title: TGF-beta1-Induced MAPK activation promotes collagen synthesis, nodule formation, redox stress and cellular senescence in porcine aortic valve interstitial cells.
abstract: OBJECTIVE
  Aortic valve stenosis is a major cause of valve replacement, particularly in the elderly. TGF-beta1 is upregulated in stenotic valves and induces calcification and collagen synthesis in cultured valve interstitial cells. It has been shown previously that TGF-beta1 increases reactive oxygen species (ROS) in these cells in association with calcifying nodule formation, but the cellular signaling pathways responsible for these TGF-beta1-induced effects are not well defined.
  
  
  METHODS
  Cultured porcine aortic valve interstitial cells were used to investigate the effects of inhibitors of TGF-beta1 signaling pathways on 3H-proline incorporation into the extracellular matrix, the peak number of calcifying nodules formed, redox stress as dichlorofluorescein diacetate (DCF-DA) fluorescence, and senescence-associated beta-galactosidase staining.
  
  
  RESULTS
  Nodule formation and proline incorporation were inhibited by SB431542, implicating the Smad pathway, by SB203580, implicating the P38 MAPK pathway, and by U0126, implicating the Mekl/2/Erk1/2 pathway in both processes. Fasudil, an inhibitor of the Rho kinase pathway, was selective in inhibiting nodule formation but not proline incorporation. It was verified that Smad2 phosphorylation, Erk1/2 phosphorylation and p38 MAPK phosphorylation were all induced by TGF-beta1, with Smad 2 phosphorylation peaking at 1-2 h and MAPK phosphorylation at 24-48 h. The effect of TGF-beta1 on phosphorylation of Smad 2 was inhibited by SB431542, on the phosphorylation of p38 MAPK was inhibited by SB203580, and on the phosphorylation of Erk1/2 was inhibited by U0126. ROS generation in response to TGF-beta1, measured as 2,7-dichlorofluorescein-diacetate fluorescence, was inhibited significantly by SB203580 and U0126, implicating both the p38 MAPK and Mekl/2/Erk1/2 signaling pathways. Both pathways also mediated TGF-beta1-induced cellular senescence which was localized to cellular aggregates and mature nodules.
  
  
  CONCLUSIONS
  These data imply that the inhibition of either Smad or MAPK signaling pathways may have a therapeutic benefit in ameliorating the adverse pathological changes associated with aortic valve stenosis.
authors.affiliation: Cardiology Department, The Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, Queen Elizabeth Hospital, Woodville South, South Australia.
authors.forename: Dividutta
authors.initials: D
authors.lastname: Das
authors.affiliation: Cardiology Department, The Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, Queen Elizabeth Hospital, Woodville South, South Australia.
authors.forename: Andrew
authors.initials: A
authors.lastname: Holmes
authors.affiliation: Cardiology Department, The Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, Queen Elizabeth Hospital, Woodville South, South Australia.
authors.forename: Geraldine A
authors.initials: GA
authors.lastname: Murphy
authors.affiliation: Cardiology Department, The Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, Queen Elizabeth Hospital, Woodville South, South Australia.
authors.forename: Kumaril
authors.initials: K
authors.lastname: Mishra
authors.affiliation: Cardiology Department, The Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, Queen Elizabeth Hospital, Woodville South, South Australia.
authors.forename: Anke C
authors.initials: AC
authors.lastname: Rosenkranz
authors.affiliation: Cardiology Department, The Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, Queen Elizabeth Hospital, Woodville South, South Australia.
authors.forename: John D
authors.initials: JD
authors.lastname: Horowitz
authors.affiliation: Cardiology Department, The Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, Queen Elizabeth Hospital, Woodville South, South Australia.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Kennedy
mesh_terms: D000818:Animals; D001021:Aortic Valve / Q000378:metabolism* / Q000473:pathology*; D001024:Aortic Valve Stenosis / Q000150:complications* / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D001549:Benzamides / Q000494:pharmacology*; D002114:Calcinosis / Q000150:complications* / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D016922:Cellular Senescence; D003094:Collagen / Q000096:biosynthesis*; D004149:Dioxoles / Q000494:pharmacology*; D004195:Disease Models, Animal; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D013552:Swine; D053773:Transforming Growth Factor beta1 / Q000037:antagonists & inhibitors*
chemical_list: C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; D004149:Dioxoles; D053773:Transforming Growth Factor beta1; D003094:Collagen; D020928:Mitogen-Activated Protein Kinases