Curr Hypertens Rep (2014) 16:431 DOI 10.1007/s11906-014-0431-2

MEDIATORS, MECHANISMS, AND PATHWAYS IN TISSUE INJURY (T FUJITA, SECTION EDITOR)

Angiotensin II and Vascular Injury Augusto C. Montezano & Aurelie Nguyen Dinh Cat & Francisco J. Rios & Rhian M. Touyz

# Springer Science+Business Media New York 2014

Abstract Vascular injury, characterized by endothelial dysfunction, structural remodelling, inflammation and fibrosis, plays an important role in cardiovascular diseases. Cellular processes underlying this include altered vascular smooth muscle cell (VSMC) growth/apoptosis, fibrosis, increased contractility and vascular calcification. Associated with these events is VSMC differentiation and phenotypic switching from a contractile to a proliferative/secretory phenotype. Inflammation, associated with macrophage infiltration and increased expression of redox-sensitive pro-inflammatory genes, also contributes to vascular remodelling. Among the many factors involved in vascular injury is Ang II. Ang II, previously thought to be the sole biologically active downstream peptide of the renin-angiotensin system (RAS), is converted to smaller peptides, [Ang III, Ang IV, Ang-(1-7)], that are functional and that modulate vascular tone and structure. The actions of Ang II are mediated via signalling pathways activated upon binding to AT1R and AT2R. AT1R activation induces effects through PLC-IP3-DAG, MAP kinases, tyrosine kinases, tyrosine phosphatases and RhoA/Rho kinase. Ang II elicits many of its (patho)physiological actions by stimulating reactive oxygen species (ROS) generation through activation of vascular NAD(P)H oxidase (Nox). ROS in turn influence redox-sensitive signalling molecules. Here we discuss the role of Ang II in vascular injury, focusing This article is part of the Topical Collection on Mediators, Mechanisms, and Pathways in Tissue Injury A. C. Montezano : A. Nguyen Dinh Cat : F. J. Rios : R. M. Touyz Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK R. M. Touyz (*) Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, 126 University Place, Glasgow G12 8TA, UK e-mail: [email protected]

on molecular mechanisms and cellular processes. Implications in vascular remodelling, inflammation, calcification and atherosclerosis are highlighted. Keywords Angiotensin receptors . Inflammation . Signal transduction . Oxidative stress . Vascular remodelling . Calcification . Atherosclerosis

Introduction The renin angiotensin system (RAS) plays a major physiological role in regulating vascular function and a pathological role in vascular injury through its actions on endothelial dysfunction, vascular remodelling and vascular inflammation [1, 2]. As such, the RAS contributes to vascular damage associated with hypertension and atherosclerosis, which are important risk factors underlying cardiovascular, cerebrovascular, renal and metabolic diseases. Drugs that inhibit the RAS promote vascular health and reduce the risks of cardiovascular events [3]. Initially the RAS was identified as an endocrine system where circulating kidney-derived renin regulates cardiovascular function through Ang II binding to its AT1 and AT2 receptors on target tissues [4]. Until recently, this system was accepted as the conventional RAS. However, it is now evident that the kidney is not the only source of renin production and that angiotensin (Ang) peptides may be formed in extra-renal tissues and organs, such as the brain, adrenal gland, pituitary gland, reproductive tissues, gastrointestinal tract, haematopoietic tissue, heart and vessels [5, 6]. Functions of this local RAS remain unclear, but may be related to finetuning of Ang II at the tissue level. Vascular injury is associated with impaired endothelial cell function and alterations in vascular smooth muscle cell (VSMC) contraction/relaxation, migration, growth/apoptosis, senescence, calcification, production/degradation of

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extracellular matrix and inflammation [7–9]. Growing evidence indicates that activation of the immune system and adipokines derived from perivascular fat may also be important [10, 11]. Cellular mechanisms and signalling pathways implicated in vascular injury are complex and multifactorial, but among the numerous systems shown to be important is the RAS [12–14]. Here, we highlight molecular mechanisms whereby Ang II influences vascular cell function, and discuss the role of Ang II in vascular remodelling, inflammation, calcification and atherosclerosis.

