Sildenafil Reduces Insulin-Resistance in Human Endothelial Cells Caterina Mammi1, Donatella Pastore2, Marco F. Lombardo2, Francesca Ferrelli2, Massimiliano Caprio1, Claudia Consoli3, Manfredi Tesauro2, Lucia Gatta3, Massimo Fini3, Massimo Federici2, Paolo Sbraccia2, Giulia Donadel2, Alfonso Bellia2, Giuseppe M. Rosano 3 , Andrea Fabbri2,4, Davide Lauro2* 1 Centre for Clinical and Basic Research, IRCCS San Raffaele Pisana, Rome, Italy, 2 Department of Internal Medicine, University of Tor Vergata, Rome, Italy, 3 San Raffaele Sulmona, L’Aquila, Italy, 4 Unit of Endocrinology, Department of Internal Medicine, Ospedale S. Eugenio and CTO A. Alesini, University of Tor Vergata, Rome, Italy Abstract Background: The efficacy of Phosphodiesterase 5 (PDE5) inhibitors to re-establish endothelial function is reduced in diabetic patients. Recent evidences suggest that therapy with PDE5 inhibitors, i.e. sildenafil, may increase the expression of nitric oxide synthase (NOS) proteins in the heart and cardiomyocytes. In this study we analyzed the effect of sildenafil on endothelial cells in insulin resistance conditions in vitro. Methodology/Principal Findings: Human umbilical vein endothelial cells (HUVECs) were treated with insulin in presence of glucose 30 mM (HG) and glucosamine 10 mM (Gluc-N) with or without sildenafil. Insulin increased the expression of PDE5 and eNOS mRNA assayed by Real time-PCR. Cytofluorimetric analysis showed that sildenafil significantly increased NO production in basal condition. This effect was partially inhibited by the PI3K inhibitor LY 294002 and completely inhibited by the NOS inhibitor L-NAME. Akt-1 and eNOS activation was reduced in conditions mimicking insulin resistance and completely restored by sildenafil treatment. Conversely sildenafil treatment can counteract this noxious effect by increasing NO production through eNOS activation and reducing oxidative stress induced by hyperglycaemia and glucosamine. Conclusions/Significance: These data indicate that sildenafil might improve NOS activity of endothelial cells in insulin resistance conditions and suggest the potential therapeutic use of sildenafil for improving vascular function in diabetic patients. Citation: Mammi C, Pastore D, Lombardo MF, Ferrelli F, Caprio M, et al. (2011) Sildenafil Reduces Insulin-Resistance in Human Endothelial Cells. PLoS ONE 6(1): e14542. doi:10.1371/journal.pone.0014542 Editor: Maurizio C. Capogrossi, Istituto Dermopatico dell’Immacolata, Italy Received July 2, 2010; Accepted December 16, 2010; Published January 28, 2011 Copyright: 2011 Mammi 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 has been funded by Institutional funding from University Tor Vergata, PRIN 2007 (Progetti Ricerca Interesse Nazionale) Ministero dell’Universita` e della Ricerca, 2007, to A. F.) and by Department of Health RF 2007 (Tremoli), Fondazione Roma - Rome Foundation Research Grant 2008–2009. 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: d.lauro@med.uniroma2.it Introduction Nitric oxide (NO) is a reactive free radical gas acting as an intracellular or extracellular messenger. It is synthesized from L- arginine by a family of isoforms of NO synthases (NOS 1–3). NO generation by NOS-3 (eNOS) in endothelial cells explains the effects of endothelial-dependent vasodilators on vascular relaxa- tion and decreased platelet adhesion and aggregation. These and others effects of NO are mediated by increased cyclic GMP (c- GMP) formation due to soluble guanylyl cyclase activation. In turn c-GMP activates protein kinase G (PKG) and through the phosphorylation of its substrates can regulate many other biological processes. The increase of c-GMP is counteracted by Phosphodiesterase 5 (PDE5) activity that transformes c-GMP into GMP. e-NOS activity is regulated by agonist-induced elevation of intracellular free Ca2+ concentration with subsequent binding of Ca2+/Calmodulin (Ca2+/CaM) to e-NOS. However shear stress and isometric contraction have been shown to activate the enzyme in a Ca2+/CaM-independent manner [1], through the activation of c-GMP dependent PKG [2]. This mechanism may serve to ensure continuous production of NO, independently of fluctua- tions in intracellular Ca2+ levels. Although eNOS was initially reported to be phosphorylated exclusively on serine residues [3] other evidence has shown that eNOS is also phosphorylated in threonine- and tyrosine-domains [4]. NO production in endothe- lial cells is regulated by phosphorylation of several eNOS consensus sequence sites by means of protein kinase (PK) Akt-1, PKC and calmodulin kinase II [5]. In addition to its well known metabolic action, insulin has vasodilatory effects that depend on NO production in the vascular endothelium. It has been demonstrated that e-NOS activation induced by insulin is mediated by the PI3K-Akt pathway resulting in e-NOS phosphorylation in Ser1177 independently of Ca2+-CaM pathway [6]. Moreover insulin inhibition of PI3K/Akt-1 pathway leads to impaired NO availability [7,8]. Reduced insulin-mediated vasodilatation might contribute to vascular damage in insulin- resistant states. We already demonstrated that hyperglycemia, via hexosamine pathway activation, induces selective insulin resistance in endothelial cells and impairs e-NOS activation [9]. PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e14542