hes1-protein--human and Vascular-Calcification

Vascular calcification represents a huge clinical problem contributing to adverse cardiovascular events, with no effective treatment currently available. Upregulation of hepatocyte growth factor has been linked with vascular calcification, and thus, represent a potential target in the development of a novel therapeutic strategy. Glycomimetics have been shown to interrupt HGF-receptor signalling, therefore this study investigated the effect of novel glycomimetics on osteogenic signalling and vascular calcification in vitro.. Primary human vascular smooth muscle cells (HVSMCs) were induced by β-glycerophosphate (β-GP) and treated with 4 glycomimetic compounds (C1-C4). The effect of β-GP and C1-C4 on alkaline phosphatase (ALP), osteogenic markers and c-Met/Notch3/HES1 signalling was determined using colorimetric assays, qRT-PCR and western blotting respectively.. C1-C4 significantly attenuated β-GP-induced calcification, as shown by Alizarin Red S staining and calcium content by day 14. In addition, C1-C4 reduced ALP activity and prevented upregulation of the osteogenic markers, BMP-2, Runx2, Msx2 and OPN. Furthermore, β-GP increased c-Met phosphorylation at day 21, an effect ameliorated by C2 and C4 and the c-Met inhibitor, crizotinib. We next interrogated the effects of the Notch inhibitor DAPT and confirmed an inhibition of β-GP up-regulated Notch3 protein by C2, DAPT and crizotinib compared to controls. Hes-1 protein upregulation by β-GP, was also significantly downregulated by C2 and DAPT. GOLD docking analysis identified a potential binding interaction of C1-C4 to HGF which will be investigated further.. These findings demonstrate that glycomimetics have potent anti-calcification properties acting via HGF/c-Met and Notch signalling.

Topics: Biomimetic Materials; Bone Morphogenetic Protein 2; Cell Line; Core Binding Factor Alpha 1 Subunit; Glycerophosphates; Homeodomain Proteins; Humans; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Proto-Oncogene Proteins c-met; Receptor, Notch3; Recombinant Proteins; Signal Transduction; Transcription Factor HES-1; Transforming Growth Factor beta; Vascular Calcification

Vascular calcification is a major clinical problem and elucidating the underlying mechanism is important to improve the prognosis of patients with cardiovascular disease. We aimed to elucidate the role and mechanism of action of Hepatocyte Growth Factor (HGF)/c-Met signalling in vascular calcification and establish whether blocking this pathway could prevent mineralisation of vascular smooth muscle cells (VSMCs) in vitro.. We demonstrate increased HGF secretion and c-Met up-regulation and phosphorylation during VSMC osteogenic differentiation. Adenoviral-mediated over-expression of HGF (AdHGF) in VSMCs accelerated mineralisation, shown by alizarin red staining, and significantly increased (45)Calcium incorporation (1.96 ± 0.54-fold [P < 0.05]) and alkaline phosphatase (ALP) activity (3.01 ± 0.8-fold [P < 0.05]) compared to controls. AdHGF also significantly elevated mRNA expression of bone-related proteins, Runx2, osteocalcin, BMP2 and osterix in VSMCs. AdHGF-accelerated mineralisation correlated with increased Akt phosphorylation, nuclear translocation of Notch3 intracellular domain (N3IC) and up-regulation of the Notch3 target protein, HES1. In contrast, adenoviral-mediated over-expression of the HGF antagonist, NK4, markedly attenuated VSMC mineralisation, and reduced c-Met phosphorylation, Akt activation and HES1 protein expression compared to AdHGF-treated cells. Furthermore, the Notch inhibitor, DAPT, attenuated N3IC nuclear translocation and AdHGF-induced mineralisation.. We demonstrate HGF induces VSMC osteogenic differentiation via c-Met/Akt/Notch3 signalling, highlighting these pathways as potential targets for intervention of vascular calcification.

Topics: Adenoviridae; Alkaline Phosphatase; Basic Helix-Loop-Helix Transcription Factors; Bone Morphogenetic Protein 2; Calcium; Cell Differentiation; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Genetic Vectors; Hepatocyte Growth Factor; Homeodomain Proteins; Humans; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Osteogenesis; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Receptor, Notch3; Receptors, Notch; RNA, Messenger; Signal Transduction; Sp7 Transcription Factor; Time Factors; Transcription Factor HES-1; Transcription Factors; Transfection; Up-Regulation; Vascular Calcification