The AT2 Receptor and Inflammation

Chapter 13

The AT2 Receptor and Inflammation Veronica Valero Esquitino,* Leon Alexander Danyel*, Ulrike M. Steckelings† *Center for Cardiovascular Research (CCRI), Institute of Pharmacology, Charité-Universitätsmedizin Berlin, Germany, †IMM—Department of Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark

INTRODUCTION Inflammation is one of the first reactions of the immune system to tissue/cell damage produced by pathogens, chemicals, or physical injury.1 While acute inflammation is considered as a healthy process indicative of protection, chronic ­inflammation has been directly associated with a wide range of disorders, such as cardiovascular, renal, and autoimmune diseases. The ­renin–angiotensin system, which has been traditionally linked to blood pressure control and volume/electrolyte balance, also plays an important role in inflammatory processes and the immune response.2,3 As in most (patho)physiological settings, it is also in the context of inflammation that the two main receptor subtypes for angiotensin II (Ang II), the angiotensin type 1 (AT1) receptor (AT1R) and angiotensin type 2 (AT2) receptor (AT2R), mediate opposing actions: while AT1R activation is linked to proinflammatory effects, the AT2R exerts anti-inflammatory effects.3 Key mechanisms involved in the AT2Rmediated anti-inflammatory effects seem to be the reduction of proinflammatory cytokine release3 or of oxidative stress.4

AT2R SIGNALING IN INFLAMMATION Nuclear factor kappa-B (NF-κB) is a protein complex, which controls the transcription of different proinflammatory mediators such as cytokines, chemokines, and adhesion molecules.5 Activation of NF-κB is triggered by proinflammatory cytokines and microbial products and involves the activation of inhibitors of kappa-B kinases (IKKs), which in turn phosphorylate inhibitors of NF-κB proteins (IκB). This phosphorylation leads to the release of RelA/p50 subunits, which can then translocate into the nucleus and initiate transcription of proinflammatory mediators.5 In 2004, Wu and colleagues demonstrated that AT2R stimulation by Ang II or the AT2R agonist CGP42112A led to dephosphorylation of the I-κB molecule by Src homology protein tyrosine phosphatase-1 (SHP-1), thus preventing NF-κB activation.6 In accordance with these results, our group found that AT2R stimulation by the nonpeptide AT2R agonist compound 21 (C21) reduced TNF-α-induced IL-6 expression by inhibiting nuclear translocation of the NF-κB p50 subunit.7 This anti-­inflammatory effect was absent in the presence of an inhibitor of tyrosine phosphatases, okadaic acid, or an inhibitor of serine/threonine phosphatases, sodium orthovanadate, pointing again to the involvement of protein phosphatases in the inhibitory effect of AT2R stimulation on NF-κB activity (Figure 1). Apart from protein phosphatases, epoxyeicosatrienoic acid (EET), an ­anti-inflammatory mediator known to generally inhibit NF-κB,8 seems to play a role in AT2R-mediated NF-κB inhibition, too, because AT2R-mediated NF-κB inhibition was prevented by the blockade of EET synthesis.7 In contrast to these observations, there are also data showing an AT2R-mediated increase in NF-κB activity.9,10 These contradicting results may be due to different experimental conditions, depending on whether NF-κB mediates proinflammatory effects or—as shown by Reinecke et al.—promotes neuroregeneration.9 The JAK-STAT signaling pathway represents another molecular mechanism involved in cytokine synthesis, inflammation, and immune response.11 Horiuchi et al. showed that AT2R stimulation inactivated STAT transcription factors by tyrosine and serine dephosphorylation.12 Interestingly, AT2R-mediated inactivation of STAT seemed not dependent on the original STAT-activating stimulus, but worked for STAT activation by ANG II, IFN-γ, PDGF, or EGF.12 Oxidative stress, that is, the production and accumulation of reactive oxygen species (ROS), is another inducer of inflammation, for example, through increasing activity of transcription factors such as NF-κB or AP-1.13 Continued oxidative stress can promote chronic inflammation and thus be an underlying contributor to chronic cardiovascular, neurological, and renal diseases or diabetes. Chabrashvili et al. conducted a study in order to generally look at the impact of AT1R and AT2R The Protective Arm of the Renin–Angiotensin System (RAS). http://dx.doi.org/10.1016/B978-0-12-801364-9.00013-4 © 2015 Elsevier Inc. All rights reserved.

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AT2R Plasma membrane Phosphatases

S T A T

A T

T

P

A

Pro-inflammatory cytokines

P

T

T

S

JAK/STAT pathway

IkB

JAK P

S

P JAK

S T A T

p65

P S P

T A T

P

NF-kB

Pro-inflammatory cytokines

P S P

IkB

P

T A T

p65

Nucleus FIGURE 1  Activation of phosphatases leads to dephosphorylation of tyrosine and serine residues of components, which are essential in the signaling pathways leading to JAK-STAT and NF-κB activation. Decreased transcriptional activity of STAT and NF-κB results in reduced synthesis of proinflammatory cytokines.

on the generation of oxidative stress.4 They found that the expression of certain factors involved in the Ang II-induced, AT1R-mediated generation of ROS, namely, the NADPH subunits p22phox, Nox-1, and p67phox, is even more increased when AT2R is blocked, pointing again to an inhibitory impact of AT2R on ROS production. Specific examples of anti-inflammatory or antioxidant effects of AT2R stimulation are reviewed in the following.

