Chronic Deficiency of Nitric Oxide Affects Hypoxia Inducible Factor-1a (HIF-1a) Stability and Migration in Human Endothelial Cells Maria Grazia Cattaneo1, Elisa Cappellini1, Roberta Benfante1,2, Maurizio Ragni1, Fausta Omodeo-Sale` 3, Enzo Nisoli1, Nica Borgese1,2,4, Lucia M. Vicentini1* 1 Department of Pharmacology, School of Medicine, Universita` degli Studi di Milano, Milano, Italy, 2 CNR, Institute of Neuroscience, Milano, Italy, 3 Department of Molecular Sciences Applied to Biosystems, Faculty of Pharmacy, Universita` degli Studi di Milano, Milano, Italy, 4 Department of Pharmacobiological Science, University of Catanzaro ‘‘Magna Graecia’’, Roccelletta di Borgia (Cz), Italy Abstract Background: Endothelial dysfunction in widely diffuse disorders, such as atherosclerosis, hypertension, diabetes and senescence, is associated with nitric oxide (NO) deficiency. Here, the behavioural and molecular consequences deriving from NO deficiency in human umbilical vein endothelial cells (HUVECs) were investigated. Results: Endothelial nitric oxide synthase (eNOS) was chronically inhibited either by NG-Nitro-L-arginine methyl ester (L-NAME) treatment or its expression was down-regulated by RNA interference. After long-term L-NAME treatment, HUVECs displayed a higher migratory capability accompanied by an increased Vascular Endothelial Growth Factor (VEGF) and VEGF receptor-2 (kinase insert domain receptor, KDR) expression. Moreover, both pharmacological and genetic inhibition of eNOS induced a state of pseudohypoxia, revealed by the stabilization of hypoxia-inducible factor-1a (HIF-1a). Furthermore, NO loss induced a significant decrease in mitochondrial mass and energy production accompanied by a lower O2 consumption. Notably, very low doses of chronically administered DETA/NO reverted the HIF-1a accumulation, the increased VEGF expression and the stimulated migratory behaviour detected in NO deficient cells. Conclusion: Based on our results, we propose that basal release of NO may act as a negative controller of HIF-1a levels with important consequences for endothelial cell physiology. Moreover, we suggest that our experimental model where eNOS activity was impaired by pharmacological and genetic inhibition may represent a good in vitro system to study endothelial dysfunction. Citation: Cattaneo MG, Cappellini E, Benfante R, Ragni M, Omodeo-Sale` F, et al. (2011) Chronic Deficiency of Nitric Oxide Affects Hypoxia Inducible Factor-1a (HIF-1a) Stability and Migration in Human Endothelial Cells. PLoS ONE 6(12): e29680. doi:10.1371/journal.pone.0029680 Editor: Holger K. Eltzschig, University of Colorado Denver, United States of America Received July 12, 2011; Accepted December 1, 2011; Published December 27, 2011 Copyright: 2011 Cattaneo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by research grants from Ministero Universita` e Ricerca (PRIN 2008) to LMV, and from University of Milano (FIRST and PUR) to MGC and LMV. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lucia.vicentini@unimi.it Introduction Integrity of endothelial cells is crucial for the maintenance of vascular homeostasis. The endothelium explicates its physiological functions by producing active molecules, among which nitric oxide (NO) is particularly important. By diffusing into neighboring smooth muscle cells, endothelial-produced NO induces vasorelax- ation, thereby controlling blood pressure levels [1,2]. NO generated in the endothelium also has antiaggregant activity that protects the cardiovascular system from thrombosis and acute events [2]. Consistent with the key role of this gaseous messenger in cardiovascular physiology, NO loss is a dangerous event that is associated with endothelial dysfunction typical of diffuse patho- logical conditions like atherosclerosis and senescence [3–5]. Moreover, the deficiency of NO and endothelial nitric oxide synthase (eNOS) activity is thought to be crucial for the development and/or acceleration of the important vascular complications associated with diabetes [6]. In addition to its effect on smooth muscle cells and platelets, NO generated by the endothelium has important functions in the endothelial cells (ECs) themselves. Indeed, the gaseous messenger plays a key role in the process of angiogenesis, stimulating proliferation, migration and differentiation of ECs to form new blood vessels [7]. In particular, NO acutely produced by angiogenic factors, such as Vascular Endothelial Growth Factor (VEGF) [8–10], endothelin [11], substance P [12] and oxytocin [13] is crucial for stimulation of EC migration. Together with the stimulatory effect of acute NO on EC chemotaxis, also the concentration and timing of NO release appear to be of crucial importance in determining the final outcome on EC physiology. In particular, recent work from our laboratory has demonstrated that long term inhibition of eNOS in Human Umbilical Vein ECs (HUVECs) by exposure to the NOS inhibitor NG-Nitro-L-arginine methyl ester (L-NAME), increases the migratory behaviour of these cells in Boyden chambers assays carried out immediately after removal of the drug [14]. These PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29680