Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches

JPT-06697; No of Pages 15 Pharmacology & Therapeutics xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches Dimitris Tousoulis ⁎,1, Chryssa Simopoulou 1, Nikos Papageorgiou, Evangelos Oikonomou, George Hatzis, Gerasimos Siasos, Eleftherios Tsiamis, Christodoulos Stefanadis 1st Cardiology Department, Athens University Medical School, Hippokration Hospital, Greece

a r t i c l e

i n f o

Keywords: Atherosclerosis Cardiovascular disease Endothelial function Therapeutic approaches

a b s t r a c t The vascular endothelium not only is a single monolayer of cells between the vessel lumen and the intimal wall, but also plays an important role by controlling vascular function and structure mainly via the production of nitric oxide (NO). The so called “cardiovascular risk factors” are associated with endothelial dysfunction, that reduces NO bioavailability, increases oxidative stress, and promotes inflammation contributing therefore to the development of atherosclerosis. The significant role of endothelial dysfunction in the development of atherosclerosis emphasizes the need for efficient therapeutic interventions. During the last years statins, angiotensin-converting enzyme inhibitors, angiotensin-receptor antagonists, antioxidants, beta-blockers and insulin sensitizers have been evaluated for their ability to restore endothelial function (Briasoulis et al., 2012). As there is not a straightforward relationship between therapeutic interventions and improvement of endothelial function but rather a complicated interrelationship between multiple cellular and sub-cellular targets, research has been focused on the understanding of the underlying mechanisms. Moreover, the development of novel diagnostic invasive and non-invasive methods has allowed the early detection of endothelial dysfunction expanding the role of therapeutic interventions and our knowledge. In the current review we present the available data concerning the contribution of endothelial dysfunction to atherogenesis and review the methods that assess endothelial function with a view to understand the multiple targets of therapeutic interventions. Finally we focus on the classic and novel therapeutic approaches aiming to improve endothelial dysfunction and the underlying mechanisms. © 2013 Published by Elsevier Inc.

Contents 1. Introduction. . . . . . . . . . . . . . . 2. Pathophysiology of endothelial dysfunction 3. Assessment of endothelial function . . . . 4. Therapeutic approaches . . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . Conflict of interest statements . . . . . . . . . References . . . . . . . . . . . . . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

0 0 0 0 0 0 0

Abbreviations: ACEIs, angiotensin-converting enzyme inhibitors; ADMA, asymmetrical dimethyl arginine; ARBs, angiotensin-receptor antagonists; BH4, tetrahydrobiopterin; CAD, coronary artery disease; DDAH, dimethyl arginine dimethyl aminohydrolase; ED, endothelial dysfunction; eNOS, endothelial nitric oxide synthase; EPC, endothelial progenitor cells; FMD, flow mediated dilation; GTP, guanosine-5′-triphosphate; LDLs, low density lipoproteins; L-NMMA, NG-monomethyl-L-arginine; NADPH, nicotinamide adenine dinucleotide phosphateoxidase; NO, nitric oxide; NOS, nitric oxide synthase; oxLDLs, oxidized low density lipoproteins; ROS, reactive oxygen species; SOD, superoxide dismutase; VEGF, vascular endothelial growth factor. ⁎ Corresponding author at: Vasilissis Sofias 114, TK 115 28, Hippokration Hospital, Athens, Greece. Tel.: +30 213 2088099; fax: +30 213 2088676. E-mail address: [email protected] (D. Tousoulis). 1 These authors have equal contribution.

http://dx.doi.org/10.1016/j.pharmthera.2014.06.003 0163-7258/© 2013 Published by Elsevier Inc.

Please cite this article as: Tousoulis, D., et al., Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches, Pharmacology & Therapeutics (2013), http://dx.doi.org/10.1016/j.pharmthera.2014.06.003

2

D. Tousoulis et al. / Pharmacology & Therapeutics xxx (2013) xxx–xxx

1. Introduction The vascular endothelium is a monolayer of cells between the vessel lumen and the vascular smooth muscle cells. It plays a pivotal role in the regulation of vascular function and structure by releasing various biochemical mediators, such as nitric oxide (NO) and prostacyclin (Tousoulis et al., 2012). A growing list of factors, including the so called “cardiovascular risk factors”, such as hypertension, hypercholesterolemia, smoking, diabetes mellitus, congestive heart failure, hyperhomocysteinemia and aging process itself, are associated with impaired endothelial function (Siasos et al., 2012). Endothelial dysfunction (ED), observed in these conditions, is characterized by decreased NO bioavailability and increased oxidative stress. As a result, ED promotes inflammation, oxidation of lipoproteins, platelet aggregation and thrombus formation and thus contributes to the development of atherosclerosis (Fig. 1). Over the last years vascular endothelium has emerged as a new therapeutic target in cardiovascular disease. Several therapeutic approaches are currently available, targeting both synthesis and production of NO in human vascular endothelium, such as statins, angiotensin converting enzyme inhibitors, and thiazolidinediones and others are still under investigation (Table 1). Nevertheless, the impact of treatments in endothelial function is not constant while the underlying mechanisms are sometimes unknown. Moreover, there is a discrepancy between the achieved endothelial improvement and clinical outcome, thus further complicating the evaluation of treatment effects. In the current review we present the available data concerning the contribution of endothelial dysfunction to atherogenesis and review the methods that assess endothelial function with a view to understand the multiple targets of therapeutic interventions. Finally we focus on the classic and novel therapeutic approaches aiming to improve endothelial dysfunction and the underlying mechanisms.

2. Pathophysiology of endothelial dysfunction It is well established that vascular endothelium has a pivotal role in the modulation of vascular function and structure mainly via the formation of NO (Luscher &Barton, 1997). More specifically, NO causes vasodilatation and is responsible for the balance of endothelium-derived contracting factors, such as endothelin-1 and thromboxane A2, thus regulating the vascular tone (Vita, 2002). Besides this, it also inhibits platelet aggregation, inflammation, vascular smooth muscle cell migration and proliferation, and leukocyte adhesion and exerts antioxidant effects. Thereby, many pathophysiological states evoke endothelial

Fig. 1. The role of endothelium in atherosclerosis. From adhesion and migration of leukocytes in sub-endothelial layers and the transformation of tissue macrophages in atherosclerotic foam cells.

Table 1 Classic and novel therapeutic approaches aiming to improve endothelial function. Agent

Impact on endothelial function

Statins ↑ eNOS expression (W. Sun et al., 2006), (Matsuno et al., 2004) ↓ NF-kB, AP-1 and HIF-1 pathways ↓ endothelial cell apoptosis Angiotensin converting enzyme inhibitors ↑ NO bioavailability (Imanishi et al., 2008) Folic acid/5-methyltetrahydrofolate ↑ eNOS coupling by BH4 stabilization (Antoniades, Antonopoulos, Tousoulis et al., ↑ vascular BH4 bioavailability 2009), (Antoniades, Shirodaria et al., 2006) Thiazolidinediones ↑ eNOS activity and expression (Hwang et al., 2005), (Calnek et al., 2003) ↓ endothelial cell apoptosis ↓ NADPH & iNOS related oxidative stress Arginase antagonists ↑ NO production (Torondel et al., 2010) ↑ BH4 deficiency Endothelium-specific GTP cyclohydrolase overexpression ↑ NO production by endothelial cells (Zheng et al., 2003) Epoxide hydrolase inhibitor ↑ NO bioavailability (D. Zhang et al., 2012) eNOS: endothelial nitric oxide synthase; NO: nitric oxide; BH4: tetrahydrobiopterin; HIF1: hypoxia-inducible factor-1; AP-1: activator protein-1; NF-kB: nuclear factor-kappa B; GTP: guanosine triphosphate; NADPH: nicotinamide adenine dinucleotide phosphate. ↑: increase/improve; ↓: decrease/inhibit.

dysfunction mainly by reducing endothelium-dependent vasodilation (Tousoulis, Charakida & Stefanadis, 2006; Antoniades, Antonopoulos, Bendall & Channon, 2009). ED plays a key role in the development of atherosclerosis and cardiovascular risk factors are highly associated with impaired endothelial function (Tousoulis, Koutsogiannis et al., 2010; Tousoulis, Papageorgiou et al., 2010; Tousoulis et al., 2011). More specifically, ED is caused by an increase in reactive oxygen species (ROS) generation and a reduction of NO bioavailability in vascular endothelium, either by decreasing its synthesis and/or by increasing its oxidative inactivation (R. Lee et al., 2012). Due to the increased oxidative stress there is a decrease in NO bioavailability mediated by diverse mechanisms. Initially, superoxide anions react with existing NO producing peroxynitrite and thus reduce the concentration of NO (Channon, 2004). In addition, ROS reduce the production of NO by reducing the enzymatic activity of endothelial nitric oxide synthase (eNOS), the enzyme responsible for NO's synthesis (Guzik et al., 2002). In the vascular endothelium, NO is synthesized from L-arginine by eNOS via the transport of electrons to L-arginine. In order for this to happen, eNOS has to be bound to an essential cofactor, which is tetrahydrobiopterin (BH4). This is known as “coupling” of eNOS (Cunnington &Channon, 2010). Without BH4 eNOS becomes “uncoupled” and electrons are transported to oxygen instead of Larginine generating superoxide rather than NO (Alp et al., 2003). In atherosclerosis, BH4 levels are decreased because of their oxidative degradation by peroxynitrite and superoxide (Antoniades, Shirodaria et al., 2007). This reduction of BH4 leads to eNOS “uncoupling” and so eNOS instead of synthesizing NO produces superoxide. This in turn reacts with existing NO to produce peroxynitrite and both of them cause further oxidation and depletion of BH4 and thus enhance even more the oxidative stress and the reduction of NO bioavailability in vascular endothelial cells (Channon, 2004). Another mechanism responsible for the reduction of NO is via the regulation of asymmetrical dimethyl arginine (ADMA) levels. ROS reduce the enzymatic activity of dimethyl arginine dimethyl aminohydrolase (DDAH), which is crucial for the catabolism of ADMA, and up-regulate gene expression of protein methyl transferases, enzymes responsible for the transformation of L-arginine to ADMA. As a result, ROS induce an increase in ADMA levels. As it has been shown, ADMA inhibits and causes “uncoupling” of eNOS and thus an increase in ADMA levels is highly associated with a decrease in NO levels (Landmesser et al., 2004). Furthermore, ROS can downregulate gene expression of eNOS and thus reduce even more NO

Please cite this article as: Tousoulis, D., et al., Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches, Pharmacology & Therapeutics (2013), http://dx.doi.org/10.1016/j.pharmthera.2014.06.003

D. Tousoulis et al. / Pharmacology & Therapeutics xxx (2013) xxx–xxx

synthesis (Guzik et al., 2000). Last, NO bioavailability can also be reduced by the nitration of G-proteins by peroxynitrite (Guzik et al., 2000). The reduction of NO bioavailability plays a key role in the pathophysiology of ED, because as mentioned above, NO is crucial for vasodilatation and has anticoagulant, anti-inflammatory, antiapoptotic and antioxidant actions. It is well established that oxidative stress leads to endothelial cell apoptosis in cell culture models because of the inhibition of antioxidants, e.g. superoxide dismutase (SOD), by ROS (Recchioni et al., 2002; Yokoyama et al., 2002). The excessive oxidative stress can harm even more the endothelial function (R. Lee et al., 2012) and some types of ROS, such as hydrogen peroxide, and enhance endothelial cell apoptosis via the destruction of thioredoxin-1 (Haendeler et al., 2005). Additionally, oxidative stress enhances the caspase-induced endothelial cell apoptosis (Irani, 2000). Vascular inflammation is highly associated with ED and plays a major role in the development of atherosclerosis. In cases of ED, there is an increased expression of adhesion and other inflammatory molecules (Tousoulis, Charakida et al., 2006). Specifically, the increased oxidative stress up-regulates the expression of many adhesion molecules, such as vascular adhesion molecule-1, intracellular adhesion molecule-1, Eand P-selectin, which are all redox sensitive (Chakrabarti et al., 2007). Furthermore, it augments the secretion of many cytokines and other inflammatory molecules, which are controlled by redox-sensitive mediators (i.e. nuclear factor kappa-light-chain-enhancer of activated B cells) (Chakrabarti et al., 2007). Therefore, there is an augmented accumulation and adhesion of leukocytes, mainly macrophages and T lymphocytes, in the sites of impaired endothelial function (Tousoulis et al., 2011). CD4+ helper T lymphocytes secrete interferon-γ, which increases the expression of major histocompatibility complex class II by monocytes and, thus, the presentation of antigens from the atherosclerotic plaques. Therefore, CD4+ T cells enhance even more the vascular inflammatory response (Leon &Zuckerman, 2005). The vascular inflammation and the increased generation of ROS promote the modification of circulating low density lipoproteins (LDLs) in oxidized LDLs (oxLDLs) (Frei et al., 1988; Libby et al., 2002), which exert a crucial role in the pathophysiology of atherosclerosis (Parthasarathy et al., 1998; Zeibig et al., 2011). After oxLDLs are phagocytosed by macrophages, they turn them into foam cells and promote even more the generation of ROS (Ishigaki et al., 2009). OxLDLs also contribute to the development of ED by damaging endothelial cells and enhancing the expression of adhesion molecules (i.e. P-selectin) (Vora et al., 1997) and cytokines (i.e. chemoattractant protein-1 and macrophage colony stimulating factor) (Cushing et al., 1990; Rajavashisth et al., 1990).

3. Assessment of endothelial function As mentioned above, ED is crucial in the pathophysiology of atherosclerosis and is a prognostic marker both in subjects at high cardiovascular risk and in those with established coronary artery disease (CAD) (Table 2) (Lerman &Zeiher, 2005). Therefore, a wide range of methods are used to evaluate endothelial function.

3

3.1. Non-invasive methods 3.1.1. Flow-mediated dilation Flow-mediated dilation (FMD) is the most widely used technique for the evaluation of endothelial function of conduit arteries introduced by the Deanfield group in 1992 (J.E. Deanfield et al., 2007). It is based on the physiology of blood flow regulation in large arteries: increased blood flow in conduit arteries causes an increase in shear stress and thus, by increasing NO production, leads to vasodilatation. This response is known as FMD or endothelium-dependent vasodilatation, because it is provoked by the capacity of vascular endothelium to produce NO (Celermajer, 2005). Endothelium-independent vasodilatation can be also tested by low dose sublingual administration of nitroglycerin (Dengel et al., 2007). Brachial artery FMD has been established as the most commonly used technique (Anderson et al., 1995; D. Sun et al., 1999). A blood pressure cuff is placed on the forearm distal to the brachial artery and is inflated to supra-systolic pressure and, then, deflated 5 min later. After cuff release, reactive hyperemia results in FMD because of the local endothelial release of NO and two dimensional ultrasound images are used to measure the brachial artery's diameter. FMD is expressed as the percentage change of the artery's diameter from the baseline size. Although FMD seems to be highly reproducible, there are some difficulties that need to be overcome in order for it to be used in routine clinical practice (Corretti et al., 2002; J. Deanfield et al., 2005; J.E. Deanfield et al., 2007). More specifically, these include the need for highly trained operators, as it is a highly operator-dependent method (Celermajer, 1998, 2005; Corretti et al., 2002; Ghiadoni et al., 2008), the expensive equipment and the need to minimize environmental and patient related influences, such as mental stress, food, smoking and temperature (P. Leeson et al., 1997; Ghiadoni et al., 2000; Papamichael et al., 2005; Charakida et al., 2010). FMD is impaired by cardiovascular risk factors from the first ten years of life and decreases in proportion to risk factor burden (Celermajer et al., 1994). Therefore, it has prognostic value in patients with CAD irrespectively from the angiographic severity of the disease (Halcox et al., 2002; L. Chan et al., 2005; Frick et al., 2005). In addition, it has a predictive role in cardiovascular risk in subjects with peripheral arterial disease (Brevetti et al., 2003) and in the general population as well (Shimbo et al., 2007). FMD impairment can be quickly reversed by medical therapy, such as statin use, and by beneficial lifestyle changes (Woo et al., 2004).

