Abstract 6: A Novel in vivo Endothelial Hierarchy from Progenitor to Mature Endothelial Cells Reveals Key SoxF-Dependent Differentiation Process

Abstract

Background: The formation of new blood vessels during adult life is often explained by angiogenesis. However, an alternate proposal now suggests that neo-vessels form from endothelial progenitors able to assemble all the intimal layers of vessel structures. Our aim was to define vessel-resident endothelial progenitors in vivo in a variety of tissues in physiological (aorta, lung) and pathological (wounds, tumors) situations. Methods and Results: Using common endothelial markers (CD34, CD31, VEGFR2) with flow cytometry, three sub-populations of endothelial cells could be identified among VE-Cadherin+ and CD45- cells. These were termed an endovascular progenitor (EVP) harboring CD31lo VEGFR2lo giving rise to an intermediate CD31intVEGFR2lo transit amplifying (TA) and a definitive differentiated (D) CD31hiVEGFR2hi population. Confirmation of these populations was demonstrated via lineage tracing using Cdh5cre ERt2 /Rosa-YFP reporter mice. Importantly, EVP cells arose from vascular resident beds that could not be transferred by bone marrow transplantation, marking their distinction for hematopoietic/myeloid origin. Furthermore, EVP displayed progenitor like status with a high proportion of cells in a quiescent cell cycle phase. Only EVP cells and not TA and D cells had self-renewal capacity as demonstrated by in vitro colony forming and transplant studies in vivo in Matrigel TM plugs in recipient mice. Through whole RNA sequencing we demonstrated that EVP cells highly expressed genes related to progenitor function such as Sox9 , Il33, Egfr and Pdfgrα, whereas D cells highly expressed genes related to differentiated endothelium including Ets1&2 , Gata2 , Cd31, Vwf and Notch . We also determined the Sox18 transcription factor as having a significant role in defining the endothelial hierarchy, which we validated through lineage-tracing using S ox18Cre ERt2 /Rosa-YFP mice. In the absence of functional SOX18/SOXF, EVP progenitors were still present, but TA and D populations were significantly reduced. Conclusion: In summary, we have demonstrated the existence of an entirely novel endothelial hierarchy, from EVP to TA to D. This has been demonstrated by the self-renewal, differentiation and molecular profiling of an EVP.