FIG. 2.

(A) Representative phase-contrast microphotographs of SCAP cultures. (a) In confluency before exposure to angiogenic medium. (b) Fourteen days after seeding on collagen I-coated plates and exposure to angiogenic medium, showing the development of a capillary-like network over the dense monolayer, and (c) 14 days after exposure to angiogenic medium, followed by trypsinization and reseeding at very low density (5 × 10⁴ cells/well of six-well plates) on thick (3 mm) and dense (∼1 mg/mL) collagen gel matrices. SCAP formed typical capillary-like structures, indicative of an endothelial cell (EC)-phenotype. (B) Real-time polymerase chain reaction (PCR) analysis of the expression of several angiogenesis-related molecules, including PECAM-1, vascular endothelial growth factor (VEGF), VEGFR-1, VEGFR-2, angiopoietin-1 (Ang-1), Ang-2, Tie-1, and Tie-2, and semiquantitative reverse transcription-PCR analysis of von Willebrand factor (vWF) and VE-cadherin expression in SCAP cultures exposed to angiogenic medium for up to 28 days. Human umbilical vein endothelial cells (HUVECs) were used as positive controls and beta-2-microglobulin (B2M) and SDHA as housekeeping genes for this assay. Real-time PCR values are means ± standard deviations of three independent experiments in duplicates (*P < 0.05, **P < 0.01 compared with control untreated cultures). (C) Representative single-parameter flow cytometry diagrams showing the percentage of cell population positive for CD31 and CD105 during the course of endothelial transdifferentiation of SCAP. Cells were examined after 3, 7, 14 and 21 days of exposure to angiogenic medium (D3 to D21). Results are expressed as means ± standard deviations of three independent experiments (*P < 0.05, **P < 0.01 compared with control untreated cultures). (D) Representative western blots showing obvious increase of PECAM-1 (CD31) protein expression 14 and 21 days after exposure to angiogenic medium. GAPDH was used as loading control protein.