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The chondroitin sulfate moiety mediates thrombomodulin-enhanced adhesion and migration of vascular smooth muscle cells.

ABSTRACT: Thrombomodulin (TM), a transmembrane glycoprotein highly expressed in endothelial cells (ECs), is a potent anticoagulant maintaining circulation homeostasis. Under inflammatory states, TM expression is drastically reduced in ECs while vascular smooth muscle cells (VSMCs) show a robust expression of TM. The functional role of TM in VSMCs remains elusive.We examined the role of TM in VSMCs activities in human aortic VSMCs stimulated with platelet-derived growth factor-BB (PDGF-BB). Using rat embryonic aorta-derived A7r5 VSMCs which do not express TM, the role of the chondroitin sulfate (CS) moiety of TM in VSMCs was delineated with cells expressing wild-type TM and the CS-devoid TM mutant.Expression of TM enhanced cell migration and adhesion/spreading onto type I collagen, but had no effect on cell proliferation. Knocking down TM with short hairpin RNA reduced PDGF-stimulated adhesion and migration of human aortic VSMCs. In A7r5 cells, TM-mediated cell adhesion was eradicated by pretreatment with chondroitinase ABC which degrades CS moiety. Furthermore, the TM mutant (TMS490, 492A) devoid of CS moiety failed to increase cell adhesion, spreading or migration. Wild-type TM, but not TMS490, 492A, increased focal adhesion kinase (FAK) activation during cell adhesion, and TM-enhanced cell migration was abolished by a function-blocking anti-integrin β1 antibody.Chondroitin sulfate modification is required for TM-mediated activation of β1-integrin and FAK, thereby enhancing adhesion and migration activity of VSMCs.

SUBMITTER: Pai VC

PROVIDER: S-EPMC5809974 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundVascular remodelling is an essential pathophysiological state in many circulatory diseases. Abnormal vascular smooth muscle cell (VSMC) behaviour leads to neointimal formation and may eventually results in major adverse cardiovascular events. The C1q/TNF-related protein (C1QTNF) family is closely associated with cardiovascular disease. Notably, C1QTNF4 has unique two C1q domains. However, the role of C1QTNF4 in vascular diseases remains unclear.MethodsC1QTNF4 expression was detected in human serum and artery tissues using ELISA and multiplex immunofluorescence (mIF) staining. Scratch assay, transwell assay and confocal microscopy were used to investigate C1QTNF4 effects on VSMC migration. EdU incorporation, MTT assay and cell counting experiment revealed C1QTNF4 effects on VSMC proliferation. C1QTNF4-transgenic, C1QTNF4-/- and AAV9-mediated VSMC-specific C1QTNF4 restoration C1QTNF4-/- mouse and rat disease models were generated. RNA-seq, quantitative real-time PCR, western blot, mIF, proliferation and migration assays were used to investigate the phenotypic characteristics and underlying mechanisms.ResultsSerum C1QTNF4 levels were decreased in patients with arterial stenosis. C1QTNF4 shows colocalisation with VSMC in human renal arteries. In vitro, C1QTNF4 inhibits VSMC proliferation and migration and alters VSMC phenotype. In vivo, an adenovirus-infected rat balloon injury model, C1QTNF4-transgenic and C1QTNF4-/- mouse wire-injury models with or without VSMC-specific C1QTNF4 restoration were established to mimic the VSMC repair and remodelling. The results show that C1QTNF4 decreases intimal hyperplasia. Especially, we displayed the rescue effect of C1QTNF4 in vascular remodelling using AAV vectors. Next, transcriptome analysis of artery tissue identified the potential mechanism. In vitro and in vivo experiments confirm that C1QTNF4 ameliorates neointimal formation and maintains vascular morphology by downregulating the FAK/PI3K/AKT pathway.ConclusionsOur study demonstrated that C1QTNF4 is a novel inhibitor of VSMC proliferation and migration that acts by downregulating the FAK/PI3K/AKT pathway, thus protecting blood vessels from abnormal neointima formation. These results provide new insights into promising potent treatments for vascular stenosis diseases.

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The chondroitin sulfate moiety mediates thrombomodulin-enhanced adhesion and migration of vascular smooth muscle cells.

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