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Reactive oxygen species signaling in vascular smooth muscle cells

Roza E. Clempus, Kathy K. Griendling
DOI: http://dx.doi.org/10.1016/j.cardiores.2006.02.033 216-225 First published online: 15 July 2006


Reactive oxygen species (ROS) have been shown to function as important signaling molecules in the cardiovascular system. Vascular smooth muscle cells (VSMCs) contain several sources of ROS, among which the NADPH oxidases are predominant. In VSMCs, ROS mediate many pathophysiological processes, such as growth, migration, apoptosis and secretion of inflammatory cytokines, as well as physiological processes, such as differentiation, by direct and indirect effects at multiple signaling levels. Therefore, it becomes critical to understand the different roles ROS play in the physiology and pathophysiology of VSMCs.

  • Oxygen radicals
  • NADPH oxidase
  • Signal transduction
  • Smooth muscle

1. Introduction

Reactive oxygen species (ROS) are a class of oxygen-derived molecules, which have long been thought to have deleterious effects on cells. It is now well established that they can also act as second messengers, influencing discrete signal transduction pathways in various systems, including the cardiovascular system [1].

Vascular smooth muscle cells (VSMCs) form the medial layer of blood vessels and represent a dynamic component of the vasculature. In the normal media they have a differentiated, contractile phenotype. With pathological stimuli, they can undergo hypertrophy or adopt a “de-differentiated” phenotype, and synthesize excess extracellular matrix and inflammatory cytokines, divide and migrate towards the intima. ROS have been implicated in all of these responses.

2 Sources of ROS

VSMCs contain numerous sources of ROS, including the NADPH oxidases, xanthine oxidase, the mitochondrial respiratory chain, lipoxygenases and nitric oxide synthases [2]. In VSMCs, most investigators have focused on the mitochondria and NADPH oxidases as major sources of ROS [3-5].

2.1 Mitochondria

Mitochondria generate ROS as byproducts during ATP production via electron transfer through cytochrome c oxidases. Mitochondrial-derived ROS have been implicated in regulation of vasomotor tone (pulmonary artery vasoconstriction [6] and cerebral artery vasodilation [7]), and in the response to hypoxia, where mitochondria have been proposed to act as oxygen sensors [6,8]. The role of mitochondria in vascular biology has been thoroughly reviewed, and will not be discussed further here [5].

2.2 NADPH oxidases

NADPH oxidases (Nox) are multiprotein complexes of various compositions depending on the cell type. The enzymes, originally described in phagocytes, consist of two membrane-bound subunits (the small subunit p22phox bound to the catalytic subunit Nox2) and potentially three cytosolic subunits, Rac1 (in non-phagocytes) or Rac2 (in phagocytes), p47phox and p67phox, which are recruited upon activation to the membrane-bound Nox/p22phox complex. All VSMCs express p22phox, while the catalytic subunit is Nox1, Nox2, Nox4 and/or Nox5. The distribution of catalytic subunits in VSMCs is tissue- and species-specific. Aortic SMCs express Nox1 and Nox4 in rodents, and also Nox5 in humans (Jay D. and Griendling K.K., 2004, unpublished). In contrast, VSMCs from human resistance arteries contain Nox2 and Nox4, but no Nox1 [9]. The expression of p47phox and rac1 is well documented, whereas that of p67phox is controversial [10]. A functional role for p47phox has been shown using VSMCs from p47phox knockout mice, in which agonist stimulation of ROS is decreased [11,12]. New homologues of the p47phox and p67phox subunits, NoxO1 and NoxA1, were recently described in other cell types, but their presence and potential functional role in VSMCs remains to be confirmed [13,14]. Nox-derived ROS have been implicated in functional responses of VSMCs such as hypertrophy, proliferation, migration and inflammation [15].

3 Nox regulation

The NADPH oxidases are activated by agonists acutely (minutes), and are also chronically (hours) upregulated to further enhance ROS production. These two phases are differentially regulated.

