Downregulation of von Willebrand Factor prevents Ang II-induced endothelin-1 expression independently of eNOS activation in porcine endothelial cells

Purpose: Angiotensin II (AngII) generated under conditions of myocardial ischemia and reperfusion increases endothelial levels of endothelin (ET)-1, von Willebrand factor (vWF) and anion superoxide (O2-), which lead to progressive coronary endothelial dysfunction. Recent study described that vWF blockade improves endothelial function in coronary patients, but the mechanisms are still unknown. Our study investigated whether the downregulation of vWF modulates the ET-1 level, eNOS activity and O2- generation in porcine aortic endothelial cells (PAOECs) chronically exposed to AngII. Methods: The silencing of vWF in PAOECs was induced with selective short interference RNA. Protein expression of endothelial vWF, ET-1, eNOS and phospho-Ser1177eNOS (p-eNOS) was measured by western blotting in wild type and vWFknockdown cells exposed to vehicle or AngII (100nM for 24h). O2- formation was measured by dihydroethidium staining. In additional experiments, wild type and vWF-knockdown cells were treated with phorbol 12-myristate 13-acetate (PMA, 5nM for 48h), a nonsubtype selective agonist of protein kinase type C and inhibitor of eNOS activity. Results: Nearly 65% silencing of vWF cell viability and growth were not impaired. Levels of ET-1, phospho-Ser1177eNOS (peNOS)/eNOS ratio and O2- were unchanged in vWF-knockdown compared to wild type cells under normal conditions. Conversely, ET-1expression was reduced by 93.7±4% (P<0.0001) in the presence of normal p-eNOS/eNOS ratio in vWF-knockdown cells under oxidative microenvironment; although, the intracellular load of O2- was reduced by 33.3±2% in vWF-knockdown cells with lower level of Mn superoxide dismutase. In additional experiments, the inhibition of eNOS activity by PMA did not reverse the downregulation of ET-1. Conclusions: We demonstrated that vWF-knockdown modulates the response of PAOECs to chronic exposure to AngII by preventing cell death, reducing ET-1 and O2- production without affecting endothelial function. Our findings support the usefulness of vWF as upstream modulator of ET-1 expression under oxidative stress.