The RAS—Old and New Concepts The RAS is considered an endocrine system where Ang II is the product of sequential enzymatic cleavage of angiotensinogen. Circulating renin converts hepaticderived angiotensinogen to Ang I, which is then cleaved by angiotensin converting enzyme (ACE) to form Ang II. Ang II mediates its physiological actions through two receptors, Ang II type I receptor (AT1R) and Ang II type 2 receptor (AT2R). Until recently, Ang II was considered the major downstream peptide of the RAS. However, over the past decade, the conventional view of the RAS has undergone major change. In particular, new RAS members (renin/prorenin receptor, ACE2 and Ang II-derived peptides) have been identified (Fig. 1), and a functionally active tissue-based RAS has been characterized [15, 16]. New Members of the RAS Renin, produced by the juxtaglomerular apparatus in the kidney, acts on

Fig. 1 New components of the renin-angiotensin system (RAS) and Ang receptors. In addition to the classical RAS, where Ang II is derived from angiotensinogen by renin and ACE, Ang II can be produced through nonACE dependent systems. Ang II itself can be metabolized to biologically active small peptides, such as Ang-(1-7), Ang-(1-9), Ang III and Ang IV. PEP, prolyl-endopeptidase; AMP, aminopeptidase; PCP, prolyl-carboxypeptidase; NEP, neutral-endopeptidase; (P)RR, prorenin receptor

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angiotensinogen to form Ang I. It is the key enzyme of the RAS because of its rate-limiting hydrolytic activity [17]. Renin also acts as an agonist of the RAS by binding to the renin/ prorenin receptor [(P)RR], a new component of the RAS [18, 19]. Prorenin bound to (P)RR undergoes a conformational change and activates signalling pathways, e.g., ERK1/2 and p38MAP kinase, which promote cell growth and fibrosis independently of Ang II in cardiomyocytes, mesangial cells, podocytes, distal tubular cells, vascular endothelial cells and VSMCs [20, 21]. The exact (patho)physiological significance of this receptor remains unclear, but it may play a role in early development. Transgenic animals overexpressing (P)RR develop high BP or glomerulosclerosis, and increased expression of (P)RR has been shown in models of hypertension and kidney damage. However, definitive proof of a role for this receptor in human cardiovascular disease is still lacking [22]. Angiotensin Converting Enzyme (ACE) 2 ACE2 catalyses Ang I and Ang II to generate the Ang peptides Ang-(1-9) and Ang-(1-7), which mediate, in general, effects that are opposite to those of Ang II [23, 24]. Reduced ACE2, and consequent decreased Ang-(1-7) production, is associated with vascular injury in hypertension, diabetes, atherosclerosis and kidney disease [25, 26]. On the other hand, increased ACE2 activity promotes vasodilation, and as such is currently being tested as a potential therapeutic target [27]. Ang II-Derived Small Peptides Ang-(1-7) is formed from Ang II by prolyl endopeptidase, prolyl carboxipeptidase or ACE2, or directly from Ang I through hydrolysis by prolyl endopeptidase and endopeptidase 24.11 [28] (Fig. 1). Ang-(1-7) is present in the circulation and in tissues [29, 30], and it binds to its G protein-coupled receptor Mas [31]. Ang-(1-7) opposes Ang II effects by mediating vasodilation, growth-inhibition, anti-inflammatory responses, anti-arrhythmogenic and antithrombotic effects [32, 33]. These processes are mediated through nitric oxide synthase (NOS)-derived nitric oxide (NO) production, activation of protein tyrosine phosphatases, inhibition of MAP kinases, and inhibition of NADPH oxidase-derived generation of reactive oxygen species (ROS) [34, 35]. Ang-(1-7)-Mas can interact with AT1R, thereby inhibiting Ang II actions. The ACE2-Ang-(1-7)-Mas axis is now considered as a counter-regulatory system to the ACEAng II-AT1R axis [34]. Other Ang II-derived peptides include Ang III, which increases blood pressure, promotes vasoconstriction and stimulates aldosterone production [36]; Ang IV, which increases renal blood flow and improves cardiac function [37]; Ang-(3-7), implicated in the brain and kidney [38]; Ang-(1-9), which enhances bradykinin actions, NO production, arachidonic acid release and regulation of platelet function [39]; and Ang-(1-12), which has been implicated in cardiac function.

