Sox9 and Rbpj differentially regulate endothelial to mesenchymal transition and wound scarring in murine endovascular progenitors

Abstract

Endothelial to mesenchymal transition (EndMT) is a leading cause of fibrosis and disease, however its mechanism has yet to be elucidated. The endothelium possesses a profound regenerative capacity to adapt and reorganize that is attributed to a population of vessel-resident endovascular progenitors (EVP) governing an endothelial hierarchy. Here, using fate analysis, we show that two transcription factors SOX9 and RBPJ specifically affect the murine EVP numbers and regulate lineage specification. Conditional knock-out of Sox9 from the vasculature (Sox9fl/fl/Cdh5-CreERRosaYFP) depletes EVP while enhancing Rbpj expression and canonical Notch signalling. Additionally, skin wound analysis from Sox9 conditional knock-out mice demonstrates a significant reduction in pathological EndMT resulting in reduced scar area. The converse is observed with Rbpj conditionally knocked-out from the murine vasculature (Rbpjfl/fl/Cdh5-CreER RosaYFP) or inhibition of Notch signaling in human endothelial colony forming cells, resulting in enhanced Sox9 and EndMT related gene (Snail, Slug, Twist1, Twist2, TGF-β) expression. Similarly, increased endothelial hedgehog signaling (Ptch1fl/fl/Cdh5-CreER RosaYFP), that upregulates the expression of Sox9 in cells undergoing pathological EndMT, also results in excess fibrosis. Endothelial cells transitioning to a mesenchymal fate express increased Sox9, reduced Rbpj and enhanced EndMT. Importantly, using topical administration of siRNA against Sox9 on skin wounds can substantially reduce scar area by blocking pathological EndMT. Overall, here we report distinct fates of EVPs according to the relative expression of Rbpj or Notch signalling and Sox9, highlighting their potential plasticity and opening exciting avenues for more effective therapies in fibrotic diseases.

Endothelial Heterogeneity in Development and Wound Healing

“…Additionally, live imaging of skin wound healing studies identified the migration of tissue resident endovascular progenitors (EVP) into the centre of wound granulation tissue and subsequent differentiation. EVPs have been identified to reside in vasculature and can give rise to mature endothelial cells, forming a vascular network independent of angiogenesis in ischemic situations of skin would healing [ 7 , 81 ]. Increased angiogenesis and neovascularisation has also been observed in skin wound healing using human EPC as a therapy, selectively blocking αVβ3 integrins, endothelial extracellular vesicles, antioxidant administration such as bee venom and also the activation of the PI3-kinase/Akt pathway [ 82 , 83 , 84 , 85 , 86 , 87 , 88 ].…”

“…Endothelial cells also undergo endothelial to mesenchymal transition (EndMT) in pathological contexts, for example in response to unique signals from their niche to transdifferentiate into fibroblasts contributing to cardiac fibrosis [ 92 , 93 ]. Indeed, the EndMT transition is central to wound healing as the pro-inflammatory and hypoxic wound site triggers EndMT in migrating endothelial cells from the local vascular bed [ 81 , 94 ]. Aberrant endothelial transdifferentiation, both delayed or advanced differentiation, can give rise to unstable vessels at the wound site resulting in excess scar tissue formation [ 81 ].…”

“…Indeed, the EndMT transition is central to wound healing as the pro-inflammatory and hypoxic wound site triggers EndMT in migrating endothelial cells from the local vascular bed [ 81 , 94 ]. Aberrant endothelial transdifferentiation, both delayed or advanced differentiation, can give rise to unstable vessels at the wound site resulting in excess scar tissue formation [ 81 ].…”

“…The endothelial cell-specific knockout of the transcriptional effector of Notch signalling, Rbpj , was utilised to demonstrate the EndMT acceleration in the absence of canonical Notch signalling [ 13 ]. In an additional study, Zhao et al demonstrate that SOX9-driven EVP differentiation increases EndMT and fibrotic scarring in skin wound healing, as the SOX9 pathway acts in opposition to RBPJ–Notch signalling axis generating a double-negative feedback regulating EndMT [ 81 ]. Indeed, applying topical Sox9 siRNA treatment directly onto wounds reduced scarring by minimising EndMT and maintaining an endothelial phenotype [ 81 ].…”

“…In an additional study, Zhao et al demonstrate that SOX9-driven EVP differentiation increases EndMT and fibrotic scarring in skin wound healing, as the SOX9 pathway acts in opposition to RBPJ–Notch signalling axis generating a double-negative feedback regulating EndMT [ 81 ]. Indeed, applying topical Sox9 siRNA treatment directly onto wounds reduced scarring by minimising EndMT and maintaining an endothelial phenotype [ 81 ].…”