3.1 Nox activation

The classic mechanism of activation of the phagocytic NADPH oxidase involves stimulus-induced activation and translocation of p47phox, p67phox and Rac to the membrane. These proteins then interact with the other two subunits, p22phox and Nox2, resulting in production of superoxide by the sequential transfer of two electrons from NADPH to molecular oxygen. In non-phagocytic cells, this paradigm has also been demonstrated, although the intimate mechanisms, the upstream players and the intracellular compartments where activation occurs are cell- and agonist-specific.

In contrast to the phagocytic oxidase, which is inactive in resting neutrophils, the vascular oxidases have basal activity as well as agonist-stimulated activity [16]. Nox4 is constitutively active in VSMCs [17], and stimulation with PMA, arachidonic acid or calcium ionophore does not further increase its activity [18]. It is, however, activated by TGF-β in fibroblasts [19]. In contrast, Nox1 and Nox2 are activated in response to PMA via protein kinase C (PKC)-dependent recruitment of p47phox to the plasma membrane (see below) [9,20]. Additionally, one report suggested that NoxA1 is important for Nox1-dependent release of ROS in VSMCs [21]. All Nox enzymes appear to require p22phox for activity, except for Nox5 [22,23], which is activated directly by calcium.

3.1.1 Rac1

Rac1 is critical for Nox1 and Nox2 activation. In rat aortic SMCs, Ang II-induced ROS production occurs in two phases: a low magnitude burst that peaks in seconds, and a greater, sustained release that lasts for hours [24]. The initial burst of superoxide appears to be critical for the subsequent, long-term release of ROS in response to Ang II. Recently, our understanding of NADPH oxidase activation was facilitated by elucidation of proximal Ang II signaling in VSMCs [25,26] (Fig. 1).

Fig. 1

Pathways of NADPH oxidase activation in response to Ang II in VSMCs. See text for details. Final proof for proteins with dotted line has yet to be obtained.

Ang II receptor (AT1-R) signaling is strictly compartmentalized to microdomains in the plasma membrane called lipid rafts. After ligand binding, AT1-R is tethered to caveolin-1, which moves the receptor into lipid rafts/caveolae and also recruits and transactivates the epidermal growth factor receptor (EGFR) [27]. EGFR transactivation mediates many ROS-sensitive downstream pathways, including activation of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway, which is required for Ang II-induced protein synthesis in VSMCs. Eventually, EGFRs migrate with tyrosine-phosphorylated caveolin to focal adhesions. All of these steps have been demonstrated to require NADPH oxidase-mediated release of ROS [28].

The mechanism of rapid ROS release is still unclear, but it is known that it requires PKC-dependent activation of p47phox (see below) [29]. ROS then activate the non-receptor tyrosine kinase c-Src, which subsequently orchestrates the signaling in lipid rafts. Inhibition of c-Src mitigates most downstream ROS-sensitive signaling and long-term activation of the NADPH oxidase. To maintain NADPH oxidase activity, the small G protein Rac1 must be activated by a Rac-guanine nucleotide exchange factor (GEF) and translocated to caveolae in a manner that requires active remodeling of the cytoskeleton [30]. How Src recruits and activates Rac1 is unclear. However, Ushio-Fukai et al. [31] recently demonstrated that within 1min of Ang II stimulation, c-Src binds to and activates c-Abl. c-Abl then tyrosine-phosphorylates SOS-1 (a RacGEF) and translocates Rac1 to lipid rafts, where it is activated by SOS-1 [27] and stimulates NADPH activity.

