Abstract

Fetuin-A (FetA) is a plasma glycoprotein important for the regulation of calcium and phosphate homeostasis. It is also an agonist to toll-like receptor 4 (TLR4) and is related to insulin resistance and inflammation. FetA has also been associated with endothelial dysfunction, which is regulated by oxidative stress. Mechanisms whereby FetA influences vascular function are unknown. We hypothesised that FetA through TLR4 and ROS production induces vascular dysfunction. Mesenteric arteries and vascular cells from WKY rats were studied. Vascular function was analysed by wire myography in the presence or absence of FetA (50 ng/mL) and/or CLI095 (CLI – 10^-6M – TLR4 inhibitor). Reactive oxygen species (ROS) were measured by chemiluminescence, Amplex Red (H₂O₂) and ELISA to nitrotyrosine levels (peroxynitrite – ONOO⁻). Protein oxidation and levels were measured by immunoblotting. WKY vessels exposed to FetA were less sensitive to ?acetylcholine (Ach)-induced and sodium nitroprusside (SNP)-induced relaxation, while sensitivity to phenylephrine was increased by FetA; an effect blocked by N-acetylcysteine (antioxidant) and ML171 (Nox1 inhibitor). Inhibition of TLR4 blocked FetA effects on endothelial-dependent relaxation and contraction, but not on endothelial-independent relaxation. FetA increased ROS production (1 fold), but decreased H₂O₂ intracellular levels (0.5 fold); and increased gene levels of IL6 (2 fold), IL1Ã&noentity;² (1 fold), RANTES (1 fold) and MMP2/9 (2 fold) in endothelial cells (EC) (p<0.05); an effect blocked by CLI095. ROS production (0.5 fold), as well as, H₂O₂ (0.5 fold) and ONOO^- (1 fold) levels, were increased by FetA in VSMCs (p<0.05). Protein oxidation was also increased by FetA in VSMCs (1 fold, p<0.05). In EC, eNOS inactivation (1 fold) and JNK activation (0.5 fold) were increased by FetA (p<0.05). In VSMCs, Rho kinase activity was increased (2 fold, p<0.05) at 30 min; while myosin light chain (MLC) activation was only increased (0.5 fold) at 15 min. In summary, FetA seems to influence vascular function through Nox1-ROS dependent mechanisms, where only endothelial dysfunction and contractile responses were mediated by TLR4 activation. Identification of FetA as a ligand of TLR4 and further characterisation of FetA-induced signalling may be of importance to the development of new therapy for treatment of hypertension.