The overall goal of our laboratory is to understand the molecular mechanisms by which reactive oxygen species regulate vascular smooth muscle cell (VSMC) growth. We have a particular emphasis on angiotensin II, and are investigating the signaling pathways that are redox sensitive.
Several years ago, we showed that angiotensin II increases superoxide production in VSMCs by activating a membrane-associated NAD(P)H oxidase [Griendling 1994]. It turned out that angiotensin II-induced hypertrophy required activation of this enzyme [Griendling 1994, Ushio-Fukai 1996, Zafari 1998]. We then began studying the structure of the oxidase, starting from the premise that it would be structurally similar to the neutrophil respiratory burst NADPH oxidase. This enzyme consists of four major subunits: a plasma membrane spanning cytochrome b558 comprised of the catalytic moiety gp91phox and a smaller, regulatory p22phox protein and two cytosolic components p47phox and p67phox. The small molecular weight G protein rac2 (in some cells rac1) participates in assembly of the active complex. We successfully cloned p22phox [Fukui 1995] and showed that antisense inhibition of this protein blocked activation of the NAD(P)H oxidase by angiotensin II and inhibited hypertrophy of VSMCs [Ushio-Fukai 1996]. We found, however, that the catalytic moiety, gp91phox, is not expressed in smooth muscle. Subsequent collaboration with Dr. David Lambeth led to the cloning of nox1, a gp91phox homologue [Suh 1999]. We showed that nox1 is expressed in VSMCs, is regulated by growth factors, and, using antisense technology, we found that nox1mediates angiotensin II-induced superoxide formation [Lassègue 2001]. Very recent work has shown that VSMCs also express nox4, another gp91phox homologue, at much higher levels than nox1 [Lassègue 2001]. The function of nox4 in these cells is not known, but is under investigation.
Given that reactive oxygen species are essential to growth, we are currently investigating the molecular pathways activated by angiotensin II that are redox-sensitive, and are trying to understand whether p22phox and the novel gp91phox homologues expressed in VSMCs interact to form a functional enzyme. As an early step in activation of many tyrosine kinase pathways, angiotensin II transactivates the EGF receptor, in part by stimulating c-Src. We have shown that both of these events are mediated by reactive oxygen species [Ushio-Fukai 2001]. Furthermore, p38MAPK and Akt/protein kinase B are redox-sensitive, while ERK1/2 is not [Ushio-Fukai 1998, Ushio-Fukai 1999]. We are now investigating tyrosine kinases and phosphatases that are further downstream in the protein synthesis and cell cycle pathways.
Another project in the laboratory is designed to understand the mechanisms by which angiotensin II activates the NAD(P)H oxidase. Activation appears to occur acutely, by as yet unknown mechanisms, and chronically, by upregulation of enzyme components [Lassègue 2001], Protein kinase C may be involved in both of these processes.
We have recently found that the NAD(P)H oxidase is important not only in growth, but also in other pathophysiological processes such as migration and inflammation. We are now studying the role of the nox enzymes and their targets in PDGF-induced migration and induction of inflammatory genes. In addition, work by others at Emory has shown that the NAD(P)H oxidase is not confined to VSMCs, but is functionally expressed in endothelial cells as well. Importantly, NAD(P)H oxidase activity is regulated by shear stress [De Keulenaer 1998], and we are currently investigating the molecular identity of the responsible oxidase.
A major thrust of the laboratory is to investigate
the role of the nox enzymes in vascular disease. Several
years ago, in collaboration with Dr. David Harrison,
we found that hypertension caused by angiotensin II
infusion is accompanied by upregulation of p22phox
and increased NAD(P)H oxidase-derived superoxide formation [Rajagopalan 1996]. More
recently, we studied the regulation of these enzymes during
restenosis. In the rat carotid injury model, superoxide
production in all layers of the vessel wall is increased
from 3-15 days, and nox1 and nox4 are differentially
upregulated [Szöcs
2002] . We are currently constructing transgenic
and knockout mice in order to specifically manipulate
the expression of these two proteins and to determine
their role in hypertension, restenosis, and atherosclerosis.