Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferation

Objective: Reactive oxygen species (ROS) produced by NAD(P)H oxidases (Nox) play a significant role in the pathophysiology of cardiovascular diseases. Expression and activity of NAD(P)H oxidases are regulated by growth factors such as angiotensin 11 and platelet-derived growth factor (PDGF). We characterized the effects of the novel Nox inhibitor VAS2870 on PDGF-dependent ROS liberation and cellular events in vascular smooth muscle cells (VSMC). Methods and results: PDGF-BB increased NAD(P)H oxidase activity (lucigenin-enhanced chemiluminescence) and intracellular ROS levels (detected by confocal laserscanning microscopy using 2,7-DCF) to 229 +/- 9% and 362 +/- 54% at 1 and 2 h, respectively. Preincubation with VAS2870 (10 and 20 mu M) 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 promoted a 4.2 +/- 0.2-fold increase of VSMC migration (modified Boyden chamber, p < 0.01) and increased DNA synthesis by maximally 3.2 +/- 0.4-fold (BrdU incorporation, p < 0.01) in a concentration-dependent manner. Preincubation with VAS2870 (0.1-20 mu M) did not affect PDGF-induced cell cycle progression. However, it abolished PDGF-dependent chemotaxis of VSMC in a concentration-dependent manner (100% inhibition at 10 mu M). 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 blocked PDGF-dependent activation of Src, but not of Akt and Erk, in a concentration-dependent manner. \ Conclusions: VAS2870 effectively suppresses growth factor-mediated ROS liberation in VSMC. Furthermore, it completely inhibits PDGF-dependent VSMC migration, whereas it does not affect DNA synthesis. These divergent effects reflect 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. (c) 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.