Although not directly addressed in these studies, Nox1 is the most likely candidate for the NADPH oxidase that mediates these events in rat aortic SMCs, because of its location in caveolae [32] and its requirement for Rac1 activation, and because depletion of Nox1 prevents Ang II-induced activation of the ROS-sensitive Akt and p38MAPK [20]. Other work has shown a requirement for PI3K in Nox1 activation, because PI3K inhibitors prevent Ang II-induced ROS release in VSMCs [24]. Furthermore, in Caco2 cells, Nox1 is recruited to EGFR and activated in response to EGF by binding to another Rac-GEF, β-PIX, which is activated via PI3K [33].

3.1.2 p47phox

Ang II also activates three classes of phospholipases (PL), PLC, PLD and PLA2 [34] that are capable of inducing NA(P)DH oxidase activation in VSMCs. PLA2 releases arachidonic acid and lysophospholipids in response to Ang II [35]. Arachidonic acid metabolites mediate Ang II-induced ROS production in rat aortic SMCs [36]. Also, exogenous lysophosphatidylcholine triggers rapid translocation of p47phox to the membrane, resulting in increased NADPH oxidase activity, ERK1/2 activation and growth [37]. PLCs produce IP3, which increases calcium influx from sarcoplasmic reticulum, and diacylglycerol (DAG), a potent PKC activator. PLD generates phosphatidic acid (PA), which can either directly bind to p47phox, increasing its membrane affinity [38], or can act indirectly through production of DAG. Of importance, addition of exogenous PA increases oxidase activity [3,39], and inhibition of endogenous PLD activity decreases ROS production by Ang II [39]. In VSMCs, PLD is the most potent activator of PKC [40], which is involved in the phosphorylation of p47phox. This triggers a conformational change in the autoinhibitory loop, exposing the Phox homology and SH3 domains and allowing them to interact with membrane phospholipids and p22phox [38]. Inhibition of PKC profoundly inhibits the early activation phase of NADPH oxidase in response to Ang II in VSMCs, while the later phase is only partially inhibited [24]. In SMC from resistance vessels, p47phox is phosphorylated and translocates to the membrane in response to short term treatment with Ang II [9]. Recent evidence suggests that the PKC-β isoform mediates oxidase activation, since antisense oligonucleotides and a PKC-β-specific inhibitor block Ang II-induced production of ROS in VSMCs [41].

Another mediator of p47phox phosphorylation and translocation to membranes in VSMCs is c-Src [29]. Since Src is also required for cytoskeletal reorganization, it may exert its effects on p47phox by modulating the actin cytoskeleton. Indeed, Touyz et al. [42,43] have shown that Ang II induces p47phox association with the actin cytoskeleton through interaction with cortactin (a predominant substrate of Src). This association is blocked by pre-treatment with cytochalasin B, which disrupts the actin cytoskeleton. Therefore, Src seems to be responsible for activation of both p47phox and Rac1 in VSMCs.

Many other stimuli were found to activate a p47phox-regulated NADPH oxidase in VSMCs, including PDGF [11] and phenylephrine [44], but the precise mechanisms are not yet elucidated.

3.2 Regulation of Nox expression

Agonists such as Ang II or PDGF induce Nox-dependent ROS release not only acutely for signaling events, but also over the long term. This effect is mediated by modulation of expression of Nox subunits. For example, prolonged treatment with Ang II, PDGF or serum upregulates Nox1 and downregulates Nox4 in rat aortic SMCs [20]. Both PDGF and PGF-2α upregulate Nox1 by the convergence of PKC-δ and EGFR/ERK/PI3K signaling pathways on ATF-1 transcription factor activation [45]. Conversely, Nox4 expression appears to be controlled by TGF-β1 in pulmonary artery SMCs [46] and cardiac fibroblasts [19]. Nox2 transcription is upregulated by Ang II in VSMC from small vessels [9]. Because all but Nox5 catalytic subunits require p22phox for activity, increased expression of one catalytic subunit would require excess or increased availability of p22phox. In neutrophils, p22phox interacts with Nox2 in the endoplasmic reticulum, a process that requires the incorporation of heme [47]. This interaction stabilizes the two proteins and prevents degradation via the cytosolic proteasome. Indeed, in VSMCs, expression of p22phox and Nox1 leads to increased levels of the complementary protein [22,23]. It is thus possible that induction of a Nox protein leads to a concomitant increase in p22phox simply via increased p22phox stability or processing.

