Modulation of signaling in vascular cells by reactive oxygen species (ROS) affects many aspects of cellular function, including growth, migration, and contraction. NADPH oxidases, important sources of ROS, regulate many growth-specific and migration-related signaling pathways. Identifying the precise intracellular targets of ROS enhances understanding of their role in cardiovascular physiology and pathophysiology.
An accumulating body of literature indicates that small amounts of reactive oxygen species (ROS), such as superoxide (O2·) and hydrogen peroxide (H2O2), play important roles as signaling molecules for normal cellular functions in vascular cells. ROS modulate growth factor signaling, regulate transcription factors that control gene expression associated with proliferation, differentiation, and apoptosis, and influence GTPase-dependent cytoskeletal rearrangements. ROS production and removal are tightly regulated, enabling these second messengers to have a transient mode of action and to act on adjacent targets. Under normal physiological conditions, ROS destruction by antioxidant enzymes is sufficient to maintain a controlled activation of signaling cascades. In contrast, in vascular disease, the production of ROS in excess of endogenous antioxidant capacity leads to oxidative stress, which in turn results in abnormal physiological responses. In this review, we will discuss ROS modulation of signaling cascades in vascular smooth muscle cells (VSMCs), focusing on the vasoactive hormone angiotensin II (Ang II).
Production and Metabolism of ROS
Virtually all types of vascular cells produce O2· and H2O2, two of the most significant ROS in the vessel wall (42). Production of O2· occurs via the one-electron reduction of molecular oxygen, a reaction that is mediated by several enzymatic systems and the mitochondria. Among the enzymes capable of O2· production are xanthine oxidase (XO) and the NADPH oxidases. The latter enzymes are important physiological ROS producers in the vasculature. O2· itself may modulate vascular signaling cascades, but more importantly it produces other reactive species. The reaction of O2· with nitric oxide (NO·) inactivates NO·, a primary regulator of vascular relaxation and vasodilation, causing the generation of peroxynitrite, which itself has deleterious consequences. Inactivation of NO· contributes to endothelial dysfunction in a number of diseases (106). Second, dismutation of O2· by superoxide dismutase (SOD) produces H2O2, a more stable ROS. H2O2 is implicated in the regulation of signaling pathways leading to vascular smooth muscle growth, contraction, migration, and inflammation (70). The enzymes primarily responsible for elimination of H2O2 in cells are catalase, glutathione peroxidase (GPx), and peroxiredoxins (Prx), which convert H2O2 into water and other secondary metabolites (96). Peroxiredoxins reduce H2O2 and alkylhydroperoxides with the use of reducing equivalents provided by thioredoxins (Trx) (95, 96, 124), whereas GPxs utilize glutathione (124). H2O2 can also be metabolized by myeloperoxidase, a heme enzyme produced by macrophages that converts H2O2 into reactive nitrogen and reactive chlorine (20, 51). These reactive species can attack both LDL and HDL, thus enhancing cholesterol intake and reducing cholesterol efflux, respectively, and contributing to plaque formation (84). Hormones such as Ang II, tumor necrosis factor (TNF)-α, platelet-derived growth factor (PDGF), and interleukin (IL)-1β alter the expression and activity of these antioxidant enzymes (23, 36, 102, 120). The net result is that ROS levels reflect a balance between regulated production and removal of these molecules. The oxidative state of vascular cells thus dynamically shifts between a pro-oxidant state and a more reductive environment.
Neutrophil NADPH oxidase
The vascular NADPH oxidases are closely related to the neutrophil NADPH oxidase, a multimeric enzyme that produces ROS essential to phagocyte microbicidal functions, allowing for the release of ROS from phagosomes into the extracellular space to kill invading bacteria (27, 28). The phagocytic NADPH oxidase consists of five subunits. Together, the integral membrane proteins gp91phox and p22phox comprise the cytochrome b558 membrane complex, which is localized in submembranous vesicles and in the plasma membrane. gp91phox is the catalytic subunit of the phagocyte cytochrome and binds one FAD and two heme molecules. p47phox, p67phox, and the small molecular weight G-protein Rac are cytosolically localized and do not interact with the cytochrome in resting cells. On agonist stimulation, p47phox is phosphorylated on eight to nine serines by either proline-directed kinases or protein kinase C (PKC) (32). S359 and S370 are phosphorylated first, and then S379 acquires a phosphate exposing an SH3 binding site, which interacts with the proline-rich region of p22phox and facilitates translocation to the membrane (62). Finally, S303 and S304 are phosphorylated, leading to full catalytic activity (58). p67phox then binds to the translocated p47phox, providing a binding site for activated Rac and forming the functional enzyme capable of producing O2·.
