Angiotensin II, NADPH Oxidase, and Redox Signaling in the Vasculature

Abstract

Significance: Angiotensin II (Ang II) influences the function of many cell types and regulates many organ systems, in large part through redox-sensitive processes. In the vascular system, Ang II is a potent vasoconstrictor and also promotes inflammation, hypertrophy, and fibrosis, which are important in vascular damage and remodeling in cardiovascular diseases. The diverse actions of Ang II are mediated via Ang II type 1 and Ang II type 2 receptors, which couple to various signaling molecules, including NADPH oxidase (Nox), which generates reactive oxygen species (ROS). ROS are now recognized as signaling molecules, critically placed in pathways activated by Ang II. Mechanisms linking Nox and Ang II are complex and not fully understood. Recent Advances: Ang II regulates vascular cell production of ROS through various recently characterized Noxs, including Nox1, Nox2, Nox4, and Nox5. Activation of these Noxs leads to ROS generation, which in turn influences many downstream signaling targets of Ang II, including MAP kinases, RhoA/Rho kinase, transcription factors, protein tyrosine phosphatases, and tyrosine kinases. Activation of these redox-sensitive pathways regulates vascular cell growth, inflammation, contraction, and senescence. Critical Issues: Although there is much evidence indicating a role for Nox/ROS in Ang II function, there is still a paucity of information on how Ang II exerts cell-specific effects through ROS and how Nox isoforms are differentially regulated by Ang II. Moreover, exact mechanisms whereby ROS induce oxidative modifications of signaling molecules mediating Ang II actions remain elusive. Future Directions: Future research should elucidate these issues to better understand the significance of Ang II and ROS in vascular (patho) biology. Antioxid. Redox Signal. 19, 1110–1120.

Introduction

Angiotensin II (Ang II), the major bioactive peptide of the renin–angiotensin system (RAS), plays a major role in the regulation of vascular function and structure. It is a multifunctional vasoactive peptide produced systemically and locally within the vascular wall. Ang II is a potent vasoconstrictor that also has mitogenic, proinflammatory, and profibrotic actions. These effects are elicited through myriad-signaling pathways, many of which involve reactive oxygen species (ROS), particularly superoxide anion (O₂^•−) and hydrogen peroxide (H₂O₂) (41, 47, 71). Under physiological conditions, ROS production is tightly controlled, and ROS play an important role as a signaling molecule in the control of endothelial function and vascular tone. In pathological conditions, when ROS bioavailability is increased (oxidative stress), Ang II signaling is altered, leading to endothelial dysfunction, vascular remodeling, and inflammation, important processes underlying vascular injury in cardiovascular disease.

Ang II exerts its diverse actions via two G-protein-coupled receptors, Ang II type 1 (AT₁R) and type 2 (AT₂R) receptors (27, 41, 47, 71). The AT₁R mediates most of the known actions of Ang II. The AT₂R is associated with antiproliferative, proapoptotic, and vasodilatory actions of Ang II and tends to counteract AT₁R effects. Signaling pathways elicited by Ang II/AT₁R involve interactions with several heterotrimeric G-proteins coupled to second messengers and cytosolic proteins, including phospholipase C (PLC), phospholipase A2 (PLA2), phospholipase D (PLD), and protein kinase C (PKC) (71). In addition, Ang II/AT₁R signals through activation of many receptor and nonreceptor tyrosine kinases and serine threonine kinases, important in cell growth, hypertrophy, and inflammation. Growing evidence indicates that many of the pathways through which Ang II signals involve activation of NADPH oxidase (Nox), which is a major source of ROS in vascular cells.

Nox and Ang II

Griendling and colleagues were among the first to demonstrate that Ang II activates vascular Nox (31, 75). Using rat aortic vascular smooth muscle cells (VSMCs), they showed that treatment of VSMCs with Ang II for 4–6 h caused a nearly threefold increase in intracellular O₂^•− formation as detected by the lucigenin assay (31). This, derived from activation of both NADPH and NADH oxidases (31, 75) and p22phox, was found to be obligatory in this process (75). The pathophysiological significance of these cell-based findings was confirmed in rat studies where Ang II-induced hypertension was associated with increased vascular generation of Nox-derived O₂^•− and endothelial dysfunction, effects that were blocked by losartan, an AT₁R blocker (59, 62). These early studies paved the way for the field relating to Ang II, oxidative stress, and hypertension.

