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Endothelial RIPK3 Regulation and its Effects on Vascular Permeability Following Intestinal Ischemia‐Reperfusion Injury
Author(s) -
Fernando Charmain,
Griffin Courtney
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.01680
Subject(s) - necroptosis , ischemia , reperfusion injury , vascular permeability , medicine , pathology , chemistry , programmed cell death , apoptosis , biochemistry
The cytoplasmic protein receptor‐interacting serine/threonine‐protein kinase 3 (RIPK3) is primarily known for promoting necroptosis, but more recent findings suggest that RIPK3 has additional functions separate from necroptosis. Our lab recently demonstrated that in the absence of the chromatin‐remodeling enzyme CHD4, RIPK3 is elevated in hypoxic cultured embryonic endothelial cells (ECs) and that elevated RIPK3 causes vessel fragility in midgestation mouse embryos. Our current study investigates the regulation of endothelial RIPK3 following intestinal ischemia‐reperfusion injury (IRI) in mice and its effects on vascular permeability and damage following IRI. To assess RIPK3 regulation following IRI, first‐order branches of the superior mesenteric artery were clamped for 1 hr to induce ischemia, and clamps were removed subsequently for 24 hr reperfusion (C57BL/6J mice; 10‐12 weeks old). Unclamped vessels and downstream gut tissue from the same animal were used as sham controls. RIPK3 and VE‐Cadherin expression (mean fluorescence intensity; MFI) were quantified in 1 st and 2 nd order mesenteric arteries using immunostaining of en face vessel preparations. RIPK3 was significantly elevated (P=0.0216) at 1 hr ischemia and following 24 hr IR injury (P=0.0295) compared to sham controls (7‐8 vessels/group, n=4 mice). Junctional VE‐Cadherin showed increased expression (not significant) following 1 hr ischemia. Although no changes were observed following 24 hr IRI, VE‐Cadherin expression at mesenteric arterial endothelial junctions were discontinuous and unstable suggesting a potential barrier dysfunction associated with IRI. To assess the role of RIPK3 on vascular permeability, we used Ripk3 fl/fl ;Cdh5(PAC)‐Cre ERT2 mice ( Ripk3 endothelial cell knockout=RIPK3 ECKO ) compared to littermate controls, and vascular permeability was assessed using the Evans blue assay following 24 hr intestinal IRI. Gut vascular permeability was increased following IRI in both RIPK3 ECKO (n=4 mice)and littermate controls (n=3 mice), however permeability was also significantly increased (P=0.02) in sham gut regions and the livers (P=0.04) of RIPK3 ECKO mice unlike in controls. Collectively, these data suggest that deletion of endothelial RIPK3 increases vascular damage downstream of injury sites and points toward a protective role for RIPK3 in the vasculature following IRI. Future studies evaluating mechanisms of RIPK3 protective effects in endothelial cells following IRI will provide insight into therapeutic targets for vessel recovery from IRI.