Spatiotemporal molecular changes of the cutaneous wound after endothelial Sox9 inactivation

Summary: Abstract Body: Cutaneous expression of SRY-Box transcription factor 9 (Sox9) is tightly regulated during skin homeostasis and is re-activated in fibroblasts and endothelial cells (ECs) upon injury. Notably, EC Sox9 expression has emerged as one of the contributing factors to cutaneous and cardiac fibrosis via the endothelial to mesenchymal transition (EndMT). Abrogating Sox9 activity in ECs using the endothelial-specific Cdh5CreERT/Sox9fl/fl/Rosa-YFP (Sox9EKO) mouse model reduced wound scaring and improved closure rate. However, the underlying molecular mechanism remains unknown. In this study, using spatial transcriptomic profiling (10X Visium), we investigated the immune landscape and spatial molecular changes of the day 7 wildtype (Cdh5CreERT/Rosa-YFP) and Sox9EKO skin wounds. Spatial deconvolution using public wound sc-RNA sequencing datasets identified 20 major cell populations. We observed fewer C1qHi macrophages and more T cells within the EC niche in the Sox9EKO wounds. In concordance, gene set enrichment analysis showed signatures of increased T cell activation/differentiation, hypoxia, angiogenesis, and apical junction. Reduced expression of inflammation signalling (e.g., type II interferon) within the EC niche has suggested a less inflammatory EC phenotype, probably due to reduced apoptosis and autophagy, contributing to reduced EndMT, evident by lower TGFβ and PDGFR signalling. Ligand-receptor analysis showed reduced Caveolin 1 (Cav1) interaction in EC spots. Cav1 is required for oxLDL uptake and was shown to induce EC stiffening upon exposure, one factor that triggers EndMT. Overall, our data indicate that changes in the immune milieu might favour a less inflammatory EC phenotype or vice versa in the Sox9EKO wounds and potentially contribute to less EndMT and rapid wound closure. In conclusion, loss of Sox9 in the endothelium accelerates wounds and reduces fibrosis through the modulation of inflammation and a reduction of endothelial to mesenchymal transition. Seen Ling Sim¹, Chenhao Zhou¹, Sam Tan¹, Simranpreet Kaur¹, Laura Sormani¹, Edwige Roy¹, Abbas Shafiee¹, Kiarash Khosrotehrani¹ 1. Frazer Institute, The University of Queensland, Brisbane, QLD, Australia. Stem Cell Biology, Tissue Regeneration and Wound Healing