Stimulation of a vascular smooth muscle cell NAD(P)H oxidase by thrombin. Evidence that p47(phox) may participate in forming this oxidase in vitro and in vivo.
Thrombin is a potent vascular smooth muscle cell (VSMC) mitogen. Because recent evidence implicates reactive oxygen intermediates (ROI) in VSMC proliferation in general and atherogenesis in particular, we investigated whether ROI generation is necessary for thrombin-induced mitogenesis. Treatment of human aortic smooth muscle cells with thrombin increased DNA synthesis, an effect that was antagonized by diphenyleneiodonium but not by other inhibitors of cellular oxidase systems. This effect of thrombin was accompanied by increased O-2 and H2O2 generation and NADH/NADPH consumption. ROI generation in response to thrombin pretreatment could also be blocked by diphenyleneiodonium, suggesting that the NAD(P)H oxidase was necessary for ROI generation and thrombin-induced mitogenesis. Because of observed differences between the VSMC and neutrophil oxidase, we examined whether the cytosolic components of the phagocytic NAD(P)H oxidase were present in VSMC. p47(phox) and Rac2 were present in VSMC. Furthermore, thrombin increased expression of p47(phox) and Rac2 and stimulated their translocation to the cell membrane. We examined whether p47(phox) might be similarly regulated in vivo in a rat aorta balloon injury model and found that p47(phox) protein was increased after injury. Immunocytochemistry localized expression of p47(phox) to the neointima and media of injured arteries. Our data demonstrate that generation of O-2 and H2O2 is required for thrombin-mediated mitogenesis in VSMC and that p47(phox) is regulated by thrombin in vitro and is associated with vascular lesion formation in vivo.
Division of Cardiology and Sealy Center for Molecular Cardiology, University of Texas Medical Branch, Galveston, Texas 77555-1064, USA. cpatters@utmb.edu
C
C
Patterson
J
J
Ruef
N R
NR
Madamanchi
P
P
Barry-Lane
Z
Z
Hu
C
C
Horaist
C A
CA
Ballinger
A R
AR
Brasier
C
C
Bode
M S
MS
Runge
D000818:Animals; D001011:Aorta; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008934:Mitogens / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D019255:NADPH Oxidases; D010750:Phosphoproteins / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*; D020744:rac GTP-Binding Proteins
D008934:Mitogens; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D009243:NAD; D013481:Superoxides; D009249:NADP; D006861:Hydrogen Peroxide; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D013917:Thrombin; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins