RIPK3 Protects Against Endothelial Activation and Vascular Permeability in a Mouse Model of Ischemia-Reperfusion Injury

BACKGROUND: RIPK3 (receptor-interacting protein kinase 3) has context-specific roles that are frequently associated with cellular damage and death. We previously found that hypoxia can trigger elevated levels of RIPK3 in endothelial cells (ECs), which contributes to lethal vascular rupture during mouse embryonic development. However, it is unknown whether elevated RIPK3 likewise compromises endothelial barrier function in adult vasculature under hypoxic conditions such as ischemia-reperfusion (I/R) injury. METHODS: Twelve-week-old male and female littermate control or inducible EC-specific Ripk3 knockout ( Ripk3 iECKO ) mice were exposed to surgical intestinal I/R injury. Clodronate liposomes were used to reduce circulating monocytes in vivo. Immortalized murine EC (MS1 [mile sven 1) and murine macrophage (BMA3.1A7 [bone marrow A clone 3.187]) lines were used for in vitro experiments. RESULTS: Ripk3 iECKO mice displayed an unexpected increase in small intestinal vascular permeability after I/R injury, rather than the decrease we predicted. Subsequent analyses using multiplex cytokine assays revealed significantly elevated levels of IL-6 (interleukin-6) in the serum and small intestinal tissue of I/R-injured Ripk3 iECKO mice. Upon TNFα (tumor necrosis factor-alpha) stimulation of immortalized murine Ripk3 knockout ECs grown in vitro, we found increased transcription and secretion of IL-6. These cells also expressed elevated levels of VCAM-1 (vascular cell adhesion molecule-1), which was likewise upregulated in the small intestines of Ripk3 iECKO mice. Using an IL-6 neutralizing antibody, we found that IL-6 triggered VCAM-1 elevation in Ripk3 knockout cells. This VCAM-1 expression correlated with enhanced macrophage binding to Ripk3 knockout cells and increased accumulation of leukocytes in Ripk3 iECKO small intestines following I/R injury. Importantly, reduction of circulating monocytes with clodronate liposomes led to rescue of IR injury–induced vascular permeability in Ripk3 iECKO mice. CONCLUSIONS: Endothelial RIPK3 suppresses EC activation and inflammation associated with IL-6 and VCAM-1 elevation to protect the vascular barrier in the context of intestinal I/R injury. Thus, endothelial RIPK3 plays surprisingly beneficial roles that reduce I/R injury–induced vascular dysfunction.