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CircDLPAG4/HECTD1 mediates ischaemia/reperfusion injury in endothelial cells via ER stress.

ABSTRACT: Background: Vascular endothelial cell dysfunction, characterized by cell apoptosis and migration, plays a crucial role in ischaemia/reperfusion (I/R) injury, a common aspect of cardiovascular diseases. Recent studies have suggested that non-coding RNAs, such as circular RNAs (circRNA), play a role in cell dysfunction in I/R injury, although the detailed mechanism is unclear.Methods: Human umbilical vein endothelial cells (HUVECs) were used for in vitro I/R model. Protein expression was detected by western blotting (WB) and immunocytochemistry. The CRISPR/Cas9 system, WB, cell viability assays, Hoechst staining and a 3D migration model were used to explore functional changes. RNA expression was evaluated using quantitative real-time PCR and a FISH assay combined with lentivirus transfection regulating circRNAs and miRNAs. A mouse myocardial I/R model using C57 mice was established to confirm the in vitro findings.Results: In HUVECs, I/R induced a significant time-dependent decrease in HECTD1 associated with an approximately 45% decrease in cell viability and increases in cell apoptosis and migration, which were attenuated by HECTD1 overexpression. I/R-induced upregulation of endoplasmic reticulum stress was also attenuated HECTD1 overexpression. Moreover, miR-143 mimics inhibited HECTD1 expression, which was restored by circDLGAP4 overexpression, providing insight as to the molecular mechanism of I/R-induced HECTD1 in endothelial cell dysfunction.Conclusion: Our results suggest a critical role for circDLGAP4 and HECTD1 in endothelial cell dysfunction induced by I/R, providing novel insight into potential therapeutic targets for the treatment of myocardial ischaemia.

SUBMITTER: Chen L

PROVIDER: S-EPMC6973311 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background & aimsMitochondrial permeability transition pore (mPTP) opening is critical for mediating cell death during hepatic ischaemia-reperfusion injury (IRI). Blocking mPTP opening by inhibiting cyclophilin D (CypD) is a promising pharmacological approach for the treatment of IRI. Here, we show that diastereoisomers of a new class of small-molecule cyclophilin inhibitors (SMCypIs) have properties that make them attractive candidates for the development of therapeutic agents against liver IRI.MethodsDerivatives of the parent SMCypI were synthesised and evaluated for their ability to inhibit CypD peptidyl-prolyl cis-trans isomerase (PPIase) activity and for their mitoprotective properties, evaluated by measuring mitochondrial swelling and calcium retention capacity in liver mitochondria. The ability of the selected compounds to inhibit mPTP opening was evaluated in cells subjected to hypoxia/reoxygenation using a calcein/cobalt assay. Their ability to inhibit cell death was evaluated in cells subjected to hypoxia/reoxygenation by measuring lactate dehydrogenase (LDH) release, propidium iodide staining, and cell viability. The compound performing best in vitro was selected for in vivo efficacy evaluation in a mouse model of hepatic IRI.ResultsThe two compounds that showed the strongest inhibition of CypD PPIase activity and mPTP opening, C105 and C110, were selected. Their SR diastereoisomers carried the activity of the racemic mixture and exhibited mitoprotective properties superior to those of the known macrocyclic cyclophilin inhibitors cyclosporin A and alisporivir. C105SR was more potent than C110SR in inhibiting mPTP opening and prevented cell death in a model of hypoxia/reoxygenation. Finally, C105SR substantially protected against hepatic IRI in vivo by reducing hepatocyte necrosis and apoptosis.ConclusionsWe identified a novel cyclophilin inhibitor with strong mitoprotective properties both in vitro and in vivo that represents a promising candidate for cellular protection in hepatic IRI.Impact and implicationsHepatic ischaemia-reperfusion injury (IRI) is one of the main causes of morbidity and mortality during or after liver surgery. However, no effective therapies are available to prevent or treat this devastating syndrome. An attractive strategy to prevent hepatic IRI aims at reducing cell death by targeting mitochondrial permeability transition pore opening, a phenomenon regulated by cyclophilin D. Here, we identified a new small-molecule cyclophilin inhibitor, and demonstrated the enhanced mitoprotective and hepatoprotective properties of one of its diastereoisomers both in vitro and in vivo, making it an attractive lead compound for subsequent clinical development.

Project description:Background and aimsAlthough type 2 innate lymphoid cells (ILC2s) were originally found to be liver-resident lymphocytes, the role and importance of ILC2 in liver injury remains poorly understood. In the current study, we sought to determine whether ILC2 is an important regulator of hepatic ischaemia/reperfusion injury (IRI).MethodsILC2-deficient mice (ICOS-T or NSG) and genetically modified ILC2s were used to investigate the role of ILC2s in murine hepatic IRI. Interactions between ILC2s and eosinophils or macrophages were studied in coculture. The role of human ILC2s was assessed in an immunocompromised mouse model of hepatic IRI.ResultsAdministration of IL-33 prevented hepatic IRI in association with reduction of neutrophil infiltration and inflammatory mediators in the liver. IL-33-treated mice had elevated numbers of ILC2s, eosinophils, and regulatory T cells. Eosinophils, but not regulatory T cells, were required for IL-33-mediated hepatoprotection in IRI mice. Depletion of ILC2s substantially abolished the protective effect of IL-33 in hepatic IRI, indicating that ILC2s play critical roles in IL-33-mediated liver protection. Adoptive transfer of ex vivo-expanded ILC2s improved liver function and attenuated histologic damage in mice subjected to IRI. Mechanistic studies combining genetic and adoptive transfer approaches identified a protective role of ILC2s through promoting IL-13-dependent induction of anti-inflammatory macrophages and IL-5-dependent elevation of eosinophils in IRI. Furthermore, in vivo expansion of human ILC2s by IL-33 or transfer of ex vivo-expanded human ILC2s ameliorated hepatic IRI in an immunocompromised mouse model of hepatic IRI.ConclusionsThis study provides insight into the mechanisms of ILC2-mediated liver protection that could serve as therapeutic targets to treat acute liver injury.Impact and implicationsWe report that type 2 innate lymphoid cells (ILC2s) are important regulators in a mouse model of liver ischaemia/reperfusion injury (IRI). Through manipulation of macrophage and eosinophil phenotypes, ILC2s mitigate liver inflammation and injury during liver IRI. We propose that ILC2s have the potential to serve as a therapeutic tool for protecting against acute liver injury and lay the foundation for translation of ILC2 therapy to human liver disease.

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CircDLPAG4/HECTD1 mediates ischaemia/reperfusion injury in endothelial cells via ER stress.

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