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Tissue-Based RAS A tissue system is characterized by the presence of all RAS components, including angiotensinogen, renin, ACE, Ang I, Ang II, and Ang II receptors. Such a local system is present in the heart, vessels, kidney, adrenal gland, pancreas, central nervous system, reproductive system, lymphatic and adipose tissue [40]. Components of the RAS have also been identified in the eye, which may be important physiologically in maintaining ocular pressure [41], and pathologically in retinopathies associated with hypertension and diabetes [42]. Increased activation of tissue RAS has been demonstrated in cardiovascular diseases such as atherosclerosis, myocardial infarction, cardiac failure, diabetes and kidney disease [43], independently of blood pressure (BP) elevation.

Ang II in the Vascular System—Molecular Mechanisms Angiotensin Receptors and Signalling Ang II mediates effects via complex intracellular signalling pathways that are stimulated following binding of the peptide to its cell-surface receptors [44, 45] AT1R and AT2R [46, 47]. AT1R and AT2R are members of the seven transmembrane-domain, G proteincoupled receptor (GPCR) superfamily, and are distinguished pharmacologically by their affinities for antagonists. Ang II binding to the AT1R results in coupling of G proteins (Gq/G11 and/or Gi/Go) to the C-terminal of the receptor and to stimulation of many intracellular signalling pathways, including PLC, Ca2+ channels, PLD, PLA2, adneylate cyclase, MAP kinases, JAK-STAT pathway and NADPH oxidase, amongst others [48]. AT1R mediates most of the pathophysiological effects of Ang II, including vasoconstriction, inflammation growth, and fibrosis, whereas AT2R may counteract many of the AT1R-mediated actions [49, 50]. GPCR interact with G proteins as well as with accessory proteins, termed GPCR interacting proteins (GIP). GIP specifically associated with AT1R, called AT1R-associated protein (ATRAP), acts as a negative regulator of AT1R [51]. ATRAP-transgenic mice exhibit reduced neointimal formation, and decreased inflammatory responses and oxidative stress in response to Ang II, supporting the inhibitory role of ATRAP in cardiovascular remodelling [52]. Moreover, in ATRAP-/- mice, blood pressure and plasma volume are increased [53]. Another GIP has also been identified, ARAP1 (AT1R-associated protein 1), which is involved in the increase in membrane AT1R number following Ang II stimulation, and in the recycling of the receptor [54]. GPCR interacting proteins have also been associated with AT2R, including AT2R-interacting protein (ATIP), which is involved in transinactivation of receptor tyrosine kinases and growth inhibition [55]. ATIP is also known as mitochondrial tumor suppressor gene 1 (MTUS1) and AT2R binding protein of 50 kDa (ATBP50).