Mechanisms whereby Ang II regulates Nox are complex and occur at the gene, transcriptional, and post-transcriptional levels and involve numerous intermediate signaling molecules (e.g., c-Src, PKC, receptor tyrosine kinases, protein disulfide isomerase, and polymerase delta-interacting protein 2 [Poldip2]) and scaffolding proteins/platforms (e.g., lipid rafts, caveolae, actin, caveolin, and cortactin) (23, 36, 46). More recently, it has been shown that Nox-derived ROS, in turn, regulate Ang II receptors (58). Hence, there is a feed-forward system where Ang II regulates ROS-generating Nox, which regulates expression and activation of AT₁R (Fig. 1).

FIG. 1.

Role of Nox-derived ROS in Ang II-mediated effects in vascular cells. Ang II binds to its AT₁R, which couples to heterometric Gq proteins, to activate PLC, leading to generation of IP3 and DAG, resulting in increased [Ca²⁺]_i that triggers phosphorylation of MLC₂₀ and stimulation of contraction. Ang II also induces contraction through the RhoA/Rho-kinase pathway that increases Ca²⁺ sensitivity by inhibiting MLCP. Ang II/AT₁R stimulates Nox-derived ROS formation, which regulates MAPKs, tyrosine kinases, PTPs, and transcription factors. Formation of ROS through Ang II/AT₁R further regulates AT₁R through a feed-forward mechanism (dashed lines). Ang II, angiotensin II; AT₁R, Ang II type 1 receptor; [Ca²⁺]_i, intracellular free-calcium concentration; DAG, diacylglycerol; IP3, inositol-3-phosphate; MAPKs, mitogen-activated protein kinases; MLCK, myosin light-chain kinase; MLCP, myosin light-chain phosphatase; Nox, NADPH oxidase; PLC, phospholipase C; PTPs, protein tyrosine phosphatases; ROS, reactive oxygen species.

Nox Family Oxidases

Nox was originally considered to be expressed only in phagocytic cells involved in host defense and innate immunity. The prototypical reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase, Nox2, that is found in phagocytes, comprises five components, (phox for phagocyte oxidase), p47phox, p67phox, p40phox, p22phox, and gp91phox (4), and the small G protein Rac 1/2. It is now clear that there is a family of NAD(P)H oxidases, called Noxs, the primary function of which is to catalyze the transfer of electrons from NADPH to molecular oxygen via the catalytic subunit (Nox). The mammalian Nox family comprises seven members: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1, and Duox2 (28, 67). All are transmembrane proteins that have a core catalytic subunit (Nox) and numerous regulatory subunits. Unlike phagocytic NAD(P)H oxidase, which is activated only upon stimulation and which generates O₂^•− in a burst-like manner extracellularly, nonphagocytic Noxs are constitutively active, produce O₂^•− intracellularly in a slow and sustained fashion, and act as intracellular signaling molecules, influencing not only transcription factors but also other molecules involved in inflammation, cell growth, and contraction, such as mitogen-activated protein (MAP) kinases, tyrosine kinases, and protein phosphatases (15, 16).

Nox1, Nox2, Nox4, and Nox5 have been identified in cardiovascular and renal tissue (39). Although all Noxs have the same function to generate ROS, mechanisms of activation, subunit requirements, and intracellular distributions vary between isoforms (Fig. 2). Nox1 and Nox2 are constitutively associated with p22phox, and the full activation of Nox1/p22phox and Nox2/p22phox requires interaction with other cytosolic subunits, including p47phox (or its homolog NADPH organizer 1 [NoxO1]), p67phox (or homolog NADPH activator 1 [NoxA1]), and Rac (21, 42, 65). Nox4 is constitutively active and associates with p22phox, but does not require other oxidase subunits for its activation, whereas Nox5 functions independently of any Nox subunits and is activated in a calcium–calmodulin-dependent manner (21, 65).

