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Department of Physiology (L.H.), Faculty of Medicine, Semmelweis University, H-1444 Budapest, Hungary; and Endocrinology and Reproduction Research Branch (K.J.C.), National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510
Address correspondence and requests for reprints to: Kevin J. Catt, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, Building 49, Room 6A36, National Institutes of Health, Bethesda, Maryland, 20892-4510. E-mail: cattk{at}mail.nih.gov.
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ABSTRACT |
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B pathway. The AT1R also signals via Gi/o and G11/12 and stimulates G protein-independent signaling pathways, such as ß-arrestin-mediated MAPK activation and the Jak/STAT. Alterations in homo- or heterodimerization of the AT1R may also contribute to its pathophysiological roles. Many of the deleterious actions of AT1R activation are initiated by locally generated, rather than circulating, Ang II and are concomitant with the harmful effects of aldosterone in the cardiovascular system. AT1R-mediated overproduction of reactive oxygen species has potent growth-promoting, proinflammatory, and profibrotic actions by exerting positive feedback effects that amplify its signaling in cardiovascular cells, leukocytes, and monocytes. In addition to its roles in cardiovascular and renal disease, agonist-induced activation of the AT1R also participates in the development of metabolic diseases and promotes tumor progression and metastasis through its growth-promoting and proangiogenic activities. The recognition of Ang II’s pathogenic actions is leading to novel clinical applications of angiotensin-converting enzyme inhibitors and AT1R antagonists, in addition to their established therapeutic actions in essential hypertension. |
INTRODUCTION |
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Recent studies using gene targeting to delete or enhance specific components of the RAS have confirmed its physiological roles and its novel functions in the development and maintenance of renal vascular and papillary structure (11, 12, 13). They have also highlighted the importance of paracrine and autocrine effects of local RAS in several tissues. Clinical trials using ACE inhibitors and AT1R blockers have shown that the actions of this system extend far beyond blood pressure control (14). It has become clear that the deleterious actions of local RAS on cardiovascular remodeling and renal function account for the beneficial effects of these agents in patients with hypertension, left ventricular hypertrophy, heart failure, and diabetic nephropathy (14). The RAS has also been proposed to exert intracrine effects due to the intracellular formation of Ang II (15, 16). However, kinetic studies suggest that intracellular Ang II is largely derived from receptor-mediated uptake of the extracellular peptide (17).
Animal models and clinical data have also helped to establish that inhibition of Ang II action in nonclassical target sites, such as immune cells, explains some of the unanticipated therapeutic effects of ACE inhibitors and AT1R blockade (14). Parallel studies on the molecular mechanism of action of Ang II have revealed that its main target, the AT1R, is one of the most versatile members of the G protein-coupled receptor (GPCR) family. Ang II exerts several cytokine-like actions via the AT1R and can stimulate multiple signaling pathways, transactivate several growth factor receptors, and promote the formation of reactive oxygen species (ROS) and other proinflammatory responses. Furthermore, recent evidence indicates that the AT1R may serve as a "stretch sensor" because mechanical stress can activate the AT1R without the involvement of Ang II (18, 19). Many of these effects are highly cell specific, and numerous studies are currently under way to identify the roles of these signaling pathways in the physiological and pathophysiological actions of Ang II. This review focuses on the implications of recent findings on the Ang II-induced signaling pathways and functions of the AT1R, which mediates the main physiological and pathological effects of the RAS. The biological actions and signaling mechanisms of AT2 receptors, Ang IV and Ang(1–7) are described in recent reviews (10, 20, 21, 22).
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THE RENIN-ANGIOTENSIN SYSTEM |
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). Angiotensin I is further cleaved by ACE, a dipeptidyl carboxypeptidase, to produce the circulating Ang II octapeptide that is the main effector hormone of the RAS. The ectoenzyme, ACE, is expressed at the surface of endothelial cells, where it catalyzes the conversion of Ang I to Ang II. ACE also cleaves and inactivates bradykinin and thus contributes to the therapeutic effects (and side-effects) of ACE inhibitors. The ACE gene has an insertion/deletion polymorphism in which a 287-bp deletion in intron 16 (ACE-D) leads to increased tissue and circulating levels of the mutant enzyme. This deletion has been reported to be a risk factor for certain cardiovascular and renal diseases and the progression of sarcoidosis (23, 24).