Vascular NADPH oxidases: new players and new insights
It was recently discovered that gp91phox is but one member of a new family of homologous proteins referred to as Nox proteins (for NADPH oxidase) (8, 67). Most vascular cells express multiple Nox proteins, including gp91phox (also known as Nox2) as well as Nox1, Nox4, and Nox5 (22) (FIGURE 1⇓). Nox1 is expressed at low levels in VSMCs from conduit vessels, endothelial cells, and fibroblasts (40). Nox4 is highly expressed in all cells in the vascular wall, especially in the cerebral vasculature (82), whereas gp91phox is predominantly expressed in VSMCs from resistance arteries and endothelial and adventitial cells. Nox5 is found in human, but not rodent, VSMCs and endothelial cells. The expression of multiple Nox homologs in individual cells and their differential regulation and subcellular localization (53) suggests that these oxidases serve distinct functions (70).
Although Nox1, Nox4, and Nox5 mediate ROS formation, until very recently, their interactions with regulatory proteins were poorly defined. It is now clear that both Nox1 and Nox4 directly interact with p22phox and that p22phox stabilizes the enzyme structure (3, 46, 64). Mutations in the proline-rich region of p22phox inhibit Nox1, but not Nox4, activity (64), suggesting the importance of this region of p22phox for binding Nox1 regulatory subunits. The significance of p47phox and its requirement for Nox1 oxidase activity in response to Ang II and PDGF is clearly established, both in vivo and in vitro (41, 47, 71, 73). Nox1 can interact with the phagocyte subunits p22phox (46), p47phox, and p67phox, as well as two novel homologs of p47phox and p67phox, known as Noxo1 and Noxa1, respectively (7, 107). Of importance, the classical subunits and novel homologs are capable of interacting with one another to regulate NADPH oxidase activity (107), suggesting that each tissue may have functional NADPH oxidase complexes of unique molecular compositions. The Noxo1 and Noxa1 homologs do, however, exhibit differences from the phagocytic NADPH oxidase subunits. These divergences include the lack of an autoinhibitory domain and the PKC phosphorylation sites in Noxo1 that are found in p47phox, as well as the absence of the first p67phox Src homology domain and the presence of a hydrophobic stretch in Noxa1 (7). Recently, Noxa1 was described as a critical component responsible for vascular oxidase activity (4). Thus, based on available current evidence, Nox1 activity in VSMCs appears to be regulated by p47phox and Noxa1 (4, 14, 69, 73), as well as the small molecular weight G-protein Rac (101). In contrast, neither Noxa1, p47phox, Noxo1, p67phox, nor Rac are required for ROS generation by Nox4 (78). Although Nox4 may not require the previously identified cytosolic oxidase regulatory subunits for its enzymatic activity, whether it has unique regulatory partners remains to be determined. In contrast to the other Noxes, Nox5 does not require p22phox and is activated directly by calcium (9, 10).
Cellular localization of NADPH oxidases in VSMCs
The subcellular localization of Nox1 and Nox4 is distinct in VSMCs, which may reflect specific cellular functions. Hilenski et al. (53) found by immunocytochemistry that Nox1 co-localizes with caveolin in punctate patches on the surface and along the cellular margins, whereas Nox4 is found in the nucleus and co-localized with vinculin in the focal adhesions. Interestingly, p22phox co-localizes in similar patterns with Nox1 and Nox4 (3, 53).
Activity of NADPH oxidases
Nox1 and Nox4 are also differentially responsive to agonists. Stimuli that promote the formation of O2· in aortic VSMCs, such as Ang II, PDGF, and phorbol 12-myristate 13-acetate (PMA), all induce upregulation of nox1 mRNA (71, 104). In contrast, agonists increase Nox2 expression in VSMCs from resistance arteries (112). Nox4 is regulated by transforming growth factor (TGF)- β 1 and serum withdrawal, but its regulation by stimuli such as Ang II, serum, and PDGF is controversial (25, 71, 103, 112). Increased expression of these catalytic subunits is universally accompanied by increased NADPH oxidase activity (70).