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ATIP is expressed in tissues in which AT2R is abundant, including the uterus and adrenal tissue [56]. Both receptors play a role in regulating VSMC function, although they differ in their actions (Fig. 1). Whereas the AT1R is associated with growth, inflammation and vasoconstriction, the AT2R is generally associated with apoptosis and vasodilation [57, 58]. In pathological conditions, AT2R has also been shown to exert hypertrophic and pro-inflammatory actions. Ang II Signalling Through Mitogen-Activated Protein (MAP) Kinases, Tyrosine Kinases and RhoA/Rho Kinase Of the many growth-signalling pathways through which Ang II signals, is the MAP kinase family [59]. In VSMCs, Ang II activates all four of the major MAP kinases, including extracellular signal-regulated kinases (ERK1/2), p38MAP kinase, c-Jun N-terminal kinases (JNK) and ERK5 [60]. ERK1/2, phosphorylated by MEK1/2 (MAP/ERK kinase), is a key growth signalling kinase, whereas JNK and p38MAP kinase, phosphorylated by MEK4/7 and MEK3/6, respectively, influence cell survival, apoptosis, differentiation and inflammation. ERK5 is involved in protein synthesis, cell cycle progression and cell growth. Ang II induces phosphorylation of multiple tyrosine kinases, including c-Src, janus family kinases (JAK), focal adhesion kinase (FAK), Pyk2, p130Cas and phosphatidylinositol 3-kinase (PI3K) [61–63]. In human and rat VSMCs, c-Src is critically involved in trophic and contractile actions of Ang II [64, 65], effects that are augmented in vascular injury associated with hypertension [66, 67]. Ang II also activates receptor tyrosine kinases, even though it may not bind directly to these receptors. This process of transactivation has been demonstrated for epidermal growth factor (EGF) receptor, platelet-derived growth factor (PDGF) receptor, subtype β, and insulin-like 1 growth factor (IGF-1) receptor [68, 69]. Mechanisms whereby Ang II-induces receptor tyrosine kinase transactivation include activation of tyrosine kinases (Pyk2 and Src), redox-sensitive processes, and through a metalloprotease-dependent shedding of heparin-binding EGF-like growth factor (HB-EGF) [70]. The metalloprotease responsible for this process is ADAM17 [70]. Ang II also increases production of vasoactive hormones and growth factors, which could promote cell proliferation, protein synthesis and fibrosis, further contributing to growth processes in arterial remodelling. Activation of RhoA and its downstream target Rho-kinase play an important role in Ang II-mediated vascular contraction and growth [71, 72]. RhoA, a member of the Rho family of small GTPase binding proteins, is abundantly expressed in vascular smooth muscle cells and participates in vasoconstriction via phosphorylation of myosin light chain (MLC) and sensitization of contractile proteins to Ca2+. In VSMCs, Ang II/AT1R increases RhoA activity [73, 74] via the G12/13 family

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of G proteins as well as Gq [157]. RhoGEFs, which catalyse the exchange of GDP for GTP on RhoA, are sensitive to G12/ 13. In Ang II-infused rodents, vascular injury and hypertension are associated with increased vascular RhoA/Rho kinase activation, effects that are ameliorated by the Rho kinase inhibitors, fasudil or Y27632 [75, 76]. Ang II Signalling Through Reactive Oxygen Species in Vascular Cells Ang II mediates many of its molecular and cellular effects by stimulating production of ROS, which are highly reactive, bioactive, short-lived molecules derived from a reduction of molecular oxygen [77]. Of the many types of ROS generated, superoxide anion (•O2-), hydrogen peroxide (H2O2), nitric oxide (NO) and peroxynitrite (ONOO-) are particularly important in the cardiovascular system [78]. Vascular ROS are produced in endothelial, adventitial and VSMCs and generated mainly from NAD(P)H oxidase, a multi-subunit enzyme [79–81] that catalyses the production of •O2- by the one electron reduction of oxygen using NAD(P)H as the electron donor: 2O2 + NADPH→2O2- + NAD(P)H + H+. The prototypical NADPH oxidase (Nox) is phagocytic NADPH oxidase, which comprises five components: (phox for PHagocyte OXidase), p47phox, p67phox, p40phox, p22phox and gp91phox and the small G proteins Rac 1/2. The Nox family comprises seven isoforms, Nox1-5, Duox1 and Duox2. Unlike phagocytic NAD(P)H oxidase, which is activated only upon stimulation and which generates •O2- in a burst-like manner extracellularly, vascular oxidases (Nox1, Nox4, Nox5) are constitutively active, produce •O2- in a slow and sustained fashion and act as intracellular signalling molecules, influencing not only transcription factors, but other molecules involved in cell growth, contraction, migration, fibrosis and inflammation, such as MAP kinases, tyrosine kinases and protein phosphatases [82–84]. Nox1 is implicated in cell growth, inflammation and fibrosis and plays an important role in the development of atherosclerosis [85]. Nox4 has vasodilatory functions, is implicated in fibrosis and inflammation and may be both vasoprotective and vaso-injurious. [86, 87]. Nox5 is implicated in cell growth and inflammation and is upregulated in human atherosclerosis [88]. Increased Nox5 activity has recently been shown to be important in renal injury, proteinuria and hypertension [89]. All vascular Nox isoforms, including Nox1, Nox2, Nox4 and Nox5, are regulated by Ang II. The functional significance of different Nox isoforms still awaits clarification, but their differential tissue distribution, cellular localization and subcellular compartmentalization probably play a major role in Nox-specific actions. Ang II is a potent stimulator of vascular NAD(P)H oxidase [90, 91]. It induces activation of the enzyme, increases expression of NAD(P)H oxidase subunits and stimulates ROS production in cultured VSMC and intact arteries. Mechanisms linking Ang II to the enzyme and upstream signalling molecules modulating vascular Nox activity include PLD, PKC, c-

