglitazone provided a similar degree of protection against adriamycin-induced renal damage as ramipril (Ochodnicky et al., 2014), suggesting that both RAS inhibition and PPAR-γ agonism may contribute to a beneficial effect. Telmisartan attenuated the increase in doxorubicin-induced increase in plasma creatinine and blood urea nitogen (Rashikh et al., 2014). In a rat model telmisartan also attenuated cisplatin-induced nephropathy, as assessed by serum creatinine and blood urea nitrogen, a tubular injury score and tubular apoptosis (Maoik et al., 2015). In a rat model of bromoethylamine-induced papillary necrosis doses of irbesartan, enalapril, diltiazem and a cocktail of hydralazine, reserpine and hydrochlorothiazide in doses yielding similar BP lowering the two RAS inhibitors reduced albuminuria whereas the two RAS-independent treatments did not; attenuation of the decrease in creatinine clearance was observed with enalapril but not irbesartan (Garber et al., 2001). Ochratoxin A reduces GFR and renal plasma flow in rats, and this was markedly attenuated by pretreatment with losartan or enalapril (Gekle & Silbernagl, 1993). To explore beneficial ARB effects against renal damage induced by X-ray contrast media, rats received a glycerine injection follow by oral telmisartan and concomitantly an i.v. injection of a high and a low osmolar contrast agent, diatrizoate and iohexol, respectively; telmisartan prevented observed increases in serum creatinine, renal ANG levels and caspase-3 activity (Duan et al., 2009). Finally, in a mouse model of cadmium-induced nephrotoxicity pre-treatment with telmisartan attenuated the increase in blood urea nitrogen and serum creatinine and histologically assessed renal damage (Fouad & Jresat, 2011). Taken together these data demonstrate that ARBs as a class provide nephroprotection in a wide range of rodent models. A reduction in proteinuria has also been reported in dogs upon telmisartan treatment (Bugbee et al., 2014), indicating that it may apply to non-rodent species as well. In most cases such nephroprotection was similar to that provided by other RAS inhibitors but typically was not mimicked by agents lowering BP independent of the RAS.

8. Conclusions ARBs as a class are effective in the prevention of hypertension and in reducing BP in established HT across a wide range of animal models representing various types of underlying pathophysiology, except those with a markedly suppressed RAS activity. They exhibit beneficial effects on possible sequelae of long-standing hypertension, which typically exceed those exhibited by RAS-independent types of anti-hypertensive medication. This includes effects on atherosclerosis, cardiac and renal function. Such beneficial effects were reported in preventive and treatment settings. They were often observed in doses lower than those yielding appreciable BP lowering. Moreover, they also were observed in vascular, cardiac and renal pathologies in non-hypertensive models, emphasizing an at least partly BP-independent effect that very often is shared by other types of RAS inhibitors. 119

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ARBs typically have little effect on basal glucose and insulin levels, particularly in lean animals, but nonetheless often (but not consistently) improve glucose and insulin sensitivity, particularly in obese animals and/or models of type 2 diabetes. Similarly, reports on lowering of triglycerides and cholesterol were largely limited to rodents with obesity and/or type 2 diabetes, and even within that group reported only inconsistently. However, ARBs were quite consistently reported to reduce liver weight in animal models where that was increased and to reduce adipose tissue mass, features also not shared by RAS-independent BP-lowering drugs.

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ARBs exhibiting additional properties including as PPAR-γ partial agonism such as telmisartan may mediate some of their effects at least partly via this mechanism. However, AT1R antagonism and PPAR-γ agonism in most cases modulate a given phenotype into the same direction, which makes it difficult to interpret the relative importance of the two pathways. This is particularly true when ARBs not exhibiting additional molecular targets and studied for comparison were administered in doses not yielding maximum AT1R blockade. Therefore, the clinical relevance of additional properties of some ARBs remains to be determined in dedicated clinical studies. Conflict of interest: MCM is a present employee of Boehringer Ingelheim. HR has been a consultant to practically all ARB manufacturers in the past but reports no present conflict of interest. CF is a retired employee of Boehringer Ingelheim. YH reports no conflict of interest.

