title: miRNA-141 is a novel regulator of BMP-2-mediated calcification in aortic stenosis.
abstract: OBJECTIVE
  Bone morphogenetic protein-2 (BMP-2) is a major regulator of aortic valve calcification. MicroRNAs (miRNAs) are essential post-transcriptional modulators of gene expression and miRNA-141 is a known repressor of BMP-2-mediated osteogenesis. We hypothesized that miRNA-141 is a key regulator of aortic valve calcification.
  
  
  METHODS
  Porcine valvular interstitial cells were isolated, transfected with miRNA-141 or control, and stimulated with transforming growth factor-β. The BMP-2, extracellular signal-regulated kinase 1/2, and runt-related transcription factor 2 levels were determined by immunoblotting and reverse transcriptase polymerase chain reaction. To determine the role of miRNA-141 in bicuspid aortic valve disease, human bicuspid (n = 19) and tricuspid (n = 17) aortic valve leaflets obtained intraoperatively were submitted for GenoExplorer human microRNA array, immunoblotting, and histologic and immunohistochemical analyses.
  
  
  RESULTS
  Stimulation of porcine aortic valvular interstitial cells with transforming growth factor-β induced morphologic alterations consistent with myofibroblastic transformation, BMP-2 signaling, and calcification. Transfection with miRNA-141 restored transforming growth factor-β-induced valvular interstitial cell activation, BMP-2 signaling, and alkaline phosphatase activity (3.55 ± 0.18 vs 4.01 ± 0.21, P < .05), suggesting upstream regulation by miRNA-141. miRNA microarray demonstrated differential expression of 35 of 1583 miRNA sequences in the bicuspid versus tricuspid aortic valve leaflets, with a 14.5-fold decrease in miRNA-141 in the bicuspid versus tricuspid leaflets (P < .05). This was associated with significantly increased BMP-2 protein expression in bicuspid aortic valve compared with the tricuspid aortic valve leaflets (P < .001).
  
  
  CONCLUSIONS
  We report a completely novel role of miRNA-141 as a regulator of BMP-2-dependent aortic valvular calcification and demonstrate marked attenuation of miRNA-141 expression in patients with bicuspid aortic valve-associated aortic stenosis. Therapeutic targeting of miRNA-141 could serve as a novel strategy to limit progressive calcification in aortic stenosis.
authors.affiliation: Division of Cardiac Surgery, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Bobby
authors.initials: B
authors.lastname: Yanagawa
authors.forename: Fina
authors.initials: F
authors.lastname: Lovren
authors.forename: Yi
authors.initials: Y
authors.lastname: Pan
authors.forename: Vinay
authors.initials: V
authors.lastname: Garg
authors.forename: Adrian
authors.initials: A
authors.lastname: Quan
authors.forename: Gilbert
authors.initials: G
authors.lastname: Tang
authors.forename: Krishna K
authors.initials: KK
authors.lastname: Singh
authors.forename: Praphulla C
authors.initials: PC
authors.lastname: Shukla
authors.forename: Nikhil P
authors.initials: NP
authors.lastname: Kalra
authors.forename: Mark D
authors.initials: MD
authors.lastname: Peterson
authors.forename: Subodh
authors.initials: S
authors.lastname: Verma
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001021:Aortic Valve / Q000378:metabolism*; D001024:Aortic Valve Stenosis / Q000378:metabolism*; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism; D002114:Calcinosis / Q000378:metabolism*; D016009:Chi-Square Distribution; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D035683:MicroRNAs / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D013552:Swine; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology
chemical_list: D055396:Bone Morphogenetic Protein 2; D050659:Core Binding Factor Alpha 1 Subunit; C533895:MIRN141 microRNA, human; D035683:MicroRNAs; D016212:Transforming Growth Factor beta; D000469:Alkaline Phosphatase