A recent elegant study by Janiszewski et al. [48] suggested a novel role for protein disulfide isomerase (PDI) in the regulation of Nox activity. These authors showed that PDI associates with Nox1, Nox2 and Nox4, and that PDI inhibitors diminish basal and Ang II-stimulated oxidase activity. The mechanism by which PDI regulates NADPH oxidase activity is unclear, but could involve stabilization of the intermolecular complex, mediation of the redox regulation of NADPH oxidase activity (as shown for H2O2 [49]), or regulation of protein processing or trafficking between endoplasmic reticulum and the cell membrane.

4 Role of ROS in VSMC physiology and pathophysiology

ROS are involved in many of the physiological and pathophysiological processes of VSMCs: growth, migration, secretion of inflammatory cytokines, extracellular matrix and matrix metalloproteinases, contraction, differentiation, and death (apoptosis). In this review, we will concentrate on growth, differentiation and migration of VSMCs, as the other processes have been reviewed recently elsewhere [50].

4.1 ROS signaling related to growth and survival

Growth of VSMCs involves both hypertrophy and hyperplasia. Many growth-related signaling pathways are shared by multiple agonists; therefore, we will focus on Ang II as a prototypical hypertrophic agent and PDGF as a model for proliferative agonists (Fig. 2).

Fig. 2

ROS signaling related to growth of VSMCs. See text for details.

4.1.1 Angiotensin II Non-receptor and receptor tyrosine kinases

Many of the growth pathways triggered in response to Ang II are mediated by ROS. As noted above, after binding to AT1 receptors, Ang II stimulates acute production of ROS by NADPH oxidase and activation of c-Src. It is not known exactly how ROS affect Src activity in VSMCs. Src activity is modulated by kinases (Csk, Chk, PDGFR, ErbB2/HER2, PKC, PKA, CDK1) and phosphatases (PTP1B, SHP1, SHP2, CD45, PTP-BL), as well as through redox modification of cysteine residues [51]. Src signals to a variety of downstream effectors including EGFR, p85 (the regulatory subunit of PI3K), RasGAP, Shc, several integrin signaling proteins (tensin, vinculin, cortactin, talin, and paxillin), focal adhesion kinase, PLCγ, Janus kinase (JAK)-2 [51,52], phosphoinositide-dependent kinase-1 (PDK1), and c-Abl.

Transactivation of tyrosine kinase receptors by G protein-coupled receptors is a common pathway for further transmission of ROS-sensitive signals. Ang II transactivates the EGFR [28], IGF-1R [53], and PDGFR [54], of which EGFR and PDGFR transactivation are redox-sensitive. Ang II-mediated transactivation of PDGFR is through Shc [54], whereas EGFR transactivation appears to involve Src-mediated matrix-metalloproteinase (MMP)-activated release of the heparin-binding EGF-like growth factor (HB-EGF) [55]. The transactivated EGFR perpetuates the ROS production from Nox in response to Ang II in a feed-forward mechanism [24]. S-glutathiolation and activation of Ras

The precise mechanism by which ROS directly modify specific signaling proteins is an active area of investigation. Proteins containing cysteine residues in their active sites are potential candidates for H2O2, whereas those containing Fe–S centers are potential targets for superoxide [56]. The small GTPase Ras is one of these proteins, whose activation is dependent upon S-glutathiolation at Cys118 and is redox-sensitive in response to Ang II in VSMCs [57]. This is a mechanism by which Ras confers ROS-sensitivity to downstream signaling molecules such as p38MAPK and Akt, leading to hypertrophy. S-glutathiolation, together with S-nitrosylation and disulfide modifications, are general mechanisms for redox-regulation of proteins. For a better understanding of these modifications, please refer to an excellent recent review [58]. PI3K, MAPKs and JAKs

