Abstract 1534: Inhibition of ROS Liberation Attenuates PDGF-Dependent Chemotaxis, but not Proliferation in Vascular Smooth Muscle Cells - Critical Role of Src Kinase
Reactive oxygen species (ROS) play a significant role in the pathogenesis of cardiovascular disease. They contribute to various cellular functions and are involved in the regulation of vascular tone, migration, and apoptosis. NAD(P)H oxidases are the major source of ROS in the vascular wall. The activity of these enzymes is regulated by growth factors such as angiotensin II and platelet-derived growth factor (PDGF). Recently, novel homologues of the NAD(P)H oxidase subunit gp91phox have been identified (nox1–5). We characterize the novel nox inhibitor VAS2870 in VSMC. PDGF-BB increased NAD(P)H oxidase-activity (measured as lucigenin-enhanced chemiluminescence) and intracellular ROS levels (detected by confocal laserscanning microscopy using 2,7 DCF staining) to 229±9% and 362±54% at 1 and 2h, respectively. Preincubation with VAS2870 (10 and 20 μM) completely abolished PDGF-mediated NAD(P)H oxidase activation and ROS production. Since ROS are involved in various growth factor-induced cellular functions, the influence of VAS2870 on PDGF-induced DNA synthesis and chemotaxis was determined. PDGF increased VSMC migration to 4,2±0,2-fold (modified Boyden chamber, p<0,01) and concentration-dependently enhanced DNA synthesis to maximally 3,2±0,4-fold (BrdU incorporation, p<0,01). VAS2870 preincubation (0,1–20 μM) had no effect on PDGF-induced cell cycle progression. However, it concentration-dependently abolished PDGF-dependent chemotaxis (100 % inhibition at 10 μM). These findings were related to PDGF-dependent signaling events. Western blot analyses using phospho-specific antibodies revealed that the downstream signaling molecules Akt, Erk, and Src were activated by PDGF. However, VAS2870 concentration-dependently blocked PDGF-dependent activation of Src, but not of Akt and Erk. Our data indicate that the novel nox inhibitor VAS2870 effectively suppresses growth factor-mediated ROS liberation in VSMC. Furthermore, it abolishes PDGF-dependent VSMC migration, whereas it does not affect DNA synthesis. These divergent effects demonstrate the critical role of Src activity, which - in contrast to Akt and Erk - appears to be redox-sensitive and is absolutely required for PDGF-induced chemotaxis, but not cell cycle progression.