FIG. 2.

Nox activation and differences between Nox homologs. Nox comprises a complex of membrane and cytosolic subunits. Nox2 is the classical prototype. Membrane proteins are p22phox and the Nox subunit, and form a noncovalent heterodimer. These proteins possess the electron transport apparatus and may act as a physical conduit for the electron transfer that occurs across the membrane. The cytosolic proteins (p47phox, p67phox, NoxO1, NoxA1, and rac 1/2) are cofactors for enzymatic activity and are used to initiate and/or regulate electron transfer. To Nox2 be activated, p47phox is phosphorylated and translocates from the cytosol to the membrane with the other cytosolic subunits (p67phox and rac1/2). Nox1 can also be activated in a similar way to Nox2, but possesses other cytosolic subunits such as NoxO1 and NoxA1. Nox4 does not require any cytosolic subunit to be activated. Nox4 is constitutively active in cells, and its activity is controlled by Poldip2. On the other hand, Nox5 activation is not dependent on any subunit, but because of calcium-binding domains (EF hands), its activity is controlled by calcium and calmodulin. NoxA1, NADPH activator 1; NoxO1, NADPH organizer 1; Poldip2, polymerase (DNA-directed) delta-interacting protein 2.

Hyperactivation of Noxs leads to excessive ROS generation that disrupts redox networks, normally regulated by thiol-dependent antioxidant systems. This results in oxidative stress, triggering molecular processes, which, in the vasculature, contributes to vascular damage (Fig. 3). All vascular cell types, including endothelial cells, VSMCs, and adventitial cells (fibroblasts and adipocytes), are capable of producing ROS through Noxs. Noxs have been extensively reviewed (21, 37, 39, 65), and only an overview of recent developments related to Ang II is discussed here.

FIG. 3.

Ang II-stimulated redox-sensitive pathways that promote vascular remodeling. Generation of ROS by plasma membrane-associated Noxs and cytosolic Noxs in response to Ang II-AT₁R signaling leads to activation of multiple pathways that promote vascular injury and remodeling. e⁻, electron; NO, nitric oxide; SOD, superoxide dismutase.

Regulation of Noxs and Nox Subunits by Ang II

Ang II is functionally associated with Nox1, Nox2, and Nox5 and variably with Nox4 in the vasculature. The biological significance of different Noxs being activated by the same vasoactive agent still awaits clarification, but their differential tissue distribution, cellular localization, and subcellular compartmentalization probably play a major role in Nox-specific actions elicited by Ang II (5). Ang II-activated Nox1 appears to be important in VSMCs from large arteries, but Nox2 may be more important in small-resistance arteries, especially in humans (31, 68, 75). Nox4 abundance is greater in endothelial cells than in VSMCs, at least in basal conditions, although there is probably important cross-talk between Noxs in different cell types (Fig. 4). Regulation also seems to differ in pathological conditions. For example, while Ang II increased expression of all Noxs in VSMCs from normal Wistar Kyoto rats (WKY), only Nox1 was influenced in VSMCs from spontaneously hypertensive rats (SHR) (7, 19, 66). This may be important in vascular dysfunction associated with hypertension. In transgenic mice, in which Nox1 is overexpressed in vascular smooth muscle (SMCnox1), ROS production is enhanced in response to Ang II, causing endothelial nitric oxide synthase (NOS) uncoupling and decreased nitric oxide bioavailability, with resultant impaired vasorelaxation (19).

FIG. 4.

Distribution of vascular Noxs in endothelial and VSMCs and downstream signaling pathways regulated by Nox-derived ROS. In basal conditions, Nox4 expression is greater in endothelial cells than in VSMCs. Nox1 and Nox2 are expressed in VSMCs from small arteries, whereas Nox1, but not Nox2, is expressed in VSMCs from large arteries. These Noxs are upregulated in pathological conditions. The exact distribution of vascular Nox isoforms in vivo still awaits confirmation. VSMCs, vascular smooth muscle cells.

