Experimental Biology and Medicine 231:237-251 (2006)
© 2006 Society for Experimental Biology and Medicine
MINIREVIEW
Hydrogen Peroxide as a Paracrine Vascular Mediator: Regulation and Signaling Leading to Dysfunction
Noelia Ardanaz and
Patrick J. Pagano1
Hypertension and Vascular Research Division, Henry Ford Health System, Detroit, Michigan 48202-2689
1To whom requests for reprints should be addressed at Hypertension and Vascular Research Division, RM 7044, E&R Building, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, MI 48202-2689. E-mail: ppagano1{at}hfhs.org
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Abstract
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Numerous studies have demonstrated the ability of a variety of vascular cells, including endothelial cells, smooth muscle cells, and fibroblasts, to produce reactive oxygen species (ROS). Until recently, major emphasis was placed on the production of superoxide anion (O2–) in the vasculature as a result of its ability to directly attenuate the biological activity of endothelium-derived nitric oxide (NO). The short half-life and radius of diffusion of O2– drastically limit the role of this ROS as an important paracrine hormone in vascular biology. On the contrary, in recent years, the O2– metabolite hydrogen peroxide (H2O2) has increasingly been viewed as an important cellular signaling agent in its own right, capable of modulating both contractile and growth-promoting pathways with more far-reaching effects. In this review, we will assess the vascular production of H2O2, its regulation by endogenous scavenger systems, and its ability to activate a variety of vascular signaling pathways, thereby leading to vascular contraction and growth. This discussion will include the ability of H2O2 to (i) initiate calcium flux as well as (ii) stimulate pathways leading to sensitization of contractile elements to calcium. The latter involves a variety of protein kinases that have also been strongly implicated in vascular hypertrophy. Previous intensive study has emphasized the ability of NADPH oxidase-derived O2– and H2O2 to activate these pathways in cultured smooth muscle cells. However, growing evidence indicates a considerably more complex array of unique oxidase systems in the endothelium, media, and adventitia that appear to participate in these deleterious effects in a sequential and temporal manner. Taken together, these findings seem consistent with a paracrine effect of H2O2 across the vascular wall.
Key Words: reactive oxygen species • hydrogen peroxide • blood vessel • signaling • hypertrophy • hypertension
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Vascular Generation of H2O2
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Reactive oxygen species (ROS) are a class of molecules that are derived from the metabolism of oxygen and include free radical and nonradical species that are generally capable of oxidizing molecular targets. H2O2 is a cell-permeant and highly stable ROS generated mainly by dismutation of superoxide (O2–) by superoxide dismutases (SOD). Although H2O2 is defined as an ROS, unlike O2– it is not a free radical, in that it does not possess an unpaired electron in its outer shell. This renders H2O2 more stable and less reactive with other tissue radicals and, thus, a more likely paracrine ROS. In the presence of iron, intracellular H2O2 can be converted to hydroxyl radical, which can oxidize molecular targets and cause lipid peroxidation and this may account for some of its local biological effects (1, 2). O2– is considered the precursor of all ROS because in most cases it is the first ROS produced by mammalian oxidases. This is clearly true in the vasculature, where a primary source of ROS has been identified as NAD(P)H oxidase (3–10). Our laboratory and those of Griendling and Wolin initially identified this major vascular source of ROS in vascular fibroblasts, vascular smooth muscle cells (VSMCs), and endothelial cells (3, 11–14). Our findings indicate that a substantial percentage of all O2–-generating activity in rabbits, rats, and mice is traceable to adventitial fibroblast NAD(P)H oxidase, which contains all four major neutrophil-like NAD(P)H oxidase components: nox2 (previously termed gp91-phox), p22phox, p47phox, and p67phox (14). More recently, nox4 was found to be present in the adventitia (15). NAD(P)H oxidase in VSMCs has been extensively described and implicated in cell growth and impairment of endothelium-dependent relaxation (3, 12). VSMCs express critical phagocyte-like NAD(P)H oxidase components (9, 16–18) as well as homologues of essential phagocyte nox2, nox1, and nox4 that participate in O2– production (9, 19, 20). Whereas the rat and mouse aorta apparently possess a functional nox1 and nox4 but little or no nox2, nox2 appears to predominate in resistance artery smooth muscle cells (9). Likewise, endothelial cells contain phagocyte-like components including nox2 and at least one of its homologues, nox4, which are functionally involved in O2– production (11, 21–25) and endothelial dysfunction (25). While most studies confirm that NAD(P)H oxidases produce O2–, some contend that these enzymes can also produce H2O2 (Table 1
; Refs. 26–29). Furthermore, rat and human fat cells possess a membrane-bound NAD(P)H oxidase that produces H2O2 as its initial product (27). Generally speaking, however, most H2O2 is derived from dismutation of O2–.