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Acknowledgements: The authors gratefully acknowledge the comments of Prof. Walter Raasch (Lübeck, Germany) on the metabolism chapter of this manuscript.

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Table 1: ARB effects on BP in normotensive and hypertensive animal models. *: claim based on limited data, for instance a single study or a single ARB only. For details and references, see section 2.

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Animal models in which ARBs effectively lower BP - Normotensive animals o Monkey - Normotensive but sodium-depleted animals o Rat o Dog* - Hereditary hypertensive rat models o SHR o Stroke-prone SHR o Stroke-resistant SHR o SHR with concomitant type 1 diabetes o SHR with cyclosporine A treatment and high-salt diet o New Zealand genetically hypertensive rat* - Renal hypertensive models o Renovascular hypertension (two-kidney-one-clip or one-kidney-one-clip)  Mouse*  Rat  Hamster*  Dog o Hypertension with reduced renal mass or parenchymal renal disease  Rats with various degrees of nephrectomy  Rats injected with anti-Thy-1 monoclonal antibody  Rats with polycystic kidney disease - Hypertension with obesity o High-fat diet  Mouse*  Rat o High-fructose diet in rat o Hypertension in genetically determined obesity  db/db mouse  KK/Ta mouse*  SHRSP.Z-Leprfa/IzmDmcr rat*  Cohen-Rosenthal diabetic hypertensive rat  Koletsky rat (fak/fak) rat*  SHRcp rat  Otsuka Long-Evans Tokushima fatty rat  Zucker diabetic fatty rat - Hypertension caused by STZ injection (type 1 diabetes) in rat - Hypertension in genetically modified animals o Mouse  Transgenic harboring human renin and angiotensinogen*  Transgenic harboring C-reactive protein*  Endothelial NO synthase knock-out*  Endothelin-1 knock-out*  Hypoxia-inducible factor-1α knock-out* 121

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o (mRen-2)27 rat Chronic cold exposure in rat* Cardiac pressure overload (aortic banding) in rat Sino-aortic denervation in rat* Kidney allograft transplantation in rat* Pharmacological intervention o L-NAME treatment in rat o Cylcosporin A treatment in rat* o Chronic isoprenaline infusion in rat*

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Animal models in which ARBs have only small if any effects on BP - Normotensive healthy animals o Mouse o Rat o Rabbit o Sheep* o Dog - Animals on high-salt diet o SHR and stroke-prone SHR o DOCA-treated o Diabetic Goto-Kakizaki rat* - Monoamine uptake inhibitor treatment (tesofensine)

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Animal models with equivocal data - KK-Ay mouse - Dahl S rats on high-salt diet

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ARB

reference

Stroke-prone SHR, salt-loaded

Candesartan

(Bennai et al., 1999)

Eprosartan

(Barone et al., 2001)

Telmisartan

Candesartan

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Stroke-prone SHR, salt-loaded plus ANG infusion

Telmisartan

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Aneurysm in ApoE knock-out mice Irbesartan with intra-aortic elastase followed Telmisartan by ANG infusion

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(Webb et al., 1998a) (Dong et al., 2011; Takai et al., 2009; Webb et al., 1998a) (Kishi et al., 2014) (Kishi et al., 2014) (Iida et al., 2012) (Iida et al., 2012)

Telmisartan

(Matsumoto et al., 2010)

Mice with myocardial infarction

Olmesartan

(Chen et al., 2014)

Rats with myocardial infarction

Olmesartan

(Koike et al., 2001)

SHR with myocardial infarction

Irbesartan

(Richer et al., 1999)

CHF in mice due to transgenic calsequestrin expression

Losartan

(Günther et al., 2010)

