Figure 3. hPLG stimulates ECFC proliferation.

(A) 12,000 ECFCs/well were seeded on collagen-coated plastic and hPLG and cultured for 24 h in complete culture medium before being PFA-fixed and nuclear-stained with Hoechst 33342 (in blue). (B) ECFCs were cultured either on rat collagen I or hPLG and incubated with Pazopanib (20 μg/mL), Ki8751 (100 nM) or Tivozanib (1 μM). The number of cell per mm² was then estimated by microscopy. Data show mean ± SEM and are representative of three independent experiments. Statistical significance was tested by one-way ANOVA with Bonferroni’s post-test to compare different inhibitors against the control conditions (*P ≤ 0.05). (C) Activation of ERK1/2 by contact with hPLG versus rat collagen I coating (incubation 1 hour in complete medium) and its inhibition by 50 μM PD98059. ERK1/2 phosphorylation was tested by phospho-specific immunoblotting. Last lane demonstrates expression but no phosphorylation of ERK1/2 from platelets in hPLG (in the absence of ECFCs). (D) ERK inhibition by 50 μM PD98059 significantly inhibits ECFC proliferation on hPLG but not on rat collagen I coating. Data show mean ± SEM and are representative of six independent experiments. ECFC proliferation was also tested in 2D cultures on collagen and fibrin gels with hPL provided as a source of extra GFs. Representative pictures are shown in (E) and quantitative analysis is shown in (F). As normal distribution and equal variance of the samples were confirmed using the Shapiro-Wilk normality test and Bartlett’s homoscedasticity test (Supplementary Table 2), statistical significance was tested using one way ANOVA with Bonferroni post-test (*P ≤ 0.05, n = 6).