3.1.2. Pulse wave analysis The arterial waveform gives information about the stiffness of conduit arteries and the wave reflection in the arterial system (Wilkinson &McEniery, 2004), which happens mainly at branch points and is measured by the augmentation index, which is the difference between the first and second systolic peaks (Nichols et al., 2008). Wave reflection is mainly affected by changes in the impedance of small arteries, which is much more dynamic than the one of the conduit and relatively static arteries. Specifically, vasoconstriction increases wave reflection

Table 2 The prognostic role of endothelial function for cardiovascular disease. Study

Type

Population

Number of subjects

Duration

Prognosis

(Fathi et al., 2004) (Suwaidi et al., 2000) (Hollenberg et al., 2001) (Targonski et al., 2003) (Perticone et al., 2001) (Schachinger et al., 2000) (Schindler et al., 2003)

Prospective Prospective Prospective Prospective Prospective Retrospective Prospective

High risk patients Coronary artery disease Heart transplants Non-obstructive coronary artery disease Hypertensives Coronary artery disease Normal coronary angiograms

444 157 73 503 225 147 130

24 months 28 months 32 months 90 months 32 months 7.7 years 45 months

No prognostic value Positive prognostic value Positive prognostic value Positive prognostic value Positive prognostic value Positive prognostic value Positive prognostic value

Please cite this article as: Tousoulis, D., et al., Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches, Pharmacology & Therapeutics (2013), http://dx.doi.org/10.1016/j.pharmthera.2014.06.003

4

D. Tousoulis et al. / Pharmacology & Therapeutics xxx (2013) xxx–xxx

and, thus, augmentation index, whereas vasodilatation has the opposite effect (Nichols et al., 2008). As NO is an important regulator of vascular tone, glyceryl trinitrate reduces wave reflection by causing vasodilatation, in an endotheliumindependent way (R.P. Kelly et al., 1990), whereas LG-monomethyl Larginine, a NOS inhibitor, increases it by causing vasoconstriction (Wilkinson, MacCallum et al., 2002). Arterial stiffness and wave reflection can be also reduced by the β2 agonist salbutamol that causes vasodilatation via an endothelium-dependent way (Wilkinson, Hall et al., 2002). Therefore, endothelial function can be evaluated by recording the shape of the arterial waveform after the sublingual administration of glyceryl trinitrate that acts as an endothelium-independent stimulus and after the inhaled administration of salbutamol that acts as an endothelium-dependent stimulus (Chowienczyk et al., 1999). Some clinical studies have recorded the shape of the arterial waveform after inhaled salbutamol administration. Impaired endotheliumdependent response was observed in conditions traditionally related to ED, such as CAD (Hayward et al., 2002), peripheral arterial disease (Kals et al., 2006), hypercholesterolemia (Wilkinson, Hall et al., 2002) and diabetes mellitus (Chowienczyk et al., 1999). The endotheliumindependent response was usually unimpaired. Nevertheless, at present, there are no data relating this method to the effects of medical therapy. 3.1.3. Gauge-strain plethysmography This non-invasive technique was proposed by Hokanson et al. for the assessment of endothelial function of brachial artery (Hokanson et al., 1975). Electrically calibrated plethysmography, based on the tissue volume change, assesses the changes in forearm blood flow during reactive hyperemia, caused by the inflation of a blood pressure cuff placed proximally to the forearm. The cuff is inflated up to the pressure where arterial inflow is allowed but venous outflow is impeded and thus, the tissue volume is increased in proportion to the rate of the arterial inflow. This method measures the % change of blood flow (ml/min/ 100 ml tissue) from baseline to maximum flow after 5 min of ischemia. Both the maximum flow and the overall time-flow curves give important information for the endothelial function (Woodcock, 1985; Antoniades, Tousoulis et al., 2006; Antoniades, Tousoulis et al., 2007). Although this technique is less accurate than FMD, it is preferred in many cases because it is simple, reproducible, less observerdependent and does not require highly trained personnel (Tousoulis et al., 2011). 3.1.4. Peripheral arterial tonometry This method evaluates peripheral endothelial function through measurement of pulse amplitude in the fingertip at rest and during reactive hyperemia. This is done with the EndoPAT device (Itamar Medical Caesarea, Israel), which consists of two fingertip probes that are placed on one finger of each hand and sense volume changes in the digit with each arterial pulsation. A blood pressure cuff is also placed on one arm (study arm), whereas the other is used for the control of any systemic effects (control arm). During reactive hyperemia, digital pulse amplitude and so peripheral arterial tonometry signal amplitude increases. This has been attributed partially to NO synthesis (Nohria et al., 2006). Three phases take place at this test: baseline, occlusion and hyperemia. In the beginning, baseline data are recorded and then, the cuff is inflated up to suprasystolic pressures for 5 min. During this occlusion period, there are no signals from the study finger, but there are still signals from the control finger. After the deflation of the cuff, there is reactive hyperemia in the study arm and an increase in the pulse amplitude of the finger, which is recorded, digitized and analyzed. It has been suggested that this technique may be able to identify patients at early stages of CAD, as the digital reactive hyperemia measured in this test is decreased in subjects with coronary ED (Bonetti et al., 2004). It is also reduced in patients with low FMD and ED in comparison to patients with higher FMD responses and a more preserved

endothelial function (Kuvin et al., 2003). Cardiovascular risk factors, such as obesity and diabetes mellitus, have also been linked to reduced responses (de Jongh et al., 2004; Mitchell et al., 2005). However, there are still not enough clinical studies to evaluate the value of this method in risk stratification of individuals. 3.1.5. Laser Doppler flowmetry (LDF) This method evaluates skin microcirculation (Gush &King, 1991) based on the assumption that the responses in microcirculation are indicative of the endothelial function in the rest of the arterial system (Khan et al., 2008). In this technique, endothelium-dependent vasodilators, usually acetylcholine, are directly delivered to the skin through iontophoresis. Laser Doppler probes located on the skin of the internal face of the forearm to measure the hyperemic responses, which give important information about the endothelium function of skin microcirculation. However, the acetylcholine induced response is not mediated by NO, but by prostanoids (Kruger et al., 2006). Acetylcholine induced vasodilatory response is impaired in subjects with cardiovascular risk factors, such as diabetes mellitus (Caballero et al., 1999; Khan et al., 2000), hypercholesterolemia and hypertension (Abularrage et al., 2005) and can be improved after medical therapy (Kubli et al., 2001; Settergren et al., 2008; Rossi et al., 2009). However, this impaired response cannot be used as a treatment indication. Nevertheless, laser Doppler flowmetry is still a simple and cost-effective method for the evaluation of endothelial function. 3.1.6. Magnetic resonance imaging Magnetic resonance imaging is a very useful and effectual method in the assessment of both peripheral and central measures of endothelial function, such as distensibility and pulse wave velocity (Wiesmann et al., 2004; J.M. Lee et al., 2008), and can be used for imaging of brachial artery and assessment of FMD. The procedure is the same as ultrasoundbased measurements, with endothelial function being evaluated after a five-minute occlusion of the forearm, distal to the imaging site, and after sublingual administration of nitroglycerine. The main disadvantage of magnetic resonance imaging compared to ultrasound-based techniques is its high cost. Nevertheless, it has many advantages, such as its superior reproducibility for FMD measurements compared to ultrasound, mainly due to its low operator-dependence (C.P. Leeson et al., 2006). Furthermore, it performs cross-sectional measurements, which are more sensitive in changes of size after reactive hyperemia compared to ultrasound measurements of brachial artery diameter (C.P. Leeson et al., 2006). Additionally, as mentioned above, it allows the evaluation of both endothelial function and arterial stiffness and, thus, it is a very useful method for risk stratification and control of vascular response to medical therapy. 3.1.7. Positron emission tomography This non-invasive method is very useful in the evaluation of myocardial perfusion and viability. Therefore, it indicates the development of atherosclerosis and allows the assessment of endothelial function in coronary microcirculation (Tamaki et al., 1994). 3.2. Invasive methods 3.2.1. Quantitative coronary angiography with intracoronary infusion of vasoactive agents Endothelial function in coronary circulation is associated with endothelial function in peripheral arteries and so, its assessment has predictive value for patients with established CAD (Schachinger et al., 2000; Suwaidi et al., 2000). Despite its invasive nature, angiography is still the gold standard for the evaluation of endothelial function in epicardial coronary arteries. It is usually combined with intracoronary infusion of substances that regulate either in an endothelium-dependent or in an endothelium-independent way vascular tone. The most commonly used is acetylcholine, which causes vasodilatation in normal epicardial

Please cite this article as: Tousoulis, D., et al., Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches, Pharmacology & Therapeutics (2013), http://dx.doi.org/10.1016/j.pharmthera.2014.06.003

D. Tousoulis et al. / Pharmacology & Therapeutics xxx (2013) xxx–xxx

arteries with intact endothelium via a receptor-mediated enhancement of NO synthesis by endothelial cells (Drexler et al., 1991; Widlansky et al., 2003). However, if epicardial arteries have impaired endothelial function, acetylcholine leads to vasoconstriction because of its direct impact on the underlying vascular smooth muscle cells (Tousoulis &Davies, 1998). In order to estimate the NO-dependent component of endothelium-dependent vasodilatation and evaluate NO availability, it is common to repeat acetylcholine infusion with simultaneous intracoronary infusion of NG-monomethyl-L-arginine (L-NMMA), an NOS inhibitor that impedes the vasodilatory effect of acetylcholine (Tousoulis et al., 1995; Tousoulis et al., 1996). Instead of acetylcholine, it is common to use other substances, such as adenosine, bradykinin, serotonin or substance P, which also enhance NO synthesis. Finally, it is typical to use nitroglycerin, an exogenous NO donor, in order to assess endothelium-independent vasodilatation and compare its effect with the response of endothelium-dependent drugs (Halcox et al., 2002; Targonski et al., 2003). This technique can also be used for the assessment of endothelial function in coronary microcirculation by measuring coronary blood flow with Doppler flow wire. The evaluation of coronary endothelial function both in epicardial arteries and in microcirculation is very important, because if it is impaired, it has prognostic value for acute coronary syndromes even when there are no angiographically detectable lesions (Halcox et al., 2002; Targonski et al., 2003). Furthermore, it plays a crucial role in heart transplant recipients, as the endothelium is an early target of immunological, pharmacological and ischemic graft injury and its impairment has been related to the development of graft atherosclerosis (Vassalli et al., 1997). 3.2.2. Intrabrachial infusion of vasoactive agents As mentioned above, endothelial function in coronary circulation is a window towards endothelial function in peripheral arteries and, thus, infusion of vasoactive agents can also be applied in the brachial artery (Faulx et al., 2003), which is easier and less dangerous. It is a minimally invasive, modified strain-gauge plethysmography method that measures the changes in forearm blood flow (ml/min/100 ml tissue) during the intra-arterial infusion of acetylcholine followed by nitroprusside in order to evaluate both endothelium-dependent and endotheliumindependent vasodilatation in microcirculation (Tousoulis, Antoniades, & Stefanadis, 2005). The simultaneous infusion of acetylcholine and LNMMA allows the evaluation of NO availability (Tousoulis et al., 1997). Ideally, the forearm blood flow at the contralateral arm must be also measured in order to control for any systemic hemodynamic effects, caused by the drug infusion. According to clinical studies, changes in forearm blood flow after the induction of acetylcholine are impaired in subjects with cardiovascular risk factors (Panza et al., 1990; Chowienczyk et al., 1992). The assessment of endothelial function with this method has been shown to have prognostic value for cardiovascular events in patients with CAD (Heitzer et al., 2001; Fichtlscherer et al., 2004) and patients with essential hypertension (Perticone et al., 2001). 3.3. Circulating markers of endothelial function The evaluation of endothelial function by using circulating markers has been already proposed (J.E. Deanfield et al., 2007; Versari et al., 2007). Circulating markers of endothelial function that can be used are molecules that associate with endothelial injury and/or repair, or products of endothelial cells that change after impairment of endothelium, such as adhesion molecules and cytokines. 3.3.1. Asymmetrical dimethyl arginine (ADMA) ADMA is an endogenous NOS inhibitor that increases in patients with cardiovascular risk factors and is related to reduced NO bioavailability (Vallance &Leiper, 2004). Since ADMA levels in plasma have been associated with atherosclerosis, it may be useful for the evaluation

5

of endothelial function and risk stratification of patients, but, at present, it still does not represent a reliable measure (Boger et al., 2005). 3.3.2. Soluble (s) E-selectin This endothelial leukocyte adhesion molecule reflects activation of the endothelium and has been found to be increased in patients with essential hypertension (Blann et al., 1994; DeSouza et al., 1997). However, there has not been found any correlation between increased levels of sE-selectin and reduced endothelium-dependent vasodilatation, which is also present at hypertensive patients (De Caterina et al., 2001). However, a relation was found with the endothelium-independent vasodilatation suggesting that increased concentration of sE-selectin is mainly associated with structural, rather than functional vascular alterations. 3.3.3. Thrombomodulin Thrombomodulin is a molecule with anticoagulant activities released by damaged endothelial cells. Soluble thrombomodulin is a marker of endothelial injury and it has been related to CAD, peripheral arterial disease and stroke. This marker is not increased in asymptomatic and healthy subjects (Ishii et al., 1991). 3.3.4. von Willebrand factor (vWF) von Willebrand factor is a very important ligand for the adhesion of platelets to the endothelium (Vlot et al., 1998) and is released by injured endothelial cells. Its plasma levels are increased in patients with cardiovascular disease and it has prognostic value for CAD and stroke. Therefore, it is considered as a very useful measurement for the assessment of endothelial function (Lip &Blann, 1997). 3.3.5. Circulating endothelial cells and circulating endothelial progenitor cells (EPC) Repair of the damaged endothelium may be a prerequisite for the prevention of ED and there is a growing body of evidence suggesting that circulating EPC may play a vital role (Asahara et al., 1999). Circulating endothelial cells and circulating EPC are novel markers that could be used for the assessment of endothelial damage and repair (Goon et al., 2005) and they have also been linked to other measures of endothelial function and to cardiovascular events (Werner et al., 2005). However, at present, there are not still enough data associating these markers with the extent of ED and so they have not been established at clinical use yet. 3.3.6. Endothelial microparticles (EMP) Microparticles are vesicles released from plasma membranes after cell activation or apoptosis (Hugel et al., 2005). In plasma, there are microparticles from different cells, which increase in atherothrombotic diseases. Microparticles from endothelial cells, which normally represent a small percentage of the total amount of microparticles, increase in case of ED suggesting that circulating endothelial microparticle levels may be a marker of ED. Clinical studies have shown that plasma levels of endothelial microparticles increase in cardiovascular and atherothrombotic diseases (Boulanger et al., 2006), in diabetes mellitus (Esposito et al., 2007) and in obesity (Esposito et al., 2006). Finally, it has been found that increased circulating endothelial microparticle concentration is also highly associated with the extent of ED in cases of acute coronary syndromes (Mallat et al., 2000). 4. Therapeutic approaches As ED is a key event in the development of atherosclerosis and its evaluation is a major predictor of clinical outcome in the general population, therapeutic interventions have been developed aiming to improve endothelial function. Statins, angiotensin-converting enzyme inhibitors (ACEIs), angiotensin-receptor antagonists (ARBs), antioxidants, beta-blockers and insulin sensitizers have been shown to fulfill this goal. Other substances, such as L-arginine, tetrahydrobiopterin

Please cite this article as: Tousoulis, D., et al., Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches, Pharmacology & Therapeutics (2013), http://dx.doi.org/10.1016/j.pharmthera.2014.06.003

6

D. Tousoulis et al. / Pharmacology & Therapeutics xxx (2013) xxx–xxx

Fig. 2. Potential therapeutic approaches in endothelial dysfunction. Abbreviations: NO: nitric oxide; BH4: tetrahydrobiopterin; ACEIs: angiotensin converting enzyme inhibitors; ARBs: angiotensin receptor blockers; Vit C: vitamin C; 5-MTHF: 5-methyltetrahydrofolate.