In addition to regulation of expression, vasoactive agonists such as PDGF and PGF-2α also acutely activate NADPH oxidases (71, 79, 94). Nox1 is activated within minutes of Ang II and PDGF stimulation (71, 101, 129), and treatment of VSMCs with nox1 antisense inhibits O2· production and ROS-dependent activation of signaling molecules (71) by these agonists. It has not been determined what agonists stimulate Nox4 activity in VSMCs; however, TGF- β1 is a potent activator of Nox4 in human cardiac fibroblasts (25) and pulmonary artery SMCs (103). Agonists such as thrombin and TNF-α also activate NADPH oxidase-dependent O2· production in VSMCs, but the responsible catalytic subunit has not been determined (26).
AT-1 Receptor-Mediated NADPH Oxidase Activation
Multiple signaling pathways contribute to activation of vascular NADPH oxidases over a broad time frame, beginning with receptor stimulation. Of these, the best-studied in VSMCs are those by which Ang II activates Nox1 and Nox2. Ang II not only stimulates NADPH oxidase-dependent O2· production in VSMCs but in endothelial cells and adventitial fibroblasts as well (88, 132).
Initial stimulation of the AT1 receptor (AT1R) by Ang II results in rapid activation of phospholipase C (PLC) enzymes via the α-subunit of heterotrimeric G proteins (Gα) or tyrosine phosphorylation (117). PLC produces inositol trisphosphate (IP3) and diacylglycerol (DAG). DAG and the release of calcium from the sarcoplasmic reticulum by IP3 activate PKC, leading to phosphorylation of p47phox and initial activation of the NADPH oxidase (101, 112) (FIGURE 2⇓). PKC activation is also mediated by phospholipase D (PLD), which forms phosphatidic acid (PA) and serves as a source for DAG (68, 69, 114). In addition, calcium activates phospholipase A2 (PLA2), which cleaves phosphatidylcholine to produce lysophosphatidylcholine (LPC) and arachidonic acid (AA). Both LPC and AA enhance NADPH oxidase activity (130).
Nearly simultaneously with phospholipase stimulation, Ang II also activates Src kinase via the βγ-subunits of the heterotrimeric Gq protein (117). c-Src transactivates the epidermal growth factor receptor (EGFR) by phosphorylating two specific tyrosines on the cytoplasmic domain of the receptor: Tyr 1068 and Tyr 1173 (118). c-Src also activates c-Abl, which can then phosphorylate the Rac guanine nucleotide exchange factor (GEF) Sos (son-of-sevenless) (133). The EGFR in turn activates PI3-kinase, which produces phosphatidylinositol 3,4,5-trisphosphate (PIP3), a lipid important in recruiting PH-domain-containing proteins to the membrane. PIP3 is involved in Rac1-mediated activation of the NADPH oxidase, presumably via stimulation of a Rac-GEF such as Sos. Rac activation is required for Nox1 stimulation by Ang II (101). In a self-sustained activation loop, ROS released by the oxidase activate c-Src and thus further increase NADPH oxidase-derived ROS production, enabling prolonged ROS generation in response to Ang II stimulation. In physiological conditions, this increased intracellular ROS production does not alter the cell’s redox state due to the presence of large reserves of reducing agents such as glutathione and endogenous antioxidant defense mechanisms such as SOD, catalase, and peroxidases (35).
Modulation of Signaling by ROS
ROS modulate signaling cascades and thus the ultimate physiological response by modifying specific residues or heme groups on proteins. Each ROS has a specific mechanism of action. The most well-known target of O2· is NO· (see above). O2· also reacts with iron-sulfur (Fe-S) centers of heme-containing molecules, resulting in alteration of their function. One example is inactivation of aconitase, leading to inhibition of mitochondrial function (38). H2O2, in turn, can oxidize Cys residues in proteins, allowing for ROS-specific effects. H2O2 appears to specifically target Cys residues in the thiolate form, oxidizing them to cysteine sulfenic acid (Cys-SOH), and depending on the presence of Trx and oxygen, ultimately to sulfinic (Cys-SO2H) and sulfonic (Cys-SO3H) forms. The first two of these reactions are reversible, whereas the latter is irreversible (95). Reversible modifications have been shown to regulate the activity of protein tyrosine phosphatases, peroxiredoxins (see above), and transcription factors (e.g., OxyR) (34, 127). These are likely to be but a few of the known substrates for H2O2 and O2·, and further investigations in the future should illuminate others.
