Novel role of gp91(phox)-containing NAD(P)H oxidase in vascular endothelial growth factor-induced signaling and angiogenesis.
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 O2*- 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(phox-/-) 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.
Ushio-Fukai
Masuko
M
Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Ga 30322, USA. mfukai@emory.edu
Tang
Yan
Y
Fukai
Tohru
T
Dikalov
Sergey I
SI
Ma
Yuxian
Y
Fujimoto
Mitsuaki
M
Quinn
Mark T
MT
Pagano
Patrick J
PJ
Johnson
Chad
C
Alexander
R Wayne
RW
D000818:Animals; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D016228:Endothelial Growth Factors / Q000494:pharmacology / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D016166:Free Radical Scavengers / Q000494:pharmacology; D005799:Genes, Dominant; D017930:Genes, Reporter; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology / Q000502:physiology*; D008222:Lymphokines / Q000494:pharmacology / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013347:Subcellular Fractions / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D042442:Vascular Endothelial Growth Factors; D020830:rac1 GTP-Binding Protein / Q000096:biosynthesis / Q000235:genetics
D016228:Endothelial Growth Factors; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D008562:Membrane Glycoproteins; D016376:Oligonucleotides, Antisense; D017382:Reactive Oxygen Species; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D013481:Superoxides; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D040301:Vascular Endothelial Growth Factor Receptor-2; D020830:rac1 GTP-Binding Protein