Loss of Endothelial Cell RIPK3 Leads to Increased Vascular Permeability Following Intestinal Ischemia‐Reperfusion Injury

Abstract

The receptor‐interacting serine/threonine‐protein kinase 3 (RIPK3) is primarily known for promoting necroptosis, but recent findings suggest that RIPK3 has non‐necroptotic roles as well, particularly in endothelial cells (ECs). Our lab has reported both beneficial and detrimental roles for endothelial RIPK3 during vascular development, which has prompted us to study RIPK3 in the context of different postnatal vascular pathologies. Our current study focuses on roles for endothelial RIPK3 following intestinal ischemia‐reperfusion (I/R) injury in mice. First‐order branches of the superior mesenteric artery that feeds the ileum were clamped for 1 hr to induce ischemia, and clamps were removed subsequently to allow 24 hr reperfusion in tamoxifen‐induced Ripk3;Cdh5(PAC)‐CreERT2 mice (RIPK3ECKO) compared to wildtype littermate control mice (WT) (males and females, 12 weeks old). Unclamped small intestinal regions were considered as sham control in the same mouse. An Evans blue permeability assay was utilized in assessing small intestinal and distal organ vascular permeability. Small intestinal vascular permeability directly downstream of I/R injury was significantly increased in both RIPK3ECKO and WT mice (n=6 mice/group), however permeability was also significantly increased in sham small intestinal regions and in livers of RIPK3ECKO unlike in WT controls (n=6 mice/group). No difference in vascular permeability was observed between RIPK3ECKO and WT mice (n=5 mice/group) at baseline without I/R injury in all organs assessed. However, in uninjured RIPK3ECKO mice we do observe an increase in the CD68+ proinflammatory macrophage marker in the liver and submucosal layer of the small intestine compared to uninjured WT mice (n=5 mice/group). When clodronate liposomes were administered to deplete macrophages and determine whether this CD68+ macrophage population was contributing to the increased permeability in RIPK3ECKO mice following I/R injury, we observed a permeability rescue in the small intestine but not in the liver (n=4‐5 mice/group). This finding suggests that there are organ‐specific differences in macrophage contribution to vascular permeability following I/R injury. Our current studies are evaluating circulating and tissue specific cytokines and endothelial cell adhesion molecules to understand why RIPK3ECKO vasculature becomes more permeable in response to I/R injury. Discovering cellular mechanisms for vascular endothelial RIPK3 following I/R injury could provide novel therapeutic targets to promote vessel recovery after I/R injury.