Vegf rhvegf165

SCAP (3×10⁵) were seeded on 0.1% gelatin-coated 6-well plates in α-MEM and incubated at 37°C in a humidified atmosphere with 5% CO₂. At 40–50% cell confluence, the culture medium was changed to EGM-2 (Lonza, Walkersville, MD, USA), plus 50 ng/mL VEGF (rhVEGF165, Peprotech, NJ, USA), 20 ng/mL BMP-4 (R&D Systems, Minneapolis, MN, USA) and the chemical cocktail 0.5 mM VPA (Sigma-Aldrich, St. Louis, MO, USA), 3 μM CHIR99021 (Stemgent, Beltsville, MD, USA), 1 μM Repsox (Selleck, Houston, TX, USA), 10 μM Forskolin (Caymen, Ann Arbor, MI, USA), 5 μM Y-27632 (Sigma-Aldrich) for a 4-day induction, and then BMP-4 was replaced by 100 μM 8-Br-3,5-cAMP (Sigma-Aldrich) in the next 4 days (Appendix Table 1). The medium was changed every 2 days. The obtained cells are passaged and cultured in a maintenance medium composed of EGM-2 and 0.5 mM VPA, 3 μM CHIR99021, 1 μM Repsox, 10 μM Forskolin and 5 μM Y-27632.

The ability of the cells to produce nitric oxide (NO) was assessed by measuring the concentration of NO in the culture medium containing 2 ng/mL VEGF (rhVEGF165, Peprotech) and 1μM Calcium Ionophore A23187 (Sigma-Aldrich) with the use of the NO detection kit (Invitrogen, Waltham, MA) according to the manufacturer’s instructions. The amount of nitrate was determined by converting it to nitrite, followed by the colorimetric determination of the total concentration of nitrite as a coloured azo dye product of the Griess reaction that absorbed visible light at 540 nm with the use of a microplate reader (MTX Lab Systems, WA).