ROS-Dependent Signal Transduction in VSMC Growth and Hypertrophy
De-differentiated VSMCs are prominent in cardiovascular diseases such as hypertension, atherosclerosis, and restenosis after balloon angioplasty. These altered VSMCs exhibit phenotypic changes that support growth, and ROS production is intimately involved in many processes leading to both proliferation and hypertrophy. Not only are receptor-associated signals modulated by ROS, but those further downstream that regulate translation initiation and gene expression are modified as well. The short half-life of O2· limits the likelihood of this ROS serving a paracrine role in the vasculature; however, its more stable metabolite, H2O2, is a more viable candidate for this role due to its ability to diffuse more freely across the vascular wall (6). Therefore, H2O2 produced by cells other than VSMCs, including endothelial cells and adventitial fibroblasts, can also potentially modulate hypertrophic and proliferative pathways (6). The regulation of growth-related signaling by ROS is known for multiple growth factors and hormones, but the two best studied are Ang II and PDGF. Therefore, Ang II will be discussed as a prototypical hypertrophic agent, whereas PDGF will be used as a model proliferative agent.
In VSMCs, hypertrophic agents such as Ang II activate both ROS-sensitive and ROS-insensitive signaling pathways, leading to translation initiation and increased protein synthesis (FIGURE 2A⇑). Ang II-induced VSMC hypertrophy is inhibitable by the flavin oxidase inhibitor diphenylene iodonium (DPI), p22phox antisense, and catalase (41, 119, 129), thus implicating NADPH oxidase-derived H2O2 in the growth response.
An early ROS-dependent signal activated by Ang II is c-Src (101). On parallel Ca2+-mediated activation of proline-rich tyrosine kinase 2 (Pyk2), c-Src and Pyk2 form a complex that binds to and phosphorylates the EGFR and c-Abl (118), creating binding sites for downstream signaling molecules. Ang II also activates two adapter molecules, p130Cas, which forms a complex between Pyk2 and PI3-K (97), and Gab1, which binds to the p85 subunit of PI3-K and translocates it to the membrane (44). One consequence of PI3-K activation is translocation of Akt to the membrane, where 3-phosphoinositide-dependent protein kinase-1 (PDK-1) phosphorylates Akt at Thr308 (109). In parallel, Nox-dependent generation of ROS triggers the S-glutathiolation of Ras on Cys118 and a subsequent increase in Ras activity leading to p38MAPK phosphorylation (2). Akt and p38MAPK exist in a complex, and on stimulation of p38MAPK (115), MAPKAPK-2 is recruited to this complex and phosphorylated, where it phosphorylates Akt on Ser473 (108), leading to full Akt activation. Another mechanism contributing to ROS sensitivity of p38MAPK is the regulation of its upstream kinase, apoptosis signal-regulating kinase 1 (ASK1) by Trx. Trx, in a reduced form, binds to and inhibits ASK1-mediated signaling by promoting ASK1 ubiquitination and degradation. ROS activate ASK1 in part by oxidizing Trx and forming an intramolecular disulfide bond between C32 and C35, thereby releasing Trx from ASK1 (43).
Simultaneously with p38MAPK/Akt activation, Ang II stimulates extracellular signal-regulated kinase 1/2 (ERK1/2) via the association of Shc (Src homology complex) with the EGFR and recruitment of Grb2 (growth factor receptor-bound protein-2) and Sos to activate Raf-1 (12). The redox sensitivity of this step is controversial, but most laboratories find that ERK activation is not dependent on ROS (45, 97, 113). Both ROS-sensitive and ROS-insensitive signaling pathways are required for hypertrophy and converge at the level of translation initiation. The rate-limiting step in the initiation of protein translation is phosphorylation of PHAS-1 and subsequent release of eukaryotic initiation factor-4E (eIF4E). PHAS-1 is phosphorylated on multiple sites, two of which are Thr70 and Ser65. Before Ser65 phosphorylation, PHAS-1 must be phosphorylated on Thr70 (50, 98), which involves the non-ROS-sensitive ERK1/2 and PI3-kinase signaling pathways (98). Subsequently, Ser65 is phosphorylated by Akt in a manner inhibited by antioxidants (116). ROS also contribute to Ser65 phosphorylation by inhibiting protein phosphatase 2A (PP2A) (92, 98). These reports implicate PHAS-1 as a single molecule that serves to integrate ROS-sensitive and ROS-insensitive signaling pathways in VSMCs.
