NADPH Oxidase 4 Promotes Endothelial Angiogenesis Through Endothelial Nitric Oxide Synthase Activation

Abstract

Background Reactive Oxygen Species (ROS) serve signaling functions in the vasculature, and hypoxia has been associated with increased ROS production. NADPH oxidase 4 (Nox4) is an ROS-producing enzyme that is highly expressed in the endothelium, yet its specific role is unknown. We sought to determine the role of Nox4 in the endothelial response to hypoxia. Methods and Results Hypoxia induced Nox4 expression both in vitro and in vivo and overexpression of Nox4 was sufficient to promote endothelial proliferation, migration, and tube formation. To determine the in vivo relevance of our observations, we generated transgenic mice with endothelial-specific Nox4 overexpression using the VE-cadherin promoter (VECad-Nox4 mice). In vivo, the VECad-Nox4 mice had accelerated recovery from hind limb ischemia and enhanced aortic capillary sprouting. Because endothelial nitric oxide synthase (eNOS) is involved in endothelial angiogenic responses and eNOS is activated by ROS, we probed the effect of Nox4 on eNOS. In cultured ECs overexpressing Nox4 we observed a significant increase in eNOS protein expression and activity. To causally address the link between eNOS and Nox4 we crossed our transgenic Nox4 mice with eNOS-/- mice. Aorta from these mice did not demonstrate enhanced aortic sprouting and VECad-Nox4 mice on the eNOS-/- background did not demonstrate enhanced recovery from hind limb ischemia. Conclusions Collectively, we demonstrate that augmented endothelial Nox4 expression promotes angiogenesis and recovery from hypoxia in an eNOS-dependent manner.

Deficient angiogenesis in redox-dead Cys17Ser PKARIα knock-in mice

“…Our observations are also consistent with recent observations that Nox4 KO mice are deficient in their angiogenic response to the same hind-limb ischaemia implemented to RIa redox-dead mice [9][10][11] . Discussion Nox-derived oxidants can act as powerful mediators of growth factor signalling 10,11,20 . However, the targets of these oxidants and the mechanism by which they enhance vessel growth have remained elusive.…”

“…Recently three independent studies have specifically implicated Nox4 as an isozyme of NADPH oxidase that coupled to angiogenesis [9][10][11] . Consequently, we compared VEGF-induced RIa oxidation in the aorta from Nox4 knockout (KO) mice with littermate WTs, finding the former were substantially deficient (Fig.…”

“…Several studies have identified nicotinamide adenine dinucleotide phosphate-oxidase (Nox) enzymes as a major source of oxidants required for angiogenesis [9][10][11] . Although oxidase enzymes mediate angiogenesis, little is known about the molecular targets that sense and transduce the growth-stimulating 'oxidant signal'.…”

Hemorrhagic Transformation after Ischemic Stroke in Animals and Humans

“…Delayed Hemorrhagic Transformation: Role of Reactive Oxygen Species Reactive oxygen species have an important role in vascular remodeling and angiogenesis, 117 and thus likely contribute to delayed HT. They have regulatory effects on many angiogenic factors including VEGF, hypoxia inducible factor-1a signaling, MMP activation, NFkB activity, protein kinase C activity, ERK1/2 activity, and p38 MAP kinase activity.…”

“…NOX4-induced ROS has been shown to promote endothelial cell migration and proliferation as well as promote blood flow recovery after ischemia. 117 When NOX1 is knocked out in a mouse, angiogenic activity is impaired. 120 Likewise, in the NOX2 mouse knockout or when NOX2 is inhibited, ischemia-induced neovascularization is impaired.…”