The Cytosolic NADPH Oxidase Subunit NoxO1 Promotes an Endothelial Stalk Cell Phenotype

Abstract

Objective— Reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases contribute to angiogenesis and vascular repair. NADPH oxidase organizer 1 (NoxO1) is a cytosolic protein facilitating assembly of constitutively active NADPH oxidases. We speculate that NoxO1 also contributes to basal reactive oxygen species formation in the vascular system and thus modulates angiogenesis. Approach and Results— A NoxO1 knockout mouse was generated, and angiogenesis was studied in cultured cells and in vivo. Angiogenesis of the developing retina and after femoral artery ligation was increased in NoxO1 −/− when compared with wild-type animals. Spheroid outgrowth assays revealed greater angiogenic capacity of NoxO1 −/− lung endothelial cells (LECs) and a more tip-cell–like phenotype than wild-type LECs. Usually signaling by the Notch pathway switches endothelial cells from a tip into a stalk cell phenotype. NoxO1 −/− LECs exhibited attenuated Notch signaling as a consequence of an attenuated release of the Notch intracellular domain on ligand stimulation. This release is mediated by proteolytic cleavage involving the α-secretase ADAM17. For maximal activity, ADAM17 has to be oxidized, and overexpression of NoxO1 promoted this mode of activation. Moreover, the activity of ADAM17 was reduced in NoxO1 −/− LECs when compared with wild-type LECs. Conclusions— NoxO1 stimulates α-secretase activity probably through reactive oxygen species–mediated oxidation. Deletion of NoxO1 attenuates Notch signaling and thereby promotes a tip-cell phenotype that results in increased angiogenesis.

Context‐dependent function of ROS in the vascular endothelium: The role of the Notch pathway and shear stress

“…Although available data are not sufficient to depict a comprehensive picture of the mechanisms that allow interactions between Notch and ROS in the endothelium, there are some clues that allow some speculations. As mentioned above, Nfr2 is activated in response to oxidative stress and its modulation of Notch is well established but also a direct regulation of Notch processing enzymes by ROS has been reported . Therefore, it appears possible that the Notch pathway can be altered by Nfr2 to protect cells from endogenous or exogenous stressors, or precisely regulated, through production of H 2 O 2 in response to a physiological stimulus, by targeted oxidization of a specific Notch ligand, receptor or processing enzyme.…”

“…Similarly, it has been recently reported that Nox2‐mediated ROS production promotes arterial EC specification in mouse‐induced pluripotent stem cells (miPSCs) and contributes to the angiogenic potency of transplanted miPSC‐derived EC by activating the Notch signaling . Furthermore, Brandes et al have shown that ROS act as regulators of angiogenesis by modulating the Notch signaling. In this study it has been demonstrated that LECs (lung endothelial cells) knockout for the Nox organizer protein 1 (NoxO1) exhibit attenuated Notch signaling as a consequence of a diminished NoxO1‐dependent ROS activation of the metalloprotease ADAM17.…”

Roles of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase in Angiogenesis: Isoform-Specific Effects

“…NoxO1, the homologue to p47 phox , facilitates the assembly of NADPH oxidase subunits and, therefore, promotes NADPH oxidase activation mainly through NOX1 [79]. NoxO1 −/− mice had faster retinal vascular development with less avascular retinal area at both postnatal day 5 (p5) and p7 and increased capillary density at p7 [80]. In the hindlimb ischemic-reperfusion model, NoxO1 −/− mice had faster restoration of blood flow and capillary density with increased VEGF expression in the muscles of the ischemic legs [80].…”

“…NoxO1 −/− mice had faster retinal vascular development with less avascular retinal area at both postnatal day 5 (p5) and p7 and increased capillary density at p7 [80]. In the hindlimb ischemic-reperfusion model, NoxO1 −/− mice had faster restoration of blood flow and capillary density with increased VEGF expression in the muscles of the ischemic legs [80]. Lung ECs isolated from NoxO1 −/− mice had increased angiogenic capacity and expression of VEGFR2 and the tip cell marker, Notch ligand Delta-like 4 (DLL4) [80].…”

“…In the hindlimb ischemic-reperfusion model, NoxO1 −/− mice had faster restoration of blood flow and capillary density with increased VEGF expression in the muscles of the ischemic legs [80]. Lung ECs isolated from NoxO1 −/− mice had increased angiogenic capacity and expression of VEGFR2 and the tip cell marker, Notch ligand Delta-like 4 (DLL4) [80]. These findings suggest that activation of NoxO1 inhibits angiogenesis in physiologic vascular development and vascular recovery from ischemic insults.…”