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Src, PI3K and Rac [92•, 93, 94]. In hypertension, diabetes and atherosclerosis these processes are augmented, contributing to increased activation of the oxidase and consequent oxidative stress [95–97]. Superoxide and H2O2 activate multiple signalling molecules, including MAP kinases (p38MAP kinase, JNK, ERK-5 and ERK1/2), non-receptor tyrosine kinases (Src, JAK2, STAT, p21Ras, Pyk2 and Akt), receptor tyrosine kinases (EGFR, IGFR and PDGFR), protein tyrosine phosphatases, calcium channels and redox-sensitive transcription factors (NF-κB, AP-1 and HIF-1) [98–100]. Activation of these molecules participates in cell growth, migration, expression of pro-inflammatory genes, production of extracellular matrix proteins and contraction. All of these processes play important roles in vascular injury associated with cardiovascular diseases. Mechanisms whereby ROS modify signalling molecules are complex, but oxidative modification of proteins, e.g., protein tyrosine phosphatases (PTP), is important [101]. Inhibition of Nox is now being considered as a possible therapeutic target in patients. [102•]. Noxs may also be targets of conventional cardiovascular drugs such as ACE inhibitors, AT1 receptor blockers and calcium channel blockers, and effects of these agents may be mediated, in part, by decreasing Nox-derived ROS generation. Clinical studies support a direct antioxidant effect of AT1 receptor blockers, as demonstrated in hypertensive patients treated with candesartan, where oxidative stress and inflammation were reduced independently of blood pressure-lowering actions [103, 104].

Angiotensin II, Vascular Remodelling and Inflammation Vascular smooth muscle cells are critically involved in maintaining vascular integrity and tone. They are the main constitutive cells of the vascular wall, assuming various functional and structural functions. Vascular smooth muscle cells are dynamic, highly plastic, multifunctional cells that contribute to arterial remodelling by influencing cell growth (hyperplasia and hypertrophy), apoptosis, cell elongation, reorganization, altered production of extracellular matrix proteins, fibrosis, migration and inflammation [105, 106] (Fig. 2). Moreover, quiescent contractile VSMCs can be differentiated into a proliferative phenotype under pathological conditions. Such plasticity and phenotypic switching are major contributors to vascular injury in various cardiovascular diseases, and the RAS is critically involved in these processes. Ang II, through the AT1R, promotes a phenotypic switch from a contractile to a proliferative and synthetic phenotype of VSMCs, leading to changes in the contractile machinery, vascular hypertrophy/proliferation and secretion of contractile, mitogenic, inflammatory and pro-fibrotic mediators [8]. These processes are controlled by transcriptional factors

