Novel Role of gp91 ^phox -Containing NAD(P)H Oxidase in Vascular Endothelial Growth Factor–Induced Signaling and Angiogenesis

Abstract

Abstract-Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell proliferation and migration, primarily through the receptor tyrosine kinase VEGF receptor2 (Flk1/KDR). Reactive oxygen species (ROS) derived from NAD(P)H oxidase are critically important in many aspects of vascular cell regulation, and both the small GTPase Rac1 and gp91 phox are critical components of the endothelial NAD(P)H oxidase complex. A role of NAD(P)H oxidase in VEGF-induced angiogenesis, however, has not been defined. In the present study, electron spin resonance spectroscopy is utilized to demonstrate that VEGF stimulates O 2 ·Ϫ production, which is inhibited by the NAD(P)H oxidase inhibitor, diphenylene iodonium, as well as by overexpression of dominant-negative Rac1 (N17Rac1) and transfection of gp91 phox antisense oligonucleotides in human umbilical vein endothelial cells (ECs). Antioxidants, including N-acetylcysteine (NAC), various NAD(P)H oxidase inhibitors, and N17Rac1 significantly attenuate not only VEGF-induced KDR tyrosine phosphorylation but also proliferation and migration of ECs. Importantly, these effects of VEGF are dramatically inhibited in cells transfected with gp91 phox antisense oligonucleotides. By contrast, ROS are not involved in mediating these effects of sphingosine 1-phosphate (S1P) on ECs. Sponge implant assays demonstrate that VEGF-, but not S1P-, induced angiogenesis is significantly reduced in wild-type mice treated with NAC and in gp91 phoxϪ/Ϫ mice, suggesting that ROS derived from gp91 phox -containing NAD(P)H oxidase play an important role in angiogenesis in vivo. These studies indicate that VEGF-induced endothelial cell signaling and angiogenesis is tightly controlled by the reduction/oxidation environment at the level of VEGF receptor and provide novel insights into the NAD(P)H oxidase as a potential therapeutic target for angiogenesis-dependent diseases.

Hemorrhagic Transformation after Ischemic Stroke in Animals and Humans

“…117 ROS also promote EPC mobilization, differentiation, and homing and thus contribute to vascular remodeling and angiogenesis. 119 NADPH oxidase (NOX) is an important source of superoxide and ROS. NOX4-induced ROS has been shown to promote endothelial cell migration and proliferation as well as promote blood flow recovery after ischemia.…”

Vascular endothelial growth factor in eye disease

“…Increases in retinal nitrotyrosine levels and lipid peroxidation have been shown to correlate with VEGF overexpression and breakdown of the blood-retinal barrier, and treatments that reduce peroxynitrite formation have been shown to block the early signs of diabetic retinopathy (El-Remessy, 2003b;El-Remessy et al, 2006). Moreover, VEGF itself has been shown to promote the formation of superoxide anion and peroxynitrite in cultured endothelial cells (El-Remessy et al, 2007;Ushio-Fukai et al, 2002). Furthermore, formation of both oxidants appears to be required for transduction of VEGF's angiogenic signal.…”

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…Additionally, NOX-mediated effects on e.g. cell proliferation or migration also depend on strict spatial localization of NOX activation in association with the cytoskeleton (70,71) or in membrane rafts (43,72,73), and on direct interactions with target proteins (3,43,55). This allows for effective oxidative signaling in the presence of abundant cytosolic antioxidant mechanisms (Cu/Zn SOD, GSH peroxidase), and assures confined oxidant production and target specificity.…”