Angiogenesis is involved in embryonic development and wound repair, as well as in pathological conditions such as cancer, diabetic retinopathy and inflammation of atherosclerosis. Angiogenesis occurs through degradation of extracellular matrix, endothelial cell migration, proliferation and organization into tube network structure, followed by lumen formation. With increasing insight into the role of angiogenesis in various pathologies, modulation of vascular outgrowth is now regarded as a potential therapeutic target. Vascular endothelial growth factor (VEGF) is one of the most potent angiogenesis growth factors so far identified and stimulates endothelial cell proliferation and migration in vitro and angiogenesis in vivo. In endothelial cells VEGF binds to two tyrosine kinase receptors, VEGF receptor-1 (also termed Flt-1) and VEGFR2 (also termed KDR/Flk1). The angiogenic effects of VEGF in endothelial cells are mediated mainly through VEGFR2. VEGF binding initiates tyrosine phosphorylation of VEGFR2, which results in the activation of downstream signaling enzymes including MAP kinases such as ERK1/2 and p38MAP kinase, and cell survival kinase Akt. The molecular mechanisms regulating these events are incompletely understood.

Recent evidence suggests that reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide (H 2O 2) are involved in the signaling pathways mediating many stress and growth responses, including angiogenesis. There is, however, relatively little information concerning the molecular pathways involved in regulating reduction/oxidation (redox)-sensitive signaling events in angiogenesis. We have recently demonstrated that VEGF stimulation with cultured endothelial cells induces robust ROS production mainly through activation of NAD(P)H oxidase, a major source of ROS in the vessel wall. We also found that gp91phox (Nox2)-derived ROS play an important role in VEGF-induced autophosphorylation of VEGFR2 and angiogenic responses such as cell migration and proliferation in endothelial cells. We also showed that neovascularization in response to VEGF and to hindlimb ischemia are impaired in Nox2 -/- mice.

Thus, we are currently investigating 1) how NAD(P)H oxidase is activated, 2) how ROS are involved in VEGFR2 autophosphorylation and downstream signaling cascades leading to angiogenesis, 3) how ROS signals are specifically transferred from the receptor to the nucleus to induce redox-sensitive gene expression, 4) what is the source of cells (inflammatory cells or endothelial cells) that produce ROS to induce neovascularization in response to tissue ischemia, 5) whether ROS are involved in mobilization of bone marrow-derived endothelial progenitor cells (EPC) as well as its homing to the injury site, which contribute to postnatal neovascularization in response to hindlimb ischemia. The overall objective is to gain mechanistic insights into how ROS derived from NAD(P)H oxidase are involved in angiogenesis signaling, proliferation, migration in endothelial cells and EPCs as well as neovascularization in response to ischemic injury. Understanding these mechanisms may reveal that the components of NAD(P)H oxidase and their regulators as potential therapeutic targets for treatment of angiogenesis-dependent diseases and for promoting neovascularizaion in ischemic heart and limb diseases.

Another project is focusing on "IQGAP1", a novel VEGF type 2 receptor (VEGFR2) binding protein which we identified using yeast two-hybrid system. IQGAP1 is an F-actin binding scaffold protein that regulates cell motility and morphogenesis by interacting directly with cytoskeletal and cell-cell adhesion molecules including active Rac1 and VE-cadherin/ b -catenin. We found that: 1) IQGAP1 expression is dramatically increased in the newly-formed capillary endothelial cells in a mouse hindlimb ischemia model of angiogenesis; 2) Overexpression of IQGAP1 increases ROS production and cell migration in endothelial cells; 3) VEGF promotes recruitment of activated VEGFR2 and Rac1 to the IQGAP1-VE-cadherin complex at adherens junctions, which promotes ROS-dependent loss of cell-cell contacts and subsequent EC migration; 4) Wound assays reveal that IQGAP1 colocalizes with active VEGFR2 and gp91phox of NAD(P)H oxidase at the leading edge in active migrating endothelial cells. Thus, we are currently testing hypothesis that IQGAP1 may function as a VEGFR2 binding scaffold protein to couple ROS-dependent signaling to VEGF-mediated endothelial migration, which may contribute to neovascularization. These studies should facilitate the development of new therapeutic strategies to modulate endothelial repair in response to injury and neovascularization.

Finally, our laboratory is studying angiotensin II (Ang II) signaling in vascular smooth muscle cells. Ang II is a potent mediator of vascular hypertrophy, a hallmark of hypertension and atherosclerosis. These effects are mediated through the Ang II type 1 receptor (AT 1R). Major outputs of the AT 1R are dependent upon the transactivation (tyrosine phosphorylation) of the EGF receptor (EGF-R) whose response is mediated through reactive oxygen species (ROS) derived from NAD(P)H oxidase and caveolae/lipid rafts. Caveolae/lipid rafts are cholesterol-enriched, specialized membrane microdomains where multimolecular complexes of signaling molecules such as EGF-R and Src are compartmentalized via interacting with caveolin-1. We are currently testing hypothesis that caveolin-1 plays an essential role in AT 1R trafficking into caveolin-enriched lipid rafts and NAD(P)H oxidase activation, thereby regulating ROS-dependent EGF-R transactivation and its downstream redox signaling, which may contribute to vascular hypertrophy using siRNA and knockout mice for caveolin-1.

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