623 Changing endothelial cell fate in wound healing through modulation of Sox9 to reduce scarring

Abstract

Endothelial cells that line the vascular endothelium undergo a variety of cellular transitions, with one such transition known as endothelial to mesenchymal transition (EndMT). We identified transcription factor Sox9 to be highly expressed in a progenitor population in the endothelium. We here aimed to investigate the potential molecular regulation of EndMT by the transcription factor Sox9 as well as its interaction with Notch signaling. A vascular-specific Sox9 knock-out (Sox9fl/flCdh5CreERt2Rosa-YFP (Sox9eKO)) and a vascular specific rbpj knock-out mouse model were used to assess EndMT. Wild-type mice were used for Sox9 siRNA knock-down. Loss of rbpj resulted in an increase in Sox9 expression in the vasculature. Full thickness excisional large wounds were used to assess fibrotic scar area following Sox9eKO or Sox9 siRNA knock-down. Full thickness small wounds 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 YFP labelled endothelial cells, vessels stained for endothelial (CD31) and mesenchymal/EndMT (α-SMA, FSP-1 and SLUG) markers across time-points assessed (D3 to D7), in both Sox9eKO and Sox9 siRNA knock-down. Flow cytometry analysis showed a significant reduction in the intensity of the pro-fibrotic marker CD26 in both Sox9eKO and Sox9 siRNA knock-down (***P<0.001). Loss of rbpj resulted in an increase in Sox9, EndMT and fibrosis. 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.