Abstract
Expression of endothelin (ET)-1 is increased in endothelial cells exposed to angiotensin II (Ang II), leading to endothelial dysfunction and cardiovascular disorders. Since von Willebrand Factor (vWF) blockade improves endothelial function in coronary patients, we hypothesized that targeting endothelial vWF with short interference RNA (siRNA) prevents Ang II-induced ET-1 upregulation. Nearly 65 ± 2% silencing of vWF in porcine aortic endothelial cells (PAOECs) was achieved with vWF-specific siRNA without affecting cell viability and growth. While showing ET-1 similar to wild type cells at rest, vWF-silenced cells did not present ET-1 upregulation during exposure to Ang II (100 nM/24 h), preserving levels of endothelial nitric oxide synthase activity similar to wild type. vWF silencing prevented AngII-induced increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity and superoxide anion (O2−) levels, known triggers of ET-1 expression. Moreover, no increase in O2− or ET-1 levels was found in silenced cells treated with AngII or NOX-agonist phorbol ester (PMA 5 nM/48 h). Finally, vWF was required for overexpression of NOX4 and NOX2 in response to AngII and PMA. In conclusion, endothelial vWF knockdown prevented Ang II-induced ET-1 upregulation through attenuation of NOX-mediated O2− production. Our findings reveal a new role of vWF in preventing of Ang II-induced endothelial dysfunction.
Highlights
Endothelin (ET)-1, a potent vasoconstrictor and pro-inflammatory peptide, maintains the vascular tone in healthy humans[1,2], but its expression is upregulated in various cardiovascular disorders like systemic and pulmonary hypertension[3,4], myocardial infarction[5,6] and fibrosis[7]
Since endothelial function directly depends on the cell phenotype[17], we hypothesized that von Willebrand factor may modulate ET-1 expression during angiotensin II (Ang II) stimulation, despite being so far mostly a hallmark of endothelial phenotype mainly involved in hemostasis[18]
RT-PCR analysis with efficiency in the range of 95–105% was performed to assess the von Willebrand factor (vWF) gene expression in porcine aortic endothelial cells (PAOECs) transfected with short interfering RNA (siRNA)-vWF. vWF gene expression was markedly reduced in transfected PAOECs and the expression of the corresponding reference gene was revealed
Summary
Endothelin (ET)-1, a potent vasoconstrictor and pro-inflammatory peptide, maintains the vascular tone in healthy humans[1,2], but its expression is upregulated in various cardiovascular disorders like systemic and pulmonary hypertension[3,4], myocardial infarction[5,6] and fibrosis[7]. High levels of angiotensin II (Ang II), a biologically active hormone, increase ET-1 expression in endothelial cells[8,9], impair endothelial nitric oxide synthase (eNOS) activity[10] and promote endothelial oxidant stress, which together contribute to the development of endothelial dysfunction[11]. Unraveling how endothelial vWF prevents the onset of endothelial dysfunction could provide new avenues for protection against Ang II-induced cardiovascular injury. We tested the effects of short interfering RNA (siRNA)-mediated gene silencing of endothelial vWF during chronic exposure to high levels of Ang II in primary porcine aortic endothelial cells (PAOECs), a well-established in vitro culture system for studying the molecular mechanisms underlie alterations of vWF expression[27] and endothelial function in general[28]. Our findings reveal a hitherto unsuspected role of endothelial vWF downregulation in preventing Ang II-induced ET-1 upregulation through reduced NADPH oxidase (NOX)-mediated oxidative stress, without impairing nitric oxide (NO) production
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