Stimulation of a Vascular Smooth Muscle Cell NAD(P)H Oxidase by Thrombin

doi:10.1074/jbc.274.28.19814

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**

From the Division of Cardiology and Sealy Center for Molecular Cardiology, University of Texas Medical Branch, Galveston, Texas 77555-1064 and the ^§Division of Cardiology, University of Heidelberg, D-69115 Heidelberg, Germany

Abstract

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^phoxmight 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.

Footnotes

↵* This work was supported by the Sealy and Smith Foundation, National Institutes of Health (NIH) Grant HL57352 (to M. S. R.), and NIH Grants HL03658 and AG15234 (to C. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ These authors contributed equally to this work.
↵¶ To whom correspondence should be addressed: Division of Cardiology, University of Texas Medical Branch, 9.138 Medical Research Bldg., 301 University Blvd., Galveston, TX 77555-1064. Tel.: 409-772-1631; Fax: 409-772-0682; E-mail: cpatters@utmb.edu.
Abbreviations:

ROI

reactive oxygen intermediate(s)

VSMC

vascular smooth muscle cell(s)

PDGF

platelet-derived growth factor

HASMC

human aortic smooth muscle cell(s)

DPI

diphenyleneiodonium chloride

NBT

nitro blue tetrazolium

DCFDA

2′,7′-dichlorofluorescein diacetate

PCR

polymerase chain reaction

RT-PCR

reverse transcriptase-PCR
- Received November 9, 1998.
- Revision received March 23, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.