Human endothelial colony forming cells (ECFCs) require endothelial protein C receptor (EPCR) for cell cycle progression and angiogenic activity

Vascular repair and regeneration are critical for tissue homeostasis. Endothelial colony forming cells (ECFCs) are vessel-resident progenitors with vasoreparative capacity and they offer an important avenue for allogeneic cytotherapy to achieve perfusion of ischemic tissues. Endothelial Protein C Receptor (EPCR) has been proposed as a reliable marker for vascular endothelial stem cells, but its precise role in ECFC biology remains unknown. The current study has investigated the biological relevance of EPCR in ECFC function. Our data shows that over 95% of ECFCs exhibit high EPCR expression. These levels surpassing CD34 and CD157, positions EPCR as a new robust ECFC immunophenotypic marker, alongside established markers CD31 and CD105. Functionally, depleting ECPR expression in ECFCs significantly diminished angiogenic activity, including proliferation, migration and tube formation. This knockdown also altered normal ECFC barrier function. Transcriptomic analysis indicated that knockdown of EPCR led to enrichment of gene signatures for cell cycle, TGF beta, and focal adhesion kinases and cell cycle arrest in G1 was confirmed ECFCs in which EPCR was depleted. Mechanistically, EPCR knockdown led to increased release of TGFB2 and SMAD2/3 activation, coupled with increased p21, decreased pFAK, and increased transgelin. Additionally, we showed that quiescent ECFCs showed significantly lower EPCR expression when compared to proliferating ECFCs. In agreement with this, cell sorting experiments demonstrated that ECFCs with the highest EPCR expression exhibited the highest clonogenic capacity. In summary, our findings highlight that EPCR expression in ECFCs is critical for their angiogenic activity, by modulating processes such as cell cycle progression, barrier function, and endothelial to mesenchymal transition. ECFCs were Transfected at 70% confluency with siRNA for PROCR or control for 16 hrs. Cells were passaged and used for experiments after 48 hrs. 6 clones per condition (paired) were profiled for RNAseq