Despite the presence of vitamin D receptor (VDR) in endothelial cells, the effect of vitamin D on endothelial function is unknown. An unbalanced production of vasoactive endothelial factors such as nitric oxide (NO) or endothelin-1 (ET-1) results in endothelial dysfunction, which can alter the normal cardiovascular function. Present experiments were devoted to assess the effect of active vitamin D (calcitriol) on the synthesis of endothelial vasoactive factors. The results were that, in cells, calcitriol increased ET-1 and NO productions, which were measured by ELISA and fluorimetric assay, respectively. Calcitriol also increased endothelin-converting enzyme-1 (ECE-1) and endothelial-nitric oxide synthase (eNOS) activities, their mRNA (qPCR), their protein expressions (Western-blot), and their promoter activities (transfection assays). Calcitriol did not change prepro-ET-1 mRNA. The effect was specific to VDR activation because when VDR was silenced by siRNA, the observed effects disappeared. Mechanisms involved in each upregulation differed. ECE-1 upregulation depended on AP-1 activation, whereas eNOS upregulation depended directly on VDR activation. To evaluate the in vivo consequences of acute calcitriol treatment, normal Wistar rats were treated with a single ip injection of 400 ng/kg calcitriol and euthanized 24 h later. Results confirmed those observed in cells, that production and expression of both factors were increased by calcitriol. Besides, calcitriol-treated rats showed a slight rise in mean blood pressure, which decreased when pretreated with FR-901533, an ECE-1 antagonist. We conclude that calcitriol increases the synthesis of both ET-1 and NO in endothelial cells. However, the ET-1 upregulation seems to be biologically more relevant, as animals acutely treated with calcitriol show slight increases in blood pressure.

Objective-Endothelial function depends on the equilibrium in the synthesis of vasoactive endothelial factors. It is well known that endothelin and nitric oxide (NO) exhibit reciprocal regulation. We assessed the ability of NO to regulate endothelin-converting enzyme-1 (ECE-1) expression in vascular endothelial cells. Methods and Results-Bovine aortic endothelial cells were incubated with 2 different NO donors as well as with a cyclic-GMP analog, dibutyryl-cGMP (dB-cGMP). ECE-1 protein content and mRNA expression were evaluated by Western blot and Northern blot, respectively, promoter activity by transfection experiments, ECE-1 activity by ELISA, and cGMP production by radioimmunoassay. Both NO donors decreased ECE-1 protein content, mRNA expression, and ECE-1 activity. ODQ, an inhibitor of soluble guanylyl cyclase, blocked those effects. NO donors raised cGMP levels, and dB-cGMP mimicked their effects on ECE-1 expression, which were blocked by KT5823, a nonspecific PKG inhibitor. The changes on ECE-1 expression were due to a destabilization on 3Ј-untranslated region (3Ј-UTR) of this mRNA, because the activity of a luciferase reporter construct containing the 3Ј-UTR of the ECE-1 gene was reduced by dB-cGMP in a PKG-dependent manner. The biological relevance of this regulation was confirmed in bovine aortic endothelial cells coincubated with macrophages in the presence of lipopolysaccharide, in eNOS-deficient mice, and in Wistar rats treated with NO donors. In every case, an inverse relationship was observed between NO and ECE-1 protein content. Conclusion-Our

In pathological situations such as chronic inflammation, TWEAK could be more harmful through this effect at endothelial level. Pharmacological blockade of this cytokine could prevent the haemodynamic and structural changes related to an increased ET-1 synthesis.

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