Another important mediator of Ang II responses in VSMCs is PI3K, which can be activated by Src via EGFR transactivation and Ras. The products of PI3K, PtdIns, activate Rac1 and subsequent ROS release from NADPH oxidases [24]. PtdIns also target PDK1, a signal integrator that activates the AGC family of serine/threonine kinases, including Akt, p70S6 kinase, PKC, PRK, and p21-activated protein kinase-1 (PAK1). However, recent evidence from Taniyama et al. [59] shows that in VSMCs, Ang II triggers a Pyk-2- and Src-dependent, but PI3K-independent, phosphorylation of PDK1 at multiple tyrosine residues. The phosphorylation at Tyr9 is ROS-sensitive and is required for the subsequent phosphorylation at Tyr373/376 [59,60]. Tyrosine phosphorylated PDK1 modulates the formation of focal adhesions in response to Ang II, possibly by regulating paxillin phosphorylation [59]. Another target of PI3K is Akt, which is an important integrator of Ang II responses leading to hypertrophy and survival [61]. ROS sensitivity of Akt is conferred by the phosphorylation of MAPKAPK-2 by p38MAPK (another redox-sensitive kinase [62]), leading to recruitment of MAPKAPK-2 to the constitutively associated Akt–p38MAPK complex and phosphorylation of Akt specifically on Ser473 [63]. Downstream, Akt inhibits glycogen synthase kinase-3, and activates p70S6 kinase and the transcription factors AP-1 and E2F [64].

Besides p38MAPK and MAPKAPK-2, other mitogen-activated protein kinases (MAPKs), are involved in Ang II-stimulated growth pathways and are sensitive to ROS. c-Jun NH2 terminal kinase (JNK) activation in response to Ang II is blocked by several antioxidants, demonstrating its redox sensitivity [65]. The ROS sensitivity of extracellular regulated kinase1/2 (ERK1/2) has been subject to controversy, since some groups have found it sensitive [66,67] and others have found it insensitive to ROS in VSMCs [53,62,65]. Of note, Ang II induces nitration and activation of MEK1 and ERK1/2, the latter being dependent upon NADPH oxidase and iNOS release of ROS [67], implicating a potential crosstalk between the two ROS generators. Another ROS-sensitive MAPK, ERK5, appears to be activated primarily by Nox in response to Ang II, and by mitochondrial-derived ROS in response to endothelin-1 [68].

Janus tyrosine kinases (JAKs) activate ERK1/2 and STATs in VSMCs that are required for both Ang II and PDGF-induced growth. Schieffer et al. [69] demonstrated that JAK2 activation in response to Ang II is attenuated by NADPH oxidase inhibitors. The mechanism of activation of JAK2 by oxidants in VSMCs is unclear, although in fibroblasts, it is dependent on Fyn kinase and the Shc-Grb2-Sos complex [70]. Another possible mechanism involves modulation of phosphatases such as SHP1 and SHP2 [41]. Downstream consequences of ROS-mediated JAK activation include STAT1 and STAT3 phosphorylation and nuclear translocation, increased expression of heat shock protein-70, and activation of ERK1/2 [71]. Transcription factors

Many of the effects of Ang II involve transcription of growth-related genes. One of the most well studied ROS-sensitive transcription factors is AP-1, a heterodimer of Fos and Jun. Its activation is dependent upon p22phox, inasmuch as the antisense p22phox oligonucleotides inhibit AP-1 binding to DNA in response to Ang II and PDGF-AA [72]. AP-1 regulates diverse biological functions, including cell proliferation, protein synthesis, apoptosis and secretion of inflammatory and profibrotic factors. Some of these downstream events such as MMP-1 and endothelin-1 expression are ROS-sensitive in response to Ang II [73]. Another transcription factor that is central to smooth muscle cell proliferation, survival and induction of inflammatory cytokines is NFκB. Its ROS sensitivity in VSMC has been suggested by a few studies [73,74] and is well established in other cell types [75]. Of importance, NFκB regulates a set of genes which inhibit further ROS production, with subsequent inhibition of apoptotic cascades [76]. Other ROS-sensitive transcription factors include cyclic AMP response element-binding protein (CREB) [77], hypoxia-inducible factor-1α (HIF-1α) [78] and the growth arrest homeobox gene, Gax [79].

