Figure 6. BNP stimulation of endothelial cell proliferation.

(A) Representative pictures of the ZI+BZ and RZ of C57BL/6 infarcted hearts, 10 days after surgery, treated or not with BNP and stained with DAPI (nuclei in blue) and antibodies against CD31 protein (green) and BrdU (pink). Scale bars: 100 µm. (B) Percentage of proliferating endothelial cells/per pictures in each area of the infarcted hearts (number of CD31⁺BrdU⁺ cells/CD31⁺ cells). Results in BNP-treated hearts related to those obtained in saline-treated hearts. At least 10 different pictures evaluated per mouse and per area. n = 10–9 mice per group 1–3 days after MI, n = 6 mice per group 10 days after MI. Individual values are represented. (C) Quantitative relative expression of mRNA coding for VEGF-A in ZI+BZ and RZ 3 and 10 days after MI. Results expressed as fold-increase above the levels in the hearts of saline-injected mice. n = 11–14 hearts per group. (B–C) Results are means ± SEM (represented in red). # p<0.05 for different variance between groups, *p≤0.05 using unpaired T tests with or without Welch’s corrections. (D) NMCs isolated from unmanipulated Cdh5:ROSA26 mice injected 2 weeks before with tamoxifen. GFP⁺ cells were sorted and stimulated immediately with or without BNP (5 µg/ml) during 1h30 at room temperature. Western bot analysis was then performed on these cells to evaluate p38 MAP kinase activation. Blots were stained with antibodies against phospho p38 (pp38) (43 kDa), p38 (43 kDa) and Tubulin (55 kDa). Quantification of the pp38/p38 ratio obtained from six independent cell sorting experiments. Results are means ± SEM, *p<0.05 using paired T test. (E) Representative pictures of BNP-treated infarcted hearts 10 days after surgery and stained with antibody against pp38. GFP⁺ cells represent either endothelial cells (in Cdh5:ROSA mice, picture at the top) or WT1⁺ cells (in WT1:ROSA mice, picture at the bottom).