doi: 10.1161/01.ATV.0000077235.97226.82
© 2003 American Heart Association, Inc.
Editorials |
c-Src and Smooth Muscle NAD(P)H Oxidase
Assembling a Path to Hypertrophy
From the Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, Mich.
Correspondence to Dr Patrick Pagano, Henry Ford Hospital, Hypertension and Vascular Research Division, Room 7044, E&R Bldg, 2799 W Grand Blvd, Detroit, MI 48202-2689. E-mail ppagano1{at}hfhs.org
Early studies of NAD(P)H oxidase in vascular tissue focused on the regulation of this complex enzyme and suggested its deleterious role in end-organ damage in hypertension and atherosclerosis.1,2 The preponderance of data regarding the regulation of this enzyme and its similarity to the phagocyte prototype that mediates antimicrobial properties contributed to an impression that NAD(P)H oxidase played a similar toxic role in the vascular wall. Increasingly, reports challenge this notion and ascribe a very different role to the oxidase and ROS, demonstrating participation by angiotensin II–induced superoxide anion (O2-) in central nervous system signaling, and NAD(P)H oxidase as a critical player in growth factor–induced angiogenesis.3,4
See page 981
Significant strides have been made recently in our understanding of the regulation of cardiovascular NAD(P)H oxidases, revealing elaborate control of these low-capacity NAD(P)H oxidases as generators of important signaling agents in cardiovascular disease including O2- and hydrogen peroxide (H2O2). At the focus of the discussion, endothelial and adventitial cells appear to contain a functional phagocyte-like NAD(P)H oxidase including p22phox, gp91phox, p47phox, and p67phox.5–7 In contrast, vascular smooth muscle cells (VSMCs) appear to vary substantially in terms of the existence of homologues of gp91phox (nox-1 and nox-4) and the absence of p67phox.8–10 The upregulation of vascular p22phox, p47phox, p67phox, and nox-2 has suggested that active de novo synthesis and assembly occur during hormonal stimulation.8,11,12 Angiotensin II (AngII) has been shown to cause phosphorylation and translocation of p47phox in VSMCs, resulting in enhanced oxidase activity.13 Recently, substantial insight was gained from a thorough examination of the oxidase in human resistance VSMCs, which illustrated a striking homology with the phagocyte oxidase including the presence of classical gp91phox.8
Various studies have addressed upstream regulation of NAD(P)H oxidase in animal models. AngII-induced NAD(P)H oxidase activity has been explained by phospholipase D activation,14 and as with its phagocyte counterpart, this is likely to be related to direct activation of the enzyme by arachidonic acid or eicosanoids.15,16 In phagocytes, protein kinase C (PKC) is an important mediator of p47phox phosphorylation and oxidase activation,17 and recent data suggest direct involvement of PKC in vascular cell oxidase activation.10 Perhaps the best evidence of receptor-associated stimulation of the oxidase is the involvement of an essential GTP-bound subunit called Rac1.17,18 Recently, Seshiah et al10 reported intriguing results in rat aortic VSMCs, suggesting that NAD(P)H oxidase activation initiates a feed-forward mechanism involving activation of c-Src by small amounts of ROS from NAD(P)H oxidase and thereby leads to sustained oxidase activation. This study gave the most complete picture thus far of the upstream pathways leading from receptor to NAD(P)H oxidase activation in vascular tissue.
