Sildenafil Effect in HUVECs eNOS, anti-iNOS. The primary antibody was visualized using horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies and enhanced chemiluminescence. foetal corpora cavernosa (hfCC) cells used as positive control (Fig. 1A). Furthermore, Western Blot studies showed a significant expression of PDE-5 protein (Fig. 1B). NO production determination The FACScalibur (Fluorescent Activated Cell Sorting, Becton Dickinson) was used to quantify NO production. Endothelial cells were washed with PBS and starved for 1 h at 37uC in M199 phenol free medium plus 0.1% BSA. After 30 min. different inhibitors were added when required (LY 294002 30 mM, L- NAME 1 mM). Then 30 min. later sildenafil (1 mM) was supplemented when demanded. Arginine (100 mM) was incubated 1h later and after 1h diaminofluoresceine-2 diacetate (DAF-2DA, 5 mM) probe was added. After 20 min. insulin (100 nM) has been added for 1h. Finally endothelial cells were collected and analyzed with FACS, by the Cellquest program (Becton Dickinson). Statistical analysis of data Data are representative of three or more independent experiments. Results are reported as mean 6 SE or as percent increase respect to control where appropriate. Differences between treatments were analysed by ANOVA followed by Student’s test and Mann Whitney test. A p value,0.05 was considered significant. Results PDE5 expression in HUVECs PDE-5 mRNA expression, measured in HUVECs cell line by RT-PCR, was detectable compared to the expression in human Insulin up-regulates eNOS and PDE5, whereas sildenafil modulates only eNOS expression in HUVECs Treatment with sildenafil (1 mM/5 h), insulin (100 nM/ 30 min), alone and in combination induced a 2-fold increase of eNOS but not iNOS mRNA expression, that remained roughly unchanged after treatment. iNOS protein expression was also unchanged in the same conditions (Fig. 1D). Interestingly, the positive effect of sildenafil on eNOS expression was not amplified by co-treatment with insulin. PDE-5 mRNA expression was up regulated (,3 fold) by insulin alone or in combination with sildenafil (,2.5 fold, Fig. 1C), whereas sildenafil alone did not significantly modify it. These data show that in our cellular model PDE5 expression is responsive to insulin treatment. Also, HUVEC cells respond to sildenafil in terms of eNOS expression in accordance with previous data, which showed that sildenafil increases eNOS expression in pulmonary vessels [27]. Sildenafil increases Akt-1 phosphorylation in insulin- resistance conditions In order to validate our model of insulin resistance, we measured Akt-1 Ser473 phosphorylation in HUVECs cultured in HG and Gluc-N for 72 h. In keeping with previous observations, Akt-1 phosphorylation induced by insulin is impaired in HUVECs exposed to HG (30 mM) or Gluc-N (10 mM) [28,29] respectively of ,27% and ,37% (Figure 2). Interestingly, in the same Figure 3. eNOS Ser1177 phosphorylation after sildenafil treatment. Sildenafil chronic treatment (1 mM/72 h) enhances eNOS phosphorylation in basal condition and after HG treatment, but not significantly after Gluc-N treatment. Full bars represent conditions without sildenafil, dashed bars represent sildenafil treatment. Ctrl was expressed as 100%. Means 6 S.E. (n = 5). *p,0.05 vs Ctrl, { p,0.05 vs insulin; { p,0.05 vs HG; {{ p,0.05 vs HG + insulin. doi:10.1371/journal.pone.0014542.g003 PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e14542