PDGF-BB promotes vascular development and sprouting angiogenesis. (A) Embryoid bodies were cultured in 15% FBS for 6 or 8 days in the absence (basal) or presence of 30 ng/mL PDGF-BB. Endothelial cells were visualized by immunohistochemical staining for CD31 (red). Arrows in the larger panels at the left (bars represent 300 μm) indicate areas shown at higher magnification in the smaller panels to the right (bars represent 100 μm). For the 8-day PDGF-BB–treated culture, a higher magnification is also shown at the bottom far right to demonstrate sprouting and a tip cell extending filopodia (bar represents 20 μm). For all conditions, 6 to 8 bodies were analyzed in 3 or more independent experiments. (B) Quantification of the mean area and length estimated in 10 individual embryoid bodies for each condition, stained immunohistochemically for CD31. Results are expressed as fold induction over basal (mean ± SD). (C) FACS analysis of CD31 and VE-cadherin–expressing cells in collagenase-dispersed embryoid bodies treated with PDGF-BB (BB) or not treated (basal). FL5 (CD31), FL1 (VE-cadherin). Blue indicates reactivity of the secondary antibody alone. (D) Real-time PCR analysis of VEGF-A, PDGF-B, CD31, VEGFR-2, VE-cadherin (VE-cad), PDGFR-α, PDGFR-β, RGS-5, and α-SMA expression in embryoid bodies cultured for 8 days in the absence (basal) or presence of PDGF-BB. Data are given as mean ± SD and are based on analyses of 6 individual RNA preparations. *P < .001, paired Student t test. (E) Immunoblotting for expression of CD31 and α-SMA in R1 embryoid bodies cultured for 8 days in the absence (–) and presence (+) of PDGF-BB.