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Osteoprotegerin (OPG), an inhibitor of vascular calcification, has pleiotropic vascular effects independently of its actions on calcification. OPG has been associated with vascular inflammation and remodelling and may be important in cardiovascular disease where OPG levels may be elevated. Molecular mechanisms and functional consequences of OPG stimulation in the vasculature are unclear. We propose that syndecan-1, a membrane glycoprotein, may be important and that reactive oxygen species (ROS) play a role in OPG signalling. Vascular reactivity of resistance arteries from WKY rats was studied by wire myography in the presence or absence of OPG (50 ng/mL) and/or synstatin (SSNT – 10-6M – syndecan-1 inhibitor). Rat endothelial cells (EC) and vascular smooth muscle cells (VSMC) were studied. Levels of ROS were measured by chemiluminescence, Amplex Red (H2O2) and ELISA (nitrotyrosine; peroxynitrite – ONOO-). Protein oxidation and levels were measured by immunoblotting. Exposure of resistance arteries to OPG induced endothelial (decreased relaxation to acetylcholine) and VSMC (decreased relaxation to sodium nitroprusside – SNP) dysfunction, as well as, increased contraction to phenylephrine. All responses were blocked by SSNT, N-acetylcisteine (antioxidant) and ML171 (Nox inhibitor). In EC, OPG-induced ROS production (240±46.1% increase vs. veh, p<0.05) was blocked by SSNT. OPG decreased H2O2 production/release (61±5.4% vs. veh) and increased eNOS Thr 495 phosphorylation (inhibitory site) (100±24% vs. veh, p<0.05). In VSMC, OPG increased H2O2 (69±3%) and ONOO- (43±12%) levels, protein oxidation (61±15%), Rho kinase (200±39%) and myosin light chain activation (55±3%) (all vs. veh, p<0.05). Increase in OPG-induced ONOO- levels was exacerbated by SNP (130±16% vs. veh, p<0.05), a nitric oxide donor. In conclusion, vascular dysfunction elicited by OPG is mediated by syndecan-1 and ROS. Whether syndecan-1 also impacts on OPG-sensitive calcification is unclear. Our data identify a novel molecular mechanism through syndecan- 1/ROS that may underlie injurious effects of OPG.