Anti kdr

Transfection of mimic-miR-130a (10 nM) was performed on ECs using HiPerfect Transfection method (Qiagen, Valencia, USA)^{63 (link)}. Total RNA from untreated ECs, ECs transfected with mimic-miR-130a, treated with sEV with high (D17, D18, D20) or low miR-130a content (D1, D2, D4) was extracted using All-in-One Purification kit (Norgen, Thorold, ON Canada). miR-Scramble (Scr) was used as transfection reference sample. The expression of miR-130a was evaluated by RT-PCR^{63 (link)}. miR-130a targets were analyzed by Western Blot analysis. Proteins were obtained using RIPA buffer, as previously described^{63 (link)}. Anti-KDR (1:200, Santa Cruz), anti-ROCK1 (1:200, Abcam), and anti-HOXA5 (1:200, Abcam) antibody, together with anti-βactin, were used (1:200, Santa Cruz).

All tissue culture reagents were obtained from Euroclone, except ECGS and heparin (Sigma Aldrich) and EGM-2 (Lonza, Bend, OR, USA). Carboxymethylcellulose (M0512) was obtained from Merck KGaA, Darmstadt, Germany and type 1 rat tail collagen from Serva Electrophoresis GmbH, Heidelberg, Germany. The primary antibodies used were rabbit polyclonal anti-KDR (Santa Cruz Biotechnology, Heidelberg, Germany, sc-504), and mouse monoclonal anti-eNOS (BD Transduction Laboratories, La Jolla, CA, USA, 610296). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Dako (Agilent, Santa Clara, CA, USA, #P0399 and #P0260 for swine anti-rabbit and rabbit anti-mouse antibodies, respectively). Proteins in Western blot were detected by the LiteAblot Turbo Extra-Sensitive Chemiluminescent Substrate (Euroclone, Pero, Italy).