Oral Presentation Australasian Society for Dermatology Research 2022 Annual Scientific Meeting

Working towards a uniform definition for endothelial progenitor cells in murine aorta and wound healing contexts (#158)

Cassandra Styke 1 , Simranpreet Kaur 1 2 , Jilai Zhao 1 , Seen-Ling Sim 1 , Abbas Shafiee 1 3 , Chenhao Zhou 1 , Ho Yi Wong 1 , Kiarash Khosrotehrani 1
  1. The University of Queensland Diamantina Institute, Woolloongabba, Queensland, Australia
  2. Mater Research, Woolloongabba, Queensland, Australia
  3. Queensland University of Technology, Brisbane, QLD, Australia

Targeting endothelial progenitor cells (EPCs) for clinical therapeutics, including vascularization in wound healing, has been hindered by the lack of a uniform EPC definition. Various putative EPC populations have been characterized using several genes/surface markers; we hypothesized that overlapping these markers would allow a consensus definition to be validated functionally. Here we examined the overlap of several markers from across the field with EVPs (VE-Cadherin(VECad)+Lineage(Lin)negCD34+CD31lo) in homeostatic murine aorta and an injury model of full-skin excisional wounds, hoping to narrow a consensus definition. Flow cytometry revealed that among markers tested, Protein C Receptor (Procr) and Platelet-derived growth factor receptor alpha (PDGFRα) were consistently more likely to be co-expressed with EVPs (78.04%, 82.28%) than with mature differentiated endothelial (D) cells (VECad+LinnegCD34+CD31+; 54.22%, 24.65%) in the aorta of adult C57Bl/6 mice (n=5; p<0.001). Single-cell RNA-sequencing confirmed clustering of Procr and PDGFRα with EVPs, while other markers studied clustered with D cells. Immunofluorescent staining confirmed Procr+ EVPs co-localized with YFP+ endothelium in the aortae of Cdh5-CreERT2/Rosa-EYFP reporter mice. Aortic Procr+ EVPs had greater endothelial colony forming capability when cultured in vitro (2.74%) than either Procrneg EVPs (0.69%) or Procr+/neg D cells (0%; n=3), and greater engraftment when implanted in vivo (5.67% vs. 0.79%; n=3; p<0.05). Lineage tracing via flow cytometry of developing PDFGRα-MerCreMer/Rosa-EYFP mice showed that aortic YFP+ EVPs at day 0 (D0) differentiated into D cells by D84 (D0 0.74% D cells, D84 4.67%; n=5; p<0.001). This result was then validated in adult full-skin excisional wounds from the same mice, showing that YFP+ EVPs at D0 differentiated into D cells by D5 (D0 0.04%, D5 1.19%; n=7; p<0.005), suggesting that mesenchymal marker PDGFRα marks an EPC population capable of endothelial fate in both homeostasis and injury. The characteristics displayed by Procr+ and PDGFRα+ EVPs suggest these may mark a true EPC population.