(BH4) and folic acid, are also under investigation for their contribution in the amelioration of ED. Furthermore, other forms of therapy, such as gene and epigene-based therapies and therapy with EPCs, are being developed with promising results for their effectiveness. The actions of therapeutic agents are summarized in Fig. 2 and Table 1. 4.1. Statins Statins inhibit HMG-CoA reductase, the rate limiting step in cholesterol biosynthesis, and thus reduce the level of lipids in plasma, which is considered to be the main mechanism underlying their beneficial clinical effects (Table 3) (Tamai et al., 1997). However, inhibition of HMG-CoA reductase also blocks the production of isoprenoid intermediates, which have important biological actions (Goldstein &Brown, 1990). This is considered to be related with a variety of statin properties, such as antithrombotic and anti-inflammatory effects and direct effects on endothelial function (Li &Mehta, 2003; Mehta, 2003; Wassmann &Nickenig, 2003; Tousoulis, Antoniades, Bosinakou, Kotsopoulou, Pitsavos et al., 2005; Tousoulis, Antoniades, Bosinakou, Kotsopoulou, Tsioufis et al., 2005; Tousoulis, Antoniades, Vassiliadou et al., 2005; Tousoulis, Antoniades, Katsi et al., 2006). Improvement of endothelial function is one of the earliest clinical effects after initiation of statin therapy and may occur before any significant reduction in serum cholesterol levels (O'Driscoll et al., 1997). There are several pathways through which statins improve endothelial function. Statins improve endothelial function by up-regulating the expression and activity of eNOS via two pathways. In the first pathway, statins activate protein kinase Akt (Kureishi et al., 2000) and AMP-activated protein kinase (W. Sun et al., 2006), which both regulate many cellular processes. The phosphorylation and activation of Akt are achieved

by the activation of phosphatidylinositol 3-kinase and thus, phosphatidylinositol 3-kinase inhibitors can block the effect of statins on Akt. Akt activation by statins inhibits apoptosis and causes an increase in NO production (Kureishi et al., 2000). AMP-activated protein kinase is also an important regulator of NO production and angiogenesis mediated by eNOS and is activated by statins via an increase in Threonine172 phosphorylation (W. Sun et al., 2006). In the second pathway, statins inhibit geranylgeranylation of the small G-protein Rho and thus, affect the translocation of inactive Rho from the cytosol to the membrane (Laufs &Liao, 1998). Activated Rho binds and activates Rho-associated kinases, which cause phosphorylation of myosin light chains required for the formation of actin stress fibers and focal adhesion complexes. Anchoring of mRNAs to the actin cytoskeleton is necessary for their stability and translational expression and so, Rhomediated reorganization of the actin cytoskeleton arranges the trafficking and subcellular localization of specific mRNAs. Therefore, disruption of Rho-mediated endothelial actin cytoskeleton up-regulates eNOS by increasing eNOS mRNA half-life. Statins can also improve endothelial function through their antioxidant effects. They reduce angiotensin II-induced free radical production in vascular smooth muscle cells in two ways. They have been shown to down-regulate angiotensin AT1 receptor gene expression and reduce AT1 receptor mRNA levels (Wassmann et al., 2001). Additionally, statins inhibit Rac1-mediated nicotinamide adenine dinucleotide phosphateoxidase (NADPH) by blocking the geranylgeranylation dependent translocation of Rac1 from the cytosol to the membrane, which is essential for the activation of NADPH oxidase (Wagner et al., 2000). Furthermore, it has been shown that statins up-regulate the expression of antioxidant enzymes, such as catalase and SOD (Briasoulis et al., 2012). Statins also up-regulate the expression of cyclo-oxygenase-2 and prostacyclin (Degraeve et al., 2001), which regulate endothelial function. In addition, Rho kinase inhibition caused by statins reduces preproendothelin-1 mRNA expression and endothelin-1 bioavailability (Wolfrum et al., 2003). All these, as well as the increase in NO bioavailability induced by statins, contribute to the improvement of endothelium-dependent vasodilatation. However, attenuation of ED can also be achieved by repair and regeneration of damaged endothelial cells. Specifically, it has been shown that simvastatin enhances the regeneration of endothelial cells in injured hamster carotid arteries via vascular endothelial growth factor (VEGF) secretion (Matsuno et al., 2004). It has been shown that statins up-regulate gene expression of guanosine-5′-triphosphate (GTP)-cyclohydrolase I, the rate limiting enzyme in the biosynthesis of BH4 (Antoniades et al., 2011). This increases the levels of vascular BH4, and decreases eNOS uncoupling thus improving endothelial function by reducing ROS generation and increasing NO bioavailability in the human vascular endothelium (Antoniades et al., 2011).

Table 3 Effect of statin treatment on flow mediated dilation and forearm blood flow. Study

Design

Population

Number Statin

Dose

Duration

(Tan et al., 2002)

Randomized, double blind, placebo-controlled study

Diabetes mellitus

80

Atorvastatin

12 weeks ↑ FMD

(Beishuizen et al., 2005) (Ceriello et al., 2002) (Taneva et al., 2006) (Perticone et al., 2000)

Diabetes mellitus Diabetes mellitus Hyperlipidemia Hyperlipidemia

250 30/20 33 18/12

Simvastatin Simvastatin Atorvastatin Atorvastatin

(ter Avest et al., 2005)

Randomized, placebo-controlled, double-blind Double-blind, crossover, placebo-controlled study Randomized, placebo-controlled, double-blind Randomized, double blind, placebo-controlled crossover study Double-blind randomized crossover study

10 20 20 40 80 10

Hyperlipidemia

18

Rosuvastatin

40 mg/day

(Mullen et al., 2000) (John et al., 1998) (Dupuis et al., 2005)

Double-blind, 2 × 2 factorial study Randomized, double-blind, placebo-controlled trial Randomized double-blind 2 × 2 factorial

Diabetes mellitus 84 Hypercholesterolemia 29/12 Acute coronary 50 syndromes

Atorvastatin Fluvastatin Atorvastatin/ pravastatin

40 40 40 80

mg/day or mg/day mg/day mg/day mg/day mg/day

mg/day mg/day mg/day or mg/day

24 weeks 12 weeks 6 weeks 4 weeks

Results

↔ FMD ↑ FMD ↑ FMD ↑ FBF

12 weeks ↔ FMD ↔ FBF 6 weeks ↑ FMD 6 weeks ↑ FBF 4 weeks ↑ FMD

FBF: forearm blood flow; FMD: flow-mediated dilation. ↑: Increase; ↔: neutral effect.

Please cite this article as: Tousoulis, D., et al., Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches, Pharmacology & Therapeutics (2013), http://dx.doi.org/10.1016/j.pharmthera.2014.06.003

D. Tousoulis et al. / Pharmacology & Therapeutics xxx (2013) xxx–xxx

It has been proposed that treatment with statins attenuates the apoptosis of EPCs (Lavi et al., 2010) and increases their number in a dose-dependent way (Bellia et al., 2010; Hong et al., 2010). According to some reports, this increase is independent of the reduction in serum cholesterol levels induced by statins and is associated with the improvement of endothelial function (Pirro et al., 2009; Higashi et al., 2010). However, other studies have not shown any change in the number of EPCs during statin treatment (Yoshida et al., 2010). 4.2. Inhibitors of the rennin angiotensin aldosterone system Angiotensin converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) are antihypertensive agents. ACEIs block the formation of angiotensin II by inhibiting angiotensin converting enzyme and impede its activation of angiotensin I receptors in the adrenal cortex. Therefore, they reduce vasoconstriction and aldosterone and its effects on vessels. They also inhibit the metabolism of bradykinin, which causes NO/endothelium derived relaxing factor mediated vasodilatation (Tousoulis, Antoniades, Koumallos et al., 2006). Angiotensin II receptor blockers (ARBs) displace angiotensin II from the angiotensin I receptor and reduce blood pressure by preventing angiotensin I receptor induced vasoconstriction, aldosterone, catecholamines and arginine vasopressin release, water intake and hypertrophic responses. However, it has been shown that ACEIs and ARBs do not only reduce blood pressure, but also exert cardio and renal protective effects and improve endothelial function in patients with hypercholesterolemia and coronary artery disease independently to their actions on arterial pressure control (Hornig et al., 2001) by several different mechanisms (Tables 4, 5). ACEIs and ARBs contribute to the reversal of ED through their antioxidant effects. Angiotensin II activates NADPH oxidase via angiotensin I receptor stimulation (Griendling et al., 2000) and increases its activity. ACEIs and ARBs impede the activation of NADPH oxidase (Warnholtz et al., 1999) and down-regulate its expression. Therefore, they attenuate O− 2 generation by vascular endothelium (Nickenig &Harrison, 2002). This is also attributed to the ability of ARBs to increase vascular BH4 levels and restore eNOS uncoupling (Imanishi et al., 2008; Nussberger et al., 2008). In addition, it has been shown that ACEIs containing a sulfhydryl group have the ability to scavenge superoxide anion (Liu et al., 1992) and non-superoxide radicals (Mak et al., 1990). ACEIs inhibit the metabolism of bradykinin and increase its concentration in the vicinity of endothelial bradykinin receptors 1 and 2. This leads to an increased production of vasoprotective antithrombotic and anti-inflammatory NO and prostaglandin I2 by the endothelium (Faggiotto &Paoletti, 1999). It has also been shown that ACEIs and some ARBs act as allosteric enhancers, induce a conformational change in ACE and increase bradykinin receptor 2 functions. Furthermore, ACEIs can act as allosteric agonists of bradykinin receptor 1. This enhancement of bradykinin receptors 1 and 2 signaling induced by ACEIs leads to an increased NO production and may partially explain the ability of ACEIs to improve endothelial function (Erdos et al., 2010). ACEIs and ARBs also improve endothelial function through their effects on ADMA and DDAH. It is known that ADMA is increased in ED,

7

that DDAH is the enzyme responsible for its catabolism and that DDAH activity is diminished by ROS. The exact pathways through which ACEIs and ARBs regulate ADMA metabolism are still being evaluated. However, it is possible that these drugs may reduce ADMA levels by enhancing DDAH activity induced by their ability to decrease ROS generation by vascular endothelium (Antoniades, Shirodaria et al., 2009). Indeed, in some studies serum markers of oxidative stress have been reduced after treatment with these drugs (Ito et al., 2001; Fu et al., 2005). According to the available literature, both ACEIs and ARBs do not provide a complete endothelial protection. This might be due to the nonabsolute blockade of the renin–angiotensin–aldosterone system by both classes. A solution to this might be given by aliskiren, a non-peptide renin inhibitor that blocks the first enzyme of the renin–angiotensin– aldosterone system and reduces the circulating levels of angiotensin II (Staessen et al., 2006). Theoretically, aliskiren provides a better blockade of the RAAS as compared to ACEIs and ARBs. Recently, it was shown that therapy with aliskiren increased acetylcholine-induced vasodilatation and basal plasma NO levels in an animal model of atherosclerosis (Imanishi et al., 2008). However, at present, there is no available information for the effects of aliskiren on humans. 4.3. Insulin sensitizers Several recent studies have shown that ED and insulin resistance coexist (Steinberg et al., 1996). Besides its metabolic functions, insulin also has vascular actions. Specifically, it interacts with the endothelium leading to NO release and thus, vasodilatation of skeletal muscle vessels (Baron &Clark, 1997). Obese individuals with insulin resistance have impaired endothelial function (Steinberg et al., 1996). Therefore, it has been proposed that drugs that increase insulin sensitivity may improve endothelial function. Most studies have used thiazolidinediones in order to investigate this hypothesis. Thiazolidinediones, also known as glitazones, are activators of peroxisome proliferator receptor-γ (PPAR-γ), which is a ligand activated nuclear transcription factor expressed in endothelial and vascular smooth muscle cells (Duan et al., 2008). It has been demonstrated that they reduce relative mRNA levels of the NADPH oxidase subunits, NADPH oxidase 1 (nox-1), gp91phox (NOX2) and NADPH oxidase 4 (NOX4), as well as NADPH-dependent O− 2 production in human umbilical vein endothelial cells and that they up-regulate expression and activity of SOD (Hwang et al., 2005). They also increase endothelial NO release without altering eNOS expression (Calnek et al., 2003). Additionally, they also improve endothelial function by inhibiting the expression of adhesion molecules, such as vascular adhesion molecule-1, intracellular adhesion molecule-1 and E-selectin in activated endothelial cells and so, by reducing chemotaxis of monocytes/macrophages to the endothelium (Jackson et al., 1999; Pasceri et al., 2000). There have been several clinical studies demonstrating the improvement of endothelial function in diabetic (Caballero et al., 2003) and nondiabetic (Watanabe et al., 2000) subjects after 4–12 weeks of therapy with thiazolidinediones.

Table 4 Impact of angiotensin converting enzyme inhibitors/angiotensin receptor blockers on nitric oxide/endothelial function. Author(s)/study

Results/effects

(Gemici et al., 2010) (Virdis et al., 2011) (Blann et al., 1994), (O'Driscoll et al., 1999) (Kampoli et al., 2012) (Cousin et al., 2010) (Faggiotto &Paoletti, 1999) (Mancini et al., 1996)

↑ inducible NO synthase and cyclooxygenase-2 ↑ subcutaneous, epicardial, brachial, and renal (circulation) endothelial function ↑ stimulated and basal NO-dependent endothelial function ↑ bradykinin, ↓ apoptosis, ↑ VEGF, ↑ CD34+ mobilization, ↓ TNF-α ↑ endothelial NO synthase expression ↑ the production of NO and PGI2 ↑ endothelial cell function and vascular tone

NO: nitric oxide; VEGF: vascular endothelial growth factor; TNF-α: tumor necrosis factor alpha; prostaglandin I2. ↑: increase. ↓: decrease.

Please cite this article as: Tousoulis, D., et al., Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches, Pharmacology & Therapeutics (2013), http://dx.doi.org/10.1016/j.pharmthera.2014.06.003

8

D. Tousoulis et al. / Pharmacology & Therapeutics xxx (2013) xxx–xxx

Table 5 Representative studies focusing on the effect of ACE-inhibitors on endothelial function. Study

Type

Population number

Drug

Dose

Duration

(McFarlane et al., 1999) (Nagamia et al., 2007) (Kovacs et al., 2006)

Randomized, crossover double-blind controlled Crossover fashion with placebo Randomized, double-blind, crossover

Diabetes mellitus type 1 (20 subjects) Metabolic syndrome (44 subjects) Myocardial infarction (50 subjects)

4 mg/day 20 mg/day 10 mg/day

3 months ↔ 14 months ↑ 3 months ↑

(Asselbergs et al., 2008) (Mancini et al., 1996) (Inanc et al., 2010) (Trevelyan et al., 2005)

Double-blind, randomized, placebo-controlled Double-blind, randomized, placebo-controlled Randomized placebo Randomized, controlled, blinded end point

Albuminuria (276 subjects) Normotension with CAD (105 subjects) Behçet's disease (92 subjects) CAD for CABG (49 subjects)

Randomized, double-blind, crossover Randomized, double-blind, parallel-group

Rheumatoid arthritis (11 subjects) Diabetes mellitus type 1 (91 subjects)

20 mg/day 40 mg/day 10 mg/day 50 mg/day or 20 mg/day 2.5–10 mg/day 20 mg/day

48 months ↔ 6 months ↑ ↑ 2 months ↑

(Flammer et al., 2008) (Mullen et al., 1998)

Perindopril Quinapril Enalapril or quinapril Fosinopril Quinapril Lisinopril Losartan or enalapril Ramipril Enalapril

2 months 6 months

Endothelial function

↑ ↔

↑: positive effect, ↔: no effect, CAD: coronary artery disease, CABG: coronary artery bypass grafting.

4.4. Third generation β-blockers (nebivolol, carvedilol) Lately, attention has been given to the so-called third generation βblockers, such as nebivolol and carvedilol. Nebivolol is a selective β1adrenoreceptor antagonist and it has been reported that it increases NO release from the endothelium. Specifically, intra-arterial infusion of nebivolol leads to vasodilatation, inhibited after the infusion of LNMMA, which indicates that nebivolol causes NO-dependent vasodilatation (Cockcroft et al., 1995). In addition, nebivolol reduces oxidative stress (Cominacini et al., 2003; Fratta Pasini et al., 2005; Mason et al., 2005) by inhibiting endothelial NADPH oxidase activity (Evangelista et al., 2007) and by directly scavenging ROS (Oelze et al., 2006). Furthermore, it has been proposed that nebivolol might reduce plasma levels of ADMA possibly by up-regulating the expression of DDAH2. This was tested by Pasini et al. (2008) in 40 patients with essential hypertension treated with atenolol and nebivolol in a double-blind randomized study. In this study, it was found that only in nebivolol group, there was a significant reduction in ADMA levels, an increase in FMD and an important correlation between these two changes. It was also found that in patients treated with nebivolol and not with atenolol, there was decreased ADMA and increased DDAH2 expression and eNOS activity in human umbilical vein endothelial cells. Carvedilol is a non-selective β1-blocker with additional α1adrenoreceptor antagonist activity. It has been shown that it improves endothelial function by exerting antioxidant effects (Feuerstein &Ruffolo, 1995). A recent study has indicated that in a group of hypertensive patients with diabetes mellitus, carvedilol increased FMD and thus, improved endothelial function (Bank et al., 2007). This beneficial effect has also been confirmed in the forearm microcirculation of patients with dilated cardiomyopathy (Nishioka et al., 2007). However, further studies are needed in order to clarify the exact mechanism(s) through which carvedilol exerts its antioxidant effects.

