Figure 4. Increased number of endothelial cells in vitro after BNP treatment.

(A–B) Quantitative relative expression of mRNAs coding for endothelial cell specific proteins CD31 (pecam1 gene), von Willbrand factor (vwf gene), Ve-cadherin (cdh5 gene), eNOS (nos3 gene), alpha smooth muscle actin (alpha SMA) (acta2 gene) in NMCs isolated from neonatal (A) or adult (B) hearts cultured until confluence with or without BNP. Results expressed as fold-increase above the levels in untreated cells. (C) Flow cytometry analysis to determine the percentage of CD31⁺ cells (left: Representative histograms) in untreated or BNP-treated NMCs isolated from neonatal or adult hearts. Right: Quantification of the number of CD31⁺ cells. Results expressed as fold-increase above the number obtained in untreated cells. Neonatal NMCs were isolated from heart of C57BL/6 (WT), NPR-A or NPR-B deficient pups. Adult NMCs were isolated only from C57BL/6 hearts. (D) Adult NMCs isolated from FUCCI mice and treated with or without BNP. Flow cytometry analysis (left: representative dot plots) to determine among the CD31⁺ cells, the percentages of cells in the G1 phase (Fl2+ Fl1-), in the G1- > S phase (Fl2+Fl1+) and in the G2, M phase (Fl2-Fl1+). Right: Quantification of the number of CD31⁺ cells in the different phases of the cell cycle. Results expressed as fold-increase above the numbers obtained in untreated cells. For all quantifications, the results are means ± SEM, paired T tests were used, *p<0.05.