The clinical use of endothelial colony forming cells (ECFC) is hampered by their restricted engraftment. We aimed to assess engraftment, vasculogenic and pro-angiogenic activities of ECFC in immunocompetent (C57BL/6: WT) or immunodeficient (rag1 −/−C57BL/6: Rag1) mice. In addition, the impact of host immune system was investigated where ECFC were co-implanted with mesenchymal stem/stromal cells (MSC) from adult bone marrow (AdBM-MSC), fetal bone marrow (fBM-MSC), fetal placental (fPL-MSC), or maternal placental (MPL-MSC). Transplantation of ECFCs in Matrigel plugs resulted in less cell engraftment in WT mice compared to Rag1 mice. Co-implantation with different MSCs resulted in a significant increase in cell engraftment up to 9 fold in WT mice reaching levels of engraftment observed when using ECFCs alone in Rag1 mice but well below levels of engraftment with MSC-ECFC combination in Rag1 recipients. Furthermore, MSCs did not reduce murine splenic T cell proliferation in response to ECFCs in vitro. ECFCs enhanced the murine neo-vascularization through paracrine effect, but with no difference between Rag1 and WT mice. In conclusions, the host adaptive immune system affects the engraftment of ECFCs. MSC co-implantation improves ECFC engraftment and function even in immunocompetent hosts mostly through non-immune mechanisms.

Skin wound healing in adults is characterized by a peak of angiogenesis followed by regression of the excessive vasculature in parallel with collagen deposition and fibrosis in the wound. We hypothesized that regressing vessels in healing wounds were in fact entering an endothelial to mesenchymal transition contributing to scarring. Using vascular-specific fate tracking (Cdh5-cre/ROSA-YFP mice), full-thickness excisional wounds were analyzed to reveal a time-dependent transition from endothelial phenotype characterized by vascular endothelial-cadherin, CD31, and CD34 toward a mesenchymal phenotype characterized by alpha-smooth muscle actin and fibroblast-specific protein 1 expression. We next conditionally ablated RBPJ in the vasculature (Rbpj/Cdh5-creROSA-YFP) to evaluate the role of canonical Notch signaling in this process. Endothelial to mesenchymal transition was clearly accelerated after the loss of Notch signaling within the vasculature. The acceleration of endothelial to mesenchymal transition resulted in delayed wound healing, increased fibrosis, and extensive scar tissue formation, with the rapid loss of key endothelial genes and proteins and upregulation of mesenchymal protein expression (alpha-smooth muscle actin and fibroblast-specific protein 1) in vessels. Our findings here uncover a cellular contributor to skin wound scarring through the process of endothelial to mesenchymal transition in skin wounds and demonstrate the importance of Notch signaling in regulating this critical process during healing.

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The incidence of chronic wounds is escalating, and the associated healing process is especially problematic in an aging population with increased morbidity. Targeting increased inflammation in chronic wounds is a promising but challenging therapeutic strategy. Indeed, inflammation and especially macrophages are required for wound healing. As the NLRP3 inflammasome has been implicated with various other inflammatory diseases, in this study, we used MCC950—a selective NLRP3 small molecule inhibitor—on murine models of both acute and chronic wounds. This molecule, while tested for other inflammatory conditions, has never been investigated to reduce topical inflammation driving chronic wounds. We found that there were no significant differences when the treatment was applied either topically or orally in wild-type C57Bl/6 mice and that it even impaired wound healing in obese mice. The treatment was also unable to improve re-epithelialisation or angiogenesis, which are both required for the closure of wounds. We are inclined to believe that MCC950 may inhibit the closure of chronic wounds and that it does not alter wound-associated macrophage polarisation.