In vitro studies have indicated that Ang II is a potent stimulator of vascular Noxs (70). It induces activation (phosphorylation) of the oxidase subunits; it increases expression of Nox isoforms and Nox subunits (p47phox, p67phox, and p22phox); and it stimulates ROS production in cultured VSMCs and in intact arteries (76). Mechanisms linking Ang II to the enzyme and upstream signaling molecules modulating NAD(P)H oxidase in vascular cells have not been fully elucidated, but phospholipases, PKC, Src tyrosine kinases, phosphatidylinositol 3-kinase (PI3K), and Rac may be important (8, 64). Ang II stimulates Nox activity through various phospholipases, including PLC, PLA2, and PLD (8, 64). We showed that c-Src is critically placed between the AT₁R and Nox, where it stimulates phosphorylation of p47phox and activation of the oxidase (73). These processes involve cortactin interaction with actin, which may act as a scaffolding network for Nox subunit trafficking to the cell membrane to assemble the functionally active oxidase complex (72). Lipid rafts/caveolae play an important role in agonist-stimulated activation of vascular Noxs and may act as signaling platforms to integrate redox events (13). In pathological conditions, where the RAS is upregulated, for example, hypertension, diabetes, and atherosclerosis, activation of Noxs by Ang II is augmented, leading to increased ROS generation and oxidative stress (11, 45).

Nox-Derived ROS As Signaling Molecules in the Vasculature

The dynamics and chemical properties of ROS dictate the biological role that they will play. The more reactive the species, the shorter is the half-life and the more rapidly it will interact with other molecules. ROS can also be electrically charged or electrically neutral, hydrophobic, or hydrophilic. Such characteristics determine their ability to cross membranes and/or to move in the environment between aqueous and lipophilic environments.

ROS are produced as intermediates in reduction–oxidation (redox) reactions, leading from O₂ to H₂O. The major mechanism for ROS generation begins with the reduction of O₂ by the addition of one electron, to generate O₂^•−, considered the primary ROS. O₂^•− interacts with other molecules to produce secondary ROS, directly or through enzyme- or metal-catalyzed reactions (35). Reduction of O₂^•− leads to formation of H₂O₂, which is further converted to secondary metabolites such as highly reactive hydroxyl HO^•. Although the favored reaction is the generation of H₂O₂, O₂^•− also reacts with nitric oxide (NO^•) to form peroxinitrite (ONOO⁻), with transition metals, such as iron found in iron/sulfur center-containing proteins, or it may be protonated to the hydroperoxyl radical (H₂O^•). H₂O^• is particularly important in lipid peroxidation and atherogenesis.

Of the ROS generated in vascular cells, O₂^•− and H₂O₂ appear to be especially important. In biological systems, O₂^•− is short-lived owing to its rapid dismutation to H₂O₂. Dismutation can be spontaneous (rate constant=8×10⁴/mol/s) or enzymatic via superoxide dismutase (SOD) (rate constant=2×10⁹/mol/s) (26). Superoxide reacts with NO^• to form ONOO⁻ with a rate constant of 4–16×10⁹/mol/s (3), resulting in NO^• quenching and resultant decreased in NO^• bioavailability. Redox-regulated uncoupling of NOS also contributes to decreased NO^• and increased generation of O₂^•− (Fig. 5). In addition, oxidants react with FeS₄ or with protein thiols such as cysteine residues, an effect that is increased by metabolic stress (10). Although O₂^•− has the capacity to react with many molecules, the preferred reaction is dismutation to H₂O₂ because of the fast reaction rate of SOD. Three mammalian SOD isoforms have been identified: copper/zinc SOD (SOD1), manganese-containing mitochondrial SOD (Mn-SOD, SOD2), and extracellular SOD (EC-SOD, SOD3) (48). The major vascular SOD is EC-SOD. The negative charge on O₂^•− makes it unable to cross cellular membranes, except possibly through ion channels, such as chloride channel 3 (CLC-3). CLC-3 transports O₂^•− out of endosomes into the cytoplasm in endothelial cells and has been implicated to play an important role in VSMC regulation (51). H₂O₂ has a longer lifespan than O₂^•−, is relatively stable, and is easily diffusible within and between cells. The main source of H₂O₂ in vascular tissue is the dismutation of O₂^•−: 2O₂^•−+2H⁺ → H₂O₂+O₂.

