title: Notch1 Mutation Leads to Valvular Calcification Through Enhanced Myofibroblast Mechanotransduction.
abstract: OBJECTIVE
  Calcific aortic valve disease (CAVD) is a significant cardiovascular disorder, and controversy exists as to whether it is primarily a dystrophic or osteogenic process in vivo. In this study, we sought to clarify the mechanism of CAVD by assessing a genetic mutation, Notch1 heterozygosity, which leads to CAVD with 100% penetrance in humans.
  
  
  RESULTS
  Murine immortalized Notch1(+/-) aortic valve interstitial cells (AVICs) were isolated and expanded in vitro. Molecular signaling of wild-type and Notch1(+/-) AVICs were compared to identify changes in pathways that have been linked to CAVD-transforming growth factor-β1/bone morphogenetic protein, mitogen-activated protein kinase, and phosphoinositide 3-kinase/protein kinase B-and assessed for calcification potential. Additionally, AVIC mechanobiology was studied in a physiologically relevant, dynamic mechanical environment (10% cyclic strain) to investigate differences in responses between the cell types. We found that Notch1(+/-) AVICs resembled a myofibroblast-like phenotype expressing higher amounts of cadherin-11, a known mediator of dystrophic calcification, and decreased Runx2, a known osteogenic marker. We determined that cadherin-11 expression is regulated by Akt activity, and inhibition of Akt phosphorylation significantly reduced cadherin-11 expression. Moreover, in the presence of cyclic strain, Notch1(+/-) AVICs exhibited significantly upregulated phosphorylation of Akt at Ser473 and smooth muscle α-actin expression, indicative of a fully activated myofibroblast. Finally, these Notch1-mediated alterations led to enhanced dystrophic calcific nodule formation.
  
  
  CONCLUSIONS
  This study presents novel insights in our understanding of Notch1-mediated CAVD by demonstrating that the mutation leads to AVICs that are fully activated myofibroblasts, resulting in dystrophic, but not osteogenic, calcification.
authors.affiliation: From the Department of Biomedical Engineering (J.C., L.M.R., M.K.S.-L., W.D.M.) and Divison of Cardiology, Department of Pediatrics (C.B.B., H.S.B.), Vanderbilt University, Nashville, TN; and Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Cincinnati Children's Hospital, OH (S.S.H.).
authors.forename: Joseph
authors.initials: J
authors.lastname: Chen
authors.affiliation: From the Department of Biomedical Engineering (J.C., L.M.R., M.K.S.-L., W.D.M.) and Divison of Cardiology, Department of Pediatrics (C.B.B., H.S.B.), Vanderbilt University, Nashville, TN; and Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Cincinnati Children's Hospital, OH (S.S.H.).
authors.forename: Larisa M
authors.initials: LM
authors.lastname: Ryzhova
authors.affiliation: From the Department of Biomedical Engineering (J.C., L.M.R., M.K.S.-L., W.D.M.) and Divison of Cardiology, Department of Pediatrics (C.B.B., H.S.B.), Vanderbilt University, Nashville, TN; and Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Cincinnati Children's Hospital, OH (S.S.H.).
authors.forename: M K
authors.initials: MK
authors.lastname: Sewell-Loftin
authors.affiliation: From the Department of Biomedical Engineering (J.C., L.M.R., M.K.S.-L., W.D.M.) and Divison of Cardiology, Department of Pediatrics (C.B.B., H.S.B.), Vanderbilt University, Nashville, TN; and Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Cincinnati Children's Hospital, OH (S.S.H.).
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Brown
authors.affiliation: From the Department of Biomedical Engineering (J.C., L.M.R., M.K.S.-L., W.D.M.) and Divison of Cardiology, Department of Pediatrics (C.B.B., H.S.B.), Vanderbilt University, Nashville, TN; and Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Cincinnati Children's Hospital, OH (S.S.H.).
authors.forename: Stacey S
authors.initials: SS
authors.lastname: Huppert
authors.affiliation: From the Department of Biomedical Engineering (J.C., L.M.R., M.K.S.-L., W.D.M.) and Divison of Cardiology, Department of Pediatrics (C.B.B., H.S.B.), Vanderbilt University, Nashville, TN; and Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Cincinnati Children's Hospital, OH (S.S.H.).
authors.forename: H Scott
authors.initials: HS
authors.lastname: Baldwin
authors.affiliation: From the Department of Biomedical Engineering (J.C., L.M.R., M.K.S.-L., W.D.M.) and Divison of Cardiology, Department of Pediatrics (C.B.B., H.S.B.), Vanderbilt University, Nashville, TN; and Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Cincinnati Children's Hospital, OH (S.S.H.). david.merryman@vanderbilt.edu.
authors.forename: W David
authors.initials: WD
authors.lastname: Merryman
mesh_terms: D000818:Animals; D001021:Aortic Valve / Q000378:metabolism / Q000473:pathology*; D001024:Aortic Valve Stenosis / Q000235:genetics* / Q000378:metabolism*; D015820:Cadherins / Q000378:metabolism; D002114:Calcinosis / Q000235:genetics* / Q000378:metabolism*; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D006801:Humans; D048370:MAP Kinase Kinase 2 / Q000378:metabolism; D040542:Mechanotransduction, Cellular / Q000235:genetics*; D051379:Mice; D009154:Mutation; D058628:Myofibroblasts / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051881:Receptor, Notch1 / Q000235:genetics*
chemical_list: D015820:Cadherins; D050659:Core Binding Factor Alpha 1 Subunit; D051881:Receptor, Notch1; C493716:Runx2 protein, mouse; C086960:osteoblast cadherin; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D048370:MAP Kinase Kinase 2
keywords: Notch1; aortic valve; cadherin-11; calcification