PDGF, a potent proliferative signal for VSMCs(FIGURE 3B⇓), exerts its effects by binding to a receptor tyrosine kinase, the PDGF receptor (PDGFR). This receptor consists of two single transmembrane domain proteins that dimerize to form a functional receptor, leading to the cross-phosphorylation of tyrosine sites in the cytoplasmic tails. PDGFR phosphorylation occurs independently of ROS (126), but Prx II, a cellular peroxidase that eliminates endogenous H2O2 produced in response to growth factors such as PDGF, negatively regulates PDGF signaling by interacting with the PDGFR and suppressing protein tyrosine phosphatase inactivation, suggesting a role for ROS in PDGF signaling (124). Notably, cellular deficiencies in Prx II result in increased production of H2O2, as well as enhanced activation of the PDGFR and PLC (124).
PDGF increases ROS production by activating a Nox1-based oxidase that requires p47phox (71, 73, 79). PDGFR activation stimulates the association of Shc/Grb2/Sos/Ras with the receptor (90), where Ras is activated by oxidative modification (105), leading to activation of Raf kinase, an upstream kinase for ERK1/2 (12, 13). ERK1/2 phosphorylation is blocked by catalase, implicating H2O2 in this response (105). ERK1/2 has multiple targets, but is quickly translocated to the nucleus, where it activates transcription factors such as AP-1 (Fos/Jun heterodimer) and Elk-1, leading to upregulation of growth-related genes. Of importance, AP-1 activity is critically dependent on redox factor-1 (Ref-1), a multifunctional DNA base excision repair and redox regulation enzyme (54). PDGF activates Ref-1 by changing its redox status, thereby promoting the reduction of Cys154 of Fos and Cys272 of Jun and enhancing their activity (1). Depletion of Ref-1 levels by antisense oligonucleotides inhibits PDGF-induced G0/G1 to S phase progression through the cell cycle in VSMCs (49).
An additional pathway that is crucial for PDGF-induced proliferation of VSMCs is initiated by the production of PIP3. PDGF increases PIP3 by two mechanisms: activation of PI3-kinase and oxidative inactivation of PIP3 phosphatase, known as phosphatase and tensin homolog (PTEN). PIP3 mediates Akt activation, which in turn activates the p70S6K/mTOR pathway leading to enhanced translation of cyclin D1 (83). Importantly, p70S6K is activated by H2O2, indicating that it does lie in a redox-sensitive pathway (95). Akt also phosphorylates and inactivates glycogen synthase kinase-3 (GSK-3) (24). GSK-3 functions to phosphorylate cyclin D1 on Thr286, thus targeting it for degradation and inhibiting cell cycle progression (15). Inactivation of GSK-3 by Akt therefore decreases cyclin D1 turnover and prolongs its half-life, leading to cell cycle progression.
ROS-mediated cell cycle regulation has been reviewed recently (16, 19, 99). It should be noted that VSMC growth is a balance between proliferation and apoptosis. Studies show that cell cycle arrest occurs in response to high levels of ROS (59, 100), whereas moderate levels of ROS function to coordinate a number of crucial events that promote growth. Previous studies in VSMCs indicate that H2O2 both promotes growth and induces apoptosis (75, 91). A major target of moderate ROS stimulation is cyclin D1, which is stabilized through the activation of the PI3-K/Akt pathway (32, 63). Furthermore, Nox1 expression leads to increased cyclin D1 transcription via redox-sensitive AP-1, NF-κB, and ETS proteins (16). Cyclin D1-CDK4/6 phosphorylates retinoblastoma protein (Rb), releasing E2F, a transcription factor that is normally complexed with Rb and mediates gene expression and progression to late G1 phase.
Conversely, high levels of ROS lead to cell cycle arrest. ROS-sensitive targets include p21, a CDK inhibitor, and p53, a tumor suppressor gene that regulates apoptosis and is a negative regulator of the cell cycle. H2O2 induces an increase in p53 mRNA expression (29). The protein p21, an effector of p53, binds to and inhibits cyclin D-CDK4 complexes and results in cell cycle arrest in G1. Deshpande et al. (29) also demonstrated that H2O2 increases p21 mRNA and protein levels significantly in VSMC, leading to arrest at the G0/G1 transition.