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) or the presence of the renin receptor, which binds to and activates circulating renin and prorenin, may also permit local Ang II formation. Ang II can be further metabolized by aminopeptidase A to form Ang III [Ang(2–8)] and then by aminopeptidase N to Ang IV [Ang(3–8)]. Ang II and III exert their effects via the AT1R and AT2R. Ang IV is also a biologically active peptide with low affinity for AT1R and AT2R and acts in the brain on insulin-regulated aminopeptidase. This protein is a transmembrane metalloprotease that cotranslocates in vesicles with glucose transporter 4 to the cell surface in response to insulin (29). Ang IV and the endogenous ligand, LVV-hemorphin 7, which are potent inhibitors of insulin-regulated aminopeptidase, enhance memory and learning and reverse memory deficits in amnesic animals (30). A recently identified second form of ACE (ACE-2) and other endopeptidases can cleave Ang I to produce Ang(1–7), which has vasodilator and cardioprotective effects (22). Ang(1–7) lacks the C-terminal phenylalanine8 residue of Ang II that is critical for binding and activation of AT1Rs, and has been proposed to act via another GPCR, putatively the mas oncogene (31).
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BIOLOGICAL ROLES OF LOCAL Ang II FORMATION IN TARGET TISSUES |
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CLASSICAL G PROTEIN-MEDIATED SIGNALING MECHANISMS |
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In addition to its rapid, G protein-mediated actions via second messengers and their pathways, Ang II activates secondary responses that are typically associated with stimulation of growth factor receptors, and regulate cell growth, proliferation, cell migration, apoptosis, and gene expression. These effects are often the consequence of Ang II-induced activation of cytoplasmic tyrosine kinases such as Pyk2, c-Src, Tyk2, FAK, Janus kinase 2 (Jak2), and transactivation of membrane-associated growth factor receptor tyrosine kinases (45, 46). Ang II-induced cytoplasmic Ca2+ signaling and protein kinase C (PKC) activation regulate the activities of Src, FAK, and Pyk2 (46). Activation of heterotrimeric G proteins by the AT1R also releases their ß
-subunits, which can cause further activation of tyrosine kinases (47, 48). At least one tyrosine kinase (Jak2) has been shown to interact directly with a tyrosine-containing motif in the cytoplasmic tail of the AT1R (49).
Tyrosine phosphorylation of receptors and scaffold proteins, such as growth factor receptors, integrins, GPCR kinase-interacting protein 1 (GIT1) and p130Cas, paxillin, tensin, vinculin, Grb2 and other adapters, is an important factor in the organization of signaling pathways (46, 50). For example, PLC is constitutively associated with GIT1, a GPCR kinase-interacting protein, and Ang II-induced, Src-mediated tyrosine phosphorylation of GIT1 leads to its activation (51). Although Ang II-induced stimulation of inositol triphosphate signaling is primarily caused by G protein-mediated activation of PLCß isoenzymes, tyrosine kinase-regulated activation of PLC also makes a significant contribution to this process in certain cell types (e.g. VSMCs) (5). In addition, GIT1 can serve as a scaffold protein to facilitate Ang II- and epidermal growth factor (EGF)-induced activation of MEK (MAPK kinase 1) and ERK (52). A recent study suggests that GIT1 is a scaffold for ERK1/2 activation in focal adhesions and regulates cell migration in VSMCs and other cells (53). Another recently identified, Src-regulated scaffold protein is p130Cas, which associates with focal adhesions and regulates the migration of smooth muscle cells via activation of c-Jun NH2-terminal kinase (JNK) (50).
Ang II is a potent stimulus of smooth muscle growth and migration and extracellular matrix formation in the vascular system (54). It stimulates the proliferation of human cerebral artery VSMCs through a basic fibroblast growth factor-dependent pathway, but causes hypertrophy without proliferation in VSMCs from peripheral arteries (55). Ang II also stimulates the proliferation of cultured human mesangial cells through activation of MAPKs, specifically JNK (56), and of IEC-18 intestinal epithelial cells via transactivation of the EGF receptor (EGFR) and the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin/p70S6K1 signaling pathway (57). In adrenal glomerulosa cells, for which Ang II acts as a trophic factor in vivo as well as a stimulus to aldosterone secretion, variable effects on growth and proliferation have been observed in vitro, in some cases with stimulation via calcineurin and MAPK (58). However, a recent report indicates that Ang II stimulates protein synthesis but inhibits proliferation in cultured rat adrenal glomerulosa cells (59). In the testis, Ang II stimulates contraction and growth of the peritubular myoid cells, apparently via MAPK and tyrosine phosphorylation (60). In the skin, activation of AT1R signaling promotes wound healing by stimulating the growth of keratinocytes and myofibroblasts (61).