Myocarditis in mice with encephalomyocarditis virus

Candesartan

(Tanaka et al., 1994)

Autoimmune myocarditis in rats

Telmisartan

(Sukumaran et al., 2010)

CHF in Dahl S rats on high-salt diet

Losartan

(von Lutterotti et al., 1992)

Telmisartan

(Takenaka et al., 2006)

Rats with 5/6 nephrectomy

Candesartan

(Podjarny et al., 2007)

SHR with 5/6 nephrectomy

Candesartan

(Kohara et al., 1993)

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(Takai et al., 2009)

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Minoxidil-treated rat Post-MI rat

References

Candesartan Lisinopril Telmisartan Captopril Candesartan Perindopril

ARB < ACE ARB > ACE ARB ≈ ACE

(Kohya et al., 1995) (Zhang et al., 1997) (Inada et al., 1997; Kagoshima et al., 1994) (Mori et al., 1995) (Wagner et al., 1998) (Nagai et al., 2004)

Candesartan Captopril

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(Ideishi et al., 1994)

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(Brown et al., 1999) (Mukawa et al., 1997) (Linz et al., 1991) (Brodsky et al., 1998)

ARB > ACE ARB > ACE

(Kim et al., 1997) (Ruzicka & Leenen, 1993)

Candesartan Delapril

ARB ≈ ACE

Candesartan Cilazapril Eprosartan Captopril Valsartan Captopril

ARB ≈ ACE ARB ≈ ACE ARB ≈ ACE

(Nishikimi et al., 1995b; Yamagishi et al., 1993) (Yoshiyama et al., 1999) (van Kats et al., 2000) (Zhang et al., 2008b)

Captopril Quinapril Ramipril Enalapril

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ACE inhibitors Candesartan Captopril Candesartan Derapril Candesartan Enalapril

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Telmisartan Candesartan

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Irbesartan Telmisartan Telmisartan Losartan Telmisartan Telmisartan Telmisartan

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ApoE knock-out mice PPAR-γ knock-out mice on high-sucrose diet PPAR-δ knock-out mice Mice on high-fat diet

Mice on high-fat diet + pioglitazone ApoE knock-out mice on high-fat diet

CR

Telmisartan

Unchanged References adipose tissue Mouse models (Feng et al., 2011; He et al., 2010; Kudo et al., 2009; Zidek et al., 2013) Telmisartan (Penna-de-Carvalho et al., 2014) Valsartan (Tomono et al., 2008) Telmisartan (Zidek et al., 2013)

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Table 4: Effects of treatment with an ARB on adipose tissue mass. Note that our search did not identify any studies in which an increased adipose tissue mass was reported upon ARB treatment.

Azilsartan Valsartan

Healthy rats

Telmisartan

SHR

Candesartan Telmisartan

(Feng et al., 2011; He et al., 2010) (Fujisaka et al., 2011) (Aubert et al., 2010) (Aubert et al., 2010; Fujisaka et al., 2011; Penna-de-Carvalho et al., 2014; Souza-Mello et al., 2010) (Aubert et al., 2010) (Nakagami et al., 2008) (Nakagami et al., 2008) (Rong et al., 2010) (Kudo et al., 2009)

(Iwai et al., 2007) Candesartan (Iwai et al., 2007) (Tomono et al., 2008) Rat models (Goyal et al., 2011a) Telmisartan (Rabie et al., 2015) (Chung et al., 2010) (He et al., 2010) 125

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Rats on high-fat diet with pioglitazone Rats on high-fructose diet Rats treated with olanzapine Koletsky rat Otsuka Long-Evans Tokushima Fatty rat

Telmisartan

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Guinea pigs on high-fat diet

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Valsartan Candesartan Telmisartan Telmisartan Telmisartan Telmisartan

