Fetuin‐A Induces Endothelial and Vascular Smooth Muscle Cell Dysfunction Through Nox1 and TLR4 Activation

Abstract

Introduction Although studies demonstrate an important role for fetuin-A (FetA) in the inhibition of vascular calcification, compiling evidence suggests that FetA is also an endogenous ligand to toll-like receptor 4 (TLR4) and is involved in insulin resistance, inflammation and cardiovascular damage. The present study seeks to unravel the vascular effects and associated molecular mechanisms of FetA, focusing on oxidative stress and TLR4. Methods Rat aortic endothelial cells (RAEC), mesenteric arteries and vascular smooth muscle cells (VSMC) from Wistar-Kyoto (WKY) rats and wild-type (WT) or Nox1 KO mice were studied. Vascular function was analysed by wire myography and in some experiments in the presence or absence of FetA (50 ng/mL) and/or CLI095 (CLI – 1 μM – TLR4 inhibitor) and Tempol (10 μM – SOD mimetic). Levels of reactive oxygen species (ROS) were measured by chemiluminescence, Amplex Red and ELISA in the presence or absence of FetA (50 ng/mL) and/or CLI095 (CLI – 1 μM – TLR4 inhibitor) and a Nox1 inhibitor NoxA1ds (10 μM). Activation of eNOS inhibitory site (Thr495) was assessed by immunoblotting. Results FetA decreased acetylcholine (LogEC50: 7.32±0.06 vs control: 8.02±0.08) and sodium nitroprusside (LogEC50: 6.48±0.19 vs control: 7.38±0.12) induced relaxation (p<0.05), all effects reversed by Tempol and CLI095. Similar findings were observed in arteries from WT but not Nox1 KO mice. In RAECs, FetA increased eNOS inactivation (C=100% vs. FetA=194%±32.4 vs. FetA+CLI095=88.39±3.87) and ROS production (C=100% vs. FetA=231%±49.2 vs. FetA+CLI095=85.62±15.4) via TLR4 (p<0.05). ROS production (66±12.2%), as well as, H2O2 (45±8%) and ONOO- (105±31.6%) levels, were increased by FetA in VSMCs (vs. control, p<0.05); effects blocked by NoxA1ds and CLI095. Conclusion FetA influences vascular function through Nox1-ROS dependent mechanisms. FetA-induced endothelial and VSMC dysfunction involve TLR4. Our findings identify a novel system whereby FetA differentially influences vascular function through Nox1-ROS and TLR4, which in turn may influence vascular dysfunction during cardiovascular diseases independent of vascular calcification.