Rat Cardiac Allograft Rejection Research Articles

Fingolimod (FTY720) prevents chronic rejection of rodent cardiac allografts through inhibition of the RhoA pathway

The Fingolimod (FTY720, Gilenya) is clinically approved for the treatment of multiple sclerosis (MS). Its therapeutic effect on MS is based on the ability to bind sphingosine 1-phosphate (S1P) receptors and block the exit of immune cells from the lymphoid organs, thus preventing immune cell-dependent injury to the central nervous system (CNS). We showed recently that, besides the S1P-related activity, the FTY720 also down-regulates RhoA, which is a master regulator of the actin cytoskeleton. Our previous studies showed that FTY720 also down-regulates Rictor, which is a signature molecule of mTORC2 complex, which regulates RhoA and dictates actin cytoskeleton specificity. Because, our previous studies showed that chronic rejection correlates with the upregulation of RhoA and mTORC2 components and that the inhibition of RhoA pathway prevents chronic rejection, here we studied the effect of FTY720 on the chronic rejection of rat and mouse cardiac allografts. We show that FTY720 in conjunction with the inhibitors of early T cell response, (CTA4-Ig in mice and Everolimus in rats) blocks macrophage infiltration into the grafts and prevents chronic rejection of rat and mouse cardiac transplants. This indicates that FTY720 may be repurposed from the MS application to the clinical transplantation as an anti-chronic rejection drug.

Precise treatment of acute antibody-mediated cardiac allograft rejection in rats using C4d-targeted microbubbles loaded with nitric oxide

Antibody-mediated rejection (AMR) constitutes an important cause of cardiac allograft loss; however, all current therapeutic strategies represent systemic applications with unsatisfactory efficacy. Previously, we successfully non-invasively detected C4d, a specific marker for AMR diagnosis, in allografts using C4d-targeted microbubbles (MBC4d). In this study, we extended this approach by incorporating nitric oxide (NO), as high NO levels manifest immunosuppressive and anti-thrombotic effects. We designed novel MBC4d loaded with NO (NO-MBC4d). A rat model of AMR was established by pre-sensitization with skin transplantation. Contrast-enhanced ultrasound (CEUS) images were obtained and quantitatively analyzed following NO-MBC4d injection. Allograft survival and histologic features were analyzed to evaluate the therapeutic effect and underlying mechanism of NO-MBC4d toward AMR. We successfully obtained CEUS images following NO-MBC4d injection and demonstrated that the ultrasound signal intensity of the myocardial area and clearance time of NO-MBC4d both increased with increased C4d grade, thereby realizing non-invasive diagnosis of AMR. Furthermore, allograft survival was significantly prolonged, and rejection was obviously attenuated following NO-MBC4d injection through significant suppression of thrombosis and reduction of inflammatory cell infiltrates. Overall, the therapeutic efficacy was significantly improved in the NO-MBC4d group compared with the control NO-MB group, demonstrating that precise treatment could significantly improve the therapeutic efficacy compared with that afforded by systemic applications. This study presented a novel tool to provide simultaneous non-invasive diagnosis and precise treatment of AMR using NO-MBC4d CEUS imaging, which may be expected to provide a better option for recipients with AMR in clinic.

Coinhibition of mTORC1/mTORC2 and RhoA /ROCK pathways prevents chronic rejection of rat cardiac allografts

Chronic rejection of transplanted organs remains an unresolved issue in clinical organ transplantation. The macrophages play a crucial role in the development of chronic rejection. We showed previously that macrophage-specific deletion of RhoA or RhoA/ROCK inhibition prevent macrophage movement to the cardiac allografts and abrogate chronic rejection in rodent transplantation models. Here we assessed the ability of the mTORC1 and mTORC2 inhibitor everolimus, alone or in conjunction with the RhoA/ROCK inhibitor Y27632, to inhibit chronic rejection of Wistar Furth (WF; RT1.Au) rat cardiac allografts heterotopically transplanted into ACI (RT1.Aa) recipients. The transplanted hearts were analyzed for vessel occlusion and tissue fibrosis. T cell and macrophage subsets infiltration was analyzed by immunostaining with T cell and macrophage molecular markers. We found that a combination of the mTOR inhibitor everolimus and the RhoA/ROCK inhibitor Y27632 prolonged allograft survival; decreased vessel occlusion, collagen deposition, and macrophage infiltration; and inhibited the chronic rejection of rat cardiac allografts. These results indicate that coinhibition of the mTORC1/mTORC2 and RhoA pathways in transplant recipients abrogates chronic rejection of rat cardiac allografts, and will aid in the development of clinically applicable anti-chronic rejection therapies.