In many tissues, stimulation by Ang II leads to MAPK activation by transactivating growth factor receptors, including EGF, platelet-derived growth factor, and IGF receptors, and Axl (62, 63, 64), but in some cells [e.g. human embryonic kidney (HEK) 293 cells], PKC activation can directly activate the pathway leading to ERK activation (65) (Fig. 2). Transactivation of the EGFR and other growth factor receptors is an important component of AT1R-mediated signaling via MAPK pathways in cardiac and smooth muscle cells. This transactivation pathway is mediated by Ca2+, PKC, and Src-dependent transactivation of transmembrane matrix metalloproteases, which cause release of heparin-binding EGF from its precursor in the cell membrane and activation of the EGFR (66, 67, 68). In hepatic C9 cells Ang II-induced activation of endogenous AT1Rs mediates ERK1/2 activation by a similar mechanism (65, 69, 70). In C9 cells, agonist activation of endogenous AT1R and EGFR induced the caveolin-dependent formation of a multireceptor complex containing both the AT1R and the transactivated EGFR. Conversely, activation of EGFR caused phosphorylation, internalization, and desensitization of AT1Rs, via a PLC, PI3K-, and PKC-mediated mechanism (71) (Fig. 2).
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G PROTEIN-INDEPENDENT EFFECTS OF Ang II |
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). Arrestins were initially defined as molecules that bind to agonist-activated and phosphorylated GPCRs and cause homologous desensitization of these receptors by uncoupling them from their cognate G proteins. Like many other activated GPCRs, the AT1R binds ß-arrestins, which have clathrin and activator protein (AP) 2 adapter-binding domains that target them to endocytosis via clathrin-coated vesicles. Recent studies also demonstrated that ß-arrestins can also serve as scaffold proteins that organize signaling complexes for the activation of MAPKs (73, 74, 75, 76, 77). As in other GPCRs, activation of the AT1R leads to its phosphorylation by specific receptor kinases and binding of ß-arrestin, which uncouples it from G proteins and also serves as an adaptor for clathrin-mediated internalization. The AT1R belongs to the class B group of GPCRs, which bind ß-arrestins tightly due to the presence of a defined serine-threonine-leucine motif (78, 79) in their cytoplasmic tail (80, 81). This tight binding causes ß-arrestin to remain associated with the receptor during its internalization into endosomes (77). ß-Arrestins also serve as scaffolds to organize the formation of signaling complexes (74) that mediate AT1R-mediated activation of ERK and JNK3 MAPKs (77). Studies on a mutant AT1R that cannot couple to G proteins have shown that ß-arrestin2-mediated ERK activation can occur in the absence of G protein activation (82). Although the biological role of this process has not been defined, it could promote the phosphorylation of cytosolic targets of ERK, which when activated by this mechanism cannot enter the nucleus due to their association with the receptor, and are mostly located on the surface of endosomes (74). Studies on PAR2 receptors have shown that ß-arrestin-mediated MAPK activation does not stimulate DNA synthesis or cell division but can promote reorganization of the actin cytoskeleton and chemotaxis and may contribute to migration of cancer cells (83, 84, 85). A recent report suggests that Ang II can also induce chemotaxis by a G protein-independent, ß-arrestin-mediated mechanism (86). Additional studies are needed to elucidate the exact biological function(s) of these mechanisms after AT1R activation.
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Studies with another mutant AT1R, with deletions of Ala221 and Leu222, suggest that Ang II-induced, Cdc42-mediated activation of JNK can occur in a mutant receptor that is unable to activate Gq-mediated signaling (90, 91). The latter mutant was also able to stimulate Rap1-dependent ERK1/2 activation, but the physiological relevance of this pathway needs further studies because it was not activated by the wild-type receptor.
Activation of the Jak/Signal Transducer and Activator of Transcription (STAT) Signaling Pathway
In cardiac fibroblasts, aortic smooth muscle cells, and the kidney, Ang II-induced AT1R activation also stimulates the Jak/STAT pathway (Fig. 3), a characteristic signal transduction mechanism of cytokine receptors (5). This pathway has been linked to several of the pathophysiological effects of Ang II, including cardiac hypertrophy, dysfunction and heart failure (92), vascular inflammation and smooth muscle cell growth (93), and renal tubulointerstitial fibrosis (94). Jak/STAT activation also provides a positive feedback pathway by up-regulating angiotensinogen formation, leading to increased production of Ang II (95). The ability of cyclical stretch to induce matrix metalloproteinase expression in cardiac myocytes is also mediated by the Ang II-Jak/STAT pathway (96).
