AP-1 and STAT-1 decoy oligodeoxynucleotides attenuate transplant vasculopathy in rat cardiac allografts.

AIMS: Cardiac allograft vasculopathy (CAV) continues to be an unsolved clinical problem requiring the development of new therapeutic strategies. We have previously demonstrated that ex vivo donor allograft treatment with decoy oligodeoxynucleotides (ODN) targeting the transcription factors, activator protein-1 (AP-1) or signal transducer and activator of transcription-1 (STAT-1), delays acute rejection and prolongs cardiac allograft survival. Here, we investigated whether this treatment regime also prevents the occurrence of CAV in a fully allogeneic rat heart transplantation model. METHODS AND RESULTS: Wistar-Furth rat cardiac allografts were perfused ex vivo with AP-1 decoy ODN, STAT-1 decoy ODN, or buffer solution and transplanted into the abdomen of Lewis rats immunosuppressed with cyclosporine. Treatment with both decoy ODNs but not vehicle significantly attenuated the incidence and severity of CAV. Laser-assisted microdissection/real-time polymerase chain reaction as well as immunohistochemistry analyses revealed a significant increase in CD40 abundance in the coronary endothelial cells and medial smooth muscle cells on day 1 post transplantation which was virtually abolished upon AP-1 or STAT-1 decoy ODN treatment. While the AP-1 decoy ODN primarily attenuated basal CD40 expression, the STAT-1 decoy ODN suppressed tumour necrosis factor-alpha-/interferon-gamma-stimulated expression of CD40 in rat native endothelial cells. CONCLUSION: Treating donor hearts with decoy ODNs neutralizing AP-1 or STAT-1 at the time of transplantation prevents upregulation of CD40 expression in the graft coronary arteries and effectively inhibits CAV.

STAT-1 and AP-1 decoy oligonucleotide therapy delays acute rejection and prolongs cardiac allograft survival.

OBJECTIVE: Acute myocardial rejection is a cell-mediated process characterized by increased leukocyte recruitment into the graft myocardial tissue. Transcription factors like STAT-1 and AP-1 are critically involved in this process by regulating vascular adhesion molecule expression. The aim of our study was to investigate the effect of decoy oligodeoxynucleotide (dODN) treatment targeting transcription factors AP-1 and STAT-1 on acute cardiac allograft rejection in a rat transplant model. METHODS: Wistar-Furth (WF) cardiac allografts were transplanted into Lewis (LEW) rats after perfusion with STAT-1 or AP-1 dODN solution (5 micromol/l), buffer or the corresponding mutant control ODNs. Grafts were harvested and processed for histologic and immunohistochemical evaluation. RESULTS: As demonstrated by fluorescence dye-labelled dODN, exposure of the grafts to the dODNs during 45 min of warm ischemia resulted in a dominant uptake of naked DNA by the graft endothelium. Treatment with AP-1 and STAT-1 dODNs, but not with vehicle or the control dODNs, significantly prolonged cardiac allograft survival by approximately 40% from 5.6+/-0.5 days to 7.8+/-1.3 days and 7.4+/-0.5 days, respectively (mean+/-S.D., p<0.01, n=5 in each group). Immunohistochemical examination on days 1, 3 and 6 revealed a marked reduction of infiltrating leukocytes (AP-1 dODN: 85%, STAT-1 dODN: 50%), namely T-cells, in the dODN-perfused grafts at day 3 post transplantation. In addition, as demonstrated by immunohistochemical analysis, endothelial expression of ICAM-1 and VCAM-1 was found to be markedly reduced in dODN-treated grafts. CONCLUSION: Both AP-1 and STAT-1 dODN treatments suppress graft endothelial adhesion molecule expression, reduce graft infiltration and in turn significantly delay acute rejection. The utilization of dODNs in the cardioplegic solution might be a novel strategy to protect transplanted organs from early damage during transplantation, to preserve organ function and bridge the critical phase after transplantation when standard immunosuppression is not yet completely effective.

Effect of dopamine on inflammatory status in kidneys of brain-dead rats.

BACKGROUND: Brain death has been identified as an independent risk factor for chronic allograft dysfunction. In two independent retrospective clinical studies, we showed that dopamine treatment of brain-dead donors improves long-term kidney graft survival. The mechanisms underlying the protective effects of dopamine treatment in vivo have not been identified. To elucidate the mechanisms underlying the protective effect of dopamine on kidneys of brain-dead donors, we studied a model for brain death in rats. METHODS: In F344 rats, brain death was induced by epidural inflation of a 3F Fogarty catheter. Apneic animals were mechanically ventilated, and clinically relevant dosages of dopamine (2, 6, 10, or 14 microg/kg/min) were given for 6 hr from the onset of brain death. Ventilated, non-brain-dead animals served as controls. RESULTS: Dopamine significantly reduced renal monocyte infiltration and major histocompatibility class II and P-selectin expression in brain-dead animals. It also prevented further up-regulation of the inflammatory markers tumor necrosis factor-alpha and monocyte chemoattractant peptide-1. Concomitantly, the presence of inducible anti-oxidant heme oxygenase-1, known for its cytoprotective effects, was strongly increased by dopamine. CONCLUSION: We identified several mechanisms underlying the protective effects of dopamine treatment on kidney grafts. The identification of these mechanisms may help to design more effective future strategies for treatment of cadaveric kidney donors.

3-Deazaadenosine prevents leukocyte invasion by suppression of adhesion molecule expression during acute cardiac allograft rejection: involvement of apoptotic cell death.

BACKGROUND: In the initial phase after cardiac transplantation, mononuclear cells infiltrate the graft, initiating a relevant impulse for rejection. 3-Deazaadenosine (c3Ado), an analog of adenosine, has proven anti-inflammatory properties both in vitro and in vivo. We hypothesized that c3Ado can serve as a therapeutic tool to reduce cellular infiltration in cardiac allograft transplantation. METHODS: Using the Wistar-Furth-to-Lewis rat cardiac allograft model, animals were treated with 5 mg c3Ado subcutaneously twice per day. Allografts of untreated animals served as controls. Grafts were harvested on Days 1, 3 and 6 after transplantation for further examination (n = 4 per group and timepoint). RESULTS: Immunohistochemical examination of c3Ado-treated grafts revealed up to 80% reduction of infiltrating major histocompatability complex (MHC) II-positive cells and T-cell-receptor-positive cells (R73) as well as ED1-positive monocytes and macrophages at Days 3 and 6 after transplantation. Adhesion molecule (ICAM-1 and VCAM-1) expression at Days 1 and 3 was almost completely abolished in c3Ado-treated grafts. However, c3Ado treatment did not prevent apoptotic cell death (TUNEL assay, DNA laddering) at Day 6, nor did it prolong allograft survival. As in controls, grafts were rejected at Day 7. CONCLUSION: c3Ado significantly reduces graft infiltration by preventing leukocyte invasion, most likely through suppression of adhesion molecule expression. Although graft survival was not prolonged, treatment with c3Ado may still serve as a strategy to protect hearts from early damage after transplantation. Further studies will show whether peri-operative use of c3Ado can bridge the critical phase after transplantation when standard immunosuppression is not yet completely efficacious.