INFLAMMATION IN CARDIOVASCULAR DISEASE AND THE ROLE OF THE AT2 RECEPTOR Atherosclerosis AT2Rs are expressed in atherosclerotic plaques and modify the inflammatory component of atherosclerosis.14 This antiinflammatory effect was shown, for example, in ApoE−/− mice on a cholesterol-rich diet with a targeted overexpression of AT2Rs in the vascular smooth muscle cells, in which atherosclerotic lesion development was significantly attenuated through a kinin/NO-dependent mechanism.15 In contrast, AT2R deficiency in ApoE−/− mice aggravated atherosclerotic lesions by increasing superoxide production and macrophage infiltration.14,16 AT2R stimulation by Ang II in ApoE−/− mice deficient of AT1R reduced atherogenesis, but had no effect on macrophage/T-cell infiltration and markers of oxidative stress.17 However, direct AT2R stimulation with the peptide AT2R agonist CGP42112 in ApoE−/− mice in a study by Kljajic et al. not only significantly reduced atherosclerotic lesion progression but also resulted in improved plaque stability, reduced oxidative stress, reduced vascular cell adhesion, and improved endothelial function.18

Myocardial Infarction: Heart Failure Cardiac tissue injury after myocardial infarction (MI) occurs in several phases: initial acute ischemia and related cell death and local proteolysis, an inflammatory response with massive invasion of inflammatory cells and eventually a fibrotic response, which involves not only the infarcted but also the peri-infarct area.19 The location and size of the scar and the course of remodeling determine whether chronic heart failure will develop as a result of MI. A beneficial effect of AT2R stimulation (directly by AT2R agonists or indirectly by ARBs) on cardiac function post-MI has been demonstrated in a number of studies.20 However, while most studies focused on cardiac function, hypertrophy, and fibrosis, only few studies looked at the effect on MI-related inflammation. In a study by our group, treatment of rats with C21 for 1 week after MI resulted in significantly improved cardiac function, and this coincided with a strong and significant attenuation in elevated levels of proinflammatory cytokines IL-1β, IL-2, and IL-6 in the peri-infarct zone.21 Moreover, oxidative stress was reduced as shown by lowered plasma levels of myeloperoxidase. These effects were blocked by the AT2R antagonist PD 123319 pointing to AT2R specificity of the C21 effects.21 In addition to this classical anti-inflammatory effect, AT2R stimulation may further act by modifying the cellular immune response in ischemic heart injury. Curato et al. isolated CD8+ T cells from the peri-infarct area of rat hearts and

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found that a subpopulation of these cells (CD8+AT2R+) expressed AT2Rs and at the same time changed phenotype to being noncytotoxic.22 These cells were further characterized by increased levels of IL-10 and decreased levels of IL-2 and INF-γ when compared with CD8+AT2R− T cells. The CD8+AT2R+ subpopulation was enhanced upon AT2R stimulation in vivo. Furthermore, intramyocardial transplantation of CD8+AT2R+ T cells after MI led to a decrease in infarct size. In addition to this CD8+AT2R+ cell population, a population of c-Kit+AT2R+ cells was also found to be increased in hearts of infarcted rats treated with C21 and to act antiapoptotic on cardiomyocytes.23

Stroke Therapeutic measures in relation to stroke can be either the prevention of occurrence of stroke, for example, through control of hypertension or antithrombotic therapy; acute recanalization by thrombolysis, which however is only possible in 5–7% of patients; or reduction of ischemic tissue injury and attenuation of neurological deficits. Stroke prevention is successfully performed in current cardiovascular medicine, whereas an effective neuroprotective treatment is still a huge, unmet therapeutic need. In stroke, ischemic injury leads to excitotoxicity and oxidative damage followed by disintegration of the blood–brain barrier and a pronounced inflammatory response.24 This chain of events determines the actual amount of permanent damage, that is, therapeutic measures to interrupt this fatal cascade may diminish neuronal loss and long-term neurological deficits. Several studies with various experimental approaches have shown that AT2R stimulation is able to reduce infarct size and neurological deficits after stroke and that the attenuation of inflammation and oxidative stress seem to be major protective mechanisms in this context.25 For example, in AT2R-knockout mice undergoing permanent middle cerebral artery occlusion (MCAO), a greater ischemic area and more severe neurological deficits (compared with wild-type mice) were associated with increased superoxide anion production and NADPH oxidase activity.26 The reverse experimental approach in the same model, which was AT2R stimulation by systemic application of C21 initiated after stroke, resulted in a marked attenuation of superoxide anion production and of proinflammatory cytokine expression including monocyte chemoattractant protein 1 and tumor necrosis factor-α.27 Similar results were obtained in another stroke model and another species, which was transient, endothelin-induced MCAO in rats.28 In this study, peripheral and central administration of C21 reduced infarct size and neurological deficits, which coincided with an anti-inflammatory effect as shown by reduced levels of chemokine CCL2 and its receptor CCR2 and of inducible nitric oxide synthase. Microglia, the macrophages of the brain, are rapidly activated after stroke. They not only are essentially involved in stroke-induced inflammation but also can have neuroprotective actions by the release of BDNF. Microglia express AT2Rs. However, data are controversial currently about the effect of AT2R stimulation on microglia activation in the context of stroke, since both a decrease and an increase in microglia markers have been observed.28–30 Interestingly, enhanced AT2Rmediated microglia activation coinciding with a diminished infarct size mainly seems to involve the protective phenotype of microglia.30