Other enzymes appear to be involved in the vascular production of O2–, although their contribution to vascular generation of ROS seems significantly lower than that of NAD(P)H oxidase. Even so, the activity of these other sources appears to be accentuated under pathological conditions. For instance, nitric oxide synthase (NOS) can generate not only NO but also O2– when concentrations of substrate or cofactors are compromised, leading to uncoupled NOS and increased O2– production (39, 40). The mitochondria seem to be a major source of O2– and H2O2 in the lung and heart via the mitochondrial respiratory chain complex I and III (41–43). Mitochondria do not appear to contribute significantly to total vascular ROS production (13, 14). This is most likely attributable to the relative metabolic inactivity of quiescent blood vessels. However, during oxygen delivery, rapid remodeling, or tissue damage, mitochondria would be expected to play a more significant role. Interestingly, the metabolic abnormalities seen in diabetes reveal a role for mitochondrial ROS in endothelial cells (44, 45).
Xanthine dehydrogenase is converted to xanthine oxidase by thiol oxidation or irreversible proteolytic cleavage, and its activity in endothelial cells seems to be increased in ischemia-reperfusion (46–48). One recent study showed that xanthine oxidase activity is upregulated in response to oscillatory shear stress, possibly implicating ROS derived from this source in the development of dysfunction and plaque formation at vascular regions of disturbed flow (49). Interestingly, in that same study (49), NADPH oxidase was described as an important modulator of xanthine oxidase expression and activity, suggesting a positive feedback loop between the two enzymes. Another intriguing study posits a link between xanthine oxidase and activation of cyclooxygenase 2, indicating a positive association in inflammation (50). Since cyclooxygenase has been described as a generator of vascular ROS, this interaction may participate in feed-forward generation of ROS, as suggested generally for NADPH oxidases (5, 8). Other studies have shown that cytochrome P450 and lipoxygenases are capable of releasing O2–, which may play an important role in vascular function (51, 52). Finally, other enzymes, including heme oxygenases and peroxidases, have also been implicated in O2– production (6). Although the preponderance of evidence appears to support a dominant role for NAD(P)H oxidases in the production of vascular ROS—given that inhibitors of other enzymatic sources appear to have negligible effects under most conditions—these enzymes in combination may contribute significantly to total ROS levels and alterations in tone and remodeling. Regardless of its source, O2– is spontaneously or efficiently converted to the stable and tissue-permeant H2O2 by abundant and ubiquitous intracellular and extracellular SODs (53, 54). The relevance of each class of SOD will depend, of course, on the location of O2– production in the cell. That is, manganese SOD (Mn-SOD) found in the mitochondria is critical to the proper handling of O2– derived from mitochondrial electron transport (55). Cytosolic copper/zinc (Cu/Zn)-SOD dismutes O2– derived from a variety of above-mentioned cytosolic oxidases, including VSMC NADPH oxidases (20, 56). Finally, the fate of extracellular production of O2– by leukocytes (and suggested for adventitial fibroblasts) is determined by the extracellular tethered form of Cu/Zn-SOD (ec-SOD) (57, 58). It has been suggested that endothelial cells can also produce extracellular O2– (59). As the expression of SOD is enhanced by inflammatory cytokines, in hypertension and in direct response to angiotensin II (Ang II) (60, 61), the conversion of O2– to H2O2 appears to be favored in cardiovascular disease (see Fig. 1). Given the stability of H2O2 and its plausibility as a paracrine mediator of vascular dysfunction, in this review we will focus on the regulation and biological activity of this important vascular ROS.
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Figure 1. Scheme showing possible mechanisms of regulation of H2O2 in cardiovascular disease. Under normal conditions (A), constitutively active oxidases produce O2– that is rapidly converted by abundant superoxide dismutases to H2O2. Catalase and glutathione peroxidase inactivate H2O2. In hypertension, multiple oxidases leading to the production of O2– as well as SOD appear to be upregulated (B). Additionally, factors in cardiovascular disease causing reduced catalase and/or Gpx activity are expected to contribute to an increased steady-state level of H2O2.