In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Touyz et al19 convincingly demonstrate in human resistance artery VSMCs that c-Src regulates activation of NAD(P)H oxidase. This is the first report to our knowledge of c-Src involvement in NAD(P)H oxidase activation in human blood vessels, and the findings shed important new light on AngII signaling mechanisms in resistance arteries. Because most reports on the vascular oxidase have focused on large conduit arteries, this study represents a significant advance in our understanding of the role of NAD(P)H oxidase in the regulation of vascular tone in hypertension. The article clearly shows that AngII-induced p47phox phosphorylation and translocation, processes characteristic of phagocyte NAD(P)H oxidase activation,12 are completely blocked by c-Src inhibition in human VSMCs. Furthermore, the data demonstrate that c-Src activation by AngII contributes to chronic elevation of protein expression of p22phox, gp91phox, and p47phox, a strong indication of c-Src involvement in long-term oxidase activation. These findings are corroborated in c-Src -/- versus wild-type mice in which the authors show that AngII-stimulated oxidase activity was significantly reduced, with no elevation in subunit expression compared with controls. These data are consistent with another report showing that c-Src activates NAD(P)H oxidase in the rat.10 Thus, at this juncture, there appears to be substantial homology between the human vascular and phagocyte oxidase as well as those characterized in other mammalian species. Previously reported interactions of the c-Src-NAD(P)H oxidase pathway with PIP3 may suggest acute involvement in vascular contraction.10
On a more chronic level, the clear demonstration of oxidase involvement in c-Src signaling pathways in both rats and humans20 suggests a conserved involvement of the oxidase in cell growth. Moreover, the demonstration that c-Src mediates upregulation of oxidase subunits suggests chronic involvement of the oxidase in constriction and long-term development of vessel hypertrophy. Indeed, there is a growing body of evidence showing that NAD(P)H oxidase is part of a signaling cascade leading to cell de-differentiation, proliferation, and migration. VSMC migration and proliferation have long been implicated in narrowing of the arterial lumen in atherosclerosis and response to injury. While the mechanisms involved are not fully understood, AngII, growth factors, and proto-oncogenes have been suggested as mediators in this process.21 Recently, cyclic strain has been shown to cause oxidative stress in vascular cells.22–24 Souza et al elegantly demonstrated rapid and massive increases in NAD(P)H oxidase-derived O2- in response to cyclic stretch25 and upregulation of nox-1, nox-2, and nox-4 in response to carotid balloon injury suggests that all three nox homologues play a role in this process.26 In fact, We27 showed that a NAD(P)H oxidase assembly inhibitor significantly blocked this increase in the rat carotid artery, suggesting activation of de novo NAD(P)H oxidase assembly in response to injury, which is essential to hyperplasia. Cyclic strain activates rabbit aortic extracellular signal-regulated kinase 1/2 (ERK 1/2) in an O2--dependent manner, suggesting that this kinase is a link between stretch-induced oxidase activity and cell proliferation.28 In the rat, p38 mitogen-activated protein kinase (MAPK), which is rapidly activated by ROS, has been implicated as a mediator of proliferation.29
On the other hand, NAD(P)H oxidase has been implicated in the hypertrophic phenotype of type 1 VSMCs. In cultured smooth muscle cells, AngII has clearly been shown to induce hypertrophy,30,31 which is mediated by activation of NAD(P)H oxidase-derived H2O2 that in turn can activate proto-oncogene expression, MAP kinases (p38 MAPK), and Akt/PKB, leading to a growth response.32,33 Two recent reports verify the functional involvement of gp91phox (nox-2)-based oxidase in medial hypertrophy.34,35 However, molecular evidence suggests a far more complex signaling cascade involving vascular NAD(P)H oxidase-derived ROS. For example, one study showed that epidermal growth factor receptor (EGF-R) and platelet-derived growth factor receptor (PDGF-R) transactivation by AngII involves NAD(P)H oxidase-derived ROS.36 The resulting tyrosine phosphorylation causes activation of Shc-Grb2-Sos, which stimulates ras, leading to downstream activation of MAPKs and transcription factors involved ultimately in a growth program which includes hypertrophy, cell proliferation, and migration (Figure).37 Redox-sensitive kinases playing a role in this cascade are ERK 1/2, c-Jun N-terminal kinases (JNK), big MAPK, p38 MAPK,38–40 and Akt/PKB.32,33 Because these pathways overlap in their involvement in proliferation and hypertrophy, it is unclear how selectivity is achieved.
In this regard, involvement of the proproliferative c-Src pathway in the regulation of VSMC NAD(P)H oxidase raises many more questions that need to be addressed. The relatively advanced differentiation state of the cell is expected to play a major role in attenuating the ability of VSMCs to divide.41 Moreover, even in the face of full activation of c-Src and oxidase pathways, ROS-induced expression of cyclin-dependent kinase inhibitors such as p27Kip1 could prevent cell cycle progression to mitosis.42 The precise location of the oxidase downstream from c-Src may play an important role in selective stimulation of redox-sensitive kinases involved in both the hypertrophic phenotype and regulation of cyclin-dependent kinases. In this light, oxidase activation, although part of the c-Src pathway normally viewed as proproliferative, could provide a "braking" mechanism suspending VSMCs in a hypertrophic state (Figure). Careful analysis of the unique signaling profile activated in hypertrophic versus proliferative cells will be necessary to clarify this issue.
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