4.5. Antioxidants Many antioxidant vitamins, such as vitamin C, vitamin E and beta carotene, have been evaluated as a treatment option for cardiovascular disease (Reaven et al., 1993; Jialal et al., 1995). They exert their effects through different mechanisms in order to prevent oxidant-induced cell damages. They can scavenge ROS, attenuate their production or interfere with ROS-induced alterations. Vitamins C and E may inhibit LDL oxidation by scavenging ROS and thus blocking lipid peroxidation chain reaction (Carr et al., 2000). They also increase NO bioavailability. Specifically for vitamin C has been shown to scavenge ROS that reacts with NO (Padayatty et al., 2003) and possibly stabilizes BH4 (Heller et al., 2001) ameliorating thus eNOS coupling. Vitamin E has been shown to attenuate the inhibitory role of oxLDL on eNOS expression (Carr et al., 2000). Several small clinical trials have demonstrated that oral administration of vitamin C and/or E improves endothelial function (Antoniades

et al., 2003; Tousoulis, Antoniades, Tentolouris et al., 2003; Tousoulis, Antoniades, Tountas et al., 2003). Additionally, antioxidants may also exert beneficial effects on the thrombosis/fibrinolysis system, which is known to play a crucial role in atherosclerosis. Indeed, it has been demonstrated that short-term treatment with high doses of vitamin C (Tousoulis, Antoniades, Tountas et al., 2003) as well as coadministration of high doses of vitamins C and E (Antoniades et al., 2003) not only improved reactive hyperemia but also reduced plasma levels of pro-thrombotic molecules, such as von Willebrand factor (vWF), and antithrombotic molecules, such as tissue plasminogen activator. Furthermore, we (Tousoulis, Antoniades, Tentolouris et al., 2003) have also demonstrated that co-administration of high doses of vitamins C and E also exerts non-specific anti-inflammatory effects, as it reduces plasma levels of tumor necrosis factor alpha, soluble vascular cell adhesion molecule-1, soluble intracellular adhesion molecule-1 and the expression of pro-inflammatory cytokines, such as interleukin-1b and interleukin-6. Although short-term treatment with antioxidants improves endothelial function, it is uncertain if long-term treatment can maintain this result (Tousoulis, Antoniades, Tentolouris et al., 2003). Indeed, large randomized clinical trials, such as the HOPE study (Effects of ramipril on cardiovascular and microvascular outcomes in people with diabetes mellitus: results of the HOPE study and MICRO-HOPE substudy. Heart Outcomes Prevention Evaluation Study Investigators, 2000), the GISSI-prevenzione trial (Dietary supplementation with n−3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico, 1999) and the Heart Protection Study (MRC/BHF Heart Protection Study of antioxidant vitamin supplementation in 20,536 high-risk individuals: a randomized placebo-controlled trial, 2002), have not confirmed the beneficial effects of antioxidants on endothelial function observed in small clinical trials. Furthermore, it has also been reported that oral administration of vitamins attenuates the beneficial effects of statins on cholesterol lowering and on endothelial function (Tousoulis, Antoniades, & Stefanadis, 2006). Additionally, it is possible that only early lesions in the vascular endothelium, and not advanced ones, are responsive to treatment with antioxidants (Siekmeier et al., 2007). Conclusively, despite the apparent short term beneficial effects, oral administration of antioxidant vitamins does not improve long term cardiovascular risk. Recently, new antioxidant therapeutic approaches have been proposed targeting specific subcellular sources of oxidative stress. Given the essential role of mitochondria in ROS generation, these new therapeutic approaches focus on increasing the antioxidant ability of mitochondria and so, protecting it from oxidative damage. These approaches depend on the drug being selectively taken up by mitochondria within the vascular endothelium. MitoQ10 is a mitochondria targeted ubiquinone that belongs to this new category of antioxidants. It has been shown to improve endothelial function and protect against the development of hypertension in young stroke-prone spontaneously

Please cite this article as: Tousoulis, D., et al., Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches, Pharmacology & Therapeutics (2013), http://dx.doi.org/10.1016/j.pharmthera.2014.06.003

D. Tousoulis et al. / Pharmacology & Therapeutics xxx (2013) xxx–xxx

hypertensive rats (Graham et al., 2009). One other new antioxidant is Szeto–Schiller (SS)-31 which is an aromatic cationic peptide that targets the mitochondrial inner membrane. It has been demonstrated that SS-31 reduces angiotensin induced NADPH oxidase 4 (NOX4) upregulation and mitochondrial oxidative damage in mice (Dai et al., 2011). This new category of antioxidants has given hope in the antioxidant treatment for ED, but clinical studies need to be done. 4.6. L-Arginine L-Arginine, a semi-essential amino acid, is the substrate for eNOS and the precursor molecule for the synthesis of NO. Recently, it has been proposed that changes in eNOS dimerization and competitive inhibition of eNOS by endogenous inhibitors, such as ADMA, may lead to eNOS “uncoupling”, in which ROS are released instead of NO (Channon &Guzik, 2002). Therefore, L-arginine may exert beneficial effects on endothelial function by competing with the harmful results of ADMA on eNOS function. This antagonism between L-arginine and ADMA may also explain why L-arginine supplementation increases NO synthesis when administered to patients with high ADMA levels at baseline (Boger, 2006). Interestingly, it has no effect when administered to healthy subjects (Gates et al., 2007) or to older patients with coronary artery disease and renal dysfunction (Jahangir et al., 2009). L-Arginine also affects endothelial function in many ways. Besides its antagonism with ADMA, it can further regulate endothelial function by stimulating the secretion hormones, such as insulin and growth hormone (Heffernan et al., 2010). Growth hormone up-regulates gene expression of eNOS, Mn-SOD, Zn-SOD, increases the expression of eNOS mRNA, reduces ADMA, decreases generation of ROS and increases endothelial progenitor cells (Thum et al., 2007; Csiszar et al., 2008). Furthermore, L-arginine has antioxidant and antiapoptotic abilities, reduces the expression of adhesion molecules, chemotactic peptides and endothelin-1, inhibits platelet aggregation and increases smooth muscle cells relaxation (Lerman et al., 1998). However, despite the positive results from small case control trials where L-arginine supplementation increases NO bioavailability in vascular endothelium, there are conflicting findings about its efficacy and it is still unclear whether chronic administration has beneficial effects on clinical outcome in patients with CAD.

4.7. Tetrahydrobiopterin (BH4) BH4 is an essential cofactor for eNOS that regulates endothelial function through its effect on NO synthesis. Its deficiency, caused by the reaction of BH4 with ROS, leads to eNOS “uncoupling”, converting the enzyme to a source of superoxide instead of NO (Channon &Guzik, 2002; Kuzkaya et al., 2003). Given the pivotal role of BH4 in the regulation of endothelial function, it has been proposed that BH4 supplementation may be a rational therapeutic approach to reverse ED. In humans, acute intra-coronary or intra-brachial infusion of BH4 leads to an increase in NO-mediated effects on forearm blood flow and short term improvements in endothelial function in subjects with established CAD (Maier et al., 2000), diabetes (Nystrom et al., 2004), or other cardiovascular risk factors, such as smoking (Heitzer et al., 2000), hypertension (Higashi et al., 2002) and hypercholesterolemia (Fukuda et al., 2002). However, despite these promising findings and the indisputable importance of BH4 in NO mediated endothelial function, it still remains unclear whether BH4 can be an effective therapeutic target because of the lack of evidence as far as long term efficacy is concerned.

9

2002). Therefore, it has been demonstrated that high plasma levels of homocysteine are related with cardiovascular risk (Wald et al., 2002). Folic acid and 5-methyltetrahydrofolate, which is its circulating metabolite, have been shown to directly affect vascular endothelium, independently of their actions on plasma levels of tHcy (Antoniades, Antonopoulos, Tousoulis et al., 2009). Indeed, intra-arterial infusion of 5-methyltetrahydrofolate improved FMD in patients with CAD before any decrease in plasma homocysteine was detected (Doshi et al., 2002). Antoniades, Shirodaria et al. (2006) have shown that 5methyltetrahydrofolate directly affects vascular endothelium by improving endothelial NO bioavailability and by reducing vascular superoxide generation both in vivo and ex vivo. This is due to the ability of 5-methyltetrahydrofolate to increase eNOS activity, to improve the dimer/monomer ratio in human vessels in vivo (Antoniades, Shirodaria et al., 2006) as well as eNOS coupling (Stroes et al., 2000) and to scavenge peroxynitrite radicals leading to a significant increase in vascular BH4 bioavailability. However, despite these findings, prospective randomized clinical trials are required before folic acid or 5-methyltetrahydrofolate supplementation proves to be an effective therapeutic strategy against ED. 4.9. Gene therapy 4.9.1. Vascular endothelial growth factor (VEGF) At present, the most widely targeted gene for therapeutic use in cases of ED is VEGF, which plays a pivotal role in angiogenesis (Holmes &Zachary, 2005) and regulates endothelial function by stimulating the production of NO and prostacyclin (Wheeler-Jones et al., 1997; Holmes &Zachary, 2005). Preclinical animal studies have supported VEGF-stimulated angiogenesis as a beneficial therapeutic for ischemic heart disease (Holmes &Zachary, 2005), but as far as human clinical trials are concerned the findings for the beneficial effects of VEGF gene delivery are conflicting and several ones are still in progress. Rajagopalan et al. (2001) demonstrated that adenoviral VEGF gene delivery into the skeletal muscle of the lower limbs in subjects with peripheral arterial disease improved lower limb flow reserve and endothelial function after four weeks, which is also supported by animal studies (Bauters et al., 1995; Takeshita et al., 1998). However, in a murine model with VEGF genetic deletion, VEFG gene delivery has not rescued the degeneration of endothelial cells and showed that in vivo autocrine VEGF signaling is required for endothelial cell survival (S. Lee et al., 2007). Concerns have also been raised about the possible adverse effects of VEGF gene transfer, as it can lead to edema formation, induce neovascularization in plaques and change their stability or induce neovascularization in tissues in which this might be harmful, such as tumors (Epstein et al., 2001) or in the retina of diabetic patients.

4.8. Homocysteine, folate and 5-methyltetrahydrofolate (5-MTHF)

4.9.2. Endothelial nitric oxide synthase (eNOS) Given the role of NO in endothelial homeostasis and the role of eNOS in NO synthesis, gene therapy has focused on up-regulating eNOS expression. In a leporine model, adenoviral gene transfer of eNOS improved endothelial dependent relaxation of carotid arteries and increased cyclic guanosine-monophosphate levels. This showed that eNOS gene delivery to a healthy vessel can increase NO synthesis and thus, regulate vascular tone (Kullo et al., 1997). Additionally, it has been demonstrated in animal studies that adenoviral eNOS gene transfer improves blood flow in mice with hind limb ischemia (Smith et al., 2002), increases endothelial proliferation in murine models of myocardial infarction (Smith et al., 2005) and reduces restenosis after balloon angioplasty (Hayashi et al., 2004) and coronary artery stenting (Sharif et al., 2008).

It is generally thought that homocysteine leads to increased intracellular oxidative stress in vascular endothelium by increasing oxidative inactivation of NO, the generation of peroxynitrite and by reducing NO production (Verhaar et al., 1999; Doshi et al., 2002; Hyndman et al.,

4.9.3. Dimethyl arginine dimethyl aminohydrolase (DDAH) As mentioned above, DDAH metabolizes ADMA, which affects eNOS coupling and thus, leads to reduced NO and increased superoxide generation. There are two isoforms of DDAH: DDAH1, found in tissues

Please cite this article as: Tousoulis, D., et al., Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches, Pharmacology & Therapeutics (2013), http://dx.doi.org/10.1016/j.pharmthera.2014.06.003

10

D. Tousoulis et al. / Pharmacology & Therapeutics xxx (2013) xxx–xxx

expressing neuronal NOS, and DDAH2, found in tissues expressing eNOS. According to studies in rabbits, adenoviral DDAH2 gene transfer to rabbit aortas increased DDAH expression and improved endothelial dependent relaxation (Feng et al., 2010; Hayashi et al., 2004; Sharif et al., 2008; Lu et al., 2010). One other study in human umbilical vein endothelial cells infected with recombinant DDAH1 and DDAH2 adenovirus revealed an increase in DDAH expression and a decrease in ADMA levels. In this study, incubation of DDAH1+/− murine carotid arteries led to increase NO synthesis and improved acetylcholine induced relaxation (Torondel et al., 2010). 4.9.4. Guanosine-5′-triphosphate cyclohydrolase 1 (GTPCH1) eNOS uncoupling is a major source of superoxide and occurs when there is a decrease in BH4. Guanosine-5′-triphosphate cyclohydrolase 1 is the rate limiting enzyme of BH4 synthesis. Therefore, given its role in BH4 synthesis, guanosine-5′-triphosphate cyclohydrolase 1 can be used as a target for gene therapy. Adenoviral guanosine-5′-triphosphate cyclohydrolase 1 gene delivery to carotid arteries of deoxycorticosterone acetate–salt hypertensive rats increased BH4 levels and basal NO release, reduced superoxide and improved endothelial-dependent relaxation (Zheng et al., 2003). 4.9.5. Extracellular superoxide dismutase (SOD) Extracellular SOD is an important scavenger of superoxide extracellular SOD. Thus gene delivery is considered as a promising method to attenuate oxidative stress (Qin et al., 2008). According to studies in rats, the delivery of replication deficient recombinant adenovirus encoding human extracellular SOD led to a reduction of superoxide levels and improved acetylcholine induced endothelial relaxation, showing improved endothelial function (Lund et al., 2004; Iida et al., 2005; Brown et al., 2006). Furthermore, extracellular SOD transfer has also led to a reduction of blood pressure in spontaneously hypertensive rats (Chu et al., 2003) and an increase in NO bioavailability in carotid arteries of spontaneously hypertensive rats (Fennell et al., 2002).

of neovascularization or re-endothelialization (Asahara et al., 1999). It has been shown that in cases of increased cardiovascular risk, there is decreased EPC bioavailability (Hill et al., 2003). EPC injection into sites of injury, such as in ischemic tissues, has given promise for the growth of new vessels (Masuda &Asahara, 2003). Animal studies have demonstrated that genetically-modified EPC transplantation is very promising in enhancing re-endothelialization and thus, improving endothelial function (Kong et al., 2004; Cui et al., 2011). Particularly interesting is the overexpression of eNOS in EPCs by retroviral transduction, which has been shown to increase endothelial-dependent vasodilatation (Cui et al., 2011) and inhibit neointimal hyperplasia (Kong et al., 2004). 5. Conclusions The role of vascular endothelium in atherosclerosis is well established. In addition, several methods have been developed to evaluate endothelial function which is a well known determinant of atherogenesis. However, among the available methods evaluating endothelial function, there is no method combining sufficient sensitivity and specificity in order to be used in clinical practice. Despite the efforts in developing therapeutic strategies that improve endothelial function, only few of them have shown to have long term clinical benefits. Currently, statins, ACEIs/ARBs and thiazolidinediones have given the most promising results, and besides the treatment of underlying cardiovascular risk factors, they also affect directly the vascular endothelium and improve its function. Gene therapy is also another area gaining significant interest over the last years, but still under investigation. Therefore, it has become evident that further large scale studies are required to evaluate in depth the available methods and strategies aiming to monitor and improve endothelial dysfunction. Conflict of interest statements The authors declare that there are no conflicts of interest.

4.10. Epigenetics

References

Epigenetics has gained significant interest in the past few years. Studies in this area have progressed our knowledge on how the environment affects the genome at a molecular level (Matouk &Marsden, 2008). It refers to changes in heritable chromatin affecting gene expression that do not come from alterations in the DNA sequence itself and include mechanisms, such as DNA methylation, histone density and posttranslational modification and RNA-based mechanisms (T.K. Kelly et al., 2010). The controlled expression of eNOS has revealed the significant role of epigenetic regulation in the vascular endothelium (Yan et al., 2010). eNOS expression is highly controlled by its chromatin accessibility, with the chromatin modulation of its promoter being different between endothelial cells that express eNOS and non-endothelial cells (Matouk &Marsden, 2008). These differences include increased methylation of histone amino terminal tails and posttranslational acetylation and decreased DNA methylation in endothelial cells (Y. Chan et al., 2004; Fish et al., 2005). According to animal studies, endothelial expression of eNOS can be down-regulated by methylation inhibitors and RNA-based mechanisms that change DNA methylation and promote histone alteration (Y. Chan et al., 2004; Fish et al., 2005; M.X. Zhang et al., 2005; M.X. Zhang et al., 2008). Understanding the epigenetic pathways that control endothelial response to redox stimuli will provide an opportunity for epigene-based therapy to become a therapeutic strategy against ED.

