Data di Pubblicazione:
2014
Citazione:
EFFECTS OF CHRONIC NITRIC OXIDE DEPRIVATION ON ENDOTHELIAL CELL BEHAVIOUR / E. Cappellini ; tutor: L. Vicentini ; supervisore: M. G. Cattaneo ; coordinatore: A. Panerai. DIPARTIMENTO DI BIOTECNOLOGIE MEDICHE E MEDICINA TRASLAZIONALE, 2014 Jan 16. 26. ciclo, Anno Accademico 2013. [10.13130/cappellini-elisa_phd2014-01-16].
Abstract:
The endothelium explicates its physiological functions by producing active molecules, among which nitric oxide (NO) is particularly important. It is well known that endothelial dysfunction (ED), i.e. an impaired function of the endothelium coupled with a reduced release of NO, is a risk factor for atherosclerosis together with a list of conditions such as hypertension, hypercholesterolemia, smoking, diabetes, and the aging process itself. These conditions are also associated with a significant increase in Reactive Oxygen Species (ROS) in the vascular wall that may contribute to the establishment of ED and to the development of its late effect on cardiovascular system.
In the present study, the behavioural and molecular consequences deriving from chronic NO deprivation were investigated in human primary endothelial cells (human umbilical vein endothelial cells, HUVECs). To inhibit NO formation, endothelial nitric oxide synthase (eNOS) was chronically inhibited by treatment with L-NG-Nitroarginine methyl ester (L-NAME), a structural analogue of L-arginine that competitively block the active site of the enzyme, or by transfection with a siRNA specific for eNOS.
We observed that a 48-h L-NAME treatment induced in HUVECs a higher migratory capability (evaluated by chemotaxis assays in Boyden’s chamber) which was independent of the reduced activity of the cyclic GMP/protein kinase G pathway present in chronically NO deprived HUVECs. In the attempt to explain the mechanism(s) through which NO deficiency enhances migration, we investigated if chronic L-NAME treatment affected the expression and production of Vascular Endothelial Growth Factor (VEGF) and of its receptor KDR. RT-qPCR analyses, accompanied by ELISA assays and western blot analyses, demonstrated that both VEGF and KDR mRNAs and proteins were significantly augmented in L-NAME treated cells, thus suggesting the establishment of an autocrine loop responsible for the increased migration.
Increased VEGF production and cell motility are typical events occurring in hypoxic cancer cells, due to the accumulation of hypoxia-inducible factor-1α (HIF-1α), which plays a major role in the transcriptional activation of genes encoding angiogenic factors. Similarly, induction of VEGF expression during hypoxia has been described in endothelial cells (ECs). Interestingly, we observed a significant nuclear accumulation of HIF-1α in HUVECs chronically treated with L-NAME. Moreover, the transcriptional activity of HIF-1α was responsible for the increases in VEGF/KDR expression and migration since the transfection with ΔARNT (a dominant negative form of the HIF-1β subunit that maintains the capacity of forming an heterodimer but cannot bind DNA) is able to totally blunt both the effects in L-NAME treated HUVECs, thus confirming the involvement of an autocrine loop in the pro-migratory effect induced by NO deprivation. The dependence of HIF-1α stabilization from NO deficiency was confirmed by using the NO donor DETA/NO. Very low doses of DETA/NO reverted both the HIF-1α accumulation and the consequent increases in VEGF expression and cell motility induced by L-NAME treatment. Furthermore, to investigate whether the observed effects were due to the specific inhibitory effect of L-NAME on eNOS activity, we knocked-down the enzyme by using RNA interference methodology. In eNOS silenced cells, HIF-1α accumulated in the nucleus and VEGF production was enhanced thus confirming the dependence of the observed effects on eNOS inhibition. All these results suggest that basal release of NO may act as a negative controller of HIF-1α levels and cell motility in HUVECs with important consequences on ECs physiology.
In the attempt to unravel the pathway(s) linking NO deficiency to H
Tipologia IRIS:
Tesi di dottorato
Keywords:
nitric oxide ; endothelium ; Reactive Oxygen Species ; Hypoxia Inducible Factor-1 ; NF-E2-related factor-2
Elenco autori:
E. Cappellini
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