(Müller-Fielitz et al., 2012) (de las Heras et al., 2009) (Rosseli et al., 2009) (Goyal et al., 2011a; Miesel et al., 2012; Müller-Fielitz et al., 2012; Müller-Fielitz et al., 2014; Rosseli et al., 2009; Sugimoto et al., 2006; Wang et al., 2012c) Telmisartan (Zanchi et al., 2007) (Guo et al., 2008; Sugimoto et al., 2006) (Chung et al., 2010) (He et al., 2010) (Zanchi et al., 2007) (Rabie et al., 2015; Ramesh Petchi et al., 2012) (Ramesh Petchi et al., 2012) Azilsartan (Zhao et al., 2011) Olmesartan (Harada et al., 1999) (Zhao et al., 2013) Guinea pig models (Xu et al., 2015)

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Figure legends

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Figure 1: Effect of irbesartan on the development of hypertension in SHR with treatment from the age of 4-20 weeks, followed by observation without further treatment for another 8 weeks, i.e. irbesartan treatment was stopped at week 20. Data are means of 115 rats per group and have been drawn based on data published by (Vacher et al., 1995). Values on irbesartan treatment reported to significantly differ from those in control for all time points except 4 weeks, i.e. prior to start of treatment.

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Figure 2: Mechanisms involved in ANG-induced BP elevation. See section 2.10 for details.

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Figure 3: Acetylcholine-induced vasodilatation of aortic rings from 30 week old WKY, SHR and SHR treated with irbesartan in weeks 16-30. Data are means ± SD of 20 animals. Drawn based on data published by (Zalba et al., 2001).

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Figure 4: Effects of a 2 week treatment of 20 week old SHR with single daily s.c. injections of saline (control), losartan or hydralazine on vascular hypertrophy. Data are means ± SD of 510 animals. Calculated and drawn based on data published by (Soltis, 1993).

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Figure 5: Effect of genetic ablation or pharmacological inhibition on atherosclerosis in the murine aortic arch. Upper panel: ApoE knock-out mice were compared to ApoE knock-out mice with additional AT1R knock-out were fed normal chow and killed at age of 24 weeks (n = 4 each). Lower panel: 6 week old ApoE knock-out mice were fed a Western-type diet and received either hydralazine (30 mg/kg/day, n = 3) or telmisartan (10 mg/kg/day, n = 4) for 18 weeks. In both experiments, extent of atherosclerosis was assessed as en face Sudan IV staining. Data are mean ± SD; *: p < 0.05 vs. ApoE knock-out or vs. ApoE knock-out with hydralazine treatment. Drawn based on data published by (Fukuda et al., 2010).

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Figure 6: Schematic drawing of the roles of cardiomyocytes and cardiac fibroblasts in stretchinduced hypertrophy and fibrosis. Production of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was used as indicator of hypertrophic phenotype. Note that coculture alone already increased ANP/BNP production, and that stretch and ANG caused further increases in co-culture. For references, see section 4.2.1. and specifically (Harada et al., 1998b). Figure 7: Effect of a 3 week oral treatment, starting immediately after the coronary artery ligation, with an ARB (valsartan) and an ACE inhibitor (temocapril) in a rabbit model of myocardial infarction. Data are means ± SD of 5 animals. Calculated and drawn based on data published by (Makino et al., 2003). Figure 8: Effect of an ARB (valsartan) and an ACE inhibitor (perindopril) on basal plasma glucose and i.v. glucose tolerance and insulin release tests in a rat model of type 2 diabetes. Diabetes had been induced by a high-fat diet plus a single low-dose injection of streptozotocin. One week after start of the diet, pharmacological treatment started and was continued for another 8 weeks. Data are means ± SD of 8-10 animals. Calculated and drawn based on data published by(Yuan et al., 2010). Figure 9: Effects of an ARB (telmisartan), an ACE inhibitor (enalapril) and a direct renin inhibitor (aliskiren) on triglycerides, total cholesterol and LDL cholesterol in a rat model of type 2 diabetes. Rats were put on a high-fat diet, followed by a single low-dose injection of 127

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Figure 10: Effect of ARB treatment on liver mass in mice and rats. Studies involved different ARBs and healthy animals as well as those from various models of metabolic disease; for details, see section 6.6. Each data point represents the mean data from one study, expressed as % of the value observed in the control group not receiving an ARB. Mean ± SD of all studies within a species are indicated.