MRC/BHF Heart Protection Study of antioxidant vitamin supplementation in 20,536 highrisk individuals: a randomised placebo-controlled trial. Lancet 360. (2002)., 23–33. Effects of ramipril on cardiovascular and microvascular outcomes in people with diabetes mellitus: results of the HOPE study and MICRO-HOPE substudy. Heart Outcomes Prevention Evaluation Study Investigators. Lancet 355. (2000)., 253–259. Dietary supplementation with n−3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico. Lancet 354. (1999)., 447–455. Abularrage, C. J., Sidawy, A. N., Aidinian, G., Singh, N., Weiswasser, J. M., & Arora, S. (2005). Evaluation of the microcirculation in vascular disease. J Vasc Surg 42, 574–581. Alp, N. J., Mussa, S., Khoo, J., Cai, S., Guzik, T., Jefferson, A., et al. (2003). Tetrahydrobiopterin-dependent preservation of nitric oxide-mediated endothelial function in diabetes by targeted transgenic GTP-cyclohydrolase I overexpression. J Clin Invest 112, 725–735. Anderson, T. J., Uehata, A., Gerhard, M.D., Meredith, I. T., Knab, S., Delagrange, D., et al. (1995). Close relation of endothelial function in the human coronary and peripheral circulations. J Am Coll Cardiol 26, 1235–1241. Antoniades, C., Antonopoulos, A. S., Bendall, J. K., & Channon, K. M. (2009). Targeting redox signaling in the vascular wall: from basic science to clinical practice. Curr Pharm Des 15, 329–342. Antoniades, C., Antonopoulos, A. S., Tousoulis, D., Marinou, K., & Stefanadis, C. (2009). Homocysteine and coronary atherosclerosis: from folate fortification to the recent clinical trials. Eur Heart J 30, 6–15. Antoniades, C., Bakogiannis, C., Leeson, P., Guzik, T. J., Zhang, M. H., Tousoulis, D., et al. (2011). Rapid, direct effects of statin treatment on arterial redox state and nitric oxide bioavailability in human atherosclerosis via tetrahydrobiopterin-mediated endothelial nitric oxide synthase coupling. Circulation 124, 335–345. Antoniades, C., Shirodaria, C., Crabtree, M., Rinze, R., Alp, N., Cunnington, C., et al. (2007). Altered plasma versus vascular biopterins in human atherosclerosis reveal relationships between endothelial nitric oxide synthase coupling, endothelial function, and inflammation. Circulation 116, 2851–2859. Antoniades, C., Shirodaria, C., Leeson, P., Antonopoulos, A., Warrick, N., Van-Assche, T., et al. (2009). Association of plasma asymmetrical dimethylarginine (ADMA) with elevated vascular superoxide production and endothelial nitric oxide synthase uncoupling: implications for endothelial function in human atherosclerosis. Eur Heart J 30, 1142–1150. Antoniades, C., Shirodaria, C., Warrick, N., Cai, S., de Bono, J., Lee, J., et al. (2006). 5Methyltetrahydrofolate rapidly improves endothelial function and decreases

4.11. Endothelial progenitor cells (EPC) Another recent and interesting area is gene-therapy towards EPCs with subsequent genetically-altered EPC transplantation. EPCs come from the bone marrow and differentiate into endothelial cells at sites

Please cite this article as: Tousoulis, D., et al., Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches, Pharmacology & Therapeutics (2013), http://dx.doi.org/10.1016/j.pharmthera.2014.06.003

D. Tousoulis et al. / Pharmacology & Therapeutics xxx (2013) xxx–xxx superoxide production in human vessels: effects on vascular tetrahydrobiopterin availability and endothelial nitric oxide synthase coupling. Circulation 114, 1193–1201. Antoniades, C., Tousoulis, D., Marinou, K., Papageorgiou, N., Bosinakou, E., Tsioufis, C., et al. (2007). Effects of insulin dependence on inflammatory process, thrombotic mechanisms and endothelial function, in patients with type 2 diabetes mellitus and coronary atherosclerosis. Clin Cardiol 30, 295–300. Antoniades, C., Tousoulis, D., Marinou, K., Stefanadi, E., Ntarladimas, I., Latsios, G., et al. (2006). Effects of lipid profile on forearm hyperemic response in young subjects. Hellenic J Cardiol 47, 152–157. Antoniades, C., Tousoulis, D., Tentolouris, C., Toutouza, M., Marinou, K., Goumas, G., et al. (2003). Effects of antioxidant vitamins C and E on endothelial function and thrombosis/fibrinolysis system in smokers. Thromb Haemost 89, 990–995. Asahara, T., Masuda, H., Takahashi, T., Kalka, C., Pastore, C., Silver, M., et al. (1999). Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 85, 221–228. Asselbergs, F. W., van der Harst, P., van Roon, A.M., Hillege, H. L., de Jong, P. E., Gans, R. O., et al. (2008). Long-term effects of pravastatin and fosinopril on peripheral endothelial function in albuminuric subjects. Atherosclerosis 196, 349–355. Bank, A. J., Kelly, A. S., Thelen, A.M., Kaiser, D. R., & Gonzalez-Campoy, J. M. (2007). Effects of carvedilol versus metoprolol on endothelial function and oxidative stress in patients with type 2 diabetes mellitus. Am J Hypertens 20, 777–783. Baron, A.D., & Clark, M. G. (1997). Role of blood flow in the regulation of muscle glucose uptake. Annu Rev Nutr 17, 487–499. Bauters, C., Asahara, T., Zheng, L. P., Takeshita, S., Bunting, S., Ferrara, N., et al. (1995). Recovery of disturbed endothelium-dependent flow in the collateral-perfused rabbit ischemic hindlimb after administration of vascular endothelial growth factor. Circulation 91, 2802–2809. Beishuizen, E. D., Tamsma, J. T., Jukema, J. W., van de Ree, M.A., van der Vijver, J. C., Meinders, A. E., et al. (2005). The effect of statin therapy on endothelial function in type 2 diabetes without manifest cardiovascular disease. Diabetes Care 28, 1668–1674. Bellia, A., Rizza, S., Galli, A., Fabiano, R., Donadel, G., Lombardo, M. F., et al. (2010). Early vascular and metabolic effects of rosuvastatin compared with simvastatin in patients with type 2 diabetes. Atherosclerosis 210, 199–201. Blann, A.D., Tse, W., Maxwell, S. J., & Waite, M.A. (1994). Increased levels of the soluble adhesion molecule E-selectin in essential hypertension. J Hypertens 12, 925–928. Boger, R. H. (2006). Asymmetric dimethylarginine (ADMA): a novel risk marker in cardiovascular medicine and beyond. Ann Med 38, 126–136. Boger, R. H., Maas, R., Schulze, F., & Schwedhelm, E. (2005). Elevated levels of asymmetric dimethylarginine (ADMA) as a marker of cardiovascular disease and mortality. Clin Chem Lab Med 43, 1124–1129. Bonetti, P. O., Pumper, G. M., Higano, S. T., Holmes, D. R., Jr., Kuvin, J. T., & Lerman, A. (2004). Noninvasive identification of patients with early coronary atherosclerosis by assessment of digital reactive hyperemia. J Am Coll Cardiol 44, 2137–2141. Boulanger, C. M., Amabile, N., & Tedgui, A. (2006). Circulating microparticles: a potential prognostic marker for atherosclerotic vascular disease. Hypertension 48, 180–186. Brevetti, G., Silvestro, A., Schiano, V., & Chiariello, M. (2003). Endothelial dysfunction and cardiovascular risk prediction in peripheral arterial disease: additive value of flow-mediated dilation to ankle–brachial pressure index. Circulation 108, 2093–2098. Briasoulis, A., Tousoulis, D., Androulakis, E. S., Papageorgiou, N., Latsios, G., & Stefanadis, C. (2012). Endothelial dysfunction and atherosclerosis: focus on novel therapeutic approaches. Recent Pat Cardiovasc Drug Discov 7, 21–32. Brown, K. A., Chu, Y., Lund, D.D., Heistad, D.D., & Faraci, F. M. (2006). Gene transfer of extracellular superoxide dismutase protects against vascular dysfunction with aging. Am J Physiol Heart Circ Physiol 290, H2600–H2605. Caballero, A. E., Arora, S., Saouaf, R., Lim, S.C., Smakowski, P., Park, J. Y., et al. (1999). Microvascular and macrovascular reactivity is reduced in subjects at risk for type 2 diabetes. Diabetes 48, 1856–1862. Caballero, A. E., Saouaf, R., Lim, S.C., Hamdy, O., Abou-Elenin, K., O'Connor, C., et al. (2003). The effects of troglitazone, an insulin-sensitizing agent, on the endothelial function in early and late type 2 diabetes: a placebo-controlled randomized clinical trial. Metabolism 52, 173–180. Calnek, D. S., Mazzella, L., Roser, S., Roman, J., & Hart, C. M. (2003). Peroxisome proliferator-activated receptor gamma ligands increase release of nitric oxide from endothelial cells. Arterioscler Thromb Vasc Biol 23, 52–57. Carr, A.C., Zhu, B. Z., & Frei, B. (2000). Potential antiatherogenic mechanisms of ascorbate (vitamin C) and alpha-tocopherol (vitamin E). Circ Res 87, 349–354. Celermajer, D. S. (1998). Testing endothelial function using ultrasound. J Cardiovasc Pharmacol 32(Suppl. 3), S29–S32. Celermajer, D. S. (2005). Brachial artery FMD with 5-minute distal cuff occlusion—a useful pathophysiological test after all! J Appl Physiol 99(1985), 1619. Celermajer, D. S., Sorensen, K. E., Bull, C., Robinson, J., & Deanfield, J. E. (1994). Endothelium-dependent dilation in the systemic arteries of asymptomatic subjects relates to coronary risk factors and their interaction. J Am Coll Cardiol 24, 1468–1474. Ceriello, A., Taboga, C., Tonutti, L., Quagliaro, L., Piconi, L., Bais, B., et al. (2002). Evidence for an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial dysfunction and oxidative stress generation: effects of short- and long-term simvastatin treatment. Circulation 106, 1211–1218. Chakrabarti, S., Blair, P., & Freedman, J. E. (2007). CD40-40L signaling in vascular inflammation. J Biol Chem 282, 18307–18317. Chan, Y., Fish, J. E., D'Abreo, C., Lin, S., Robb, G. B., Teichert, A.M., et al. (2004). The cellspecific expression of endothelial nitric-oxide synthase: a role for DNA methylation. J Biol Chem 279, 35087–35100.

11

Chan, L., Shaw, A. G., Busfield, F., Haluska, B., Barnett, A., Kesting, J., et al. (2005). Carotid artery intimal medial thickness, brachial artery flow-mediated vasodilation and cardiovascular risk factors in diabetic and non-diabetic indigenous Australians. Atherosclerosis 180, 319–326. Channon, K. M. (2004). Tetrahydrobiopterin: regulator of endothelial nitric oxide synthase in vascular disease. Trends Cardiovasc Med 14, 323–327. Channon, K. M., & Guzik, T. J. (2002). Mechanisms of superoxide production in human blood vessels: relationship to endothelial dysfunction, clinical and genetic risk factors. J Physiol Pharmacol 53, 515–524. Charakida, M., Masi, S., Luscher, T. F., Kastelein, J. J., & Deanfield, J. E. (2010). Assessment of atherosclerosis: the role of flow-mediated dilatation. Eur Heart J 31, 2854–2861. Chowienczyk, P. J., Kelly, R. P., MacCallum, H., Millasseau, S.C., Andersson, T. L., Gosling, R. G., et al. (1999). Photoplethysmographic assessment of pulse wave reflection: blunted response to endothelium-dependent beta2-adrenergic vasodilation in type II diabetes mellitus. J Am Coll Cardiol 34, 2007–2014. Chowienczyk, P. J., Watts, G. F., Cockcroft, J. R., & Ritter, J. M. (1992). Impaired endothelium-dependent vasodilation of forearm resistance vessels in hypercholesterolaemia. Lancet 340, 1430–1432. Chu, Y., Iida, S., Lund, D.D., Weiss, R. M., DiBona, G. F., Watanabe, Y., et al. (2003). Gene transfer of extracellular superoxide dismutase reduces arterial pressure in spontaneously hypertensive rats: role of heparin-binding domain. Circ Res 92, 461–468. Cockcroft, J. R., Chowienczyk, P. J., Brett, S. E., Chen, C. P., Dupont, A. G., Van Nueten, L., et al. (1995). Nebivolol vasodilates human forearm vasculature: evidence for an Larginine/NO-dependent mechanism. J Pharmacol Exp Ther 274, 1067–1071. Cominacini, L., Fratta Pasini, A., Garbin, U., Nava, C., Davoli, A., Criscuoli, M., et al. (2003). Nebivolol and its 4-keto derivative increase nitric oxide in endothelial cells by reducing its oxidative inactivation. J Am Coll Cardiol 42, 1838–1844. Corretti, M. C., Anderson, T. J., Benjamin, E. J., Celermajer, D., Charbonneau, F., Creager, M.A., et al. (2002). Guidelines for the ultrasound assessment of endothelialdependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol 39, 257–265. Cousin, M., Custaud, M.A., Baron-Menguy, C., Toutain, B., Dumont, O., Guihot, A. L., et al. (2010). Role of angiotensin II in the remodeling induced by a chronic increase in flow in rat mesenteric resistance arteries. Hypertension 55, 109–115. Csiszar, A., Labinskyy, N., Perez, V., Recchia, F. A., Podlutsky, A., Mukhopadhyay, P., et al. (2008). Endothelial function and vascular oxidative stress in long-lived GH/IGFdeficient Ames dwarf mice. Am J Physiol Heart Circ Physiol 295, H1882–H1894. Cui, B., Huang, L., Fang, Y., Guo, R., Yin, Y., & Zhao, X. (2011). Transplantation of endothelial progenitor cells overexpressing endothelial nitric oxide synthase enhances inhibition of neointimal hyperplasia and restores endothelium-dependent vasodilatation. Microvasc Res 81, 143–150. Cunnington, C., & Channon, K. M. (2010). Tetrahydrobiopterin: pleiotropic roles in cardiovascular pathophysiology. Heart 96, 1872–1877. Cushing, S. D., Berliner, J. A., Valente, A. J., Territo, M. C., Navab, M., Parhami, F., et al. (1990). Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells. Proc Natl Acad Sci U S A 87, 5134–5138. Dai, D. F., Chen, T., Szeto, H., Nieves-Cintron, M., Kutyavin, V., Santana, L. F., et al. (2011). Mitochondrial targeted antioxidant Peptide ameliorates hypertensive cardiomyopathy. J Am Coll Cardiol 58, 73–82. De Caterina, R., Ghiadoni, L., Taddei, S., Virdis, A., Almerigogna, F., Basta, G., et al. (2001). Soluble E-selectin in essential hypertension: a correlate of vascular structural changes. Am J Hypertens 14, 259–266. de Jongh, R. T., Serne, E. H., IJzerman, R. G., de Vries, G., & Stehouwer, C. D. (2004). Impaired microvascular function in obesity: implications for obesity-associated microangiopathy, hypertension, and insulin resistance. Circulation 109, 2529–2535. Deanfield, J., Donald, A., Ferri, C., Giannattasio, C., Halcox, J., Halligan, S., et al. (2005). Endothelial function and dysfunction. Part I: methodological issues for assessment in the different vascular beds: a statement by the Working Group on Endothelin and Endothelial Factors of the European Society of Hypertension. J Hypertens 23, 7–17. Deanfield, J. E., Halcox, J. P., & Rabelink, T. J. (2007). Endothelial function and dysfunction: testing and clinical relevance. Circulation 115, 1285–1295. Degraeve, F., Bolla, M., Blaie, S., Creminon, C., Quere, I., Boquet, P., et al. (2001). Modulation of COX-2 expression by statins in human aortic smooth muscle cells. Involvement of geranylgeranylated proteins. J Biol Chem 276, 46849–46855. Dengel, D. R., Kelly, A. S., Steinberger, J., & Sinaiko, A.R. (2007). Effect of oral glucose loading on endothelial function in normal-weight and overweight children. Clin Sci (Lond) 112, 493–498. DeSouza, C. A., Dengel, D. R., Macko, R. F., Cox, K., & Seals, D. R. (1997). Elevated levels of circulating cell adhesion molecules in uncomplicated essential hypertension. Am J Hypertens 10, 1335–1341. Doshi, S. N., McDowell, I. F., Moat, S. J., Payne, N., Durrant, H. J., Lewis, M. J., et al. (2002). Folic acid improves endothelial function in coronary artery disease via mechanisms largely independent of homocysteine lowering. Circulation 105, 22–26. Drexler, H., Zeiher, A.M., Meinzer, K., & Just, H. (1991). Correction of endothelial dysfunction in coronary microcirculation of hypercholesterolaemic patients by L-arginine. Lancet 338, 1546–1550. Duan, S. Z., Usher, M. G., & Mortensen, R. M. (2008). Peroxisome proliferator-activated receptor-gamma-mediated effects in the vasculature. Circ Res 102, 283–294. Dupuis, J., Tardif, J. C., Rouleau, J. L., Ricci, J., Arnold, M., Lonn, E., et al. (2005). Intensity of lipid lowering with statins and brachial artery vascular endothelium reactivity after acute coronary syndromes (from the BRAVER trial). Am J Cardiol 96, 1207–1213. Epstein, S. E., Kornowski, R., Fuchs, S., & Dvorak, H. F. (2001). Angiogenesis therapy: amidst the hype, the neglected potential for serious side effects. Circulation 104, 115–119.

