powder and dissolved in deionized H2O. The drug solution was filtered (0.45 mm pore size) and the solution was applied to a 285 mL Sephadex G-25 (superfine) column, equilibrated before using in deionized H2O at 20uC [25]. Sildenafil was eluted with 500 mL of 1% formic acid, lyophilized and resuspended in deionized H2O, and then was used to stimulate cell cultures. The Sildenafil concentration of 1mM was chosen based on data from Luedders et al. [26]. Cell culture HUVEC and HAEC were growth in EGM-2MV at 37uC in an atmosphere of 95% air–5% CO2. HfCC were growth in M199 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and antibiotics (50 U of penicillin and 50 mg of streptomycin/ml). HUVEC and HAEC from passages 3 to 6 were cultured for 72 hours in presence of 5.5 mmol/L glucose, 30 mmol/L glucose (high glucose; HG), 10 mmol/L Gluc-N with or without 1 mM of sildenafil (5 or 72 h) and/or 1027 M insulin. LY 2940002 (30 mM) was used to block the PI3K and inhibit the PI3K/Akt pathway. RT-PCR analysis The expression of endogenous PDE5 was determined by reverse transcription (RT) of total RNA followed by PCR analysis. Total RNA was extracted using TRIZOL. One microgram of total RNA was reverse transcribed using M-MLV reverse transcriptase according to the manufacturer’s protocol. PCR of the cDNA was performed using Platinium High Fidelity Taq using the following pairs of primers: 1. PDE5 sense: (59-39) ACC GCT ATT CCC TGT TCC TT 2. PDE5 antisense: (59-39) AAG GTC AAG CAG CAC CTG AT Sildenafil Effect in HUVECs PCR parameters were: 35 cycles (94uC for 45s, 58uC for 45s, and 72uC for 1 min). The PCR conditions were optimized for actin gene used as control. Real-Time Quantitative PCR Single-stranded cDNA was synthesized from 4mg total RNA samples using High-Capacity cDNA Archive Kit (Applied Biosystems). cDNA (40 ng) was amplified by RT-PCR using ABI PRISM 9700 System and TaqMan reagents. The 206 Assays-on- demand gene expression used were: PDE-5 (Hs00903251_m1), iNOS (Hs00167248_m1), and eNOS (Hs00167166_m1). Human RNA 18s were used for samples normalization. Each reaction was carried out in triplicate. Western Blot Analysis Cells were incubated for four hours in EBM-2 additioned with 0,1% BSA. After starvation cells were incubated in EGM-2MV with HG or with 10 mM Gluc-N to induce insulin resistance for 72 h with or without 1 mM sildenafil, added every 24 h. Cells were treated with 100 nM insulin during the last 30 min. of incubation. Extracts were obtained by lysing the cells in lysis buffer (50 mM Tris-HCl pH 7.6, 100 mM NaCl, 2 mM EDTA, 1 mM MgCl2, 1 mM CaCl2, 1% Triton X-100, 10% glycerol, 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 2 mM sodium ortovana- date, 5 mM sodium pyrophosphate and protease inhibitor). Protein concentration was determined by the method of Bradford, using bovine serum albumin (BSA) as a standard. 100 mg of proteins from each lysate were separated on a 10% SDS/PAGE gel and transferred to nitrocellulose membranes. Then membranes were incubated with the following antibodies: anti-Akt-1, anti- phospho Ser-473 Akt-1, anti-eNOS, anti-phospho Ser-1177 Figure 2. Akt-1 phosphorylation after sildenafil treatment. Insulin plus sildenafil treatment (1 mM/72 h) enhances Akt-1 phosphorylation in insulin resistance condition respect to the same conditions without sildenafil. 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 insulin; {p,0.05 vs HG+insulin; 1 p,0.05 vs Gluc-N+insulin. doi:10.1371/journal.pone.0014542.g002 PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e14542