Upregulation of PAI-1 is mediated through TGF-beta/Smad pathway in transplant arteriopathy.
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-beta) induces PAI-1 production via Smads.


METHODS
In vivo, TGF-beta receptors (TbetaRs), 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-beta treatment were detected by Western blot and by TaqMan real-time RT-PCR, respectively.


RESULTS
Immunohistochemical staining revealed that vascular parenchymal cells contained TbetaRI, TbetaRII, Smad2, Smad3, and Smad4, known signaling transducers for TGF-beta/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-beta treatment.


CONCLUSIONS
Phospho-Smad2 staining in aortic allografts indicates the activation of TGF-beta signaling in allo-transplantation; and co-localization of PAI-1 and phospho-Smad2 suggests that PAI-1 upregulation is mediated mainly by TGF-beta/Smad pathway in aortic allografts.
Dong
Chunming
C
Division of Cardiology, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA. dong0005@mc.duke.edu
Zhu
Shoukang
S
Wang
Tao
T
Yoon
Woohyun
W
Goldschmidt-Clermont
Pascal J
PJ
D000818:Animals; D001011:Aorta / Q000276:immunology / Q000473:pathology; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000276:immunology; D006085:Graft Survival / Q000235:genetics / Q000276:immunology; D016027:Heart Transplantation / Q000009:adverse effects*; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008297:Male; D009206:Myocardium / Q000276:immunology / Q000473:pathology; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics* / Q000276:immunology; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051785:Smad Proteins; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics* / Q000276:immunology; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000276:immunology; D015854:Up-Regulation / Q000235:genetics / Q000276:immunology; D014652:Vascular Diseases / Q000235:genetics* / Q000276:immunology
D004268:DNA-Binding Proteins; D017395:Plasminogen Activator Inhibitor 1; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051785:Smad Proteins; D051899:Smad2 Protein; C497784:Smad2 protein, rat; D051900:Smad3 Protein; C497787:Smad3 protein, rat; D051901:Smad4 Protein; C490811:Smad4 protein, rat; D015534:Trans-Activators; D016212:Transforming Growth Factor beta