FIG. 5.

Nox and eNOS by Ang II in endothelial cells. Ang II stimulates production of O₂^•− by Nox1, Nox2, and Nox5 through the AT₁R. Additionally, Ang II stimulates production of H₂O₂ directly through Nox4, and indirectly through the SOD-mediated conversion of O₂^•− produced by Nox1, 2, and 5. The O₂^•−-mediated oxidation and inactivation of the eNOS cofactor BH₄ promotes uncoupling of eNOS, leading to eNOS-mediated production of O₂^•−. Additionally, H₂O₂ inhibits DHFR, an enzyme that catalyzes the conversion of BH₂ to BH₄, which further reduces BH₄ bioavailability, leading to eNOS uncoupling. BH₂, dihydrobiopterin; BH₄, tetrahydrobiopterin; CaM, calmodulin; DHFR, dihydrofolate reductase; eNOS, endothelial nitric oxide synthase; Fe, eNOS heme domain; H₂O₂, hydrogen peroxide; O₂^•−, superoxide; p22, p22 phox (Nox subunit); p40, p40 phox (Nox subunit); p47, p47 phox (Nox subunit); p67, p67 phox (Nox subunit); Rac, Rho-like GTPase Rac.

H₂O₂ is tightly regulated by intracellular and extracellular enzymes, including catalase, glutathione peroxidase, thioredoxin, and other peroxyredoxins, which convert H₂O₂ to water and O₂ and other metabolites. Although both O₂^•− and H₂O₂ have been suggested to act as signaling molecules, it is mainly H₂O₂ that is considered a signaling molecule because of its relative stability, tight regulation, subcellular localization, and ability to react reversibly with cysteine residues (25).

ROS-specific effects are mediated in large part through the oxidative modification of thiols on cysteine residues within redox-sensitive target proteins (24). Of all the unique cysteine residues in the human genome, 20,000–40,000 are highly sensitive to oxidations (1). Oxidation of thiols leads to structural changes in target molecules that result in activation or inactivation of signaling proteins. For example, protein tyrosine phosphatases (PTPs) are inactivated by oxidation, leading to increased phosphorylation of downstream proteins. In addition to protein oxidation, ROS induce oxidative damage through lipid peroxidation of cellular membranes and DNA breaks (1). Through these mechanisms, O₂^•− and H₂O₂ influence many signaling molecules important in the regulation of vascular function, including MAP kinases, nonreceptor tyrosine kinases, receptor tyrosine kinases, PTPs, and redox-sensitive transcription factors (17, 40) (Fig. 6). Activation of these molecules participates in cell growth, migration, expression of proinflammatory genes, production of extracellular matrix proteins, and contraction, processes important in the regulation of vascular function and tone.

FIG. 6.

Ang II-mediated redox-sensitive growth signaling in VSMCs. In VSMCs, binding of Ang II to the AT₁R leads to increased ROS generation, in part, via activation of c-Src-induced Nox activation. ROS stimulate nonreceptor tyrosine kinases such as FAK, JAK and PI3K, and PLC/PKC as well as receptor tyrosine kinases, such as EGFR, IGFR, and PDGFR. Redox signaling in turn regulates downstream MEK cascades, leading to phosphorylation of MAPKs, which induce growth, apoptosis, differentiation, migration, and inflammation of VSMCs. Ang II/AT₁R also induces MMP-mediated extracellular release of HB-EGF, which then stimulates EGFR transactivation and ERK1/2 MAPK activation. EGFR, epidermal growth factor receptor; FAK, focal adhesion kinase; HB-EGF, heparin-binding epidermal growth factor; IGFR, insulin-like growth factor receptor; JAK, Janus-activated kinase; JNK, c-Jun NH2-terminal kinase; MEK, MAPK/ERK kinase; MMP, matrix metalloproteinase; p, phosphorylation; PDGFR, platelet-derived growth factor receptor; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLC, phospholipase C; STAT, signal transducers and activators of transcription.

