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Endothelial Hey2 deletion reduces endothelial-to-mesenchymal transition and mitigates radiation proctitis in mice.

ABSTRACT: The current study evaluated the role of Hey2 transcription factor in radiation-induced endothelial-to-mesenchymal transition (EndoMT) and its impact on radiation-induced tissue damage in mice. Phenotypic modifications of irradiated, Hey2 siRNA- and Hey2 vector plasmid-transfected human umbilical vein endothelial cells (HUVECs) resembling EndoMT were monitored by qPCR, immunocytochemistry and western blots. Subsequently, in mice, a Cre-LoxP strategy for inactivation of Hey2 specifically in the endothelium was used to study the biological consequences. Total body irradiation and radiation proctitis were monitored to investigate the impact of conditional Hey2 deletion on intestinal stem cells and microvascular compartment radiosensitivity, EndoMT and rectal damage severity. We found that EndoMT occurs in irradiated HUVECs with concomitant Hey2 mRNA and protein increase. While Hey2 silencing has no effect on radiation-induced EndoMT in vitro, Hey2 overexpression is sufficient to induce phenotypic conversion of endothelial cells. In mice, the conditional deletion of Hey2 reduces EndoMT frequency and the severity of rectal tissue damage. Our data indicate that the reduction in mucosal damage occurs through decline in stem/clonogenic epithelial cell loss mediated by microvascular protection. EndoMT is involved in radiation proctitis and this study demonstrates that a strategy based on the reduction of EndoMT mitigates intestinal tissue damage.

SUBMITTER: Mintet E

PROVIDER: S-EPMC5503994 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:AIMS/HYPOTHESIS:The role of non-cardiomyocytes in diabetic cardiomyopathy has not been fully addressed. This study investigated whether endothelial cell calpain plays a role in myocardial endothelial injury and microvascular rarefaction in diabetes, thereby contributing to diabetic cardiomyopathy. METHODS:Endothelial cell-specific Capns1-knockout (KO) mice were generated. Conditions mimicking prediabetes and type 1 and type 2 diabetes were induced in these KO mice and their wild-type littermates. Myocardial function and coronary flow reserve were assessed by echocardiography. Histological analyses were performed to determine capillary density, cardiomyocyte size and fibrosis in the heart. Isolated aortas were assayed for neovascularisation. Cultured cardiac microvascular endothelial cells were stimulated with high palmitate. Angiogenesis and apoptosis were analysed. RESULTS:Endothelial cell-specific deletion of Capns1 disrupted calpain 1 and calpain 2 in endothelial cells, reduced cardiac fibrosis and hypertrophy, and alleviated myocardial dysfunction in mouse models of diabetes without significantly affecting systemic metabolic variables. These protective effects of calpain disruption in endothelial cells were associated with an increase in myocardial capillary density (wild-type vs Capns1-KO 3646.14?±?423.51 vs 4708.7?±?417.93 capillary number/high-power field in prediabetes, 2999.36?±?854.77 vs 4579.22?±?672.56 capillary number/high-power field in type 2 diabetes and 2364.87?±?249.57 vs 3014.63?±?215.46 capillary number/high-power field in type 1 diabetes) and coronary flow reserve. Ex vivo analysis of neovascularisation revealed more endothelial cell sprouts from aortic rings of prediabetic and diabetic Capns1-KO mice compared with their wild-type littermates. In cultured cardiac microvascular endothelial cells, inhibition of calpain improved angiogenesis and prevented apoptosis under metabolic stress. Mechanistically, deletion of Capns1 elevated the protein levels of ?-catenin in endothelial cells of Capns1-KO mice and constitutive activity of calpain 2 suppressed ?-catenin protein expression in cultured endothelial cells. Upregulation of ?-catenin promoted angiogenesis and inhibited apoptosis whereas knockdown of ?-catenin offset the protective effects of calpain inhibition in endothelial cells under metabolic stress. CONCLUSIONS/INTERPRETATION:These results delineate a primary role of calpain in inducing cardiac endothelial cell injury and impairing neovascularisation via suppression of ?-catenin, thereby promoting diabetic cardiomyopathy, and indicate that calpain is a promising therapeutic target to prevent diabetic cardiac complications.

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Endothelial Hey2 deletion reduces endothelial-to-mesenchymal transition and mitigates radiation proctitis in mice.

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