The chronic allograft rejection, i.e. transplant vasculopathy (TV), is a major limiting factor in the long-term success of cardiac transplantation. In the present study, we investigated the role of endothelial to mesenchymal transition in the allograft chronic rejection and vasculopathy in a mouse model. In the first stage of experiments, we performed isograft and allograft using Tie1Cre;R26RstoplacZ double-trangenic mice on a B6 background as donor, and WT B6 and Bm12 mice as recipients. In the allograft model, heterotopic heart transplantation with a class II major histocompatibility difference between the donor and the recipient leads to rejection and vasculopathy. Notely vasculopathy in this model results in an accumulation of cell with the presence of lacZ expression (as detected by staining for Β-galactosidase (Β-gal) activity) in perivascular and intraluminal locations accompanied by concentric intimal hyperplastic lesion. In the isograft model, lacZ expression could be detected only in donor arterial endothelial cells without rejection and vasculopathy. To gather further evidence for the acquisition of the mesenchymal phentotype by endothelial cells, we used immune-flurorescence antibody to label the fibroblast marker FSP1 and CD31. We then compared lacZ⁺CD31⁺ and lacZ^–CD31^– cell isolated from TV and non-TV hearts. Western blotting and real-time PCR reveal expression of mRNA and protein corresponding to the mesenchymal cell markers FSP1, Α-SMA and type I collagen Α1 were substantially higher, and expression of the endothelial cell marker VE-cadherin was substantially lower, in the lacZ+CD31- and lacZ+CD31+ cell population.

To further confirm the role of endothelial to mesenchymal transition in the allograft chronic rejection and vasculopathy,in the second stage of experiments, we performed allograft using Smad3^+/+ and Smad3^+/- mice on a B6 background as donor, and Bm12 mice as recipients. In Smad3+/- mice, Smad3 mRNA and protein levels are reduced by approximately 50%.The allograft chronic rejection and vasculopathy were reduced by 50% in Smad3^+/- donor mice compared to Smad3^+/+ donor mice. Collectively these data indicate that Endothelial-to-mesenchymal transition contributes to cardiac allograft rejection and vasculopathy.

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