The distinct chemical properties between O₂^•− and H₂O₂ and their different sites of distribution mean that different species of ROS activate diverse signaling pathways, which lead to divergent, and potentially opposing, biological responses. For example, in the vasculature, increased O₂^•− levels inactivate the vasodilator NO^•, leading to endothelial dysfunction and vasoconstriction (61), whereas H₂O₂ acts as a direct vasodilator in some vascular beds, including cerebral, coronary, and mesenteric arteries (54).

The specificity of signaling through ROS is related to many factors, including subcellular localization of Nox isoforms, binding of Noxs to scaffolding proteins, and compartmentalization within intracellular organelles, for example, the mitochondria, the nucleus, and the endoplasmic reticulum (ER). Nox2 is membrane-associated, whereas Nox4 has been identified in the mitochondria, the ER, and the nucleus. Mitochondrial Nox4 expression is increased in kidneys from diabetic rats (29, 30, 78). It has been proposed that localization of Nox4 to the mitochondria creates a short paracrine loop, whereby ROS production by mitochondrial Nox4 regulates or is regulated by ROS generated by the mitochondrial respiratory chain (3). Furthermore, Nox subunits or Nox isoforms may interact with other proteins to facilitate localized ROS generation.

Ang II Signaling and ROS in the Vasculature

Ang II mediates effects via complex intracellular signaling pathways that are stimulated after binding of the peptide to its cell-surface receptors. Both receptors play a role in regulating VSMC function, although they differ in their actions.

Redox-sensitive growth signaling by Ang II in vascular cells

Ang II stimulates cell growth through phosphorylation of tyrosine kinases, activation of MAP kinases, mobilization of intracellular calcium (Ca²⁺), and production of ROS (77) (Fig. 6). Ang II, via AT₁R, induces phosphorylation of multiple tyrosine kinases, including c-Src, Janus family kinases (JAK), focal adhesion kinase (FAK), protein tyrosine kinase 2 (Pyk2), p130Cas, and PI3K (56). c-Src, which regulates Nox, is a critically important kinase involved in trophic and contractile actions of Ang II (12). c-Src is upstream from many growth-signaling molecules, including PLC-γ, Pyk2, FAK, JAK, Shc, MAP kinases, and PI3K (12).

Of the many growth-signaling molecules, the MAP kinase family is best characterized (38). In VSMCs, Ang II activates all four of the major MAP kinases, extracellular signal-regulated kinases (ERK1/2), p38MAP kinase, c-Jun N-terminal kinases (JNK), and ERK5. ERK1/2, phosphorylated by MAP kinase kinase 1/2 (MEK1/2) (MAP/ERK kinase), is a key growth-signaling 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. Activation of these MAP kinases requires ROS and Nox activation, because Nox inhibitors and various antioxidants attenuate Ang II-induced activation of MAP kinases (22, 63, 69). MAP kinase phosphorylation may not be directly influenced by ROS, but rather by changes in activation of upstream PTPs. PTPs possess highly conserved cysteine residues that are highly sensitive to oxidation (53).

Ang II transactivation of receptor tyrosine kinases: role of ROS

Ang II also activates receptor tyrosine kinases, classically linked to growth-signaling pathways, 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 receptor, subtype β, and insulin-like 1 growth factor receptor (16) (Fig. 6). Mechanisms whereby Ang II-induces receptor tyrosine kinase transactivation include activation of Pyk2 and Src, metalloprotease-dependent shedding of heparin-binding EGF-like growth factor and ROS (50). In rat VSMCs, Ang II, through increased oxidative stress and activation of c-Src, transactivates EGF-R, which leads to MAP kinase activation and cell growth, an effect that is augmented in SHR (43).