Please cite this article as: Tousoulis, D., et al., Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches, Pharmacology & Therapeutics (2013), http://dx.doi.org/10.1016/j.pharmthera.2014.06.003

12

D. Tousoulis et al. / Pharmacology & Therapeutics xxx (2013) xxx–xxx

Erdos, E. G., Tan, F., & Skidgel, R. A. (2010). Angiotensin I-converting enzyme inhibitors are allosteric enhancers of kinin B1 and B2 receptor function. Hypertension 55, 214–220. Esposito, K., Ciotola, M., Giugliano, F., Schisano, B., Improta, L., Improta, M. R., et al. (2007). Endothelial microparticles correlate with erectile dysfunction in diabetic men. Int J Impot Res 19, 161–166. Esposito, K., Ciotola, M., Schisano, B., Gualdiero, R., Sardelli, L., Misso, L., et al. (2006). Endothelial microparticles correlate with endothelial dysfunction in obese women. J Clin Endocrinol Metab 91, 3676–3679. Evangelista, S., Garbin, U., Pasini, A. F., Stranieri, C., Boccioletti, V., & Cominacini, L. (2007). Effect of DL-nebivolol, its enantiomers and metabolites on the intracellular production of superoxide and nitric oxide in human endothelial cells. Pharmacol Res 55, 303–309. Faggiotto, A., & Paoletti, R. (1999). State-of-the-art lecture. Statins and blockers of the renin\angiotensin system: vascular protection beyond their primary mode of action. Hypertension 34, 987–996. Fathi, R., Haluska, B., Isbel, N., Short, L., & Marwick, T. H. (2004). The relative importance of vascular structure and function in predicting cardiovascular events. J Am Coll Cardiol 43, 616–623. Faulx, M.D., Wright, A. T., & Hoit, B.D. (2003). Detection of endothelial dysfunction with brachial artery ultrasound scanning. Am Heart J 145, 943–951. Feng, M., He, Z. M., Zhu, Y. X., Liu, L. H., Lu, C. W., & Xiong, Y. (2010). Improvement of endothelial dysfunction in atherosclerotic rabbit aortas by ex vivo gene transferring of dimethylarginine dimethylaminohydrolase-2. Int J Cardiol 144, 180–186. Fennell, J. P., Brosnan, M. J., Frater, A. J., Hamilton, C. A., Alexander, M. Y., Nicklin, S. A., et al. (2002). Adenovirus-mediated overexpression of extracellular superoxide dismutase improves endothelial dysfunction in a rat model of hypertension. Gene Ther 9, 110–117. Feuerstein, G. Z., & Ruffolo, R. R., Jr. (1995). Carvedilol, a novel multiple action antihypertensive agent with antioxidant activity and the potential for myocardial and vascular protection. Eur Heart J 16(Suppl. F), 38–42. Fichtlscherer, S., Breuer, S., & Zeiher, A.M. (2004). Prognostic value of systemic endothelial dysfunction in patients with acute coronary syndromes: further evidence for the existence of the “vulnerable” patient. Circulation 110, 1926–1932. Fish, J. E., Matouk, C. C., Rachlis, A., Lin, S., Tai, S.C., D'Abreo, C., et al. (2005). The expression of endothelial nitric-oxide synthase is controlled by a cell-specific histone code. J Biol Chem 280, 24824–24838. Flammer, A. J., Sudano, I., Hermann, F., Gay, S., Forster, A., Neidhart, M., et al. (2008). Angiotensin-converting enzyme inhibition improves vascular function in rheumatoid arthritis. Circulation 117, 2262–2269. Fratta Pasini, A., Garbin, U., Nava, M. C., Stranieri, C., Davoli, A., Sawamura, T., et al. (2005). Nebivolol decreases oxidative stress in essential hypertensive patients and increases nitric oxide by reducing its oxidative inactivation. J Hypertens 23, 589–596. Frei, B., Stocker, R., & Ames, B. N. (1988). Antioxidant defenses and lipid peroxidation in human blood plasma. Proc Natl Acad Sci U S A 85, 9748–9752. Frick, M., Suessenbacher, A., Alber, H. F., Dichtl, W., Ulmer, H., Pachinger, O., et al. (2005). Prognostic value of brachial artery endothelial function and wall thickness. J Am Coll Cardiol 46, 1006–1010. Fu, Y. F., Xiong, Y., & Guo, Z. (2005). A reduction of endogenous asymmetric dimethylarginine contributes to the effect of captopril on endothelial dysfunction induced by homocysteine in rats. Eur J Pharmacol 508, 167–175. Fukuda, Y., Teragawa, H., Matsuda, K., Yamagata, T., Matsuura, H., & Chayama, K. (2002). Tetrahydrobiopterin restores endothelial function of coronary arteries in patients with hypercholesterolaemia. Heart 87, 264–269. Gates, P. E., Boucher, M. L., Silver, A. E., Monahan, K. D., & Seals, D. R. (2007). Impaired flow-mediated dilation with age is not explained by L-arginine bioavailability or endothelial asymmetric dimethylarginine protein expression. J Appl Physiol 102(1985), 63–71. Gemici, B., Tan, R., Ongut, G., & Izgut-Uysal, V. N. (2010). Expressions of inducible nitric oxide synthase and cyclooxygenase-2 in gastric ischemia\reperfusion: role of angiotensin II. J Surg Res 161, 126–133. Ghiadoni, L., Donald, A. E., Cropley, M., Mullen, M. J., Oakley, G., Taylor, M., et al. (2000). Mental stress induces transient endothelial dysfunction in humans. Circulation 102, 2473–2478. Ghiadoni, L., Versari, D., Giannarelli, C., Faita, F., & Taddei, S. (2008). Non-invasive diagnostic tools for investigating endothelial dysfunction. Curr Pharm Des 14, 3715–3722. Goldstein, J. L., & Brown, M. S. (1990). Regulation of the mevalonate pathway. Nature 343, 425–430. Goon, P. K., Boos, C. J., & Lip, G. Y. (2005). Circulating endothelial cells: markers of vascular dysfunction. Clin Lab 51, 531–538. Graham, D., Huynh, N. N., Hamilton, C. A., Beattie, E., Smith, R. A., Cocheme, H. M., et al. (2009). Mitochondria-targeted antioxidant MitoQ10 improves endothelial function and attenuates cardiac hypertrophy. Hypertension 54, 322–328. Griendling, K. K., Sorescu, D., & Ushio-Fukai, M. (2000). NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res 86, 494–501. Gush, R. J., & King, T. A. (1991). Discrimination of capillary and arterio-venular blood flow in skin by laser Doppler flowmetry. Med Biol Eng Comput 29, 387–392. Guzik, T. J., Mussa, S., Gastaldi, D., Sadowski, J., Ratnatunga, C., Pillai, R., et al. (2002). Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase. Circulation 105, 1656–1662. Guzik, T. J., West, N. E., Black, E., McDonald, D., Ratnatunga, C., Pillai, R., et al. (2000). Vascular superoxide production by NAD(P)H oxidase: association with endothelial dysfunction and clinical risk factors. Circ Res 86, E85–E90. Haendeler, J., Popp, R., Goy, C., Tischler, V., Zeiher, A.M., & Dimmeler, S. (2005). Cathepsin D and H2O2 stimulate degradation of thioredoxin-1: implication for endothelial cell apoptosis. J Biol Chem 280, 42945–42951.

Halcox, J. P., Schenke, W. H., Zalos, G., Mincemoyer, R., Prasad, A., Waclawiw, M.A., et al. (2002). Prognostic value of coronary vascular endothelial dysfunction. Circulation 106, 653–658. Hayashi, T., Sumi, D., Juliet, P. A., Matsui-Hirai, H., Asai-Tanaka, Y., Kano, H., et al. (2004). Gene transfer of endothelial NO synthase, but not eNOS plus inducible NOS, regressed atherosclerosis in rabbits. Cardiovasc Res 61, 339–351. Hayward, C. S., Kraidly, M., Webb, C. M., & Collins, P. (2002). Assessment of endothelial function using peripheral waveform analysis: a clinical application. J Am Coll Cardiol 40, 521–528. Heffernan, K. S., Fahs, C. A., Ranadive, S. M., & Patvardhan, E. A. (2010). L-Arginine as a nutritional prophylaxis against vascular endothelial dysfunction with aging. J Cardiovasc Pharmacol Ther 15, 17–23. Heitzer, T., Brockhoff, C., Mayer, B., Warnholtz, A., Mollnau, H., Henne, S., et al. (2000). Tetrahydrobiopterin improves endothelium-dependent vasodilation in chronic smokers: evidence for a dysfunctional nitric oxide synthase. Circ Res 86, E36–E41. Heitzer, T., Schlinzig, T., Krohn, K., Meinertz, T., & Munzel, T. (2001). Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Circulation 104, 2673–2678. Heller, R., Unbehaun, A., Schellenberg, B., Mayer, B., Werner-Felmayer, G., & Werner, E. R. (2001). L-Ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of tetrahydrobiopterin. J Biol Chem 276, 40–47. Higashi, Y., Matsuoka, H., Umei, H., Sugano, R., Fujii, Y., Soga, J., et al. (2010). Endothelial function in subjects with isolated low HDL cholesterol: role of nitric oxide and circulating progenitor cells. Am J Physiol Endocrinol Metab 298, E202–E209. Higashi, Y., Sasaki, S., Nakagawa, K., Fukuda, Y., Matsuura, H., Oshima, T., et al. (2002). Tetrahydrobiopterin enhances forearm vascular response to acetylcholine in both normotensive and hypertensive individuals. Am J Hypertens 15, 326–332. Hill, J. M., Zalos, G., Halcox, J. P., Schenke, W. H., Waclawiw, M.A., Quyyumi, A. A., et al. (2003). Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 348, 593–600. Hokanson, D. E., Sumner, D. S., & Strandness, D. E., Jr. (1975). An electrically calibrated plethysmograph for direct measurement of limb blood flow. IEEE Trans Biomed Eng 22, 25–29. Hollenberg, S. M., Klein, L. W., Parrillo, J. E., Scherer, M., Burns, D., Tamburro, P., et al. (2001). Coronary endothelial dysfunction after heart transplantation predicts allograft vasculopathy and cardiac death. Circulation 104, 3091–3096. Holmes, D. I., & Zachary, I. (2005). The vascular endothelial growth factor (VEGF) family: angiogenic factors in health and disease. Genome Biol 6, 209. Hong, S. J., Choi, S.C., Kim, J. S., Shim, W. J., Park, S. M., Ahn, C. M., et al. (2010). Low-dose versus moderate-dose atorvastatin after acute myocardial infarction: 8-month effects on coronary flow reserve and angiogenic cell mobilisation. Heart 96, 756–764. Hornig, B., Landmesser, U., Kohler, C., Ahlersmann, D., Spiekermann, S., Christoph, A., et al. (2001). Comparative effect of ace inhibition and angiotensin II type 1 receptor antagonism on bioavailability of nitric oxide in patients with coronary artery disease: role of superoxide dismutase. Circulation 103, 799–805. Hugel, B., Martinez, M. C., Kunzelmann, C., & Freyssinet, J. M. (2005). Membrane microparticles: two sides of the coin. Physiology (Bethesda) 20, 22–27. Hwang, J., Kleinhenz, D. J., Lassegue, B., Griendling, K. K., Dikalov, S., & Hart, C. M. (2005). Peroxisome proliferator-activated receptor-gamma ligands regulate endothelial membrane superoxide production. Am J Physiol Cell Physiol 288, C899–C905. Hyndman, M. E., Verma, S., Rosenfeld, R. J., Anderson, T. J., & Parsons, H. G. (2002). Interaction of 5-methyltetrahydrofolate and tetrahydrobiopterin on endothelial function. Am J Physiol Heart Circ Physiol 282, H2167–H2172. Iida, S., Chu, Y., Francis, J., Weiss, R. M., Gunnett, C. A., Faraci, F. M., et al. (2005). Gene transfer of extracellular superoxide dismutase improves endothelial function in rats with heart failure. Am J Physiol Heart Circ Physiol 289, H525–H532. Imanishi, T., Tsujioka, H., Ikejima, H., Kuroi, A., Takarada, S., Kitabata, H., et al. (2008). Renin inhibitor aliskiren improves impaired nitric oxide bioavailability and protects against atherosclerotic changes. Hypertension 52, 563–572. Inanc, M. T., Kalay, N., Heyit, T., Ozdogru, I., Kaya, M. G., Dogan, A., et al. (2010). Effects of atorvastatin and lisinopril on endothelial dysfunction in patients with Behcet's disease. Echocardiography 27, 997–1003. Irani, K. (2000). Oxidant signaling in vascular cell growth, death, and survival: a review of the roles of reactive oxygen species in smooth muscle and endothelial cell mitogenic and apoptotic signaling. Circ Res 87, 179–183. Ishigaki, Y., Oka, Y., & Katagiri, H. (2009). Circulating oxidized LDL: a biomarker and a pathogenic factor. Curr Opin Lipidol 20, 363–369. Ishii, H., Uchiyama, H., & Kazama, M. (1991). Soluble thrombomodulin antigen in conditioned medium is increased by damage of endothelial cells. Thromb Haemost 65, 618–623. Ito, A., Egashira, K., Narishige, T., Muramatsu, K., & Takeshita, A. (2001). Renin–angiotensin system is involved in the mechanism of increased serum asymmetric dimethylarginine in essential hypertension. Jpn Circ J 65, 775–778. Jackson, S. M., Parhami, F., Xi, X. P., Berliner, J. A., Hsueh, W. A., Law, R. E., et al. (1999). Peroxisome proliferator-activated receptor activators target human endothelial cells to inhibit leukocyte–endothelial cell interaction. Arterioscler Thromb Vasc Biol 19, 2094–2104. Jahangir, E., Vita, J. A., Handy, D., Holbrook, M., Palmisano, J., Beal, R., et al. (2009). The effect of L-arginine and creatine on vascular function and homocysteine metabolism. Vasc Med 14, 239–248. Jialal, I., Fuller, C. J., & Huet, B.A. (1995). The effect of alpha-tocopherol supplementation on LDL oxidation. A dose–response study. Arterioscler Thromb Vasc Biol 15, 190–198. John, S., Schlaich, M., Langenfeld, M., Weihprecht, H., Schmitz, G., Weidinger, G., et al. (1998). Increased bioavailability of nitric oxide after lipid-lowering therapy in hypercholesterolemic patients: a randomized, placebo-controlled, double-blind study. Circulation 98, 211–216.

