title: HGF/c-Met signalling promotes Notch3 activation and human vascular smooth muscle cell osteogenic differentiation in vitro.
abstract: OBJECTIVE
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  We demonstrate HGF induces VSMC osteogenic differentiation via c-Met/Akt/Notch3 signalling, highlighting these pathways as potential targets for intervention of vascular calcification.
authors.affiliation: Cardiovascular Research Group, University of Manchester, Manchester, UK.
authors.forename: Yiwen
authors.initials: Y
authors.lastname: Liu
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.forename: Jianyun
authors.initials: J
authors.lastname: Yan
authors.forename: Naomi
authors.initials: N
authors.lastname: Jiagbogu
authors.forename: Daniëlle A M
authors.initials: DA
authors.lastname: Heideman
authors.forename: Ann E
authors.initials: AE
authors.lastname: Canfield
authors.forename: M Yvonne
authors.initials: MY
authors.lastname: Alexander
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000469:Alkaline Phosphatase / Q000378:metabolism; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics; D005822:Genetic Vectors; D017228:Hepatocyte Growth Factor / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000378:metabolism* / Q000473:pathology; D010012:Osteogenesis; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D000071656:Receptor, Notch3; D051880:Receptors, Notch / Q000378:metabolism*; D015398:Signal Transduction; D000076182:Sp7 Transcription Factor; D013997:Time Factors; D000072056:Transcription Factor HES-1; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D015854:Up-Regulation; D061205:Vascular Calcification / Q000235:genetics / Q000378:metabolism* / Q000473:pathology
chemical_list: D000469:Alkaline Phosphatase; D051792:Basic Helix-Loop-Helix Proteins; D055396:Bone Morphogenetic Protein 2; D002118:Calcium; D050659:Core Binding Factor Alpha 1 Subunit; D017228:Hepatocyte Growth Factor; D018398:Homeodomain Proteins; D051057:Proto-Oncogene Proteins c-akt; D019859:Proto-Oncogene Proteins c-met; D012333:RNA, Messenger; D000071656:Receptor, Notch3; D051880:Receptors, Notch; D000076182:Sp7 Transcription Factor; D000072056:Transcription Factor HES-1; D014157:Transcription Factors; C525718:BMP2 protein, human; C066855:HGF protein, human; C499374:NOTCH3 protein, human; C493709:RUNX2 protein, human; C478163:SP7 protein, human; C078493:HES1 protein, human