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Endogenous Scavenger Systems that Regulate Vascular Levels of H2O2
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Steady-state H2O2 levels in vascular tissue are tightly regulated by its endogenous scavengers catalase and glutathione peroxidase (Gpx1) (62, 63). Gpx1 is found in cellular cytosol and mitochondria and is a key enzyme for the cellular defense against oxidative stress, using glutathione to reduce H2O2 and lipid peroxides to their respective alcohols (64). Catalase is found primarily in peroxisomes and exclusively catalyzes the conversion of H2O2 to water. Although the contribution of these enzymes seems to vary in tissue, in vascular preparations catalase possesses a higher Km for H2O2, compared to Gpx1, and may serve as an important intracellular defense against large amounts of H2O2 (63). Studies suggest that vascular Gpx1 is better suited to scavenge endogenous basal levels of H2O2 (63, 65). Moreover, the contribution of these enzymes to vascular ROS metabolism seems to change with age (66), and their relative contribution along the vasculature has not been established. Regardless of these distinctions, both enzymes are considered ubiquitously important in regulating endogenous H2O2. Interestingly, comparisons of aortas from populations of mice with high blood pressure versus normal blood pressure have revealed a reduction in catalase expression and activity concomitant with an increase in the activity of Mn-SOD and ec-SOD (67). It appears plausible, then, that an increase in the steady-state level of H2O2 in hypertension would be favored by such an imbalance (Fig. 1
). In fact, it is known that in human patients with essential hypertension, plasma levels of H2O2 do rise (68, 69). Clearly, more studies will be necessary to examine the relationship of SODs versus catalase and Gpx1 in the vasculature in various cardiovascular diseases.
A relatively new class of antioxidant enzymes named peroxiredoxins have been shown to reduce H2O2 and, more recently, peroxynitrite using thioredoxin as the immediate electron donor (70, 71). Although their catalytic efficiency is less than that of Gpx-1 or catalase, these enzymes seem to regulate H2O2 signaling generated by different growth factors (72). Interestingly, a recent study showed that peroxiredoxin plays a role in platelet-derived growth factor signaling in VSMCs and appears to attenuate neointima formation (73).
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H2O2 as a Vasoactive Substance
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Although numerous studies have demonstrated an autocrine effect of H2O2 on cultured smooth muscle cell signaling and hypertrophy (6, 74), its role in vascular tone is not well understood. Studies have demonstrated both a contractile and relaxant response to H2O2 depending on the species, vascular bed, and contractile state (75–78). Clearly, H2O2 has been shown to cause constriction in a variety of vascular beds. Under quiescent conditions, H2O2 reportedly contracted the aorta, pulmonary artery, and superior mesenteric artery of the rat (79–84), the porcine pulmonary artery (85), and the canine basilar artery (86). The mechanism involved in H2O2-induced vasoconstriction seems to be Ca2+-dependent in the rat aorta and dog basilar artery (79, 81, 86). Ca2+-dependent pathways lead to increased intracellular Ca2+ through voltage-gated Ca2+ channels as well as Ca2+ release from the sarcoplasmic reticulum (87) (Fig. 2). When Ca2+ levels are elevated above baseline, Ca2+ binds to calmodulin and activates myosin light chain kinase (MLCK), leading to phosphorylation of 20-kDa myosin light chains (MLC20) and constriction. In fact, H2O2 can increase intracellular Ca2+ by mobilizing extracellular Ca2+ via activation of voltage-operated Ca2+ channels in rat mesenteric VSMCs (88), a mechanism that is also reportedly involved in rat aortic contraction (79, 81). Likewise, H2O2 can mobilize Ca2+ from the sarcoplasmic reticulum via ryanodine receptors (89), caffeine-sensitive stores (81), and inhibition of Ca2+-ATPase (90), and by promoting inositol triphosphate (PI3)–induced calcium release (91). However, the role of both Ca2+-ATPase and ryanodine receptors in H2O2-induced vascular constriction remains unclear (92–94). Moreover, H2O2 has been shown to elicit Ca2+ release from the mitochondria (95) via the production of hydroxyl radical (96).
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Figure 2. Signal transduction mechanisms of smooth muscle contraction and proposed mechanism of modulation by H2O2. Depolarization and activation of voltage-operated calcium channels (VOCC) or ligand-receptor interaction and stimulation of Ca2+ release from intracellular stores and secondary activation of store-operated Ca2+ channels (SOCC) produce increases in intracellular calcium that lead to activation of myosin light chain 20 (MLC20) by Ca2+-calmodulin-myosin light chain kinase (Ca2+-CaM-MLCK). MLC20-P binds to actin and contraction is produced. MLC20-P is dephosphorylated by myosin light chain phosphatase (MLCP), leading to relaxation. H2O2 is able to increase intracellular calcium through VOCC and activation of Ca2+release from intracellular stores. It can also activate MAPK and Rho-kinase, leading to enhanced vascular smooth muscle contraction. Ca2+ sensitization mechanisms enhance contraction independently of changes in intracellular Ca2+. These mechanisms lead to an increase in MLC20-P through direct phosphorylation or inhibition of MLCP. Rho-kinase phosphorylates and inactivates MLCP. Mitogen-activated protein kinase (MAPK) through activation of other kinases (ILK), or zipper-interacting protein kinase (ZIP-K) can phosphorylate MLC20 and also inactivate MLCP. Moreover, MAPK also seems to be involved in the phosphorylation of caldesmon, favoring the interaction MLC20-P:actin.