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Fig. 2 Mechanisms whereby Ang II induces vascular injury. Binding of Ang II to its cell membrane receptor induces impairs NO synthesis by reducing eNOS activity and promoting NOS uncoupling. Ang II also activates NADPH oxidase (Nox), leading to increased generation of reactive oxygen species (ROS), that are proinflammatory, mitogenic, prothrombotic and pro-fibrotic. These events also contribute to

atherogenesis, through macrophage infiltration and foam cell formation. Ang II activates signaling pathways through calcium mobilization and MAP kinase activation and by inhibiting cyclic GMP (cGMP), leading to altered vascular reactivity, growth, calcification and fibrosis. Together, these processes contribute to vascular injury that underlies many cardiovascular diseases

regulated in large part by Ang II. Growing evidence also implicates microRNAs (miR) in VSMC differentiation and phenotypic switching. In particular, miR-143/145, miR-21, and miR-1 have been shown to promote the contractile phenotype, whereas miR-221, miR-146a, miR-24, and miR-26a may be important in the switch to the synthetic phenotype and cell proliferation associated with vascular injury [107]. Changes in intracellular calcium and magnesium, MAPK and Rho kinase signalling and generation of ROS, are all important in Ang II-mediated VSMC effects [8, 105–107]. Vascular fibrosis involves accumulation of extracellular matrix proteins, such as collagen, elastin, fibrillin, fibronectin and proteoglycans in the tunica media and is increased in vascular remodelling. Collagen accumulation may be a consequence of increased synthesis [108, 109], reduced degradation by matrix metalloproteinases (MMP) and/or enhanced tissue inhibitor of metalloproteinase-1 (TIMP-1) production [110]. All of these processes are influenced by Ang II. Apoptosis modulates fine-tuning of media growth and may be a growth-associated compensatory or adaptive process in Ang II-mediated vascular remodelling. An imbalance between growth and apoptosis may be important in the structural changes associated with vascular injury. Ang II-elicited growth/apoptosis and profibrotic effects are modulated, in part, by endogenous production of mitogenic factors, such as TGF-β, PDGF, EGF, IGF-1 and ET-1 [111, 112], and activation of mitogenic and pro-fibrotic pathways. We recently identified a new pathway for Ang II-mediated vascular cell growth and migration through Fat-1, an atypical cadherin, and Nox1 [113].

Vascular inflammation is characterized by recruitment of monocytes and lymphocytes into the subendothelial space, production of chemotactic cytokines, increased expression of adhesion molecules, reactive smooth muscle cell proliferation, platelet aggregation, altered extracellular matrix production and degradation. These processes, together with lipid oxidation, are pro-atherogenic, particularly in injured arteries in hypertension and diabetes. Ang II has significant proinflammatory actions inducing the production of ROS, cytokines, adhesion molecules and activation of redox-sensitive inflammatory genes in vascular cells [113–115]. Cytokines, chemokines and other pro-inflammatory mediators are also produced by circulating leukocytes and lymphocytes and by resident macrophages in the vessel wall in response to Ang II stimulation. These pro-inflammatory responses are associated with activation of the adaptive immune system. T lymphocytes, particularly effector T cells such as Thelper (Th)1 (interferon-gamma-producing) and Th2 lymphocytes [that produce interleukin (IL)-4], as well as Th17 (that produce IL-17), and T suppressor lymphocytes including regulatory T cells (Treg), which express the transcription factor forkhead box P3 (Foxp3), play critical roles in the vasculopathy associated with Ang II-dependent hypertension [116•, 117, 118]. Inflammation participates in vascular remodelling and atherosclerosis [119, 120], and may contribute to accelerated vascular damage in hypertension, diabetes, renal disease and in aging. Inflammation may activate the RAS, and thereby further contribute to vascular remodelling and hypertension. Activators of nuclear receptors, such as the peroxisome

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proliferator activated receptors (PPARs), which are hypolipidemic agents (the fibrates, PPARα agonists) or insulin sensitizers (glitazones, PPARγ agonists), downregulate the vascular inflammatory response in experimental animals and decrease serum markers of inflammation in humans [121, 122]. Thus, PPARs and vasoactive substances may be endogenous modulators of the inflammatory process involved in vascular injury. Ang II downregulates PPARs through activation of nuclear factor (NF)-κB [123].

