title: Genetic deficiency of plasminogen activator inhibitor-1 promotes cardiac fibrosis in aged mice: involvement of constitutive transforming growth factor-beta signaling and endothelial-to-mesenchymal transition.
abstract: BACKGROUND
  Elevated levels of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of urokinase plasminogen activator and tissue plasminogen activator, are implicated in the pathogenesis of tissue fibrosis. Paradoxically, lack of PAI-1 in the heart is associated with the development of cardiac fibrosis in aged mice. However, the molecular basis of cardiac fibrosis in aged PAI-1-deficient mice is unknown. Here, we investigated the molecular and cellular bases of myocardial fibrosis.
  
  
  RESULTS
  Histological evaluation of myocardial tissues derived from aged PAI-1-deficient mice revealed myocardial fibrosis resulting from excessive accumulation of collagen. Immunohistochemical characterization revealed that the levels of matrix metalloproteinase-2, matrix metalloproteinase-9, and transforming growth factor-β1/2 and the number of Mac3-positive and fibroblast specific protein-1-positive cells were significantly elevated in aged PAI-1-deficient myocardial tissues compared with controls. Zymographic analysis revealed that matrix metalloproteinase-2 enzymatic activity was elevated in PAI-1-deficient mouse cardiac endothelial cells. Real-time quantitative polymerase chain reaction analyses of RNA from myocardial tissues revealed the upregulation of profibrotic markers in aged PAI-1-deficient mice. The numbers of phosphorylated Smad2-, phosphorylated Smad3-, and phosphorylated ERK1/2 MAPK-, but not pAkt/PKB-, positive cells were significantly increased in PAI-1-deficient myocardial tissues. Western blot and immunocytochemical analysis revealed that PAI-1-deficient mouse cardiac endothelial cells were more susceptible to endothelial-to-mesenchymal transition in response to transforming growth factor-β2.
  
  
  CONCLUSIONS
  These results indicate that spontaneous activation of both Smad and non-Smad transforming growth factor-β signaling may contribute to profibrotic responses in aged PAI-1-deficient mice hearts and establish a possible link between endothelial-to-mesenchymal transition and cardiac fibrosis in PAI-1-deficient mice.
authors.affiliation: Feinberg Cardiovascular Research Institute, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA. a-ghosh2@northwestern.edu
authors.forename: Asish K
authors.initials: AK
authors.lastname: Ghosh
authors.forename: William S
authors.initials: WS
authors.lastname: Bradham
authors.forename: Linda A
authors.initials: LA
authors.lastname: Gleaves
authors.forename: Bart
authors.initials: B
authors.lastname: De Taeye
authors.forename: Sheila B
authors.initials: SB
authors.lastname: Murphy
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Covington
authors.forename: Douglas E
authors.initials: DE
authors.lastname: Vaughan
mesh_terms: D000375:Aging / Q000473:pathology*; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D003094:Collagen / Q000378:metabolism; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000473:pathology*; D005355:Fibrosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006331:Heart Diseases / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D008648:Mesoderm / Q000473:pathology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009206:Myocardium / Q000378:metabolism / Q000473:pathology; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000378:metabolism; D058258:Serpin E2; D015843:Serpins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000378:metabolism*
chemical_list: D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C470944:S100a4 protein, mouse; D058258:Serpin E2; C511869:Serpine2 protein, mouse; D015843:Serpins; D016212:Transforming Growth Factor beta; D003094:Collagen; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9