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H2O2 vasoconstriction is mediated by tyrosine kinase (79, 83, 86, 97), cyclooxygenase products (80, 82), protein kinase C (PKC) (79, 86), mitogen-activated protein kinases (MAPKs) (86, 97, 98), Rho-kinase (ROK) (99), and phosphorylated MLC20 (MLC20-P) (83), although the role of PKC, MLC20-P, and MAPK remains controversial (84, 85, 92). Some of these pathways have been described as Ca2+ sensitization pathways, defined as increases in generated force without increases in intracellular Ca2+ (100). These pathways lead to increases in MLC20-P, mainly via inhibition of MLCP, but they may also be regulated by activation of MLCK, direct regulation by heat-shock proteins, and even by mechanisms involving thin filament regulation (caldesmon, calponin) (Fig. 2). MLCP is inhibited by phosphorylation by a variety of kinases, including integrin-linked kinase (ILK) (101, 102), PKC (103), and ROK (104–106). ROK has been shown to be activated by ROS (105) and has been directly implicated in the constrictor effect of H2O2 in veins (99). Moreover, H2O2 has been demonstrated to cause vasoconstriction via MAPKs, including extracellular signal-regulated kinases (ERK1/2) and p38. In turn, ERK1/2 has been shown to modulate MLCK by increasing MLC20-P (107, 108) and acts as an upstream mediator of ILK, which has also been shown to phosphorylate MLC20 in smooth muscle (102, 109) and inactivate MCLP (110). Additionally, ERK1/2 is known to phosphorylate caldesmon, an actin-binding protein that, once phosphorylated, inhibits actin-myosin interaction and thus contraction (111, 112).
p38 MAPK is involved in H2O2 vasoconstriction in KCl-preconstricted mesenteric arteries (113). In a few key studies, p38 MAPK-mediated phosphorylation of HSP27 has been described as modulating contraction of VSMCs (114–116). In fact, this mechanism could be involved in activation of zipper-interacting–like kinase (ZIP kinase) and phosphorylation of MLC20 as well as inactivation of MLCP. The relevance of Ca2+ sensitization pathways in H2O2-induced vasoconstriction is not fully understood, but because of the direct effect of H2O2 in the activation of MAPK and ROK, they may play an important role in the overall response to H2O2.
Interestingly, some findings have also indicated that H2O2 could induce constriction via Ca2+-independent pathways in rat and rabbit pulmonary arteries (84, 85, 92). The latter reports of possible H2O2 constriction independent of calcium are provocative and may involve a direct effect of ROS on contractile elements. However, such discussion is currently beyond the scope of this review.
Lucchesi et al. (113) recently showed that H2O2 acts as a vasoconstrictor in mouse mesenteric resistance arteries depolarized with KCl. In that study, the same concentrations of H2O2 produced dilatation of phenylephrine-preconstricted vessels (113), implying that membrane potential of the vessel influences the effect of H2O2 on tone. Similarly, Sotnikova (81) found that increased extracellular potassium enhanced the vasoconstrictor effect of H2O2. We have obtained similar results in the abdominal aorta and superior mesenteric artery (i.e., when they were depolarized by KCl, H2O2-induced vasoconstriction was enhanced; unpublished data). It appears that extracellular potassium evokes two possible effects that contribute to H2O2-induced vaso-constriction: (i) blockade of K+ channels induced by high levels of extracellular KCl reveals a contractile effect of H2O2 via suppression of H2O2-induced K+ channel-mediated vasodilatation (117–120) and/or (ii) H2O2 activates Ca2+ sensitization pathways, enhancing the Ca2+-dependent contraction secondary to depolarization. With regard to the physiologic relevance of these studies, vascular smooth muscle depolarization has been reported in different models of hypertension produced at least in part by alterations in K+ channel activity (121–125). Decreased K+ channel activity may contribute to increased vessel contractility (121) that in turn could help explain the increased vasoconstrictor response to H2O2 in hypertensive models (80, 126).