Angiotensin II and Vascular Calcification Calcification of arteries predicts events in patients with cardiovascular disease [121, 122]. It is common in advanced age, chronic kidney disease, diabetes mellitus, atherosclerosis and hypertension, and is related to cardiovascular morbidity and mortality [124–126]. Vascular calcification is a complex and dynamic process involving many mechanisms that promote accumulation of calcium deposits in the vessel wall. This leads to increased arterial stiffness and decreased elasticity, which in turn impacts on hemodynamics and cardiovascular dysfunction. Vascular calcification is associated with mineralization of the internal elastic lamina and elastic fibres within the medial layer. It is a highly regulated process, not dissimilar to processes that control bone formation [127]. Factors that trigger and promote calcification include abnormalities in mineral metabolism, particularly hyperphosphatemia and hypercalcemia [129–132], which stimulate VSMC differentiation to an osteoblastic phenotype [128, 129]. This is driven by upregulation of transcription factors such as cbfa1 (core-binding factor 1α)/Runx2, MSX-2 and bone morphogenetic protein 2 (BMP-2) that are critically involved in normal bone development, and control the expression of osteogenic proteins, including osteocalcin, osteonectin, alkaline phosphatase, collagen-1 and bone sialoprotein [130, 131]. Another mechanism contributing to vascular mineralization is loss of calcification inhibitors, such as fetuin-A, matrix Gla protein, pyrophosphate and osteopontin [132, 133]. These events are influenced by Ang II, which induces upregulation of osteogenic proteins and signalling pathways. In particular, Ang II stimulates expression of bone morphogenetic protein 2 (BMP2) and the osteoblast transcription factor Runx2/Cbfa1 [134•], and it stimulates transcellular transport of cations such as Ca2+ and Mg2 [135, 136]. Ang II also inhibits matrix Gla, an important inhibitor of calcification [137•]. Targeting the RAS may have therapeutic potential in vascular calcification. AT1R blockade can inhibit arterial calcification by disrupting vascular osteogenesis, suggesting that patients with vascular calcification may benefit therapeutically from ARBs. The vasoprotective axis of the RAS Ang-(1-7)/Mas has also been shown to

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prevent vascular calcification in experimental models, by decreasing the expression of osteogenic proteins [138].

Angiotensin II and Atherosclerosis Atherosclerosis is a chronic inflammatory disease of the vascular wall characterized by plaque formation in the intimal layer. The early hallmark of atherosclerosis, so-called fatty streaks, is already evident during the first decade of life. However, with time the small lesions progress to become fibrous plaques and complicated lesions, characterized by a decrease in lumen diameter and consequent obstruction of blood flow [139]. Although the exact mechanisms that trigger atherogenesis are unclear, increasing evidence indicates that oxidation of Low Density Lipoprotein (LDL) is one of the main early events of the process [140]. LDL diffuses from the plasma to the subendothelial space, where it is oxidatively modified by ROS or enzymes [141]. Oxidized LDL (oxLDL) has pro-inflammatory properties. oxLDL interacts with different membrane and nuclear receptors, such as CD36, PPARγ, and receptor for platelet-activating factor (PAFR), which stimulate pro-inflammatory signalling pathways [142–145]. oxLDL also activates endothelial cells and stimulates the expression of adhesion molecules such as E-selectin, vascular cell adhesion protein 1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). The production of chemokines attracts monocytes and T cells to the arterial intima, promoting an intense activation of the immune response. Circulating leukocytes are continuously recruited into the vessel wall, where monocytes differentiate into macrophages. Macrophages in turn take up oxLDL through the scavenger receptors, CD36 and SR-A, and transform into foam cells that are present in every stage of atherogenesis. Continuous recruitment of circulating leukocytes into the vessel wall further promotes inflammation [146]. oxLDL also interacts with CD36 and Toll-like receptor 4 in VSMCs, enhancing the production of inflammatory cytokines (IL-1β, IL-6, IL-8, and MCP-1) and growth factors such as granulocyte-macrophage colony-stimulating factor (GMCSF) and granulocyte-colony-stimulating factor (G-CSF) that induce recruitment and differentiation of macrophages [147•, 148•]. VSMCs migrate from the media to the arterial intima, where they proliferate, differentiate and produce extracellular matrix to form the fibrous cap present in advanced atherosclerosis lesions, with a very important role in the plaque stability. Lectin-like oxidized LDL receptor-1 (LOX-1) is a scavenger receptor present in endothelial cells and VSMCs, and promotes the interaction with oxLDL. The expression of LOX-1 is induced by inflammatory stimuli, ROS, oxLDL and Ang II [149]. Moreover downstream activation of LOX1 by oxLDL results in the generation of large amounts of ROS [150] and upregulates AT1R via NADPH oxidase, MAPK and