ROS-Dependent Signal Transduction in VSMC Migration
VSMC migration is critical for a number of physiological processes, including embryonic development, inflammatory responses, and the response to injury (55). ROS have been implicated in cell migration in VSMCs stimulated with several agonists. Phenylephrine- and vascular endothelial growth factor (VEGF)-induced VSMC migration are blocked by treatment with catalase or the antioxidants N-acetyl cysteine (NAC) and pyrrolidine dithiocarbamate (85, 122). Thrombin-stimulated migration is inhibited by the NADPH oxidase inhibitors apocynin and DPI, implicating NADPH oxidase-derived ROS in this response (123). Wang et al (122) showed that VEGF treatment of human VSMC increased intracellular ROS, NF-κB activation, and IL-6 expression and migration, all of which were blocked by antioxidants. Monocyte chemotactic protein-1 (MCP-1) acts as a chemoattractant for VSMCs, and MCP-1-stimulated migration requires both ROS production and ERK1/2 activation in a positive activation loop, which may contribute to the atherogenic effects of MCP-1 (76).
Recently, a number of signal transduction pathways were identified as regulators of actin polymerization and contractility in cells, two processes integral to cell migration. However, regulation of the actin cytoskele-ton with respect to VSMC migration is not well understood. To begin to understand the role of ROS in VSMC motility, migration pathways delineated in fibroblasts will be used as a model (FIGURE 4⇓).
The process of cell migration can be essentially divided into distinct processes (55). Migration begins with an initial protrusion or extension of the plasma membrane at the front/leading edge of the cell. The formation of these protrusions is promoted by the polymerization of a network of cytoskeletal actin filaments and is stabilized through the formation of adhesive complexes within the protrusion. Next, as the cell migrates, the focal complexes at the front of the cell strengthen into larger, more organized focal adhesions that serve as points of traction over which the cell body moves. For the cell to make forward progression, it must release its rear adhesions to allow a net forward displacement.
Extension of plasma membrane at the cell’s leading edge
To establish a leading edge, the cell must first sense a gradient and establish polarity, which in turn allows the plasma membrane to extend lamellipodia in the direction of eventual movement (72, 86). In vascular injury, a gradient is established by platelet release of PDGF, such that VSMCs migrate toward the lumen (21, 72). Generation of filopodia and/or lamellipodia is driven by actin assembly in other cell types, which in turn is regulated by Rac (86). Rac stimulates actin polymerization via several mechanisms, including nucleation of new actin filaments (77, 81), extension of existing filaments (48), and activation of LIM kinase, which phosphorylates and inactivates the actin capping protein cofilin, thus preventing actin depolymerization (5, 128). Although the ROS sensitivity of actin dynamics is not fully understood in VSMCs, in endothelial cells, inhibition of NADPH oxidases or the use of SOD mimetics reduces actin monomer incorporation at the fast-growing barbed ends of filaments (37). Glutathiolation of G-actin increases the rate of actin polymerization (121). Because Rac plays multiple roles in fibroblast and platelet movement, the specific importance of Rac-mediated NADPH oxidase activity in lamellipodia formation in VSMCs requires further investigation.
Formation of focal complexes and adhesions for cell movement
As described above, stabilization of the lamellipodia at the leading edge of the cell occurs through the formation of adhesive complexes (48). These complexes are regions of the plasma membrane where integrin receptors, actin filaments, and other proteins cluster together. Integrins and related proteins become activated and bind to the extracellular matrix components (86). These become incorporated into focal complexes that contain focal adhesion kinase (FAK), vinculin, and paxillin (131). As the cell migrates, the focal complexes at the front of the cell grow and strengthen into larger, more organized focal adhesions that serve as points of traction over which the cell body moves (86). The mechanisms regulating the conversion of focal complexes to focal adhesions are unclear but appear to require Rho (86). In aorta, ROS activate the Rho pathway (61), but the relationship of this activation to focal adhesions is vague. However, recent work shows that Rho-GTPases contain a conserved redox-sensitive motif that appears to be critical for guanine nucleotide dissociation, raising the possibility of direct regulation of Rho-GTPases by ROS (52).