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Figure 11: Effects of ARBs on proteinuria in rat models of renal disease based on renal mass reduction. Each pair of data points connected by a line represents one study with the control/vehicle data shown as the left and ARB treatment data as the right symbol. Note that in one study data were reported for albumin (Tsunenari et al., 2005), in all other studies for total protein excretion. In study protein data were reported normalized for urinary creatinine (Sugimoto et al., 1999) and in another per 100 g body weight (Pollock et al., 1993a); in both cases data were back-calculated to total protein by the present authors; in all other studies absolute values were given. Based on data reported in (Cao et al., 2012; Gandhi et al., 1999; Hamaguchi et al., 1996; Hamar et al., 1999; Lafayette et al., 1992; Mackenzie et al., 1994; Noda et al., 1999; Pollock et al., 1993a; Sasser et al., 2012; Sugimoto et al., 1999; Toba et al., 2012; Tsunenari et al., 2005; Tu et al., 2008; Wong et al., 2000; Wu et al., 1997; Ziai et al., 1996). In some cases, values were estimated from printed figures, and some studies reported data for more than one ARB. Despite major differences in baseline proteinuria, start and duration of treatment, all 16 studies analyzed here have consistently reported major reductions of proteinuria upon ARB treatment.

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Figure 12: Effects of telmisartan on BP, albuminuria and glomerulosclerosis in m(Ren-2)27 transgenic rats. Note that a dose not lowering BP already is effective, but a BP lowering dose even more so. Data are means ± SD of 9-10 animals, except for BP; *: p < 0.05 vs. control. Drawn based on data published by (Böhm et al., 1995). Note that relative size of error bar suggest that the underlying data on albuminuria and glomerulosclerosis index are likely not to exhibit Gaussian distribution; hence, medians with confidence intervals may have been more appropriate for description but means have been reported in the underlying paper. Figure 13: Effects of an ARB (olmesartan), a Ca2+-entry blocker (nifedipine) and a βadrenoceptor antagonist (atenolol) on blood pressure and proteinuria in a model of spontaneously developing obesity and type 2 diabetes. Spontaneously hypertensive/NIHcorpulent (SHR/NDmcr-cp) rats aged 13 weeks were treated with olmesartan, nifedipine or atenolol for 20 weeks. Data are means ± SD of 10 animals. Calculated and drawn based on published data (Izuhara et al., 2005). Figure 14: Reduction of systolic BP and urinary protein excretion in spontaneously hypertensive/NIH-corpulent (SHR/NDmcr-cp) rats after 8 weeks of treatment. The upper panel shows dose-dependency for both effects (proteinuria in mg/day, open circles, left yaxis; systolic blood pressure in mm Hg, filled circles, right y-axis). The lower panel shows the correlation between both parameters. Data are mean ± SD of 8 rats per group. Drawn based on data published by (Tominaga et al., 2009).

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Abdelsaid, M., Coucha, M., & Ergul, A. (2014). Cerebrovasculoprotective effects of azilsartan medoxomil in diabetes. Transl Res 164, 424-432. Abe, J., Deguchi, J., Matsumoto, T., Takuwa, N., Noda, M., Ohno, M. et al. (1997). Stimulated activation of platelet-derived growth factor receptor in vivo in balloon-injured arteries: a link between angiotensin II and intimal thickening. Circulation 96, 1906-1913.

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Aikawa, R., Komuro, I., Yamazaki, T., Zou, Y., Kudoh, S., Zhu, W. et al. (1999). Rho family small G proteins play critical roles in mechanical stress-induced hypertrophic responses in cardiac myocytes. Circ Res 84, 458-466.

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