On the other hand, H2O2 elicits relaxation in agonist-constricted rat, mouse, and rabbit aortas (76, 127–129). This response has been observed in a variety of other vessels, including the human, mouse, rat, and rabbit mesenteric artery (76, 113, 120, 130, 131), porcine coronary artery (118, 132), human and porcine coronary arterioles (117, 133), canine cerebral (134) and basilar arteries (77), bovine and rabbit pulmonary arteries (135, 136), and rabbit iliac artery (137), among others; and the dilator response includes both endothelium-dependent (77, 127, 138, 139) and -independent mechanisms (75, 76, 113, 118, 133, 135). In some cases, both endothelium-dependent and -independent relaxation have been implicated in the effect of H2O2 (117, 129). The endothelium-dependent relaxation associated with H2O2 seems to be mediated by the NO/cyclic guanosine mono-phosphate (cGMP) pathway and may be related to an increase in intracellular Ca2+ (77, 127, 129, 138). P450 cytochrome metabolites are also plausible mediators of the endothelium-dependent response (117, 127). On the other hand, it has been suggested that H2O2-induced endothelium-independent relaxation is mediated through activation of guanylate cyclase and accumulation of cGMP (75, 76, 133, 135, 140), although the role of cGMP is questioned in other studies (117, 120, 134, 141). Another principal pathway that seems to be involved in H2O2 relaxation is activation of Ca2+-dependent (117–120, 132, 134, 142), ATP-sensitive (120, 141), and voltage-dependent K+ channels (120). In some cases, K+ channel activation is preceded by release of arachidonic acid metabolites (118, 130, 134), so that, eicosanoids have been implicated in this response; however, the role of these metabolites in H2O2-induced relaxation is uncertain (120, 128). An interesting article published by Sato et al. (133) indicates that exogenous and endogenous H2O2 may elicit vasodilatation by different pathways depending on the origin and localization of the peroxide. In spontaneously hypertensive rats (SHR), attenuation of the relaxant response to H2O2 was linked to alterations in K+ channel activity (126). Thus, alterations in K+ channel activation may play a role in the development of hypertension both by decreasing vasorelaxant responses and by enhancing vasoconstrictor responses.
In accord with its ability to activate K+ channels, multiple studies have proposed that H2O2 is an endothelium-derived hyperpolarizing factor (EDHF), including human and mouse mesenteric arteries (130, 131) and porcine and canine coronary arteries and arterioles (143, 144); however, some groups contradict this observation, as they were unable to demonstrate that EDHF is H2O2 (128, 137). It has been suggested that under conditions in which NO bioavailability is reduced, increased EDHF-induced relaxation compensates for the lack of response to NO and preserves endothelium-dependent relaxation (145, 146). In this regard, some authors postulate that in animal models in which endothelial NOS (eNOS) cofactors are compromised, uncoupled eNOS can serve as a source of H2O2 that becomes a compensatory response for endothelium-dependent vasodilatation (39, 40, 147). Even though this increase may result acutely in a beneficial effect, we postulate that over time the effect will become detrimental.
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H2O2 as a Mediator of Vascular Dysfunction
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Despite the body of data suggesting an "acute" relaxant effect of H2O2, the effect of prolonged elevations of H2O2 (as observed in models of hypertension) on constriction is an important scientific question with regard to the in vivo vascular effects of H2O2. In fact, the ability of chronically elevated H2O2 to impair endothelium-dependent relaxation has been supported by a few key studies. Consistent with a role for endogenous H2O2 in impaired relaxation, Gpx1-deficient mice exhibit contraction of mesenteric arteries in response to the endothelium-dependent agonist methacholine, whereas wild-type mice exhibit dilatation (148). Furthermore, methacholine-induced relaxation of mesenteric arteries was converted to contraction in normal versus homocysteinemic mice, an effect that was reversed by Gpx1 overexpression (149). These authors observed a decreased release of bioactive NO from hyperhomocysteinemic endothelial cells, attributing this to homocysteine auto-oxidation to NO-inactivating peroxide radicals and/or to a specific decrease in cellular Gpx. Thus, overexpression of Gpx1 was capable of restoring NO bioactivity. A subsequent study by Dayal et al. (150) showed that deletion of the Gpx1 gene caused impairment of ACh-induced dilatation of the aorta compared to wild-type mice, an effect that was enhanced in hyperhomocysteinemic strains. Thus, these data are consistent with endogenous vascular H2O2 leading to impairment of endothelium-dependent relaxation. Interestingly, a recent study (151) showed that in mice, vascular overexpression of human catalase decreases systolic blood pressure and vascular constriction per se, suggesting the relevance of endogenous H2O2 as a vasoconstrictor and regulator of blood pressure.
However, the mechanisms by which elevated concentrations of H2O2 lead to vascular dysfunction remain unclear. One possibility is that sustained increases in H2O2 enhance the pathways involved in smooth muscle contraction (Ca2+-dependent or sensitization pathways), leading to vascular dysfunction. Another possibility is that in pathological states such as hypertension, when reductions in K+ channel activity prevail, these conditions will favor the vasoconstrictor effect of H2O2. A few studies show that H2O2 can reduce vascular NO production. One intriguing article by Wedgwood and Black (152) suggests that endothelin 1–induced stimulation of H2O2 release in pulmonary VSMCs decreases eNOS expression and activity in pulmonary endothelial cells. Another interesting study (153) demonstrated decreased gene expression of inducible NOS. Finally, Jaimes et al. (154) showed that H2O2 decreased NO production by inactivation of eNOS cofactors without affecting eNOS activity.