However, Ang II exerts both direct and indirect actions on the Jak/STAT pathway because AT1R activation also causes the release of IL-6 and other cytokines that stimulate this pathway. Although Ang II clearly serves as a proinflammatory mediator by acting on VSMCs and endothelial cells, and also by directly activating macrophages and other immune cells, the exact role of Ang II-induced activation of the Jak/STAT pathway in immune cells has not yet been defined.
Janus kinases (Jaks) are cytoplasmic tyrosine kinases initially identified as essential components of interferon receptor signaling (97). The Jak family includes three ubiquitously expressed members (Jak1, Jak2, and Tyk2) and Jak3, which is expressed primarily in hematopoetic cells. During cytokine stimulation, activation of Jak kinases leads to tyrosine phosphorylation and activation of STAT proteins, which dimerize and translocate to the nucleus to regulate the expression of target genes. The Jak/STAT signal transduction pathway can also be activated by GPCRs, and Ang II-mediated activation of STAT1–3, STAT5, and STAT6 has been reported (92, 98, 99). The exact roles of the individual STAT proteins are currently under investigation, and it appears that activation of STAT1 and STAT3 in cardiac cells exerts opposing biological actions by inducing 1) apoptosis, proinflammatory, and antiproliferative mechanisms; and 2) cardioprotective, mostly antiinflammatory and growth-stimulatory responses, respectively (92). Interestingly, most of these GPCRs, including receptors for Ang II, thrombin, chemokines, and platelet activating factor, are found on the surface of blood cells, where the Jak/STAT pathway has a major role in cytokine signal transduction. In VSMCs, the AT1R activates Jak2 via its association with a YIPP motif in the C-terminal tail of the receptor (49). The same motif is required for PLC- activation (100) and possibly for EGFR transactivation (101), and optimal binding of the AT1R (102). Studies in VSMCs have suggested that Ang II-induced tyrosine phosphorylation of Jak2 is G protein mediated, because it is dependent on increased cytoplasmic Ca2+ concentration and activation of PKC
(103). However, other studies with mutant receptors indicate that the AT1R can also activate the Jak/STAT pathway via G protein-independent mechanisms. Substitutions of five carboxyl-terminal tyrosine residues (Tyr292, Tyr302, Tyr312, Tyr319, and Tyr339) with phenylalanine, and substitution of Asp74 with glutamic acid, led to mutant AT1Rs that were unable to activate Gq, but could induce tyrosine phosphorylation and activation of Jak2 and its effector, STAT1 (104). Such activation of the Jak/STAT pathway could stimulate cell growth in the absence of Gq-mediated signaling (104). These data suggest that the AT1R can regulate intranuclear targets via the Jak/STAT pathway in the absence of G protein activation.
AT1R-Associated Proteins
The C-terminal cytoplasmic domain of GPCRs binds to a variety of intracellular proteins involved in receptor signaling, desensitization, and endocytosis. These include proteins such as GRKs and ß-arrestins, and receptor-specific proteins that bind to individual receptors or classes thereof. Those associated with the AT1R include PLC- and Jak proteins (49, 100), and novel proteins found by two-hybrid studies, including AT1R-associated protein (ATRAP) (105), Ang II receptor-associated protein 1 (ARAP-1) (106), EP24.15 (EC 3.4.24.15, thimet oligopeptidase) (107), and GLP, a GDP/GTP exchange factor-like protein (108). PLC- and Jak2 kinase [the latter possibly via Src homology phosphatase 2 (SHP-2) as an adaptor] have been found to associate with the YIPP sequence in the proximal region of the cytoplasmic domain (49, 100). ATRAP is an 18-kDa protein with three N-terminal transmembrane domains and interacts specifically with the C-terminal region of the AT1R (105). Endogenous and transfected ATRAP is located in the plasma membrane and within intracellular compartments including endoplasmic reticulum, Golgi, and endocytic vesicles (109). Furthermore, it is associated with the AT1R at both sites and appears to influence receptor endocytosis and function in diverse cell types. In cardiomyocytes, overexpression of ATRAP reduces cell surface AT1Rs, as well as Ang II-induced signaling and protein synthesis (110). ARAP-1 is a less well-characterized 57-kDa protein that may have a role in AT1R recycling to the plasma membrane (106).
