title: Increased Smad2/3 phosphorylation in circulating leukocytes and platelet-leukocyte aggregates in a mouse model of aortic valve stenosis: Evidence of systemic activation of platelet-derived TGF-β1 and correlation with cardiac dysfunction.
abstract: BACKGROUND
  Transforming growth factor-β1 (TGF-β1) has been implicated in the pathogenesis of aortic valve stenosis (AS). There is, however, little direct evidence for a role of active TGF-β1 in AS due to the sensitivity of current assays. We searched for evidence of plasma TGF-β1 activation by assaying Smad2/3 phosphorylation in circulating leukocytes and platelet-leukocyte aggregates (PLAs) in a mouse model of AS (Reversa).
  
  
  METHODS
  Echocardiography was used to measure AS and cardiac function. Intracellular phospho-flow cytometry in combination with optical fluorescence microscopy was used to detect PLAs and p-Smad2/3 levels.
  
  
  RESULTS
  Reversa mice on a western diet developed AS, had significantly increased numbers of PLAs and more intense staining for p-Smad2/3 in both PLAs and single leukocytes (all p<0.05). p-Smad2/3 staining was more intense in PLAs than in single leukocytes in both diet groups (p<0.05) and correlated with plasma total TGF-β1 levels (r=0.38, p=0.05 for PLAs and r=0.37, p=0.06 for single leukocytes) and reductions in ejection fraction (r=-0.42, p=0.03 for PLAs and r=-0.37, p=0.06 for single leukocytes).
  
  
  CONCLUSIONS
  p-Smad2/3 staining is more intense in leukocytes of hypercholesterolemic mice that developed AS, suggesting increased circulating active TGF-β1 levels. Leukocyte p-Smad2/3 may be a valuable surrogate indicator of circulating active TGF-β1.
authors.affiliation: Allen and Frances Adler Laboratory of Blood and Vascular Biology, The Rockefeller University, New York, NY, United States.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Allen and Frances Adler Laboratory of Blood and Vascular Biology, The Rockefeller University, New York, NY, United States.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Burg
authors.affiliation: Allen and Frances Adler Laboratory of Blood and Vascular Biology, The Rockefeller University, New York, NY, United States.
authors.forename: Spandana
authors.initials: S
authors.lastname: Vootukuri
authors.affiliation: Allen and Frances Adler Laboratory of Blood and Vascular Biology, The Rockefeller University, New York, NY, United States. Electronic address: collerb@rockefeller.edu.
authors.forename: Barry S
authors.initials: BS
authors.lastname: Coller
mesh_terms: D000818:Animals; D001021:Aortic Valve / Q000378:metabolism / Q000473:pathology; D001024:Aortic Valve Stenosis / Q000378:metabolism / Q000473:pathology*; D001792:Blood Platelets / Q000378:metabolism / Q000473:pathology*; D004195:Disease Models, Animal; D007962:Leukocytes / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D010766:Phosphorylation; D051899:Smad2 Protein / Q000032:analysis / Q000378:metabolism*; D051900:Smad3 Protein / Q000032:analysis / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000032:analysis / Q000378:metabolism*
chemical_list: D051899:Smad2 Protein; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1
keywords: Aortic valve stenosis; Blood platelets; Heart failure; Leukocytes; Mouse; TGFB1 protein