Renal Injury Renal diseases of various causes such as hypertension, diabetes, and autoimmunity are associated with and driven by an inflammatory response. Several studies have found that AT2R stimulation attenuates the inflammatory response in the kidney. The first evidence pointing to an anti-inflammatory effect of the AT2R in kidney disease came from a model of ureteral obstruction, which in AT2R-deficient mice resulted in a more pronounced increase in macrophage infiltration than in wildtype mice.31 The same observation was made in a model of renal ablation in AT2R-knockout mice.32 The effect of direct AT2R stimulation was first studied in hypertension-related nephropathy in stroke-prone spontaneously hypertensive rats (SHRSP) fed a high-sodium diet.33 Six weeks after the initiation of this diet, massive inflammatory infiltration was detected in the kidneys by immunohistochemistry, and a certain fraction of proteins indicative for renal inflammation was significantly increased in urine samples of hypertensive compared with control rats. Oral treatment with the AT2R agonist C21 attenuated both the infiltration of inflammatory cells into the renal tissue and the increase in the urine markers of inflammation. While the study in SHRSP looked on a rather long-term effect of AT2R stimulation in hypertension-related nephropathy, Matavelli et al. studied the effect of an only 4-day treatment with C21 in a two-kidney, one-clip hypertension model in rats and found a significant reduction of elevated TNF-α and interleukin-6 (IL-6) levels in the renal interstitial fluid.34 The group of Tahir Hussain has performed several studies showing a renoprotective effect of AT2R stimulation in obese Zucker rats, a model of metabolic syndrome.35–37 These protective effects included AT2R-mediated anti-inflammation. For example, a 2-week subcutaneous infusion of the peptide AT2R agonist CGP42112A significantly reduced elevated TNF-α and IL-6

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protein levels in plasma and the renal cortex.36 These results could be confirmed in a similar study using the nonpeptide agonist C21.35 Based on in vivo and in vitro data, the latter study further suggested that the AT2R-mediated repression of proinflammatory cytokines is based on an increased synthesis of the anti-inflammatory cytokine interleukin-10.35

INFLAMMATION IN AUTOIMMUNE DISEASE AND THE ROLE OF THE AT2 RECEPTOR In addition to the inhibitory effect on inflammation, AT2R also seems to have an immunomodulatory role, for example, in autoimmune diseases. AT2Rs were found to be expressed in cells controlling innate and specific immune responses such as monocytes,38 macrophages,39 microglia,40 and T cells.41 Moreover, recent studies using animal models of autoimmunity have provided evidence of AT2R-mediated immunomodulatory actions. For instance, in a model of immune-mediated glomerulonephritis, indirect AT2R stimulation during AT1R blockade resulted in reduced MCP-1 expression and inhibition of ERK phosphorylation.42 These effects were absent in AT2R-deficient mice. Recent data about the effect of direct AT2R stimulation in two further autoimmune models, experimental autoimmune encephalomyelitis (EAE; a mouse model of multiple sclerosis) and collagen-induced rheumatoid arthritis, also indicate an anti-inflammatory immunomodulatory effect of the AT2R, which involves attenuated T-cell infiltration and promotion of anti-inflammatory Foxp3 regulatory T cells.43–45

CONCLUSIONS Current data overwhelmingly support that the AT2R exerts anti-inflammatory effects in a broad range of diseases, in which inflammation is a main contributor to the pathology. Such diseases comprise atherosclerosis, myocardial infarction (MI) and post-MI heart failure, stroke, renal disease, and autoimmune diseases. In autoimmune disease, AT2R stimulation seems to act additionally through modulation of T-cell differentiation and the immune response. Inhibition of NF-κB activity seems a major mechanism of action of AT2R in inflammation. All in all, AT2R stimulation may provide a new promising treatment approach for diseases in which pharmacological targeting of the inflammatory component is therapeutically effective.

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