The shortage of donors in cardiac transplantation may be alleviated by the use of allografts from donation after circulatory death (DCD) donors. We have previously shown that hearts exposed to 30 min warm ischemic time and then flushed with Celsior supplemented with agents that activate ischemic postconditioning pathways, show complete recovery on a blood-perfused ex vivo working heart apparatus. In this study, these findings were assessed in a porcine orthotopic heart transplant model. DCD hearts were preserved with either normothermic ex vivo perfusion (NEVP) using a clinically approved device, or with standard cold storage (CS) for 4 h. Orthotopic transplantation into recipient animals was subsequently undertaken. Five of six hearts preserved with NEVP demonstrated favorable lactate profiles during NEVP and all five could be weaned off cardiopulmonary bypass posttransplant, compared with 0 of 3 hearts preserved with CS (p < 0.05, Fisher's exact test). In conclusion, DCD hearts flushed with supplemented Celsior solution and preserved with NEVP display viability before and after transplantation. Viability studies of human DCD hearts using NEVP are warranted.
Donation after circulatory death (DCD) offers a potential additional source of cardiac allografts. We used a porcine asphyxia model to evaluate viability of DCD hearts subjected to warm ischemic times (WIT) of 20-40 min prior to flushing with Celsior (C) solution. We then assessed potential benefits of supplementing C with erythropoietin, glyceryl trinitrate and zoniporide (Cs), a combination that we have shown previously to activate ischemic postconditioning pathways. Hearts flushed with C/Cs were assessed for functional, biochemical and metabolic recovery on an ex vivo working heart apparatus. Hearts exposed to 20-min WIT showed full recovery of functional and metabolic profiles compared with control hearts (no WIT). Hearts subjected to 30-or 40-min WIT prior to C solution showed partial and no recovery, respectively. Hearts exposed to 30-min WIT and Cs solution displayed complete recovery, while hearts exposed to 40-min WIT and Cs solution demonstrated partial recovery. We conclude that DCD hearts flushed with C solution demonstrate complete recovery up to 20-min WIT after which there is rapid loss of viability. Cs extends the limit of WIT tolerability to 30 min. DCD hearts with 30-min WIT may be suitable for transplantation and warrant assessment in a transplant model.
TGF- is a key profibrotic factor, but targeting TGF- to prevent fibrosis also abolishes its protective anti-inflammatory effects. Here, we investigated the hypothesis that we can redirect TGF- signaling by preventing downstream profibrotic interaction of -catenin with T cell factor (TCF), thereby enhancing the interaction of-catenin with Foxo, a transcription factor that controls differentiation of TGF- induced regulatory T cells (iTregs), and thus, enhance anti-inflammatory effects of TGF- In iTregs derived from EL4 T cells treated with recombinant human TGF-1 (rhTGF-1) , inhibition of-catenin/TCF transcription with ICG-001 increased Foxp3 expression, interaction of -catenin and Foxo1, binding of Foxo1 to the Foxp3 promoter, and Foxo transcriptional activity. Moreover, the level of-catenin expression positively correlated with the level of Foxo1 binding to the Foxp3 promoter and Foxo transcriptional activity. T cell fate mapping in Foxp3 Ly5.1/5.2 mice revealed that coadministration of rhTGF-1 and ICG-001 further enhanced the expansion of iTregs and natural Tregs observed with rhTGF-1 treatment alone. Coadministration of rhTGF-1 with ICG-001 also increased the number of Tregs and reduced inflammation and fibrosis in the kidney fibrosis models of unilateral ureteric obstruction and ischemia-reperfusion injury. Notably, ICG-001 prevented the fibrosis in distant organs (lung and liver) caused by rhTGF-1. Together, our results show that diversion of -catenin from TCF- to Foxo-mediated transcription inhibits the-catenin/TCF-mediated profibrotic effects of TGF- while enhancing the -catenin/Foxo-mediated anti-inflammatory effects. Targeting-catenin/Foxo may be a novel therapeutic strategy in the treatment of fibrotic diseases that lead to organ failure.
TGF-β1 induces epithelial-mesenchymal transition (EMT) and autophagy in a variety of cells. However, the role of autophagy in TGF-β1-induced EMT has not been clearly elucidated and the underlying mechanisms are unclear. In the present study, we found that TGF-β1 induced both autophagy and EMT in mouse tubular epithelial C1.1 cells. Inhibition of autophagy by 3-methyladenine or siRNA knockdown of Beclin 1 reduced TGF-β1-induced increase of vimentin and decreased E-cadherin expression. In contrast, rapamycin-associated enhancement of TGF-β1-induced autophagy increased EMT of C1.1 cells. Serum rescue inhibited autophagy followed by reversal of EMT. Blocking of autophagosome-lysosomal but not proteosomal degradation reduced the decrease of E-cadherin, demonstrating a role for autophagy in degradation of E-cadherin during EMT. Autophagy promoted the activation of Src and Src-associated phosphorylation of β-catenin at Y-654 leading to pY654-β-catenin/p-Smad2 complex formation. Chromatin immunoprecipitation assay demonstrated binding by the pY654-β-catenin/p-Smad2 complex to ILK promoter thus increasing ILK expression. Taken together, our results demonstrate that TGF-β1-induced autophagy links β-catenin and Smad signaling to promote EMT in C1.1 cells through a novel pY654-β-catenin/p-Smad2/ILK pathway. The pathway delineated links disruption of E-cadherin/β-catenin-mediated cell-cell contact to induction of EMT via upregulation of ILK.
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