Ang II signaling, RhoA/Rho kinase, and ROS

Activation of RhoA and its downstream target Rho-kinase is increasingly being recognized as an important mechanism of vasoconstriction by Ang II and accordingly has been implicated in the pathophysiology of hypertension and other vascular diseases (44, 57). RhoA, a member of the Rho family of small GTPase-binding proteins, is abundantly expressed in VSMCs and participates in vasoconstriction via phosphorylation of myosin light chain and sensitization of contractile proteins to Ca²⁺. RhoA/Rho-kinase also influences VSMC migration, a process that is redox sensitive, since apocynin, which inhibits Nox activity and ROS generation, blocked Ang II-stimulated effects (55). In the renal vasculature, alpha(2)-adrenoceptors potentiate renal vascular responses to Ang II. This interaction, which influences vascular resistance, involves RhoA/Rho-kinase, ROS, and Nox2, since Ang II effects were attenuated by Y27632 (Rho-kinase inhibitor), tempol (superoxide dismutase mimetic), and gp91ds-tat (Nox2 inhibitor) (34).

RhoA and Nox also regulate microparticle release from Ang II-stimulated endothelial cells (9). Microparticles are submicron fragments that arise from plasma membrane blebbing and subsequently shed from activated or apoptotic cells. They impair angiogenesis, promote oxidative stress, and influence vasodilation, and they themselves produce ROS (49). Studies in cultured endothelial cells and in Ang II-infused mice demonstrated that fasudil and apocynin, inhibitors of Rho kinase and Nox, respectively, blunted microparticle formation (6). These processes have been implicated in Ang II-mediated oxidative damage of the endothelium.

Ang II, transcription factors, inflammation, and ROS

Important downstream targets of signaling pathways are transcription factors, which are highly redox sensitive. Many transcription factors important in inflammation, cell growth, and fibrosis are activated by Ang II through mechanisms that involve Nox and ROS. Among transcription factors that are activated and sensitive to both Ang II and ROS, there is the activator protein 1 (AP-1), nuclear factor kappa B, cyclic AMP response element-binding protein (CREB), and hypoxia-inducible factor 1 (HIF-1) (6, 74). Mitochondrial-generated ROS have also been shown to be essential intermediates for HIF-1 activation in VSMCs (18, 60).

Transcription factors act as signal integrators regulating processes related to vascular inflammation. Responses to proinflammatory stimuli, such as Ang II and ROS, lead to activation of transcription factors important in the production of proinflammatory cytokines and interleukins. These processes in turn influence leukocyte adherence, chemotaxis, and the inflammatory response, which underlie vascular damage in cardiovascular disease.

Ang II signaling, ROS, and vascular cell senescence

In addition to regulating cell growth, Ang II influences other components of the life cycle of cells, including senescence. Senescence is defined as a form of irreversible arrest in cell proliferation and is associated with a characteristic gene and protein phenotype different to that of proliferating cells. Senescent cells characteristically express senescence-associated β- galactosidase and pShc-66 (1, 15). As cells reach the end of their lifespan, they undergo replicative senescence, driven by telomere dysfunction, a process that occurs with normal aging. In pathological conditions, such as hypertension and atherosclerosis, vascular cells undergo premature senescence, which can be triggered by Ang II and oxidative stress (52). Such processes contribute to vascular remodeling and early vascular aging. Clinical studies using angiotensin receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEis) demonstrated protective effects against age-related cardiovascular changes (52). In vitro studies supported these findings showing a link between Ang II stimulation and vascular cell senescence, an effect blocked by ARBs and ACEis (14). In AT₁R knockout mice, lifespan was prolonged by almost 30% compared to genetically matched wild-type controls (2, 20). This enhanced longevity was associated with improved cardiovascular morphology, reduced ROS production, attenuated mitochondrial loss, and enhanced expression of survival genes (nicotinamide phosphoribosyltransferase [Nampt] and sirtuin-3 [Sirt3]) (2).

Molecular mechanisms through which Ang II regulates vascular cell senescence seem to involve signaling pathways similar to those that control cell growth. In addition, ROS are emerging as important modulators of senescence (32). This is evidenced by studies demonstrating that H₂O₂ induces vascular cell senescence, that Nox and ROS production are increased in senescent cells, and that antioxidants inhibit Ang II-induced senescence (32, 33). ROS induce DNA damage, which is critically involved in Ang II-mediated redox-triggered cell senescence. Both telomere-dependent and telomere-independent mechanisms have been linked to Ang II-mediated senescence (32). Telomere-related effects influence replicative senescence, whereas telomere-independent processes may contribute to premature senescence. The net effect of Ang II may relate to the concentration and chronicity of Ang II stimulation.

