The vascular endothelium can undergo a cellular transitions during both homeostasis and disease towards a mesenchymal fate (EndMT). Although prevalent in development and diseases such as tissue fibrosis, mechanisms that drive EndMT remain largely unknown. Recently, our laboratory was the first to identify transcription factor Sox9 to be expressed in a progenitor population in the endothelium. We here aimed to investigate the potential molecular regulation of EndMT by the transcription factor Sox9.
A vascular-specific Sox9 knock-out (Sox9fl/flCdh5CreERt2Rosa-YFP (Sox9eKO)) mouse model was used to assess EndMT. Wild-type (C57/Bl6) mice were used for Sox9 siRNA knock-down. Full thickness excisional large wounds (1cm x 1cm) were used to assess fibrotic scar area following Sox9eKO or Sox9 siRNA knock-down. Full thickness small wounds (6mm) were used to assess the changes in cellular phenotype in EndMT following Sox9eKO or Sox9 siRNA knock-down via immunofluorescence staining and flow cytometry.
A significant reduction in fibrotic scar area was seen following Sox9eKO (**P<0.01) and siRNA knock-down (*P<0.05). Immunofluorescence quantification showed a significant reduction in the number of cells and whole vessels stained for endothelial (CD31) and mesenchymal/EndMT (α-SMA, FSP-1 and SLUG) markers across time-points assessed (D3, D5 and D7), in both Sox9eKO and Sox9 siRNA knock-down. Flow cytometry analysis of both Sox9eKO and Sox9 siRNA knock-down showed a significant reduction in the intensity of the pro-fibrotic marker CD26 in both Sox9eKO and Sox9 siRNA knock-down compared to controls (***P<0.001).
In conclusion, the dramatic decrease in fibrotic scar area following both Sox9eKO and Sox9 siRNA knock-down highlights Sox9s role in scar formation through EndMT. This highlights Sox9 as a novel regulator for EndMT and contributes to both an understanding into the fundamentals of cardiovascular biology and identification of a potential therapeutic target using siRNA in reducing scar area.