Of special interest, Id3 and GKLF are two novel redox-sensitive transcription factors that mediate proliferation and growth arrest, respectively, in response to agonists such as Ang II and serum [80]. Mueller et al. [81] showed that Ang II-induced superoxide increases Id3 expression, allowing it to bind to and inactivate the basic helix-loop-helix transcription factor E2A. This results in inhibition of cell cycle proteins p21WAF1/Cip1, p27Kip1, p53 and Rb, and progression through the cell cycle. GKLF has different regulation and opposing functions to Id3: it is upregulated by hydroxyl radicals/H2O2 via p38MAPK and calcium, and promotes growth arrest by stimulating the aforementioned cell cycle proteins [80]. The idea that different ROS target different signaling pathways is appealing, and together with different localization of their sources [32] may explain the diverse outcomes of ROS production in cells. Translation initiation

As noted in the above sections, ROS mediate Ang II signaling at almost all levels, from receptors, to second messengers and kinase cascades, to transcription factors and gene expression. While gene expression starts in the nucleus, it ends in the cytosol with mRNA translation into protein. This represents an important site of convergence of both ROS-sensitive and insensitive signaling pathways leading to growth [82]. The rate-limiting step in translation initiation is represented by the dissociation of phosphorylated PHAS-1 from eIF4E, which then becomes phosphorylated and starts translation. Two PHAS-1 phosphorylation sites, Thr70 and Ser65, have a differential sensitivity to ROS [82]. The phosphorylation of ROS-insensitive Thr70 is regulated by ERK1/2 and PI3K pathways, whereas phosphorylation of ROS-sensitive Ser65 is regulated by p38MAPK, Akt and the PP2A [82].


PDGF is well known for its proliferative and migratory effects on VSMCs. The binding of PDGF to its receptor induces receptor dimerization and intrinsic tyrosine kinase activation, leading to receptor autophosphorylation, along with recruitment and phosphorylation of several target proteins, including those containing Src homology (SH2) domains [83]. PDGF-induced NADPH oxidase activation in VSMCs involves the heterotrimeric Gi1,2 protein [84] and Rac1 [85]. H2O2 may provide negative feedback by inducing an inhibitory phosphorylation of the PDGFR via Src- and PKC-δ-dependent phosphorylation of the PDGFβ-R at Tyr1021 [86]. Phosphatases

Most previous work on the ROS sensitivity of PDGF signaling has focused on phosphatases. These molecules are directly inhibited by ROS through reversible modifications of cysteine residues in their catalytic sites. Among them, the protein tyrosine phosphatases (PTPs) are important targets of ROS, since phosphorylation of proteins at tyrosine residues plays a critical role in many cellular functions. Currently, at least seven cytoplasmic PTPs are known to be expressed in VSMCs: low M(r) protein tyrosine phosphatase (LMW-PTP), SHP-2, PTP36, PTP2, PTP1B, FAP1 [87], and PTP-PEST [88]. A few studies have suggested a relationship between ROS and SHP-1 and -2 in VSMCs [41]; however, direct proof for regulation of these phosphatases remains to be established. Studies performed in adipocytes indicate that Nox4-derived ROS inhibit PTP1B activity in response to insulin [89]. Recently, Bhanoori et al. [90,91] suggested that inhibition of PTP1B by ROS production results in prolonged activation of the PDGFR and the JAK/STAT pathway, leading to inhibition of AP-1, and an increase in apoptosis. Oxidative inhibition of the LMW-PTP, whose role in VSMC includes inhibition of proliferation and migration [87], involves formation of a disulfide bond between Cys-12 and Cys-17, which protects the catalytic Cys-12 from further and irreversible oxidation [92]. Another phosphatase inactivated by PDGF is PTEN (phosphatase and tensin homologue), a lipid and protein phosphatase. PTEN is oxidized and inactivated by H2O2, most likely produced by Nox enzymes, in various cells in response to EGF, PDGF and insulin [93]. However, its regulation in VSMCs is not elucidated yet. Antioxidant molecules: thioredoxin

