Targeting endothelial progenitor cells (EPCs) for clinical therapeutics, including vascularization in wound healing, has been hindered by the lack of a uniform EPC definition. Multiple putative EPC populations have been characterized using various genes/surface markers; we hypothesized that overlapping these markers would allow a uniform definition to be validated functionally. We examined the overlap of several of these markers with recently identified endovascular progenitors (EVPs; VE-Cadherin(VECad)+Lineage(Lin)negCD34+CD31lo), shown to have functional progenitor capacity, in homeostatic murine aorta and full-skin excisional wounds. Flow cytometry revealed that EVPs highly co-expressed with Protein C Receptor (PROCR) and Platelet-derived growth factor receptor alpha (PDGFRA) as compared to mature differentiated endothelial (D) cells (VECad+LinnegCD34+CD31+) in the aorta of adult C57Bl/6 mice (p<0.001). Single-cell RNA-sequencing showed clustering of PROCR and PDGFRA with EVPs, while other markers clustered with D cells. Immunofluorescent staining confirmed PROCR+ EVPs co-localize with YFP+ endothelium in the thoracic aortae of Cdh5-CreERT2/Rosa-EYFP mice (p=0.005) with increased clonogenic capacity (p<0.01). Aortic PROCR+ EVPs had greater colony forming capacity in vitro and engraftment potential in vivo than PROCRneg EVPs or PROCR+/neg D cells (p<0.05). Lineage tracing of developing PDFGRA-MerCreMer/Rosa-EYFP mice showed aortic YFP+ EVPs at day 0 (D0) differentiated into D cells by D84 (p<0.001). This result was validated in adult full-skin excisional wounds from the same mice, showing that PDGFRA+ EVPs at D0 differentiated into D cells by D5 (p<0.05), suggesting PDGFRA exclusively marks an EPC population capable of endothelial fate in both homeostasis and injury. Overall, the characteristics displayed by PROCR+ and PDGFRA+EVPs suggest these may mark a true EPC population.

As part of tissue homeostasis, endothelial progenitors (EPCs) residing within the endothelium contribute to vascular maintenance and regeneration through self-renewal and differentiation. In various stem cell niche, macrophages (MΦ) help modulate progenitor stemness and behavior and have been reported to regulate blood vessel development and remodeling. Recently, subsets of perivascular macrophage (PVMΦ) were identified in the blood vessel vicinity during tissue homeostasis and wound healing. Nevertheless, the physiological role of these PVMΦ on EPC function in homeostasis and wound re-vascularisation remains elusive. In this study, we investigated the interaction and regulatory function of PVMΦ on EPCs' stemness in the murine aorta and skin using an endothelial-specific lineage tracing model, Cdh5CreERT/Rosa-ZsGreen. Imaging flow cytometry analyses tracking remanent PVMΦ bodies on endothelial cells revealed that PVMΦ preferentially formed physical interaction with endothelial cells that displayed mesenchymal properties in the healthy aorta (18.81% ± 13.13%) and skin (10.02% ± 0.92%). In situ characterization showed that the F4/80+Lyve1+ PVMΦ were in the adventitial area of the aorta, and resided within the lower dermis of the dorsal skin. MΦ depletion using clodronate liposomes doubled aorta EPC proportion (p=0.0079) in vivo, where it displayed increased proliferative potential with reduced clonogenic capacity in vitro. Functionally, attenuating macrophage-derived Wnt activity using Wlsfl/flLysM-Cre mice elevated EPC cell number by two-fold on day 5 post cutaneous injury without affecting other endothelial subpopulations. Overall, our data indicate that PVMΦ constitute part of the vascular niche by regulating EPCs' quiescence and self-renewal, potentially via paracrine Wnt signaling.

The endothelium possesses a profound regenerative capacity to adapt and reorganise in homeostasis and disease. The capacity to regenerate is increasingly attributed to a population of vessel-resident endovascular progenitor (EVP) cells that governs an endothelial hierarchy and have the ability to form vascular networks de novo. Using fate map analysis, we show that two transcription factors Sox9 and Rbpj specifically demarcate the EVP population and regulates progenitor fate choice differentiation. Conditional knock-out of Sox9 from the endothelium drove the depletion of EVP to a mature differentiated endothelial phenotype and enhanced Rbpj expression and Notch signalling. Additionally, skin wound analysis from Sox9 knock-out mice demonstrated a significant reduction in endothelial to mesenchymal transition (EndMT), reducing scar area. The converse was observed with Rbpj conditionally knocked-out from the vasculature, with enhanced Sox9 and key EndMT gene (Snail, Slug, Twist1, Twist 2, TGF-β) expression. Concurrently, vascular sonic hedgehog activation upregulates the expression Sox9 and is key in driving pathological EndMT and vascular fibrosis, resulting in over 3-fold increase in scar area in skin wound healing. In this scenario, we see EVP transitioning towards a mesenchymal fate; with increased Sox9, reduced Rbpj and enhanced EndMT gene expression. Importantly, using topical administration of siRNA against Sox9 on skin wounds significantly reduced scar area by blocking pathological EndMT. The understanding of how vascular resident EVP function opens exciting new avenues for more effective therapies in blocking fibrotic disease.