Objectives: The expression of endothelin (ET)-1, a potent vasoconstrictor and proinflammatory peptide, is increased in endothelial cells exposed to angiotensin II (Ang II). High levels of ET-1 progressively lead to endothelial dysfunction up to cardiovascular disorders. Since von Willebrand Factor (vWF) blockade improves endothelial function in coronary patients, we hypothesized that targeting endothelial vWF expression with small interference RNA (siRNA) prevents the Ang II-induced ET-1 upregulation. Materials and methods: see Results. Results: Nearly 65±2% silencing of vWF in porcine aortic endothelial cells (PAOECs) was achieved with selective siRNA (siRNA-vWF) without affecting cell viability and growth. At rest, vWF-knockdown PAOECs showed ET-1 levels similar to wild-type cells. Conversely, siRNA-vWF prevented the ET-1 upregulation after exposure to Ang II (100nM/24h), yet vWF levels were similar to unstressed cells. Even if siRNA-vWF significantly reduced eNOS expression, the levels of phospho-Ser1177-endothelial-nitric oxide synthase (eNOS)/eNOS ratio and nitric oxide in siRNA-vWF cells were similar to unstressed wildtype cells. Compared with wild-type cells, siRNA-vWF treatment significantly prevented the AngII-induced increase in NADPH oxidase activity and superoxide anion (O2-) levels, which mediate Ang II-induced ET-1 expression. Unraveling the mechanisms underlie vWF downregulation, the long-term treatment of cells with phorbol ester (PMA, 5nM/48h), which is a known activator of the NADPH-derived O2- production, did not increase O2- and ET-1 levels in vWF-knockdown cells. Conclusions: siRNA-based targeting of endothelial vWF prevented the Ang II-induced ET-1 upregulation through attenuation of O2- production due to inhibition of NADPH activity. Our findings reveal a hitherto unsuspected role of vWF in the prevention of Ang IIinduced endothelial dysfunction.
siRNA-mediated targeting of endothelial VWF prevents ET-1 upregulation in porcine aortic endothelial cells chronically exposed to angiotensin II
DUSHPANOVA, ANAR;AGOSTINI, Silvia;CIOFINI, ENRICA;CABIATI, MANUELA;CASIERI, VALENTINA;MATTEUCCI, Marco;CLERICO, ALDO;LIONETTI, Vincenzo
2015-01-01
Abstract
Objectives: The expression of endothelin (ET)-1, a potent vasoconstrictor and proinflammatory peptide, is increased in endothelial cells exposed to angiotensin II (Ang II). High levels of ET-1 progressively lead to endothelial dysfunction up to cardiovascular disorders. Since von Willebrand Factor (vWF) blockade improves endothelial function in coronary patients, we hypothesized that targeting endothelial vWF expression with small interference RNA (siRNA) prevents the Ang II-induced ET-1 upregulation. Materials and methods: see Results. Results: Nearly 65±2% silencing of vWF in porcine aortic endothelial cells (PAOECs) was achieved with selective siRNA (siRNA-vWF) without affecting cell viability and growth. At rest, vWF-knockdown PAOECs showed ET-1 levels similar to wild-type cells. Conversely, siRNA-vWF prevented the ET-1 upregulation after exposure to Ang II (100nM/24h), yet vWF levels were similar to unstressed cells. Even if siRNA-vWF significantly reduced eNOS expression, the levels of phospho-Ser1177-endothelial-nitric oxide synthase (eNOS)/eNOS ratio and nitric oxide in siRNA-vWF cells were similar to unstressed wildtype cells. Compared with wild-type cells, siRNA-vWF treatment significantly prevented the AngII-induced increase in NADPH oxidase activity and superoxide anion (O2-) levels, which mediate Ang II-induced ET-1 expression. Unraveling the mechanisms underlie vWF downregulation, the long-term treatment of cells with phorbol ester (PMA, 5nM/48h), which is a known activator of the NADPH-derived O2- production, did not increase O2- and ET-1 levels in vWF-knockdown cells. Conclusions: siRNA-based targeting of endothelial vWF prevented the Ang II-induced ET-1 upregulation through attenuation of O2- production due to inhibition of NADPH activity. Our findings reveal a hitherto unsuspected role of vWF in the prevention of Ang IIinduced endothelial dysfunction.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