At this juncture, it is important to discuss recent findings revealing that H2O2 stimulates eNOS and SOD, resulting in higher NO levels (155, 156). One report by Cai et al. (155) intriguingly demonstrates that concomitant induction of NOS and SOD by H2O2 could explain the preservation of NO despite increased O2– and point to a compensatory mechanism of NO protection under some conditions and in some vascular beds. However, most reports, including our own, have suggested that O2– levels often outpace elevations in NO (148–150, 157–163). One mechanism by which H2O2 promotes such an increase is through dysfunction of NOS, just as it may stimulate phagocyte-like oxidases to produce more O2–, H2O2, and other ROS (see below). Taken together, these data appear to indicate that H2O2 plays an important role in the regulation of NOS activity and in the biological fate of NO, leading to an overall reduced NO bioactivity.
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Ability of H2O2 to Activate Its Own Generation
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Li et al. (164) showed that exogenous H2O2 activates cellular NAD(P)H oxidase to produce O2– and that this effect was independent of xanthine oxidase, cyclooxygenase, eNOS, and mitochondrial activation. These findings were recently corroborated by Seshiah et al. (5), who proposed that small amounts of H2O2 derived from NAD(P)H oxidase can promote sustained activation of NAD(P)H oxidase. The ability of elevations in ROS to promote oxidase activation has also been inferred from studies showing that an increase in ROS leads to increased p22-phox expression (16, 32). H2O2 can also increase transferrin receptor-dependent iron uptake, amplifying intracellular mitochondrial H2O2 generation (165). NADPH oxidase activation is required for xanthine oxidase activation and subsequent H2O2 formation in response to oscillatory shear stress (49). Finally, as noted earlier, H2O2 appears to be involved in the reduced bioavailability of eNOS cofactors, which may induce uncoupled eNOS and lead to increased H2O2 production.
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H2O2 as a Mediator of VSMC Signal Transduction
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In addition to the well-characterized involvement of ROS-insensitive pathways in VSMC signal transduction, numerous studies have demonstrated a role for ROS in the activation of both proximal and downstream MAPKs (6). H2O2 derived from NAD(P)H oxidase has been implicated in the activation of c-Src, which in turn transactivates receptor tyrosine kinases (5, 97, 166), a process that involves activation of phosphatidyl inositol (PI)3-kinase (5). Epidermal growth factor receptor (EGF-R) and platelet-derived growth factor receptor (PDGF-R) are tyrosine kinases that are both transactivated by Ang II, a process mediated by H2O2 derived from NAD(P)H oxidases (167, 168). The resulting tyrosine phosphorylation generally leads to activation of Src homology complex-growth factor receptor–bound protein 2–son of sevenless complex that activates ras, leading to downstream activation of ROK, MAPKs, and transcription factors. Some of the key redox-sensitive kinases in these signaling pathways are extracellular-regulated kinase, c-Jun N-terminal kinases (JNK), big MAPK (ERK5), and p38 MAPK (169–172). In the case of p38 MAPK, activation of the Akt/protein kinase B pathway results in cellular hypertrophy (6, 172). H2O2 has been most clearly implicated in the activation of p38 MAPK and JNK (169, 173). In cultured SMCs, Ang II activation of ERK1/2 was shown to be H2O2–independent (172–174); however, exogenous H2O2 does modulate ERK1/2 phosphorylation in VSMCs and other tissues (97, 175, 176).
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Vascular Tone: Role of MAPKs
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Since multiple studies have confirmed that MAPK can be activated by H2O2, it seems plausible to infer that among these kinases, targets of paracrine activation by H2O2, p38, and ERK1/2 are most likely to participate in contraction. Supporting this notion are reports showing that H2O2 does in fact induce ERK1/2 activity in smooth muscle cells via a variety of mechanisms (175, 177). ERK1/2 has been proposed as a plausible MAPK mediator of vascular contraction in hypertension, since its activation has been shown to be associated with enhanced contraction in cultured vascular cells from SHR as well as aortas from SHR and DOCA-salt hypertensive rats (178). Several studies have shown that Ang II vasoconstriction is mediated by activation of ERK1/2 (179, 180). Touyz et al. (178) showed that in isolated SHR mesenteric arteries, an ERK1/2 inhibitor markedly reduced Ang II-induced contraction and ameliorated impaired endothelium-dependent relaxation (178). However, other studies demonstrate a dissociation between Ang II–induced contraction and ERK1/2 pathway activation in the rat aorta (181). On the other hand, with regard to serotonin-induced constriction of VSMCs, ERK1/2 activation, but not p38 MAPK or JNK, appears to be involved (182, 183).
p38 MAPK activity, however, does contribute to the contractile response of mesenteric and canine pulmonary arteries to catecholamines (115, 184). Both ERK1/2 and p38 MAPK partially regulate the endothelin-1–induced vaso-constriction in Wistar-Kyoto rats (185). Ushio-Fukai et al. (172) showed that NAD(P)H oxidase–derived H2O2 plays an important role in Ang II–induced p38 MAPK activation. They also demonstrated that overexpression of catalase with an adenoviral construct attenuated Ang II–induced H2O2 and p38 MAPK activation (172). These data were corroborated by Meloche et al. (116), who showed that Ang II–induced activation of p38 MAPK is H2O2-dependent and mediates vascular constriction.