ROS-generating hormones such as Ang II have profound effects both on focal adhesion formation and on enzymes/cytoskeletal-associated components found within these cell-matrix signaling domains (18). Ang II-induced H2O2 production activates c-Src (118), which is critically important for focal adhesion formation (60). c-Src phosphorylates FAK and Pyk2, the latter of which forms a binding site for PDK1. Phosphorylation of PDK1 on Tyr9 is required for Ang II-stimulated focal adhesion formation (109). Notably, PDK1 is also tyrosine phosphorylated by H2O2 (109). In addition, PDK1 phosphorylates the p21-associated kinase (PAK), which regulates cytoskeletal rearrangement. Downstream targets of PDK1 may also include FAK and Pyk2, which form multiprotein complexes with paxillin, tensin, and p130Cas (31, 74, 87). Rac, Src, PDK1, and PAK are all necessary for PDGF-induced migration of VSMC (126).
Generation of force and release of rear adhesions for forward progression
The next step in migration is the generation of force to initiate forward progression of the cell. It has been proposed that the GTPases Cdc42 and Rho regulate contractile forces by influencing myosin light chain (MLC) phosphorylation. MLC phosphorylation promotes dimerization and interaction with actin to drive contraction (55). Rho kinase, activated by Rho and ROS (61), functions to inhibit the myosin phosphatase, allowing MLCs to remain in a contractile (phosphorylated) state (55).
The release of rear adhesions and the continued forward progression of the migrating cell depends on focal adhesion turnover, which is critical for the continued reorganization of adhesion contacts during cell migration (72, 93). Cells lacking Src family kinases, FAK, and calpain exhibit migratory defects that appear to reflect an inhibition of focal adhesion turnover; however, focal adhesion formation is not impaired (36, 56, 66). Little is known about the process of focal adhesion turnover in VSMCs.
ROS-Dependent Signaling to Contraction
O2· regulates vasomotor tone mainly through reaction with and subsequent inactivation of NO· in vessels with intact endothelium (89). The direct effects of ROS on VSMC contraction, however, remain unclear. H2O2 induces both vasorelaxation and vascoconstriction, depending on the vascular bed and the preconstrictor agent. H2O2 prompts vasorelaxation of pulmonary (17), coronary (57), and cerebral (82) arteries. ROS also impair both NO-mediated responses and neurovascular coupling (65), as well as vasodilation mediated by activation of potassium channels in the cerebral and coronary vasculature (11, 33). The sensitivity of potassium channel-dependent relaxation to O2· and H2O2 depends on the gating properties of the channels, the structural components that are present, and the cell type (43). Although H2O2 is generally found to inhibit these channels, thus enhancing contraction (43), excitation of large conductance calcium-activated potassium channels by O2· has been reported to mediate relaxation (11). In the aorta, ROS are vaso-constrictive, and Ang II-induced aortic contraction is inhibited by catalase (111). ROS-induced calcium sensitization, specifically ROS generated by XO, is mediated through the activation of Rho and a subsequent increase in Rho kinase activity (61). As noted above, Rho kinase inhibits the myosin phosphatase, sustaining MLC phosphorylation. The RhoA/Rho kinase pathway partially mediates endothelin-1-induced contraction, and this is proposed to be via ROS; however, recent studies suggest that endothelin-1-induced contraction in veins is independent of H2O2 (110).
NADPH oxidase-mediated ROS generation clearly affects many aspects of VSMC function. In pathological conditions, excess ROS production or impaired antioxidant defenses lead to dysregulation of signaling, resulting in inappropriate growth and migration. Although many of the molecular studies have been made in cultured cells, transgenic and knockout animal models have confirmed that many of these observations hold true in vivo. For example, mice overexpressing the p22phox subunit of the NADPH oxidase selectively in VSMC show increased H2O2 and a potentiated aortic hypertrophic response to Ang II (125). Similar results were found utilizing transgenic mice that overexpress Nox1 selectively in VSMC (30). Conversely, in Nox1−/− animals, aortic hypertrophy is reduced (39). Although antioxidants inhibit neointimal formation (80), the specific role of NADPH oxidases in the VSMC migration and proliferation that accompanies restenosis remains to be investigated.
Conclusions and Future Directions
The past decade has afforded enormous progress in our understanding of the impact of ROS on VSMC function. The sources of ROS have been identified, and many of the signaling pathways that are modified by ROS have been studied; however, much remains to be discovered regarding the function of the individual Nox proteins in VSMCs. We also now require a better understanding of the regulation of Nox expression, how compartmentalization of ROS mediates specificity, and what protein modifications different ROS enable. Additional work is necessary to elucidate the role of ROS in VSMC migration and contraction. The development of new molecular tools and transgenic and knockout animals should greatly facilitate this work and should further our understanding of the critical pathophysiological roles of these newly defined signaling molecules.
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