ABSTRACT
Title
Multiple effects of chronic nitric oxide deprivation in human endothelial cells
Authors
M. G. Cattaneo1, E. Cappellini1, R. Benfante1,2, M. Ragni1, E. Nisoli1, N. Borgese1,2,3 and Lucia M. Vicentini1
1Dept of Pharmacology, School of Medicine, Università degli Studi di Milano;
2CNR, Institute of Neuroscience, Milano;
3Dept of Pharmacobiological Science, University of Catanzaro “Magna Graecia”.
1Dept of Pharmacology, School of Medicine, Università degli Studi di Milano;
2CNR, Institute of Neuroscience, Milano;
3Dept of Pharmacobiological Science, University of Catanzaro “Magna Graecia”.
Abstract
The endothelium explicates its physiological function by producing active molecules among which nitric oxide (NO) is endowed with very important properties. Endothelial-produced NO possesses vasorelaxant and antiaggregant activity that protects the cardiovascular system from thrombosis and acute events. Moreover, a tonic basal NO release controls blood pressure levels and maintain the endothelium in an anti-atherogenic state.Indeed, NO loss is associated with endothelial dysfunction typical of atherosclerosis, diabetes and senescence.
To reproduce the pathological situation of NO deficiency we chronically treated human umbilical vein endothelial cells (HUVECs) with NG-Nitro-L-arginine methyl ester (L-NAME), a structural analogue of L-arginine that competitively block the active site of endothelial nitric oxide synthase (eNOS), the enzyme responsible for endothelial NO production. The chronic NO deprivation induced profound modification in HUVEC behaviour, consisting in increased cell motility, enhanced expression of Vascular Endothelial Growth Factor (VEGF) and of its type 2 receptor (kinase insert domain receptor, KDR),and very interestingly, stabilization of hypoxia-inducible factor-1α (HIF-1α) (see poster by Cappellini, presented at this meeting in the section “Seminario Dottorandi”).
To investigate whether the observed effects of L-NAME treatment on HIF-1α accumulation were due to the specific inhibitory effect on the eNOS activity, expression of the enzyme was silenced in HUVECs by using RNA interference methodology. In agreement with the results obtained after long-term exposure to L-NAME, HIF-1α accumulated in the nucleus of eNOS knock-down cells, whereas cells transfected with control siRNA were unaffected.
The dependence of HIF-1αstabilization from NO deficiency was confirmed using the NO donor DETA/NO. Very low doses of DETA/NO reverted the HIF-1αaccumulation induced by L-NAME treatment, and also the consequent increases in VEGF expression and cell motility.
All these results suggested a crucial role for NO in the regulation of HIF-1α levels and cell motility in endothelial cells. Based on our results, we suggest that basal release of NO may act as a negative controller of HIF-1α levels and cell motility with important consequences for the endothelial cell physiology.
Finally, since NO has an important role in promoting mitochondrial biogenesis in different cell types and tissues, we analyzed the mitochondrial mass and function in HUVECs after NO deprivation. Long term L-NAME treatment induced a significant decrease in mitochondrial DNA mass, accompanied by a decrease in the incorporation of the metabolic indicator MTS. In agreement, oxygen consumption as well as ATP levels were reduced in L-NAME treated cells. These results showed for the first time that NO acts also in human endothelial cells as a regulator of mitochondrial mass and function.
To reproduce the pathological situation of NO deficiency we chronically treated human umbilical vein endothelial cells (HUVECs) with NG-Nitro-L-arginine methyl ester (L-NAME), a structural analogue of L-arginine that competitively block the active site of endothelial nitric oxide synthase (eNOS), the enzyme responsible for endothelial NO production. The chronic NO deprivation induced profound modification in HUVEC behaviour, consisting in increased cell motility, enhanced expression of Vascular Endothelial Growth Factor (VEGF) and of its type 2 receptor (kinase insert domain receptor, KDR),and very interestingly, stabilization of hypoxia-inducible factor-1α (HIF-1α) (see poster by Cappellini, presented at this meeting in the section “Seminario Dottorandi”).
To investigate whether the observed effects of L-NAME treatment on HIF-1α accumulation were due to the specific inhibitory effect on the eNOS activity, expression of the enzyme was silenced in HUVECs by using RNA interference methodology. In agreement with the results obtained after long-term exposure to L-NAME, HIF-1α accumulated in the nucleus of eNOS knock-down cells, whereas cells transfected with control siRNA were unaffected.
The dependence of HIF-1αstabilization from NO deficiency was confirmed using the NO donor DETA/NO. Very low doses of DETA/NO reverted the HIF-1αaccumulation induced by L-NAME treatment, and also the consequent increases in VEGF expression and cell motility.
All these results suggested a crucial role for NO in the regulation of HIF-1α levels and cell motility in endothelial cells. Based on our results, we suggest that basal release of NO may act as a negative controller of HIF-1α levels and cell motility with important consequences for the endothelial cell physiology.
Finally, since NO has an important role in promoting mitochondrial biogenesis in different cell types and tissues, we analyzed the mitochondrial mass and function in HUVECs after NO deprivation. Long term L-NAME treatment induced a significant decrease in mitochondrial DNA mass, accompanied by a decrease in the incorporation of the metabolic indicator MTS. In agreement, oxygen consumption as well as ATP levels were reduced in L-NAME treated cells. These results showed for the first time that NO acts also in human endothelial cells as a regulator of mitochondrial mass and function.