An interesting and recently identified AT1R-associated protein is the novel 531-residue protein termed GLP, which stimulates hypertrophic responses when expressed in VSMCs and immortalized renal proximal tubule cells (108). The AT1R also associates with caveolin in smooth muscle cells, and this has been proposed to coordinate Ang II-induced signaling (111). Other studies have suggested that a caveolin-binding-like motif in the receptor’s cytoplasmic tail has an important role in the recycling of internalized AT1Rs to the plasma membrane (112). Recently, caveolin was found to be essential for Ang II-stimulated targeting of the AT1R into lipid rafts and activation of Rac1, and subsequent ROS-mediated hypertrophic responses in VSMCs (113).
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MODULATION OF AT1R FUNCTION BY HOMO- AND HETERODIMERIZATION |
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Agonist-independent homodimerization of transiently expressed AT1Rs has been detected by bioluminescence resonance energy transfer studies in transfected COS-7 cells, in which dimerization of the receptor occurs during its early biosynthesis before cell surface expression (116, 117). This is consistent with a role of this process in targeting receptors to the cell surface, as reported for other GPCRs. Studies on the coexpression of mutant AT1Rs suggest that this process may have biological significance for the function of cell surface receptors. Also, the coexpression of two mutant AT1Rs with impaired Ang II binding affinity rescues the ligand binding activity of the native receptor (118). The coexpression of binding (K199A) and G protein coupling-impaired mutant (D125E/R126E/Y127A/M134A) receptors with the wild-type AT1 receptor has a dominant-negative inhibitory action on its inositol phosphate response, without affecting its agonist-induced ß-arrestin and ERK activation (117). These data are compatible with the hypothesis that AT1Rs, like rhodopsin, can exist and function as dimers.
Recently, a new mechanism and biological function for AT1R dimerization has been reported in monocytes, where cross-linking of AT1R dimers by factor XIIIA transglutaminase appears to promote enhanced signaling and desensitization in vitro and in vivo (119). Elevated levels of cross-linked AT1Rs were observed in hypertensive patients, and a role of this process in the onset of atherosclerosis was proposed (119). Additional studies are needed to confirm the general relevance of these interesting findings.
The AT1R also forms heterodimers with the AT2R, and with the B2 bradykinin and ß-adrenergic receptors (120, 121, 122). Studies on the AT1R and other GPCRs indicate that, although heterodimerization can occur between several pairs of GPCRs, it is a specific process that involves well-defined partners (115). Heterodimerization of the AT1 and B2 receptors results in increased activation of Gq and Gi proteins and changes the endocytic pathway of the receptor (120). Although the biological function of this process is not yet known, heterodimerization of these receptors was reported to occur in vivo in platelets and omental vessels and has been proposed to be related to the increased AT1R responsiveness in preeclampsia (123). Association of the AT1R with the AT2R inhibits its signaling activity and function (121), which may contribute to the antagonistic biological effects of the AT2R on the AT1R (see above). However, it is not yet known whether the AT2R has similar inhibitory actions on other GPCRs. This process merits further investigation, because it may explain the previously observed ligand-independent actions of the AT2R on apoptosis (124). However, it is important to emphasize that not all of the anti-AT1R effects of the AT2R are attributable to the heterodimerization of these receptors. The AT2R can activate phosphotyrosine and serine/threonine phosphatases that inhibit MAPK activation, including MAPK phosphatase 1, protein phosphatase 2A, and SH2 domain-containing phosphatase-1 (10). It also stimulates the formation of bradykinin, NO, and cGMP, which cause vasodilation and exert antiproliferative actions in vessels and attenuate AT1R-mediated cardiac fibrosis and antinatriuretic effects in the kidney (20).
Recent studies on the physical association of AT1Rs and ß-adrenergic receptors have clearly demonstrated the potential pharmacological importance of GPCR heterodimerization, because interactions between the two receptors lead to the formation of molecular complexes in which blockade of the AT1R can inhibit signaling via ß-adrenergic receptors, and vice versa (122). Elucidation of the functional role and biological significance of the heterodimerization of AT1 with other GPCRs will require further investigation.
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Ang II-STIMULATED ROS GENERATION |
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ROS are produced as intermediates during redox processes that lead to the formation of water from oxygen. The many enzymes that catalyze such reactions include reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, nitric oxide synthases, xanthine oxidases, cytochrome P450s, mitochondrial respiratory chain enzymes, cyclooxygenases, lipoxygenases, and myeloperoxidases. NADPH oxidases are the major source of ROS in leukocytes, but some of the latter enzymes are also important in endothelial cells. The univalent reduction of molecular oxygen by a free electron leads to the formation of highly reactive but unstable superoxide, which only crosses membranes via anion channels. Some of superoxide’s effects result from its rapid reaction with vasodilator and cytoprotective nitric oxide to form highly reactive, toxic peroxinitrate. Superoxide is also converted by dismutases to hydrogen peroxide, which is membrane permeant and has a longer half-life than superoxide. This facilitates the spread of the oxidative signal or stress to other intracellular compartments. Hydrogen peroxide is scavenged by catalase and gluthatione peroxidase but can also be reduced to generate highly active hydroxyl radicals in the presence of metal-containing molecules (e.g. Fe2+) (126).
