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Thrombomodulin is essential for maintaining quiescence in vascular endothelial cells.

ABSTRACT: Thrombomodulin (TM) is a thrombin receptor on endothelial cells that is involved in promoting activation of the anticoagulant protein C pathway during blood coagulation. TM also exerts protective anti-inflammatory properties through a poorly understood mechanism. In this study, we investigated the importance of TM signaling to cellular functions by deleting it from endothelial cells by CRISPR-Cas9 technology and analyzed the resultant phenotype of TM-deficient (TM ^-/- ) cells. Deficiency of TM in endothelial cells resulted in increased basal permeability and hyperpermeability when stimulated by thrombin and TNF-α. The loss of the basal barrier permeability function was accompanied by increased tyrosine phosphorylation of VE-cadherin and reduced polymerization of F-actin filaments at cellular junctions. A significant increase in basal NF-κB signaling and expression of inflammatory cell adhesion molecules was observed in TM ^-/- cells that resulted in enhanced adhesion of leukocytes to TM ^-/- cells in flow chamber experiments. There was also a marked increase in expression, storage, and release of the von Willebrand factor (VWF) and decreased storage and release of angiopoietin-2 in TM ^-/- cells. In a flow chamber assay, isolated platelets adhered to TM ^-/- cells, forming characteristic VWF-platelet strings. Increased VWF levels and inflammatory foci were also observed in the lungs of tamoxifen-treated ERcre-TM^f/f mice. Reexpression of the TM construct in TM ^-/- cells, but not treatment with soluble TM, normalized the cellular phenotype. Based on these results, we postulate cell-bound TM endows a quiescent cellular phenotype by tightly regulating expression of procoagulant, proinflammatory, and angiogenic molecules in vascular endothelial cells.

SUBMITTER: Giri H

PROVIDER: S-EPMC7980409 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundEndothelial progenitor cells (EPCs) contribute to reendothelialization and neovascularization and protect against vascular injury and ischemia of various organs. We have previously shown downregulation of microRNA (miR)-126 in EPCs from diabetic patients, which contributes to dysfunction of EPCs including impaired migratory ability. The aims of the present study were to examine (1) in vitro the effects of miR-126 on the homing and stemness of late outgrowth EPCs (LOCs), along with relevant signaling pathways, and (2) in vivo the effects of modulating LOCs by manipulating miR-126 expression on LOC homing and reendothelialization of injured arteries in GK rats (a non-obese diabetes model).MethodsRat bone marrow-derived LOCs were transfected with miR-126 inhibitor or lentiviral vectors expressing miR-126. LOC migration was determined by transwell migration assay. CXCR4 expression was measured by real-time PCR, Western blotting, and confocal microscopy while related signaling pathway proteins were measured by Western Blotting. Stemness gene expression, and gene and protein expression and promoter activity of KLF-8 were also measured. LOCs transfected with lenti-miR-126 or miR-126 inhibitor were injected into GK rats with carotid artery injury, and then vascular reendothelialization and the extent of intimal hyperplasia were examined.ResultsLenti-miR-126 increased while miR-126 inhibitor decreased LOC migration and CXCR4 expression on LOCs. miR-126 positively regulated p-ERK, VEGF, p-Akt, and eNOS protein expression, and inhibitors of these proteins blocked miR-126-induced CXCR4 expression and also reduced LOC migration. Overexpression of miR-126 promoted while inhibition of miR-126 suppressed stemness gene expression in LOCs. miR-126 also inhibited gene and protein expression and promoter activity of KLF-8 while shRNA-mediated knockdown of KLF-8 increased stemness gene expression. Upregulation of stemness gene expression by miR-126 overexpression was completely abrogated by co-transfection of lenti-KLF-8 and lenti-miR-126 into LOCs. In GK rats, transplantation of LOCs overexpressing miR-126 enhanced LOC homing and reendothelialization and decreased intimal hyperplasia of injured arteries.ConclusionOur results indicate that miR-126 protects against vascular injury by promoting CXCR4 expression and LOC homing via ERK/VEGF and Akt/eNOS signaling pathways and maintaining stemness via targeting KLF-8.

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Thrombomodulin is essential for maintaining quiescence in vascular endothelial cells.

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