Please cite this article as: Tousoulis, D., et al., Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches, Pharmacology & Therapeutics (2013), http://dx.doi.org/10.1016/j.pharmthera.2014.06.003

D. Tousoulis et al. / Pharmacology & Therapeutics xxx (2013) xxx–xxx Kals, J., Kampus, P., Kals, M., Pulges, A., Teesalu, R., & Zilmer, M. (2006). Effects of stimulation of nitric oxide synthesis on large artery stiffness in patients with peripheral arterial disease. Atherosclerosis 185, 368–374. Kampoli, A.M., Tousoulis, D., Tentolouris, C., & Stefanadis, C. (2012). Novel agents targeting nitric oxide. Curr Vasc Pharmacol 10, 61–76. Kelly, T. K., De Carvalho, D.D., & Jones, P. A. (2010). Epigenetic modifications as therapeutic targets. Nat Biotechnol 28, 1069–1078. Kelly, R. P., Gibbs, H. H., O'Rourke, M. F., Daley, J. E., Mang, K., Morgan, J. J., et al. (1990). Nitroglycerin has more favourable effects on left ventricular afterload than apparent from measurement of pressure in a peripheral artery. Eur Heart J 11, 138–144. Khan, F., Elhadd, T. A., Greene, S. A., & Belch, J. J. (2000). Impaired skin microvascular function in children, adolescents, and young adults with type 1 diabetes. Diabetes Care 23, 215–220. Khan, F., Patterson, D., Belch, J. J., Hirata, K., & Lang, C. C. (2008). Relationship between peripheral and coronary function using laser Doppler imaging and transthoracic echocardiography. Clin Sci (Lond) 115, 295–300. Kong, D., Melo, L. G., Mangi, A. A., Zhang, L., Lopez-Ilasaca, M., Perrella, M.A., et al. (2004). Enhanced inhibition of neointimal hyperplasia by genetically engineered endothelial progenitor cells. Circulation 109, 1769–1775. Kovacs, I., Toth, J., Tarjan, J., & Koller, A. (2006). Correlation of flow mediated dilation with inflammatory markers in patients with impaired cardiac function. Beneficial effects of inhibition of ACE. Eur J Heart Fail 8, 451–459. Kruger, A., Stewart, J., Sahityani, R., O'Riordan, E., Thompson, C., Adler, S., et al. (2006). Laser Doppler flowmetry detection of endothelial dysfunction in end-stage renal disease patients: correlation with cardiovascular risk. Kidney Int 70, 157–164. Kubli, S., Feihl, F., & Waeber, B. (2001). Beta-blockade with nebivolol enhances the acetylcholine-induced cutaneous vasodilation. Clin Pharmacol Ther 69, 238–244. Kullo, I. J., Mozes, G., Schwartz, R. S., Gloviczki, P., Crotty, T. B., Barber, D. A., et al. (1997). Adventitial gene transfer of recombinant endothelial nitric oxide synthase to rabbit carotid arteries alters vascular reactivity. Circulation 96, 2254–2261. Kureishi, Y., Luo, Z., Shiojima, I., Bialik, A., Fulton, D., Lefer, D. J., et al. (2000). The HMGCoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med 6, 1004–1010. Kuvin, J. T., Patel, A.R., Sliney, K. A., Pandian, N. G., Sheffy, J., Schnall, R. P., et al. (2003). Assessment of peripheral vascular endothelial function with finger arterial pulse wave amplitude. Am Heart J 146, 168–174. Kuzkaya, N., Weissmann, N., Harrison, D.G., & Dikalov, S. (2003). Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols: implications for uncoupling endothelial nitric-oxide synthase. J Biol Chem 278, 22546–22554. Landmesser, U., Hornig, B., & Drexler, H. (2004). Endothelial function: a critical determinant in atherosclerosis? Circulation 109, II27–II33. Laufs, U., & Liao, J. K. (1998). Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem 273, 24266–24271. Lavi, R., Zhu, X. Y., Chade, A.R., Lin, J., Lerman, A., & Lerman, L. O. (2010). Simvastatin decreases endothelial progenitor cell apoptosis in the kidney of hypertensive hypercholesterolemic pigs. Arterioscler Thromb Vasc Biol 30, 976–983. Lee, R., Channon, K. M., & Antoniades, C. (2012). Therapeutic strategies targeting endothelial function in humans: clinical implications. Curr Vasc Pharmacol 10, 77–93. Lee, S., Chen, T. T., Barber, C. L., Jordan, M. C., Murdock, J., Desai, S., et al. (2007). Autocrine VEGF signaling is required for vascular homeostasis. Cell 130, 691–703. Lee, J. M., Wiesmann, F., Shirodaria, C., Leeson, P., Petersen, S. E., Francis, J. M., et al. (2008). Early changes in arterial structure and function following statin initiation: quantification by magnetic resonance imaging. Atherosclerosis 197, 951–958. Leeson, C. P., Robinson, M., Francis, J. M., Robson, M.D., Channon, K. M., Neubauer, S., et al. (2006). Cardiovascular magnetic resonance imaging for non-invasive assessment of vascular function: validation against ultrasound. J Cardiovasc Magn Reson 8, 381–387. Leeson, P., Thorne, S., Donald, A., Mullen, M., Clarkson, P., & Deanfield, J. (1997). Noninvasive measurement of endothelial function: effect on brachial artery dilatation of graded endothelial dependent and independent stimuli. Heart 78, 22–27. Leon, M. L., & Zuckerman, S. H. (2005). Gamma interferon: a central mediator in atherosclerosis. Inflamm Res 54, 395–411. Lerman, A., Burnett, J. C., Jr., Higano, S. T., McKinley, L. J., & Holmes, D. R., Jr. (1998). Longterm L-arginine supplementation improves small-vessel coronary endothelial function in humans. Circulation 97, 2123–2128. Lerman, A., & Zeiher, A.M. (2005). Endothelial function: cardiac events. Circulation 111, 363–368. Li, D., & Mehta, J. L. (2003). 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors protect against oxidized low-density lipoprotein-induced endothelial dysfunction. Endothelium 10, 17–21. Libby, P., Ridker, P.M., & Maseri, A. (2002). Inflammation and atherosclerosis. Circulation 105, 1135–1143. Lip, G. Y., & Blann, A. (1997). von Willebrand factor: a marker of endothelial dysfunction in vascular disorders? Cardiovasc Res 34, 255–265. Liu, X., Engelman, R. M., Rousou, J. A., Cordis, G. A., & Das, D. K. (1992). Attenuation of myocardial reperfusion injury by sulfhydryl-containing angiotensin converting enzyme inhibitors. Cardiovasc Drugs Ther 6, 437–443. Lu, C. W., Guo, Z., Feng, M., Wu, Z. Z., He, Z. M., & Xiong, Y. (2010). Ex vivo gene transferring of human dimethylarginine dimethylaminohydrolase-2 improved endothelial dysfunction in diabetic rat aortas and high glucose-treated endothelial cells. Atherosclerosis 209, 66–73. Lund, D.D., Gunnett, C. A., Chu, Y., Brooks, R. M., Faraci, F. M., & Heistad, D.D. (2004). Gene transfer of extracellular superoxide dismutase improves relaxation of aorta after treatment with endotoxin. Am J Physiol Heart Circ Physiol 287, H805–H811. Luscher, T. F., & Barton, M. (1997). Biology of the endothelium. Clin Cardiol 20 (II-3-10).

13

Maier, W., Cosentino, F., Lutolf, R. B., Fleisch, M., Seiler, C., Hess, O. M., et al. (2000). Tetrahydrobiopterin improves endothelial function in patients with coronary artery disease. J Cardiovasc Pharmacol 35, 173–178. Mak, I. T., Freedman, A.M., Dickens, B. F., & Weglicki, W. B. (1990). Protective effects of sulfhydryl-containing angiotensin converting enzyme inhibitors against free radical injury in endothelial cells. Biochem Pharmacol 40, 2169–2175. Mallat, Z., Benamer, H., Hugel, B., Benessiano, J., Steg, P. G., Freyssinet, J. M., et al. (2000). Elevated levels of shed membrane microparticles with procoagulant potential in the peripheral circulating blood of patients with acute coronary syndromes. Circulation 101, 841–843. Mancini, G. B., Henry, G. C., Macaya, C., O'Neill, B. J., Pucillo, A. L., Carere, R. G., et al. (1996). Angiotensin-converting enzyme inhibition with quinapril improves endothelial vasomotor dysfunction in patients with coronary artery disease. The TREND (Trial on Reversing ENdothelial Dysfunction) Study. Circulation 94, 258–265. Mason, R. P., Kalinowski, L., Jacob, R. F., Jacoby, A.M., & Malinski, T. (2005). Nebivolol reduces nitroxidative stress and restores nitric oxide bioavailability in endothelium of black Americans. Circulation 112, 3795–3801. Masuda, H., & Asahara, T. (2003). Post-natal endothelial progenitor cells for neovascularization in tissue regeneration. Cardiovasc Res 58, 390–398. Matouk, C. C., & Marsden, P. A. (2008). Epigenetic regulation of vascular endothelial gene expression. Circ Res 102, 873–887. Matsuno, H., Takei, M., Hayashi, H., Nakajima, K., Ishisaki, A., & Kozawa, O. (2004). Simvastatin enhances the regeneration of endothelial cells via VEGF secretion in injured arteries. J Cardiovasc Pharmacol 43, 333–340. McFarlane, R., McCredie, R. J., Bonney, M.A., Molyneaux, L., Zilkens, R., Celermajer, D. S., et al. (1999). Angiotensin converting enzyme inhibition and arterial endothelial function in adults with Type 1 diabetes mellitus. Diabet Med 16, 62–66. Mehta, J. L. (2003). Pleiotropic effects of statins: how important are they in the prevention of vascular disease? Endothelium 10, 3–4. Mitchell, G. F., Vita, J. A., Larson, M. G., Parise, H., Keyes, M. J., Warner, E., et al. (2005). Cross-sectional relations of peripheral microvascular function, cardiovascular disease risk factors, and aortic stiffness: the Framingham Heart Study. Circulation 112, 3722–3728. Mullen, M. J., Clarkson, P., Donald, A. E., Thomson, H., Thorne, S. A., Powe, A. J., et al. (1998). Effect of enalapril on endothelial function in young insulin-dependent diabetic patients: a randomized, double-blind study. J Am Coll Cardiol 31, 1330–1335. Mullen, M. J., Wright, D., Donald, A. E., Thorne, S., Thomson, H., & Deanfield, J. E. (2000). Atorvastatin but not L-arginine improves endothelial function in type I diabetes mellitus: a double-blind study. J Am Coll Cardiol 36, 410–416. Nagamia, S., Pandian, A., Cheema, F., Natarajan, R., Khan, Q. A., Patel, A.D., et al. (2007). The role of quinapril in the presence of a weight loss regimen: endothelial function and markers of obesity in patients with the metabolic syndrome. Prev Cardiol 10, 204–209. Nichols, W. W., Denardo, S. J., Wilkinson, I. B., McEniery, C. M., Cockcroft, J., & O'Rourke, M. F. (2008). Effects of arterial stiffness, pulse wave velocity, and wave reflections on the central aortic pressure waveform. J Clin Hypertens (Greenwich) 10, 295–303. Nickenig, G., & Harrison, D.G. (2002). The AT(1)-type angiotensin receptor in oxidative stress and atherogenesis: part I: oxidative stress and atherogenesis. Circulation 105, 393–396. Nishioka, K., Nakagawa, K., Umemura, T., Jitsuiki, D., Ueda, K., Goto, C., et al. (2007). Carvedilol improves endothelium-dependent vasodilation in patients with dilated cardiomyopathy. Heart 93, 247–248. Nohria, A., Gerhard-Herman, M., Creager, M.A., Hurley, S., Mitra, D., & Ganz, P. (2006). Role of nitric oxide in the regulation of digital pulse volume amplitude in humans. J Appl Physiol 101(1985), 545–548. Nussberger, J., Aubert, J. F., Bouzourene, K., Pellegrin, M., Hayoz, D., & Mazzolai, L. (2008). Renin inhibition by aliskiren prevents atherosclerosis progression: comparison with irbesartan, atenolol, and amlodipine. Hypertension 51, 1306–1311. Nystrom, T., Nygren, A., & Sjoholm, A. (2004). Tetrahydrobiopterin increases insulin sensitivity in patients with type 2 diabetes and coronary heart disease. Am J Physiol Endocrinol Metab 287, E919–E925. O'Driscoll, G., Green, D., Maiorana, A., Stanton, K., Colreavy, F., & Taylor, R. (1999). Improvement in endothelial function by angiotensin-converting enzyme inhibition in non-insulin-dependent diabetes mellitus. J Am Coll Cardiol 33, 1506–1511. O'Driscoll, G., Green, D., & Taylor, R. R. (1997). Simvastatin, an HMG-coenzyme A reductase inhibitor, improves endothelial function within 1 month. Circulation 95, 1126–1131. Oelze, M., Daiber, A., Brandes, R. P., Hortmann, M., Wenzel, P., Hink, U., et al. (2006). Nebivolol inhibits superoxide formation by NADPH oxidase and endothelial dysfunction in angiotensin II-treated rats. Hypertension 48, 677–684. Padayatty, S. J., Katz, A., Wang, Y., Eck, P., Kwon, O., Lee, J. H., et al. (2003). Vitamin C as an antioxidant: evaluation of its role in disease prevention. J Am Coll Nutr 22, 18–35. Panza, J. A., Quyyumi, A. A., Brush, J. E., Jr., & Epstein, S. E. (1990). Abnormal endotheliumdependent vascular relaxation in patients with essential hypertension. N Engl J Med 323, 22–27. Papamichael, C. M., Aznaouridis, K. A., Karatzis, E. N., Karatzi, K. N., Stamatelopoulos, K. S., Vamvakou, G., et al. (2005). Effect of coffee on endothelial function in healthy subjects: the role of caffeine. Clin Sci (Lond) 109, 55–60. Parthasarathy, S., Santanam, N., & Auge, N. (1998). Oxidized low-density lipoprotein, a two-faced Janus in coronary artery disease? Biochem Pharmacol 56, 279–284. Pasceri, V., Wu, H. D., Willerson, J. T., & Yeh, E. T. (2000). Modulation of vascular inflammation in vitro and in vivo by peroxisome proliferator-activated receptor-gamma activators. Circulation 101, 235–238. Pasini, A. F., Garbin, U., Stranieri, C., Boccioletti, V., Mozzini, C., Manfro, S., et al. (2008). Nebivolol treatment reduces serum levels of asymmetric dimethylarginine and improves endothelial dysfunction in essential hypertensive patients. Am J Hypertens 21, 1251–1257.

Please cite this article as: Tousoulis, D., et al., Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches, Pharmacology & Therapeutics (2013), http://dx.doi.org/10.1016/j.pharmthera.2014.06.003

14

D. Tousoulis et al. / Pharmacology & Therapeutics xxx (2013) xxx–xxx

Perticone, F., Ceravolo, R., Maio, R., Cloro, C., Candigliota, M., Scozzafava, A., et al. (2000). Effects of atorvastatin and vitamin C on endothelial function of hypercholesterolemic patients. Atherosclerosis 152, 511–518. Perticone, F., Ceravolo, R., Pujia, A., Ventura, G., Iacopino, S., Scozzafava, A., et al. (2001). Prognostic significance of endothelial dysfunction in hypertensive patients. Circulation 104, 191–196. Pirro, M., Schillaci, G., Romagno, P. F., Mannarino, M. R., Bagaglia, F., Razzi, R., et al. (2009). Influence of short-term rosuvastatin therapy on endothelial progenitor cells and endothelial function. J Cardiovasc Pharmacol Ther 14, 14–21. Qin, Z., Reszka, K. J., Fukai, T., & Weintraub, N. L. (2008). Extracellular superoxide dismutase (ecSOD) in vascular biology: an update on exogenous gene transfer and endogenous regulators of ecSOD. Transl Res 151, 68–78. Rajagopalan, S., Shah, M., Luciano, A., Crystal, R., & Nabel, E. G. (2001). Adenovirusmediated gene transfer of VEGF(121) improves lower-extremity endothelial function and flow reserve. Circulation 104, 753–755. Rajavashisth, T. B., Andalibi, A., Territo, M. C., Berliner, J. A., Navab, M., Fogelman, A.M., et al. (1990). Induction of endothelial cell expression of granulocyte and macrophage colony-stimulating factors by modified low-density lipoproteins. Nature 344, 254–257. Reaven, P. D., Khouw, A., Beltz, W. F., Parthasarathy, S., & Witztum, J. L. (1993). Effect of dietary antioxidant combinations in humans. Protection of LDL by vitamin E but not by beta-carotene. Arterioscler Thromb 13, 590–600. Recchioni, R., Marcheselli, F., Moroni, F., & Pieri, C. (2002). Apoptosis in human aortic endothelial cells induced by hyperglycemic condition involves mitochondrial depolarization and is prevented by N-acetyl-L-cysteine. Metabolism 51, 1384–1388. Rossi, M., Carpi, A., Di Maria, C., Franzoni, F., Galetta, F., & Santoro, G. (2009). Skin blood flowmotion and microvascular reactivity investigation in hypercholesterolemic patients without clinically manifest arterial diseases. Physiol Res 58, 39–47. Schachinger, V., Britten, M. B., & Zeiher, A.M. (2000). Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease. Circulation 101, 1899–1906. Schindler, T. H., Hornig, B., Buser, P. T., Olschewski, M., Magosaki, N., Pfisterer, M., et al. (2003). Prognostic value of abnormal vasoreactivity of epicardial coronary arteries to sympathetic stimulation in patients with normal coronary angiograms. Arterioscler Thromb Vasc Biol 23, 495–501. Settergren, M., Pernow, J., Brismar, K., Jorneskog, G., & Kalani, M. (2008). Endothelin-A receptor blockade increases nutritive skin capillary circulation in patients with type 2 diabetes and microangiopathy. J Vasc Res 45, 295–302. Sharif, F., Hynes, S. O., Cooney, R., Howard, L., McMahon, J., Daly, K., et al. (2008). Geneeluting stents: adenovirus-mediated delivery of eNOS to the blood vessel wall accelerates re-endothelialization and inhibits restenosis. Mol Ther 16, 1674–1680. Shimbo, D., Grahame-Clarke, C., Miyake, Y., Rodriguez, C., Sciacca, R., Di Tullio, M., et al. (2007). The association between endothelial dysfunction and cardiovascular outcomes in a population-based multi-ethnic cohort. Atherosclerosis 192, 197–203. Siasos, G., Tousoulis, D., Michalea, S., Oikonomou, E., Kolia, C., Kioufis, S., et al. (2012). Biomarkers determining cardiovascular risk in patients with kidney disease. Curr Med Chem 19, 2555–2571. Siekmeier, R., Steffen, C., & Marz, W. (2007). Role of oxidants and antioxidants in atherosclerosis: results of in vitro and in vivo investigations. J Cardiovasc Pharmacol Ther 12, 265–282. Smith, R. S., Jr., Agata, J., Xia, C. F., Chao, L., & Chao, J. (2005). Human endothelial nitric oxide synthase gene delivery protects against cardiac remodeling and reduces oxidative stress after myocardial infarction. Life Sci 76, 2457–2471. Smith, R. S., Jr., Lin, K. F., Agata, J., Chao, L., & Chao, J. (2002). Human endothelial nitric oxide synthase gene delivery promotes angiogenesis in a rat model of hindlimb ischemia. Arterioscler Thromb Vasc Biol 22, 1279–1285. Staessen, J. A., Li, Y., & Richart, T. (2006). Oral renin inhibitors. Lancet 368, 1449–1456. Steinberg, H. O., Chaker, H., Leaming, R., Johnson, A., Brechtel, G., & Baron, A.D. (1996). Obesity/insulin resistance is associated with endothelial dysfunction. Implications for the syndrome of insulin resistance. J Clin Invest 97, 2601–2610. Stroes, E. S., van Faassen, E. E., Yo, M., Martasek, P., Boer, P., Govers, R., et al. (2000). Folic acid reverts dysfunction of endothelial nitric oxide synthase. Circ Res 86, 1129–1134. Sun, D., Huang, A., Smith, C. J., Stackpole, C. J., Connetta, J. A., Shesely, E. G., et al. (1999). Enhanced release of prostaglandins contributes to flow-induced arteriolar dilation in eNOS knockout mice. Circ Res 85, 288–293. Sun, W., Lee, T. S., Zhu, M., Gu, C., Wang, Y., Zhu, Y., et al. (2006). Statins activate AMPactivated protein kinase in vitro and in vivo. Circulation 114, 2655–2662. Suwaidi, J. A., Hamasaki, S., Higano, S. T., Nishimura, R. A., Holmes, D. R., Jr., & Lerman, A. (2000). Long-term follow-up of patients with mild coronary artery disease and endothelial dysfunction. Circulation 101, 948–954. Takeshita, S., Isshiki, T., Ochiai, M., Eto, K., Mori, H., Tanaka, E., et al. (1998). Endotheliumdependent relaxation of collateral microvessels after intramuscular gene transfer of vascular endothelial growth factor in a rat model of hindlimb ischemia. Circulation 98, 1261–1263. Tamai, O., Matsuoka, H., Itabe, H., Wada, Y., Kohno, K., & Imaizumi, T. (1997). Single LDL apheresis improves endothelium-dependent vasodilatation in hypercholesterolemic humans. Circulation 95, 76–82. Tamaki, N., Takahashi, N., Kawamoto, M., Torizuka, T., Tadamura, E., Yonekura, Y., et al. (1994). Myocardial tomography using technetium-99m-tetrofosmin to evaluate coronary artery disease. J Nucl Med 35, 594–600. Tan, K. C., Chow, W. S., Tam, S.C., Ai, V. H., Lam, C. H., & Lam, K. S. (2002). Atorvastatin lowers C-reactive protein and improves endothelium-dependent vasodilation in type 2 diabetes mellitus. J Clin Endocrinol Metab 87, 563–568. Taneva, E., Borucki, K., Wiens, L., Makarova, R., Schmidt-Lucke, C., Luley, C., et al. (2006). Early effects on endothelial function of atorvastatin 40 mg twice daily and its withdrawal. Am J Cardiol 97, 1002–1006.

