Abstract 571: Osteogenic Factors Regulate Vascular Function Through Reactive Oxygen Species And Toll-Like Receptor 4 Activation: Role In Hypertension.

Abstract

Osteogenic factors (OF), such as osteoprotegerin (OPG) and fetuin-A (FTA), play a role in regulating vascular calcification. Plasma levels of OPG and FTA are elevated in metabolic diseases and correlate with endothelial dysfunction, a hallmark of hypertension. Molecular mechanisms for this are elusive and the association with hypertension is unclear. Here, we postulated that OFs influence vascular function. Mesenteric arteries and VSMCs from WKY and SHRSP rats were studied. Vascular function was analyzed by wire myography. ROS production was assessed by chemiluminescence. Protein expression was analyzed by immunoblotting. Calcification was assessed by Von Kossa staining. WKY vessels exposed to OPG and FTA (50 ng/mL-30 min) were less sensitive to acetylcholine (Ach)-induced relaxation; an effect blocked by N-acetylcysteine (NAC-antioxidant), tempol (SOD mimetic) and ML171 (Nox1 inhibitor). Sensitivity to phenylephrine was increased by OPG and FTA, an effect mediated by ROS and Nox1. FTA has been characterized as an endogenous ligand to toll-like receptor 4 (TLR4). Inhibition of TLR4 with CLI095 blocked effects of FTA in WKY vessels. Stimulation with OPG (100ng/5 min) and FTA (50ng/5 min) increased ROS production (≈50%) in VSMCs from WKY rats (p<0.05 vs vehicle). ROS generation induced by OPG and FTA was blocked by GKT137831 (Nox1/Nox4 inhibitor) and PP2 (c-Src inhibitor). MAPK activation was also induced by OPG (JNK: 75%, p38: 110%, ERK1/2: 100%; p<0.05 vs vehicle), but FTA only increased ERK1/2 activation (50%, p<0.05 vs vehicle) in VSMCs from WKY rats. In VSMCs from SHRSP rats, basal levels of OPG (20%) and osterix (86%) were increased. In addition, aorta from SHRSP, but not WKY, exhibited positive Von Kossa staining. Levels of OPG (75%), FTA (40%) and osterix (50%) protein levels were increased in LinA3 mice, a mouse model which has chronically elevated Ang II levels (p<0.05 vs WT). In conclusion, our data suggest that in hypertension, OPG and FTA may influence vascular function through redox-sensitive TLR4-dependent signalling. Identification of vascular calcification-derived osteogenic factors as modulators of VSMCs biology provides new insights into molecular mechanisms of vascular dysfunction in hypertension.