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the NF-κB pathway [151], demonstrating the crosstalk between AT1R and LOX-1. LOX-1, through PKC activation, inhibits the eNOS pathway and eNOS-dependent NO production [152] and the inhibition of LOX-1 prevents endothelial dysfunction. The RAS is a key player in the pathogenesis of atherosclerosis. Expression of AT1R is increased in atherosclerosis [153], and experimental data have shown that inhibition of AT1R has protective effects on the development of atherosclerosis and metabolic disorders [154]. On the other hand, development of atherosclerosis is reduced in pro-atherogenic mice that have overexpression of AT2R [153]. Ang II stimulates expression of adhesion molecules, chemokines and cytokines, and promotes endothelial dysfunction, oxidation and uptake of LDL and proliferation of VSMCs. Many of these proatherogenic effects are mediated through Nox-derived ROS. We recently demonstrated that Nox1 plays a critical role in vascular injury associated with accelerated atherosclerosis in diabetes [85]. In advanced atherosclerosis, Ang II stimulates expression of matrix metalloproteinases (MMPs) and plasminogen activator inhibitor-1, with resultant destabilization of atherosclerotic plaques.

Page 7 of 11, 431 BHF Chair. ACM is supported by a Leadership fellowship from the University of Glasgow. Compliance with Ethics Guidelines Conflict of Interest Augusto C. Montezano, Aurelie Nguyen Dinh Cat, Francisco J. Rios, and Rhian M. Touyz declare that they have no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

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Vascular injury, characterized by endothelial dysfunction, structural remodelling and inflammation, underlies many cardiovascular disorders, including hypertension, atherosclerosis, diabetes and ischemic heart disease. At the cellular level, there is increased VSMC growth, migration, differentiation, calcification, production of extracellular matrix proteins and inflammation, stimulated in large part by activation of the RAS. Over the recent past, our views of Ang II have changed from being a simple vasoconstrictor to that of a complex growth factor that mediates effects through diverse signalling pathways involving PLC/PKC/Ca2+ mobilization, PLA2, PLD, MAP kinases, tyrosine kinases, proto-oncogene expression, RhoA/Rho kinase, and oxidative stress. Through increased Nox-derived ROS generation and activation of redoxsensitive transcription factors, Ang II promotes expression of cell adhesion molecules and induces synthesis of proinflammatory mediators and growth factors. These molecular and cellular processes facilitate increased vascular permeability, leukocyte recruitment, calcification and vascular fibrosis leading to vascular injury. Targeting some of these molecular events with RAS inhibitors may protect against vascular injury and promote vascular health. Acknowledgments Studies performed by RMT were supported by grants from the Canadian Institutes of Health Research (CIHR), JDRF and the British Heart Foundation (BHF). RMT is supported through a

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