ROS exert their effects by oxidative modification of target molecules, e.g. oxidation of cysteine residues. This is exemplified by the inactivation of protein tyrosine phosphatases by oxidation of conserved cysteines in their active sites, which catalyze the hydrolysis of protein phosphotyrosine residues by forming a cysteinyl-phosphate intermediate (127). This shifts the balance between tyrosine kinases and phosphatases toward phosphorylation, potentiating the effects of Ang II and other humoral mediators that activate tyrosine kinases. ROS also targets numerous other signaling molecules, including ion channels, MAPKs (JNK, p38 MAPK, ERK5), matrix metalloproteinases, and transcription factors [nuclear factor-B (NFB), AP1] (126). In VSMCs, Ang II-induced activation of JNK, p38 MAPK, and ERK5 is ROS-sensitive, whereas ERK1/2 activation is not (125). By these mechanisms, ROS promotes the activation of numerous signaling pathways, some of which further stimulate ROS production and exert positive actions on pathways that amplify signaling. Under physiological conditions, these pathways are controlled by antioxidant mechanisms, but in pathological states such excessive ROS accumulation causes progressive cell damage.
Ang II induces ROS generation in many target tissues, and this appears to result largely from activation of NADPH oxidases. This occurs in VSMCs (128), endothelial cells (129, 130), cardiac cells (131), phagocytic cells (132), and fibroblasts (133). NADPH oxidase was first characterized in neutrophils, where it consists of two membrane-associated (gp91phox and p22phox) and four cytosolic (p40phox, p47phox, p67phox and Rac) subunits (126). In phagocytic cells, phosphorylation of p47phox initiates the assembly of the enzyme complex and causes the respiratory burst by binding to p22phox. Homologs of gp91phox (Nox2) are also present in nonphagocytic cells. Nonphagocytic NADPH oxidases include NOX1, NOX3–5, and two related proteins, DUOX1 and DUOX2, which have NADPH oxidase and peroxidase activity. NOX1 is regulated by NOX organizer 1 and NOX activator 1, which are homologous to p47phoxand p67phox, suggesting common principles in the assembly of phagocytic and nonphagocytic NADPH oxidases (134). Endothelial cells express NOX1, 2, 4, and 5, whereas VSMCs express NOX1, 4, and 5, and possibly NOX2 (125).
During the rapid and biphasic stimulation of ROS production by Ang II in rat aortic VSMCs, an initial PKC-dependent response during the first minute of agonist stimulation is followed by a slower second increase caused by transactivation of the EGFR (135) (Fig. 4). In accordance with the role of EGFR transactivation during the sustained phase of Ang II-induced ROS generation in VSMCs, inhibition of EGFR transactivation (by AG1478 or Src kinase inhibitors) or of EGFR-mediated ROS production (by inhibitors of Rac or PI 3-kinase) diminishes the sustained increase of ROS generation. Thus, AT1R-mediated activation of Rac can mediate ROS formation, in addition to its previously reported role in JNK activation (136, 137). Studies by Touyz et al. (138, 139) have also implicated Src kinase and phospholipase D in Ang II-induced ROS generation in VSMCs. However, it is uncertain whether these effects are mediated directly by the AT1R or result from transactivation of the EGFR or other growth factor receptors. The NADPH oxidase that mediates Ang II-induced ROS formation in VSMCs has yet to be identified.
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Ang II also stimulates the formation of ROS in human neutrophils, leading to activation of MAPKs that may contribute to the pathophysiological effects of the RAS during inflammation (132). Such Ang II-induced ROS formation is mediated by activation of the AT1R via a PI 3-kinase-dependent pathway, which is also facilitated by ERK1/2 and p38 MAPKs. Ang II also increases cytosolic Ca2+ levels and stimulates calcineurin and NFB activities. Although it is clear that Ang II can regulate the function of neutrophils, further studies are required to elucidate the role(s) of AT1R-mediated second messenger generation in ROS formation and to identify the mechanisms caused by autocrine/paracrine effects of IL-6 and other cytokines released by Ang II.
