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

Cam Patterson, Johannes Ruef, Nageswara R. Madamanchi, Patricia Barry-Lane, Zhaoyong Hu, Chris Horaist, Carol A. Ballinger, Alan R. Brasier, Christoph Bode, Marschall S. Runge

Research output: Contribution to journalArticle

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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 O2- 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 O2- 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.

Original languageEnglish (US)
Pages (from-to)19814-19822
Number of pages9
JournalJournal of Biological Chemistry
Volume274
Issue number28
DOIs
StatePublished - Jul 9 1999

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NADPH Oxidase
Vascular Smooth Muscle
Thrombin
Smooth Muscle Myocytes
Muscle
Oxidoreductases
Cells
Oxygen
4-ethoxymethylene-2-phenyl-2-oxazoline-5-one
In Vitro Techniques
Neointima
Balloons
Wounds and Injuries
Cell proliferation
Cell membranes
NADP
Mitogens
NAD
Blood Vessels
Aorta

ASJC Scopus subject areas

  • Biochemistry

Cite this

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. / Patterson, Cam; Ruef, Johannes; Madamanchi, Nageswara R.; Barry-Lane, Patricia; Hu, Zhaoyong; Horaist, Chris; Ballinger, Carol A.; Brasier, Alan R.; Bode, Christoph; Runge, Marschall S.

In: Journal of Biological Chemistry, Vol. 274, No. 28, 09.07.1999, p. 19814-19822.

Research output: Contribution to journalArticle

Patterson, C, Ruef, J, Madamanchi, NR, Barry-Lane, P, Hu, Z, Horaist, C, Ballinger, CA, Brasier, AR, Bode, C & Runge, MS 1999, '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', Journal of Biological Chemistry, vol. 274, no. 28, pp. 19814-19822. https://doi.org/10.1074/jbc.274.28.19814
Patterson, Cam ; Ruef, Johannes ; Madamanchi, Nageswara R. ; Barry-Lane, Patricia ; Hu, Zhaoyong ; Horaist, Chris ; Ballinger, Carol A. ; Brasier, Alan R. ; Bode, Christoph ; Runge, Marschall S. / 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. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 28. pp. 19814-19822.
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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 O2/·- 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 O2/·- 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.",
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AU - Madamanchi, Nageswara R.

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AU - Hu, Zhaoyong

AU - Horaist, Chris

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AU - Runge, Marschall S.

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