title: Bone morphogenetic protein-4 and transforming growth factor-beta1 mechanisms in acute valvular response to supra-physiologic hemodynamic stresses.
abstract: OBJECTIVE
  To explore ex vivo the role of bone morphogenetic protein-4 (BMP-4) and transforming growth factor-beta1 (TGF-β1) in acute valvular response to fluid shear stress (FSS) abnormalities.
  
  
  METHODS
  Porcine valve leaflets were subjected ex vivo to physiologic FSS, supra-physiologic FSS magnitude at normal frequency and supra-physiologic FSS frequency at normal magnitude for 48 h in a double-sided cone-and-plate bioreactor filled with standard culture medium. The role of BMP-4 and TGF-β1 in the valvular response was investigated by promoting or inhibiting the downstream action of those cytokines via culture medium supplementation with BMP-4 or the BMP antagonist noggin, and TGF-β1 or the TGF-β1 inhibitor SB-431542, respectively. Fresh porcine leaflets were used as controls. Each experimental group consisted of six leaflet samples. Immunostaining and immunoblotting were performed to assess endothelial activation in terms of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expressions, paracrine signaling in terms of BMP-4 and TGF-β1 expressions and extracellular matrix (ECM) remodeling in terms of cathepsin L, cathepsin S, metalloproteinases (MMP)-2 and MMP-9 expressions. Immunostained images were quantified by normalizing the intensities of positively stained regions by the number of cells in each image while immunoblots were quantified by densitometry.
  
  
  RESULTS
  Regardless of the culture medium, physiologic FSS maintained valvular homeostasis. Tissue exposure to supra-physiologic FSS magnitude in standard medium stimulated paracrine signaling (TGF-β1: 467% ± 22% vs 100% ± 6% in fresh controls, BMP-4: 258% ± 22% vs 100% ± 4% in fresh controls; P < 0.05) and ECM degradation (MMP-2: 941% ± 90% vs 100% ± 19% in fresh controls, MMP-9: 1219% ± 190% vs 100% ± 16% in fresh controls, cathepsin L: 1187% ± 175% vs 100% ± 12% in fresh controls, cathepsin S: 603% ± 88% vs 100% ± 13% in fresh controls; P < 0.05), while BMP-4 supplementation also promoted fibrosa activation and TGF-β1 inhibition reduced MMP-9 expression to the native tissue level (MMP-9: 308% ± 153% with TGF-β1 inhibition vs 100% ± 16% in fresh control; P > 0.05). Supra-physiologic FSS frequency had no effect on endothelial activation and paracrine signaling regardless of the culture medium but TGF-β1 silencing attenuated FSS-induced ECM degradation via MMP-9 downregulation (MMP-9: 302% ± 182% vs 100% ± 42% in fresh controls; P > 0.05).
  
  
  CONCLUSIONS
  Valvular tissue is sensitive to FSS abnormalities. The TGF-β1 inhibitor SB-431542 is a potential candidate molecule for attenuating the effects of FSS abnormalities on valvular remodeling.
authors.affiliation: Ling Sun, Philippe Sucosky, Department of Aerospace and Mechanical Engineering, University of Notre Dame, Notre Dame, IN 46556, United States.
authors.forename: Ling
authors.initials: L
authors.lastname: Sun
authors.affiliation: Ling Sun, Philippe Sucosky, Department of Aerospace and Mechanical Engineering, University of Notre Dame, Notre Dame, IN 46556, United States.
authors.forename: Philippe
authors.initials: P
authors.lastname: Sucosky
keywords: Aortic valve; Bone morphogenetic protein; Calcification; Fluid shear stress; Transforming growth factor beta