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Ang II AND CARDIOVASCULAR DISEASE |
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Endothelial dysfunction and damage can lead to leukocyte adherence, platelet activation, and vasoconstriction, and ultimately to vascular inflammation, atherosclerosis, and thrombosis. Circulating hormones and other bioactive factors, as well as the biophysical actions of hemodynamic forces, influence the endothelium. Endothelial cells release vasoactive products that affect vasodilatation and vasoconstriction, including nitric oxide (NO), Ang II, and endothelin, both potent vasoconstrictor agents. NO not only modulates vasodilatation and vascular tone but also affects leukocyte adhesion, platelet aggregation, and apoptosis. Clinical evaluation of endothelial function is provided by measurement of brachial artery flow-mediated vasodilatation during reactive hyperemia after a short period of vascular obstruction. Ang II-induced endothelial dysfunction promotes the cytokine-mediated recruitment of monocyte/macrophages into the vascular wall by monocyte chemoattractant protein-1 and vascular cell adhesion and vascular cell adhesion molecule 1. Both ACE inhibitors and AT1R antagonists have been shown to reduce endothelial dysfunction. The deleterious effects of Ang II are corrected by treatment with PPAR activators, in part by reducing inflammatory responses mediated by NFB and AP1 (146).
Animal studies have shown that the deleterious actions of Ang II in the cardiovascular system can also be caused by treatment with aldosterone and other corticosteroids that activate the mineralocorticoid receptor (MR), particularly at levels inappropriate for the prevailing sodium status. Both hormones have been implicated in the development of endothelial dysfunction, cardiac and vascular inflammation and remodeling, and cardiac arrhythmias. The harmful effects of aldosterone in the heart were discovered in studies on the perivascular and interstitial and ventricular fibrosis associated with arterial hypertension and cardiac failure (147). The finding that Ang II and aldosterone regulate the accumulation of collagen within the rat heart led to the concept that ventricular remodeling in cardiac failure could be treated by aldosterone receptor antagonists (148). This prediction was confirmed by the RALES and EPHESUS trials, in which blockade of the MR by spironolactone and epleronone, respectively, significantly reduced mortality in patients with advanced cardiac failure (149, 150). Aldosterone has also been found to potentiate Ang II-stimulated proliferation of rat VSMCs, possibly by increasing expression of the AT1R (151, 152). Recent studies on human coronary artery VSMCs have shown that Ang II and aldosterone regulate the transcription of genes involved in vascular fibrosis, inflammation, and calcification (153).
The aldosterone responsible for these adverse cardiovascular actions has been reported to be produced in the heart and to be independent of Ang II, as indicated by its synthesis after myocardial infarction in AT1R-deficient mice (154, 155). However, in transgenic rats overexpressing human renin and angiotensinogen genes, and treated with dexamethasone and salt, the adrenal is the main source of aldosterone in the heart (156). This is consistent with the observation that angiotensin and salt-induced vascular inflammation in the heart is prevented by adrenalectomy as well as by epleronone (157). These and other findings have demonstrated that aldosterone is a critical factor in the development of Ang II-induced cardiovascular damage (158). Recent studies have shown that activation of the MR can mediate nongenomic actions of its ligands in the heart and blood vessels, and that glucocorticoid occupancy of the MR may also contribute to the development of cardiac inflammatory and fibrotic changes (159, 160).
Ang II also increases the expression of vascular endothelial growth factor (VEGF) and its receptors in aortic wall cells and causes inflammatory changes with monocyte infiltration and vascular remodeling (161). It potentiates VEGF-induced proliferation of endothelial progenitor cells and promotes their VEGF-dependent formation into networks, by increasing the expression of VEGF receptor kinase domain-containing receptors (162) as well as angiopoietin 2 and multiple growth factors (fibroblast growth factor, EGF, platelet-derived growth factor, TGF-ß). AT1R blockade inhibits angiogenesis and neoplastic cell growth factors in several tumor model systems. Ang II induces VEGF expression in stromal cells of implanted tumors in mice via PKC, AP-1, and NFB, and this appears to be a major determinant of tumor growth and the associated angiogenesis (163). All components of the RAS are expressed in the eye and are abundant in blood vessels and the ganglia cell layer of the developing rat retina (164). Studies in a murine model of ischemic retinopathy showed expression of AT1R and increased levels of ICAM-I and VEGF, changes that were reduced by treatment with telmisartan, with no effect on physiological neovascularization, by inhibition of the underlying inflammatory changes (165).