For ROS to serve as signaling molecules, both their production and removal must be regulated by agonists. In this context, Schulze et al. [94] demonstrated that activation of the reductase thioredoxin (Trx) is critical for the ROS-dependent proliferation of VSMC in response to PDGF. PDGF decreases expression of the endogenous inhibitor of Trx, thioredoxin interacting protein (Txnip), allowing Trx to be released from Txnip and to translocate to the nucleus, where it promotes transcription of growth genes. Interestingly, Txnip binds to the ROS-sensitive cysteine-sulfide center of Trx, and Txnip downregulation by PDGF is inhibited by antioxidants. This affords new insight into how oxidants and antioxidants converge to produce a physiologic response. Initially, PDGF induces an increase in oxidants at the membrane, where the NADPH oxidase is located; then, as a result of complex signaling pathways, the action of Trx in the nucleus is facilitated, to reduce and activate transcription factors such as NF-κB and redox factor-1 (Ref-1). Transcription factors

Among the ROS-sensitive transcription factors responsive to PDGF, Ref-1 is of particular interest. It is known to have two enzymatic functions, ascribed to its C- and N-terminal domains. The C-terminus has DNA repair activity, whereas the N-terminal region contains the redox regulatory domain, which reduces transcription factors such as AP-1, NF-κB, ATF/CREB and HIF1-α, increasing their binding to DNA [56]. For example, Ref-1 antisense oligodeoxynucleotides inhibit AP-1 binding to DNA and the transition of VSMCs from G0/G1 to S-phase in response to PDGF [95]. The inhibition of AP-1 binding to DNA is reversed by chemical reduction, suggesting that Ref-1 functions through reduction of AP-1.

4.2 ROS signaling related to migration

One of the most potent migratory stimuli for VSMCs is PDGF. As noted above, many of the pathways stimulated by PDGF are mediated by ROS, which is also true for migratory signals. In VSMCs, antioxidants block migration in response to PDGF [83]. Conversely, increased migration in a wound-scratch assay was observed in VSMCs extracted from p22phox-overexpressing mouse aortas [96]. Similar to Ang II, PDGF activates c-Src and Rac1 leading to NADPH oxidase release of ROS [83,85]. Upon release, ROS mediate PDGF-induced migration by activating PAK1 via tyrosine phosphorylation of PDK1 [83]. The mechanism of Rac1-Nox activation is unclear, but may involve c-Abl, because PDGF-induced activation of Rac1 is abolished in c-Abl knockout fibroblasts [97].

Several other factors have been shown to induce ROS-dependent migration of vascular SMCs, including insulin-like growth factor-1 (IGF1) [88], thrombin [98], VEGF [99] and MCP-1 [100]. Interestingly, migration in response to IGF1 is mediated by Rac1-inducible ROS and p130Cas, which can be inhibited by NO-mediated activation of PTP-PEST [88]. Another migratory agent, lysophosphatidic acid, also utilizes ROS to activate PAK1, without the involvement of transactivated EGFR or PDGFR [101]. Clearly, more work is necessary to fully understand the ROS sensitivity of the complex signaling pathways involved in migration.