Conclusions

Over the recent past, our views of Ang II have changed from being a simple vasoconstrictor to that of a complex pleiotropic factor, involved in vascular hypertrophy, fibrosis, inflammation, and aging. These effects are mediated through diverse signaling pathways involving PLC/PKC/Ca²⁺ mobilization, PLA2, PLD, MAP kinases, tyrosine kinases, proto-oncogene expression, RhoA/Rho-kinase, inflammation, and cell cycle modulation. Common to these pathways are ROS, derived in large part from vascular Noxs. Through increased generation of ROS and activation of redox-sensitive 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, structural remodeling, and premature aging. Targeting some of these molecular events with novel therapeutic strategies, possibly at the level of Nox-derived ROS, may regress or prevent arterial remodeling and aging and thereby provide important vascular protection in cardiovascular and age-related diseases.

Abbreviations Used

ACEi

angiotensin-converting enzyme inhibitor

Ang II

angiotensin II

AP-1

activator protein 1

ARB

angiotensin receptor blocker

AT₁R

Ang II type 1 receptors

AT₂R

Ang II type 2 receptors

BH₂

dihydrobiopterin

BH₄

tetrahydrobiopterin

[Ca²⁺]_i

intracellular free-calcium concentration

CaM

calmodulin

CIHR

Canadian Institute of Health Research

CLC-3

chloride channel 3

DAG

diacylglycerol

DHFR

dihydrofolate reductase

EC-SOD (SOD3)

extracellular superoxide dismutase

EGF

epidermal growth factor

eNOS

endothelial nitric oxide synthase

ER

endoplasmic reticulum

ERK1/2

extracellular signal-regulated kinase1/2

FAK

focal adhesion kinase

H₂O^•

hydroperoxyl radical

H₂O₂

hydrogen peroxide

HB-EGF

heparin-binding EGF-like growth factor

HIF-1

hypoxia-inducible factor 1

IGF-1

insulin-like 1 growth factor

IP3

inositol-3-phosphate

JAK

Janus family kinases

JNK

c-Jun N-terminal kinases

MAP

mitogen-activated protein

MAPK

mitogen-activated protein kinase

MEK1/2

MAP kinase kinase 1/2

MLC

myosin light chain

MLCP

myosin light-chain phosphatase

MLCK

myosin light-chain kinase

MMP

matrix metalloproteinase

Mn SOD (SOD2)

manganese-containing mitochondrial SOD

NAD(P)H

reduced nicotinamide adenine dinucleotide phosphate

Nampt

nicotinamide phosphoribosyltransferase

NFκB

nuclear factor kappa B

NO^•

nitric oxide

Nox

NADPH oxidase

NoxA1

NADPH activator 1

NoxO1

NADPH organizer 1

O₂^•−

superoxide anion

ONOO⁻

peroxinitrite

PDGFR

platelet-derived growth factor receptor

Phox

phagocyte oxidase

PI3K

phosphatidylinositol 3-kinase

PKC

protein kinase C

PLA2

phospholipase A2

PLC

phospholipase C

PLD

phospholipase D

Poldip2

polymerase delta-interacting protein 2

PTP

protein tyrosine phosphatases

Pyk2

protein tyrosine kinase 2

RAS

renin–angiotensin system

ROS

reactive oxygen species

SHR

spontaneously hypertensive rats

Sirt3

sirtuin-3

SMCnox1

smooth muscle cell Nox1

SOD

superoxide dismutase

SOD1

copper/zinc superoxide dismutase 1

STAT

signal transducers and activators of transcription

VSMCs

vascular smooth muscle cells

WKY

Wistar Kyoto rats

Acknowledgments

Studies performed by the author were supported by Grants 57786 and 44018 from the Canadian Institutes of Health Research (CIHR). R.M.T. was supported through a Canada Research Chair/Canadian Foundation for the Innovation award. A.C.M. was supported by a fellowship from the CIHR. D.B. was supported through a KRESCENT fellowship from the Kidney Foundation of Canada.