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

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 H 2 O 2 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 H 2 O 2 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.

Non-hypoxic pathway mediates the induction of hypoxia inducible factor 1 alpha (HIF-1 alpha ) in vascular smooth muscle cells

“…The flavoproteincontaining enzyme inhibitor Diphenyleneiodonium (DPI) has been widely used to evaluate the activity of this pathway. DPI has been shown to inhibit Ang II, thrombin and PDGF mediated induction of ROS production in VSMC (31,34,35). When cells were pretreated with 10 µM DPI prior to stimulation with Ang II, HIF-1α induction was completely inhibited (Fig.…”

“…Recent evidence has convincingly shown that in VSMC, Ang II, thrombin and PDGF can modulate the expression and activity of different elements of the NAD(P)H oxidase system (32)(33)(34)(35)(36). This system is responsible for strong increases in intracellular ROS levels found in these cells after growth factor and hormone stimulation.…”

“…Recent research has shown that the activity of the VSMC NAD(P)H system is increased by hormones and growth factors such as Ang II, thrombin and PDGF (32)(33)(34)(35)(36). The mechanism by which these compounds activate the NAD(P)H oxidase remains unclear.…”

“…Different hormones and growth factors have been shown to activate the production of ROS in VSMC cells (31,34,35). It is interesting to note that VSMC cells specifically contain homologues to the various proteins that are similar to components of the phagocytic NAD(P)H oxidase whose activation leads to the production of ROS (32)(33)(34)(35)(36).…”

Local production of O2− by NAD(P)H oxidase in the sarcoplasmic reticulum of coronary arterial myocytes: cADPR-mediated Ca2+ regulation

“…This vascular redox regulatory enzyme is of characteristics of phagocyte NOX, which is composed of two transmembrane-bounded catalytic proteins of gp91 phox and p22 phox , and three cytosol-associated subunits of p47 phox , p67 phox and p40 phox . In addition to gp91 phox named as Nox2, some other homologues of gp91 phox such as Nox1, Nox4 and Nox5 were identified in the vascular cells such as endothelial and smooth muscle cells [3,4]. NOX is now well accepted as an important enzyme that produces O 2 .− in the vasculature under physiological and pathological conditions to exert redox regulatory action on vascular function or to produce pathogenic responses.…”