4.3 ROS signaling related to differentiation

Besides mediating pathophysiological processes such as growth and migration, ROS have been implicated in physiological processes as well, such as differentiation [102]. VSMCs of the normal media of blood vessels are in a contractile, differentiated state, but are plastic, so that they can switch to a proliferative, synthetic phenotype in response to proatherogenic stimuli. Therefore, it becomes crucial to understand the different roles of ROS in physiological and pathophysiological signaling.

Su et al. [102] provided the first evidence that the expression of differentiation markers in quiescent VSMCs is dependent upon release of ROS, but their source remained obscure. Recently, Clempus et al. [103] suggested Nox4 as a possible source because siRNA against Nox4 downregulates smooth muscle-specific differentiation markers. Of interest, Nox4 changes localization from smooth muscle α-actin stress fibers in differentiated cells to focal adhesions in de-differentiated cells. The change in localization happens early in the de-differentiation process, suggesting that Nox4 might contribute to the maintenance of the differentiated phenotype by regulating the structure and/or function of the contractile apparatus. Maintenance of the polymerized actin cytoskeleton is required for the nuclear translocation of transcription factors with smooth muscle-specific activity [104]. However, regulation of differentiation by Nox4 may involve several mechanisms. Phosphatases are potential targets of Nox4-derived ROS, and indeed an association has been shown between Nox4 and PTP1B [89]. Future research is necessary to reveal the mechanisms by which ROS regulate differentiation in VSMCs.

5 Conclusion

Our understanding of the regulation of VSMC function by ROS has expanded enormously in the last 10 years. Much work remains to be done, however, especially in elucidating the precise mechanisms by which ROS modify the function of specific proteins. Additional studies on the ROS-sensitive signaling pathways involved in migration and differentiation are also necessary.

Mechanistic studies such as these will open up new avenues for therapeutic intervention targeted to specific disease processes.


We would like to acknowledge Dr. Bernard Lassègue for suggestions and critical reading of the manuscript. Authors' work is supported by NIH grants HL 58000, HL 38206, HL 58863, and HL 75209.


  • Time for primary review 20 days


  1. [1]
  2. [2]
  3. [3]
  4. [4]
  5. [5]
  6. [6]
  7. [7]
  8. [8]
  9. [9]
  10. [10]
  11. [11]
  12. [12]
  13. [13]
  14. [14]
  15. [15]
  16. [16]
  17. [17]
  18. [18]
  19. [19]
  20. [20]
  21. [21]
  22. [22]
  23. [23]
  24. [24]
  25. [25]
  26. [26]
  27. [27]
  28. [28]
  29. [29]
  30. [30]
  31. [31]
  32. [32]
  33. [33]
  34. [34]
  35. [35]
  36. [36]
  37. [37]
  38. [38]
  39. [39]
  40. [40]
  41. [41]
  42. [42]
  43. [43]
  44. [44]
  45. [45]
  46. [46]
  47. [47]
  48. [48]
  49. [49]
  50. [50]
  51. [51]
  52. [52]
  53. [53]
  54. [54]
  55. [55]
  56. [56]
  57. [57]
  58. [58]
  59. [59]
  60. [60]
  61. [61]
  62. [62]
  63. [63]
  64. [64]
  65. [65]
  66. [66]
  67. [67]
  68. [68]
  69. [69]
  70. [70]
  71. [71]
  72. [72]
  73. [73]
  74. [74]
  75. [75]
  76. [76]
  77. [77]
  78. [78]
  79. [79]
  80. [80]
  81. [81]
  82. [82]
  83. [83]
  84. [84]
  85. [85]
  86. [86]
  87. [87]
  88. [88]
  89. [89]
  90. [90]
  91. [91]
  92. [92]
  93. [93]
  94. [94]
  95. [95]
  96. [96]
  97. [97]
  98. [98]
  99. [99]
  100. [100]
  101. [101]
  102. [102]
  103. [103]
  104. [104]
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