Upregulation of PAI-1 is mediated through TGF-β/Smad pathway in transplant arteriopathy

Abstract

Background Plasminogen activator inhibitor type 1 (PAI-1) is the primary physiologic inhibitor of plasminogen activator in vivo. Increased PAI-1 expression is associated with arteriosclerosis. Transforming growth factor-beta (TGF-β) induces PAI-1 production via Smads. Methods In vivo, TGF-β receptors (TβRs), Smad2, Smad3, and Smad4, PAI-1, and Smad2 phosphorylation were examined by immunohistochemistry in 3 native aortas, 14 rat aortic syngrafts, and 19 allografts collected at 15, 30, and 45 days post-transplantation. In vitro, phosphorylation of Smad2 and induction of PAI-1 mRNA in human aortic smooth muscle cells (SMCs) in response to TGF-β treatment were detected by Western blot and by TaqMan real-time RT-PCR, respectively. Results Immunohistochemical staining revealed that vascular parenchymal cells contained TβRI, TβRII, Smad2, Smad3, and Smad4, known signaling transducers for TGF-β/Smad pathway, in all samples. Intense staining for phospho-Smad2 was observed in 94% of endothelial cells (ECs), 86% of intimal cells, 27% of medial SMCs, and 38% of adventitial cells at all 3 time points in all aortic allografts, but only in 5% of ECs in syngrafts. PAI-1 immunoreactivity was detected in similar number of cells, and from consecutive sections, phospho-Smad2 colocalized with PAI-1, in the aortic allografts. Low basal level PAI-1 expression was observed in aortic syngrafts and native vessels. Smad2 phosphorylation and time-dependent PAI-1 induction were detected in cultured SMCs upon TGF-β treatment. Conclusions Phospho-Smad2 staining in aortic allografts indicates the activation of TGF-β signaling in allo-transplantation; and co-localization of PAI-1 and phospho-Smad2 suggests that PAI-1 upregulation is mediated mainly by TGF-β/Smad pathway in aortic allografts.