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ROS-Mediated Role of PI3- and Rho-Kinase
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Studies have shown that ROS-sensitive kinases are involved in regulating PI3-kinase; thus, PI3 kinase has been described as a critical link in ROS-mediated signaling (5). The involvement of PI3 kinase in increased MLCK activity, calcium sensitivity, and the contractile state of the artery makes it an important potential upstream mediator of H2O2- dependent impaired relaxation and enhanced constriction. Vascular PI3-kinase is composed of a regulatory p85
subunit as well as various isoforms of the p110 subunit (p110, p110ß, and p110
, but not p110
), all of which are reportedly present in the vasculature and are potentially involved in pathways leading to vasoconstriction (186).
Another influential upstream ROS-sensitive inhibitor of MLCP is ROK. ROK is activated by Rho, a GTP-exchange protein, which is a member of the Ras family of signaling molecules known to be ROS-sensitive (105). ROK has been shown to mediate the maintenance (tonic) phase of contraction in response to various agonists, including phenylephrine, serotonin, and endothelin-1 (99, 187). A recent study by Jin et al. (105) showed that ROS increase membrane-bound Rho associated with ROK activation during contraction of the rat aorta, suggesting that ROK activation is associated with contraction. Importantly, ROK has been shown to inhibit MLCP. Jin et al. went on to show that a ROK inhibitor prevented ROS-elicited MLCP inhibition and hence contraction. Thus, these data suggest an important role for ROS-activated ROK in pathways leading to vascular contraction.
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Vascular Hypertrophy: Potential ROS-Mediated Role of p38 and JNK MAPKs
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The literature suggests less complex involvement of the MAPKs p38 and JNK as ROS-sensitive pathways leading to hypertrophy. As mentioned earlier, EGF-R and PDGF-R are both transactivated by Ang II, a process mediated by ROS derived from NAD(P)H oxidases (167, 168). Thus, on the cellular level, H2O2 would be expected to synergize with the effects of growth factor receptor ligands and activate pathways leading to growth. The resulting tyrosine phosphorylation of these receptors leads to activation of a series of pathways, culminating in downstream activation of the MAPKs involved in growth signaling (6, 188). The key redox-sensitive kinases playing a role in this cascade seem to be JNK and p38 MAPK (169–171), which appear to activate the Akt/protein kinase B pathway and cause cellular hypertrophy (6, 172).
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Paracrine Effect of H2O2 on Medial Smooth Muscle Hypertrophy
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Clearly, endogenous H2O2 contributes to growth-related signaling (174). Zafari et al. (189) reported that H2O2 metabolized from NAD(P)H oxidase–derived O2– mediated Ang II–induced hypertrophy of cultured smooth muscle cells. Multiple studies indicate that Ang II exerts a direct hypertrophic effect on vascular smooth muscle (190, 191). In cultured smooth muscle cell preparations, NAD(P)H oxidase–derived ROS have been implicated in hypertrophy (3, 16), activating signaling pathways and transcription factors involved in the growth response (6, 8, 74, 192, 193). Thus, ROS-dependent autocrine pathways are essential to smooth muscle growth in response to Ang II in vitro. A recent article by Zhang et al. (194) elegantly demonstrated that human catalase overexpression in VSMC decreases the hypertrophic effect of Ang II–induced hypertension, thereby indicating the important role of H2O2 in vivo. We and others have suggested a more complex in vivo scenario of paracrine, ROS-mediated influences on medial responsiveness and hypertrophy. A provocative study by Wang et al. (195) suggested a paracrine effect of ROS on medial hypertrophy. The authors showed that Ang II stimulates NAD(P)H oxidase–derived ROS in the aortic adventitia and intima, concomitant with medial hypertrophy, and that this stimulation was significantly reduced in gp91-phox–deficient mice with reduced intimal and adventitial NAD(P)H oxidase, suggesting a paracrine effect of adventitial and intimal NAD(P)H oxidase–derived ROS on medial hypertrophy (195). We postulated that adventitial NAD(P)H oxidase–derived ROS could influence medial hypertrophy. To test this hypothesis, we used an adenoviral construct targeting expression of an inhibitor of gp91-phox:p47-phox interaction (gp91ds) to the adventitia and confirmed expression of the inhibitor in adventitial fibroblasts and macrophages. Interestingly, this localized adventitial expression resulted in significant reduction in medial ROS detection and hypertrophy (196). Thus, these data support a paracrine effect of adventitial NAD(P)H oxidase–derived ROS on medial hypertrophy. It is important at this juncture to point out that in Ang II–induced hypertension, macrophages localize in the adventitia and have been implicated in medial smooth muscle hypertrophy (197), yet the contribution of ROS or cytokines derived from these cells has not been delineated (198). It is tempting to speculate that even small amounts of ROS derived from adventitial fibroblasts may be chemotactic for macrophages (199, 200), which through their larger oxidase potential exacerbate ROS levels in the adventitia and enhance prohypertrophic mechanisms (Fig. 3).
