Serelaxin blocks EndMT in a mouse model of pressure overload. (A) Immunofluorescence staining of endothelial cell marker CD31, mesenchymal marker α-SMA and DAPI. (B) Quantifications of CD31 and α-SMA positive area and ratio of CD31+α-SMA+ / CD31+ cells. Compared to sham, vehicle-treated hearts showed an increased expression of α-SMA but a decreased protein expression of CD31, while treatment with Serelaxin showed reduced α-SMA and restored CD31 protein expression. Double positive cells significantly increased in AAC-operated animals and were reduced by Serelaxin administration. (C) Immunofluorescence staining of endothelial cell marker CD31, fibrotic marker alpha-1 type I collagen (Col1α(I)) and DAPI. (D) Quantifications of CD31 and Col1α(I) positive area: Col1α(I) expression was upregulated in AAC hearts and is inhibited by Serelaxin. Compared to sham, vehicle-treated hearts showed a decreased protein expression of CD31, while treatment with Serelaxin again restored CD31 expression. (E) qPCR analysis showing the relative mRNA expression level of CD31 and EndMT key regulators Snail, Slug, and Twist in AAC hearts treated with vehicle or Serelaxin. Vehicle-treated AAC-operated hearts showed an increased expression of Snail, Slug and Twist but a decreased expression of CD31, while Serelaxin treatment reduced Snail, Slug and Twist expression and restored CD31 expression. Student t-test was used for single comparison and one-way ANOVA with Bonferroni post-hoc analysis was used for multiple group comparisons. Gene expression and associated error bars represent mean ± SEM, n≥3, n.s. no significance, * p<0.05, ** p<0.01, *** p<0.001.