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Ang II, ADIPOSE TISSUE, AND METABOLIC REGULATION |
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Adipose tissue is now recognized as an active endocrine organ that secretes a variety of hormones, cytokines, and other bioactive proteins. Adipocytes also produce angiotensinogen and other RAS components, including Ang II, and express AT1R. In addition to Ang II, they produce leptin, adiponectin, TNF
, PPAR-, and plasminogen activator inhibitor-1, or PAI-1 (171). Some of these factors act as regulators of insulin secretion and may contribute to the development of insulin resistance. Adiponectin is a potent agent in this regard, and its production is decreased in obesity-related disorders including hypertension, coronary artery disease, the metabolic syndrome, and type 2 DM (172). In adiponectin-deficient mice, pressure overload causes cardiac hypertrophy with increased ERK signaling, whereas increased adiponectin levels exert antihypertrophic actions. Adiponectin also inhibits Ang II-induced apoptosis in human endothelial cells, probably by promoting the association between endothelial nitric oxide synthase and HSP90, and thereby increasing NO production (173).
Further evidence for a role of Ang II in obesity-related disorders was provided by the beneficial effects of AT1R blockade on insulin sensitivity in adipocytes, with increased production of adiponectin and reduced expression of 11ß-hydroxysteroid dehydrogenase type 1 (174). In hypertensive hypercholesterolemic patients, combined treatment with a statin and an AT1R antagonist (losartan) improved endothelial function and inflammatory markers more effectively than either agent alone (175). Furthermore, blockade of the RAS increased circulating levels of adiponectin and improved insulin sensitivity in patients with essential hypertension (176). The ability of adiponectin to protect against vascular injury may result from its inhibitory effect on an early step in the development of atherosclerosis, the attachment of monocytes to endothelial cells (177). ACE inhibition has also been found to restore endothelial function by reducing oxidative stress, enhancing insulin resistance, in part by increasing adiponectin levels, and improving cardiac remodeling and renal function (178).
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Ang II AND DIABETES |
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Increased activity of the RAS has also been implicated in the development of diabetic cardiomyopathy and diabetic nephropathy, a major cause of end-stage renal disease. The renoprotective action of ACE inhibitors (182) is also conferred by AT1R blockers, which are recommended for treatment of type 2 DM with macroalbuminuria, nephropathy, or renal insufficiency (183). Because the prevalence of DM is reaching epidemic levels, the potential importance of these agents in the prevention and control of diabetic complications is obvious. The hyperinsulinemia in patients with type 2 diabetes could stimulate Ang II production and proliferation in VSMCs via MAPK signaling and may thereby contribute to the progression of atherosclerosis (184). Blockade of the RAS could thus reduce vascular and tissue Ang II levels and VSMC growth, with beneficial effects on cardiovascular function. In nondiabetic renal disease, combined treatment with ACE inhibition and AT1R blockade was more effective than either agent alone (185). Interestingly, RAS blockade and receptor antagonists are also effective in reducing glomerular damage and renal chemokine expression in MRL/1pr mice with lupus-like autoimmune renal disease (186).
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Ang II AND CANCER |
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Recent studies in bladder cancer cells revealed that a dominant negative ADAM17 mutant prevented Ang II-induced cleavage of heparin-binding EGF and activation of the EGFR, and inhibited cell proliferation (199). Also, ADAM15 was found to selectively mediate LPA-induced transactivation, indicating that individual ADAMs mediate specific GPCR-induced transactivation of the EGFR. Observations in AT1A receptor-deficient mice have shown the importance of the host Ang II/AT1R system in determining tumor-related growth and angiogenesis, by promoting increased macrophage infiltration and fibroblast activity in the tumor stroma, and enhanced production of VEGF (163, 200). The ability of Ang II to stimulate ROS formation may also contribute to its proneoplastic actions, because increased ROS levels induced by other GPCRs in human carcinoma cells have been found to mediate transactivation of the protooncogene MET receptor, which has been implicated in tumor invasion and metastasis (201).
These findings, derived from in vitro studies and animal models, are consistent with the clinical observation that treatment with ACE inhibitors reduced the relative risk of cancer in a retrospective study of hypertensive Scottish patients, in comparison with patients receiving other antihypertensive agents (202). More recently, an ACE polymorphism has been found to be associated with the development of endometrial carcinoma and its onset in younger women (203). These and other studies have emphasized the role of the RAS in neoplasia (204), and the potential therapeutic value of ACE inhibition and AT1R blockade, perhaps in combination with antiinflammatory agents and specific vaccines, in the treatment of cancer patients (205).
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CONCLUSIONS |
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ABSTRACT
INTRODUCTION
