Genetic Deficiency of Plasminogen Activator Inhibitor-1 Promotes Cardiac Fibrosis in Aged Mice

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. Methods and 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.