Chronic nitric oxide deprivation induces hypoxia inducible factor-1\u3b1 stabilization in human endothelial cells

The endothelium explicates its physiological functions by producing active molecules, among which nitric oxide (NO) is particularly important. By diffusing into neighbouring smooth muscle cells, endothelial-produced NO induces vasorelaxation, thereby controlling blood pressure levels. NO generated in the endothelium also has antiaggregant activity that protects the cardiovascular system from thrombosis and acute events. Consistent with the key role of this gaseous messenger in cardiovascular physiology, NO loss is a dangerous event which is associated with endothelial dysfunction typical of diffuse pathological conditions like atherosclerosis, pulmonary hypertension and senescence. In the present study, the behavioural and molecular consequences deriving from chronic NO deprivation in human primary endothelial cells (human umbilical vein endothelial cells, HUVECs) were investigated. To inhibit NO formation, endothelial nitric oxide synthase (eNOS) was chronically inhibited by treatment with NG-Nitro-L-arginine methyl ester (L-NAME), a structural analogue of L-arginine that competitively block the active site of the enzyme. After 48 h of L-NAME treatment, HUVECs displayed a higher migratory capability as evaluated by chemotaxis assays in Boyden\u2019s chamber. The possible involvement of the cyclic GMP/protein kinase G (cGMP/PKG) pathway in the observed effect was excluded, since the increased HUVEC migratory behaviour was not induced by a long term treatment with the guanylate cyclase inhibitor ODQ, indicating that cGMP and/or PKG are not involved in the regulation of cell motility. In an attempt to explain the mechanism through which NO deprivation enhances migration, we investigated by quantitative real time PCR (RT-qPCR) how chronic L-NAME treatment affects the expression level of the Vascular Endothelial Growth Factor (VEGF) receptor-2 (kinase insert domain receptor, KDR). We also analyzed VEGF itself, as endogeous production of the growth factor could potentiate migration by an autocrine loop. RT-qPCR analyses demonstrated that both VEGF and KDR mRNA levels were significantly augmented in L-NAME treated cells. Increased VEGF production and cell motility are typical events occurring in hypoxic cancer cells, due to the accumulation of hypoxia-inducible factor-1\uf061 (HIF-1\uf061), which plays a major role in the transcriptional activation of genes encoding angiogenic factors. Similarly, induction of VEGF expression during hypoxia has been described also in endothelial cells. We therefore analysed the effect of long term L-NAME treatment on HIF-1\uf061 levels in HUVECs. Most interestingly, we observed that, after 48 h of treatment, L-NAME induced in HUVECs nuclear accumulation of HIF-1\u3b1. RT-qPCR analysis revealed no significant change in HIF-1\u3b1 mRNA levels, suggesting that HIF-1\uf061 accumulation in L-NAME-treated cells was mainly due to its stabilization, as occurs under hypoxic conditions. Taken together, these data suggest that prolonged L-NAME treatment induces in endothelial cells a pseudohypoxic state, with the consequent stabilization of HIF-1\u3b1 under normoxic conditions. These events may very well occur in all of those pathological conditions where eNOS expression and/or activity are impaired thus contributing to the endothelial dysfunction typical of such diseases like hypertension and diabetes