Targonski, P. V., Bonetti, P. O., Pumper, G. M., Higano, S. T., Holmes, D. R., Jr., & Lerman, A. (2003). Coronary endothelial dysfunction is associated with an increased risk of cerebrovascular events. Circulation 107, 2805–2809. ter Avest, E., Abbink, E. J., Holewijn, S., de Graaf, J., Tack, C. J., & Stalenhoef, A. F. (2005). Effects of rosuvastatin on endothelial function in patients with familial combined hyperlipidaemia (FCH). Curr Med Res Opin 21, 1469–1476. Thum, T., Fleissner, F., Klink, I., Tsikas, D., Jakob, M., Bauersachs, J., et al. (2007). Growth hormone treatment improves markers of systemic nitric oxide bioavailability via insulin-like growth factor-I. J Clin Endocrinol Metab 92, 4172–4179. Torondel, B., Nandi, M., Kelly, P., Wojciak-Stothard, B., Fleming, I., & Leiper, J. (2010). Adenoviral-mediated overexpression of DDAH improves vascular tone regulation. Vasc Med 15, 205–213. Tousoulis, D., Antoniades, C., Bosinakou, E., Kotsopoulou, M., Pitsavos, C., Vlachopoulos, C., et al. (2005a). Effects of atorvastatin on reactive hyperemia and inflammatory process in patients with congestive heart failure. Atherosclerosis 178, 359–363. Tousoulis, D., Antoniades, C., Bosinakou, E., Kotsopoulou, M., Tsioufis, C., Tentolouris, C., et al. (2005b). Effects of atorvastatin on reactive hyperaemia and the thrombosis– fibrinolysis system in patients with heart failure. Heart 91, 27–31. Tousoulis, D., Antoniades, C., Katsi, V., Bosinakou, E., Kotsopoulou, M., Tsioufis, C., et al. (2006). The impact of early administration of low-dose atorvastatin treatment on inflammatory process, in patients with unstable angina and low cholesterol level. Int J Cardiol 109, 48–52. Tousoulis, D., Antoniades, C., Koumallos, N., Marinou, K., Stefanadi, E., Latsios, G., et al. (2006). Novel therapies targeting vascular endothelium. Endothelium 13, 411–421. Tousoulis, D., Antoniades, C., & Stefanadis, C. (2005). Evaluating endothelial function in humans: a guide to invasive and non-invasive techniques. Heart 91, 553–558. Tousoulis, D., Antoniades, C., & Stefanadis, C. (2006). Statins and antioxidant vitamins: should co-administration be avoided? J Am Coll Cardiol 47, 1237 (author reply 1237–1238). Tousoulis, D., Antoniades, C., Tentolouris, C., Tsioufis, C., Toutouza, M., Toutouzas, P., et al. (2003). Effects of combined administration of vitamins C and E on reactive hyperemia and inflammatory process in chronic smokers. Atherosclerosis 170, 261–267. Tousoulis, D., Antoniades, C., Tountas, C., Bosinakou, E., Kotsopoulou, M., Toutouzas, P., et al. (2003). Vitamin C affects thrombosis/fibrinolysis system and reactive hyperemia in patients with type 2 diabetes and coronary artery disease. Diabetes Care 26, 2749–2753. Tousoulis, D., Antoniades, C., Vassiliadou, C., Toutouza, M., Pitsavos, C., Tentolouris, C., et al. (2005). Effects of combined administration of low dose atorvastatin and vitamin E on inflammatory markers and endothelial function in patients with heart failure. Eur J Heart Fail 7, 1126–1132. Tousoulis, D., Charakida, M., & Stefanadis, C. (2006). Endothelial function and inflammation in coronary artery disease. Heart 92, 441–444. Tousoulis, D., Crake, T., Kaski, J. C., Rosen, S. D., Haider, A. W., & Davies, G. J. (1995). Enhanced vasomotor responses of complex coronary stenoses to acetylcholine in stable angina pectoris. Am J Cardiol 75, 725–728. Tousoulis, D., & Davies, G. (1998). Acetylcholine and endothelial function. Circulation 98, 1588–1589. Tousoulis, D., Davies, G., Lefroy, D. C., Haider, A. W., & Crake, T. (1996). Variable coronary vasomotor responses to acetylcholine in patients with normal coronary arteriograms: evidence for localised endothelial dysfunction. Heart 75, 261–266. Tousoulis, D., Kampoli, A.M., Papageorgiou, N., Androulakis, E., Antoniades, C., Toutouzas, K., et al. (2011). Pathophysiology of atherosclerosis: the role of inflammation. Curr Pharm Des 17, 4089–4110. Tousoulis, D., Kampoli, A.M., Tentolouris, C., Papageorgiou, N., & Stefanadis, C. (2012). The role of nitric oxide on endothelial function. Curr Vasc Pharmacol 10, 4–18. Tousoulis, D., Koutsogiannis, M., Papageorgiou, N., Siasos, G., Antoniades, C., Tsiamis, E., et al. (2010). Endothelial dysfunction: potential clinical implications. Minerva Med 101, 271–284. Tousoulis, D., Papageorgiou, N., Androulakis, E., Paroutoglou, K., & Stefanadis, C. (2010). Novel therapeutic strategies targeting vascular endothelium in essential hypertension. Expert Opin Investig Drugs 19, 1395–1412. Tousoulis, D., Tentolouris, C., Crake, T., Toutouzas, P., & Davies, G. (1997). Basal and flowmediated nitric oxide production by atheromatous coronary arteries. J Am Coll Cardiol 29, 1256–1262. Trevelyan, J., Needham, E. W., Morris, A., & Mattu, R. K. (2005). Comparison of the effect of enalapril and losartan in conjunction with surgical coronary revascularisation versus revascularisation alone on systemic endothelial function. Heart 91, 1053–1057. Vallance, P., & Leiper, J. (2004). Cardiovascular biology of the asymmetric dimethylarginine:dimethylarginine dimethylaminohydrolase pathway. Arterioscler Thromb Vasc Biol 24, 1023–1030. Vassalli, G., Gallino, A., Kiowski, W., Jiang, Z., Turina, M., & Hess, O. M. (1997). Reduced coronary flow reserve during exercise in cardiac transplant recipients. Circulation 95, 607–613. Verhaar, M. C., Wever, R. M., Kastelein, J. J., van Loon, D., Milstien, S., Koomans, H. A., et al. (1999). Effects of oral folic acid supplementation on endothelial function in familial hypercholesterolemia. A randomized placebo-controlled trial. Circulation 100, 335–338. Versari, D., Lerman, L. O., & Lerman, A. (2007). The importance of reendothelialization after arterial injury. Curr Pharm Des 13, 1811–1824. Virdis, A., Ghiadoni, L., & Taddei, S. (2011). Effects of antihypertensive treatment on endothelial function. Curr Hypertens Rep 13, 276–281. Vita, J. A. (2002). Nitric oxide-dependent vasodilation in human subjects. Methods Enzymol 359, 186–200. Vlot, A. J., Koppelman, S. J., Bouma, B. N., & Sixma, J. J. (1998). Factor VIII and von Willebrand factor. Thromb Haemost 79, 456–465.

Please cite this article as: Tousoulis, D., et al., Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches, Pharmacology & Therapeutics (2013), http://dx.doi.org/10.1016/j.pharmthera.2014.06.003

D. Tousoulis et al. / Pharmacology & Therapeutics xxx (2013) xxx–xxx Vora, D. K., Fang, Z. T., Liva, S. M., Tyner, T. R., Parhami, F., Watson, A.D., et al. (1997). Induction of P-selectin by oxidized lipoproteins. Separate effects on synthesis and surface expression. Circ Res 80, 810–818. Wagner, A. H., Kohler, T., Ruckschloss, U., Just, I., & Hecker, M. (2000). Improvement of nitric oxide-dependent vasodilatation by HMG-CoA reductase inhibitors through attenuation of endothelial superoxide anion formation. Arterioscler Thromb Vasc Biol 20, 61–69. Wald, D. S., Law, M., & Morris, J. K. (2002). Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ 325, 1202. Warnholtz, A., Nickenig, G., Schulz, E., Macharzina, R., Brasen, J. H., Skatchkov, M., et al. (1999). Increased NADH-oxidase-mediated superoxide production in the early stages of atherosclerosis: evidence for involvement of the renin–angiotensin system. Circulation 99, 2027–2033. Wassmann, S., Laufs, U., Baumer, A. T., Muller, K., Konkol, C., Sauer, H., et al. (2001). Inhibition of geranylgeranylation reduces angiotensin II-mediated free radical production in vascular smooth muscle cells: involvement of angiotensin AT1 receptor expression and Rac1 GTPase. Mol Pharmacol 59, 646–654. Wassmann, S., & Nickenig, G. (2003). Interrelationship of free oxygen radicals and endothelial dysfunction–modulation by statins. Endothelium 10, 23–33. Watanabe, Y., Sunayama, S., Shimada, K., Sawano, M., Hoshi, S., Iwama, Y., et al. (2000). Troglitazone improves endothelial dysfunction in patients with insulin resistance. J Atheroscler Thromb 7, 159–163. Werner, N., Kosiol, S., Schiegl, T., Ahlers, P., Walenta, K., Link, A., et al. (2005). Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med 353, 999–1007. Wheeler-Jones, C., Abu-Ghazaleh, R., Cospedal, R., Houliston, R. A., Martin, J., & Zachary, I. (1997). Vascular endothelial growth factor stimulates prostacyclin production and activation of cytosolic phospholipase A2 in endothelial cells via p42/p44 mitogenactivated protein kinase. FEBS Lett 420, 28–32. Widlansky, M. E., Gokce, N., Keaney, J. F., Jr., & Vita, J. A. (2003). The clinical implications of endothelial dysfunction. J Am Coll Cardiol 42, 1149–1160. Wiesmann, F., Petersen, S. E., Leeson, P.M., Francis, J. M., Robson, M.D., Wang, Q., et al. (2004). Global impairment of brachial, carotid, and aortic vascular function in young smokers: direct quantification by high-resolution magnetic resonance imaging. J Am Coll Cardiol 44, 2056–2064. Wilkinson, I. B., Hall, I. R., MacCallum, H., Mackenzie, I. S., McEniery, C. M., van der Arend, B. J., et al. (2002). Pulse-wave analysis: clinical evaluation of a noninvasive, widely applicable method for assessing endothelial function. Arterioscler Thromb Vasc Biol 22, 147–152.

15

Wilkinson, I. B., MacCallum, H., Cockcroft, J. R., & Webb, D. J. (2002). Inhibition of basal nitric oxide synthesis increases aortic augmentation index and pulse wave velocity in vivo. Br J Clin Pharmacol 53, 189–192. Wilkinson, I. B., & McEniery, C. M. (2004). Arterial stiffness, endothelial function and novel pharmacological approaches. Clin Exp Pharmacol Physiol 31, 795–799. Wolfrum, S., Jensen, K. S., & Liao, J. K. (2003). Endothelium-dependent effects of statins. Arterioscler Thromb Vasc Biol 23, 729–736. Woo, K. S., Chook, P., Yu, C. W., Sung, R. Y., Qiao, M., Leung, S. S., et al. (2004). Effects of diet and exercise on obesity-related vascular dysfunction in children. Circulation 109, 1981–1986. Woodcock, J. P. (1985). Noninvasive blood flow measurement. Crit Rev Biomed Eng 12, 201–235. Yan, M. S., Matouk, C. C., & Marsden, P. A. (2010). Epigenetics of the vascular endothelium. J Appl Physiol 109(1985), 916–926. Yokoyama, I., Negita, M., Liu, D.G., Nagasaka, T., Kobayashi, T., Hayakawa, A., et al. (2002). Prevention of free-radical induced apoptosis by induction of human recombinant Cu, Zn-SOD in pig endothelial cells. Transpl Int 15, 220–225. Yoshida, O., Kondo, T., Kureishi-Bando, Y., Sugiura, T., Maeda, K., Okumura, K., et al. (2010). Pitavastatin, an HMG-CoA reductase inhibitor, ameliorates endothelial function in chronic smokers. Circ J 74, 195–202. Zeibig, S., Li, Z., Wagner, S., Holthoff, H. P., Ungerer, M., Bultmann, A., et al. (2011). Effect of the oxLDL binding protein Fc-CD68 on plaque extension and vulnerability in atherosclerosis. Circ Res 108, 695–703. Zhang, M. X., Ou, H., Shen, Y. H., Wang, J., Coselli, J., & Wang, X. L. (2005). Regulation of endothelial nitric oxide synthase by small RNA. Proc Natl Acad Sci U S A 102, 16967–16972. Zhang, D., Xie, X., Chen, Y., Hammock, B.D., Kong, W., & Zhu, Y. (2012). Homocysteine upregulates soluble epoxide hydrolase in vascular endothelium in vitro and in vivo. Circ Res 110, 808–817. Zhang, M. X., Zhang, C., Shen, Y. H., Wang, J., Li, X. N., Chen, L., et al. (2008). Effect of 27 nt small RNA on endothelial nitric-oxide synthase expression. Mol Biol Cell 19, 3997–4005. Zheng, J. S., Yang, X. Q., Lookingland, K. J., Fink, G. D., Hesslinger, C., Kapatos, G., et al. (2003). Gene transfer of human guanosine 5′-triphosphate cyclohydrolase I restores vascular tetrahydrobiopterin level and endothelial function in low renin hypertension. Circulation 108, 1238–1245.

Please cite this article as: Tousoulis, D., et al., Endothelial dysfunction in conduit arteries and in microcirculation. Novel therapeutic approaches, Pharmacology & Therapeutics (2013), http://dx.doi.org/10.1016/j.pharmthera.2014.06.003