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Figure 3. Potential vascular sites of hydrogen peroxide and proposed paracrine effect on medial contraction and hypertrophy. NADPH oxidase in fibroblasts produces O2– that is rapidly converted to H2O2 by SOD in the adventitia and may feed forward in the upregulation of greater oxidase expression. NADPH oxidase–derived ROS are chemotactic for macrophages via the possible release of monocyte chemoattractant peptide-1 (MCP-1) and its binding to the CCR2 receptor. Recruited cells exacerbate the production of ROS. Potential sources of ROS in the endothelium include NOXes, dysfunctional eNOS, xanthine oxidase, and mitochondria. These "initiator" peroxide producers either (i) directly activate VSMC kinases, leading to hypertrophy or contraction or (ii) activate local NOX to produce H2O2, which contributes to an enhanced effect.
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We examined the effect of adventitia-targeted inhibitor expression on angioplasty-induced hyperplasia to study a potential paracrine effect of vascular ROS (201). In two consecutive studies, we compared the effect of localized adventitial expression of gp91ds and dominant negative p67-phox on neointima development in response to balloon angioplasty of the rat carotid artery (201) (unpublished observations). We found that gp91ds transfection to the adventitia inhibited neointimal hyperplasia to a greater degree than dominant negative p67-phox. These data are suggestive of a multicomponent adventitial NADPH oxidase having a paracrine effect on smooth muscle cell hyperplasia that in turn leads to neointimal growth. Since we also showed that these treatments had an inhibitory effect on fibroblast proliferation in vitro, we cannot rule out a direct role for fibroblasts in neointimal growth. However, taken together with the demonstrated importance of smooth muscle migration to neointimal growth, our findings suggest important paracrine influences of ROS, likely H2O2, compounding the effect of local medial production of ROS on vascular growth. In fact, we postulate that, as indicated in the activation of NADPH oxidase, vascular NADPH oxidases may be activated in series; that is, adventitial and endothelial NADPH oxidase–derived ROS may initiate activation of medial NADPH oxidase. Interestingly, this has been suggested by a number of authors who have shown that both the adventitia and endothelium are activated to produce greater amounts of ROS than the media under normal conditions and earlier in the disease process (14, 31, 202–205). Moreover, as mentioned above, there is burgeoning evidence of a temporal relationship among the various NADPH oxidase isoforms (206), such that one or more nox isoforms may be prominent early in the etiology of a particular disease and then taper off, whereas others predominate later. This relationship, the relative capacity of each of these isoforms to produce H2O2, and their location in the vascular wall all are likely to weigh heavily in the determination of vascular phenotype. This complex interplay among oxidase isoforms and the other metabolic pathways regulating H2O2 is expected to be the focus of intense future study.
In summary, NADPH oxidases and other ROS-generating enzymes are ubiquitous across the vascular wall. All of these sources appear to have the potential to contribute to the production of biologically active concentrations of H2O2 in muscle, which can promote vascular constriction and hypertrophy. Most certainly, under normal conditions, constitutive oxidase activities and endogenous scavenger systems, including catalase and glutathione peroxidases, maintain a homeostatic balance in favor of normal constrictor tone and wall thickness. Upon stimulation of the various oxidases by mechanical stretch and/or vasoactive hormones, increased production of peroxide and/ or a deficiency in the capacity of the peroxidases lead to elevated prevailing levels of peroxide, which we postulate are free to traverse the radius of the blood vessel. While much emphasis has been placed on NADPH oxidase and H2O2 in cultured smooth muscle, and while these studies have afforded a detailed understanding of the downstream second messenger systems affected, the physiological role of vascular ROS is expected to be considerably more complex. Multiple oxidase systems in the endothelium, media, and adventitia are all expected to produce a significant share of biologically active ROS, including H2O2, that will eventually tip the balance in favor of a biological response. With growing evidence of a feed-forward relationship among the oxidases and an appreciation of H2O2’s far-reaching vascular effects, careful study of the individual sources and their communication is expected to be an area of intense study.
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Footnotes
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This work was supported by the National Institutes of Health grants HL55425 and HL28982, American Heart Association grant 0151407Z and the Fund for Henry Ford Hospital. N.A. was also supported by an American Heart Association Fellowship 0520056Z. P.J.P. is an Established Investigator of the American Heart Association.
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Abstract
Vascular Generation of H2O2