AS2553627, a novel JAK inhibitor, prevents chronic rejection in rat cardiac allografts

Janus family kinases (JAKs) are essential molecules for cytokine responses and attractive targets for the treatment of transplant rejection and autoimmune diseases. Several JAK inhibitors have shown demonstrable effects on acute rejection in experimental cardiac transplant models. However, little is known about the potential benefits of JAK inhibitors on chronic rejection outcomes such as vasculopathy and fibrosis. Here, we examined the pharmacological profile of a novel JAK inhibitor, AS2553627, and explored its therapeutic potential in chronic rejection as well as acute rejection in a rat cardiac transplant model. AS2553627 potently inhibited JAK kinases but showed no inhibition of other kinases, including TCR-associated molecules. The compound also suppressed proliferation of IL-2 stimulated human and rat T cells. In a rat cardiac transplant model, oral administration of AS2553627 alone or co-administration with a sub-therapeutic dose of tacrolimus effectively prolonged cardiac allograft survival, suggesting the efficacy in treating acute rejection. To evaluate the effect on chronic rejection, recipient rats were administered a therapeutic dose of tacrolimus for 90 days. In combination with tacrolimus, AS2553627 significantly reduced cardiac allograft vasculopathy and fibrosis that tacrolimus alone did not inhibit. AS2553627 at the effective dose in rat transplantation models did not significantly reduce reticulocyte counts in peripheral whole blood after in vivo erythropoietin administration, indicating a low risk for anemia. These results suggest that AS2553627 may be a therapeutic candidate for the prevention of not only acute but also chronic rejection in cardiac transplantation.

Simvastatin Treatment Upregulates Anti-Fibrotic Bone Morphogenetic Protein-7 Expression at Rat Cardiac Allograft Rejection

Background: Bone morphogenetic protein (BMP)-7 mediates ischemic tolerance and anti-fibrotic effects in various organs such as kidney and heart. Recently, reno- and podocyte-protective effects of a potent HMG-CoA reductase inhibitor, pitavastatin, were accompanied by BMP-7 upregulation. Methods: Here, we investigated the effect of simvastatin treatment on BMP-7 expression in major MHC-mismatched rat cardiac allografts subjected to ischemia-reperfusion injury and adaptive immune activation at 10 days. Results: We localized Smad2 activity and Reca-1⁺ fibroblast specific protein-1⁺ immunoreactivity, suggesting endothelial-to-mesenchymal transition, at fibrotic borderline of cardiac allografts at 10 days. Simvastatin donor and recipient combination treatment significantly upregulated cardiac allograft BMP-7 expression when compared to nontreated controls at 10 days. Conclusion: The beneficial effect of statin treatment on cardiac allograft may in part be mediated through the upregulation of BMP-7.

Rat Cytomegalovirus Vaccine Prevents Accelerated Chronic Rejection in CMV-Naïve Recipients of Infected Donor Allograft Hearts

Cytomegalovirus accelerates transplant vascular sclerosis (TVS) and chronic rejection (CR) in solid organ transplants; however, the mechanisms involved are unclear. We determined the efficacy of a CMV vaccine in preventing CMV‐accelerated rat cardiac allograft rejection in naïve recipients of CMV+ donor hearts. F344 donor rats were infected with RCMV 5 days prior to heterotopic cardiac transplantation into CMV‐naïve or H2O2‐inactivated RCMV‐vaccinated Lewis recipients. Recipients of RCMV‐infected donor hearts rejected at POD59, whereas vaccinated recipients exhibited a significantly prolonged time to rejection‐POD97, similar to recipients of uninfected donor hearts (POD108). Although all of the donor hearts were preinfected, the vaccinated recipients had lower graft and PBMC viral loads at POD 7 compared to unvaccinated controls. Adoptive T cell and passive antibody transfers from vaccinated Lewis rats into naïve recipients demonstrate that both T‐cell and B‐cell arms of the adaptive immune response provide protection against CMV‐accelerated rejection. Similar findings were obtained when testing three different adjuvants in passive transfer experiments. We have determined that the timing of the vaccine prior to transplantation and the specific adjuvant play critical roles in mediating anti‐viral responses and promoting graft survival. CMV vaccination prior to transplantation may effectively increase graft survival.

Expression of HSPA12B in acute cardiac allograft rejection in rats

HSP70 may play a more important role in regulating antigen-specific immune response than other HSPs; however, HSPA12B production in transplanted heart remains obscure, which was identified as the newest member of the HSP70 family. In the current study, we performed a heart transplantation model in adult rats and investigated the dynamic changes of HSPA12B expression in the cardiac grafts. The cardiac grafts of allogeneic (Wistar–Lewis rat) and syngeneic (Lewis–Lewis rat) rat models were subjected to histopathological and immunohistochemical analyses for HSPA12B expression on days 0–7 after operation. We also examined the expression profiles of active caspase-3, whose changes were correlated with the expression profiles of HSPA12B. Our results demonstrated that HSPA12B protein exhibited biphasic patterns in transplanted heart. The first expression phase correlated with ischemical reperfusion injury over 2 days post-transplant. The second peak of HSPA12B expression was found only in allografts on day 5, concurrent with the expression of caspase-3. Immunohistochemical assay showed that compared with rare expression in isografts, there were significant protein expressions of HSPA12B and caspase-3 in heart allografts from day 5 to 7 post-transplant. Furthermore, double immunofluorescence staining for active caspase-3 and HSPA12B in isografts and allografts at day 5 post-transplant were analyzed and colocalization of HSPA12B/active caspase-3 was detected in allografts. In conclusion, this is the first description of HSPA12B expression in acute cardiac allograft rejection. Our results suggested that HSPA12B might play crucial roles in heart pathophysiology after transplantation.

Angiopoietin-2 Inhibition Prevents Transplant Ischemia-Reperfusion Injury and Chronic Rejection in Rat Cardiac Allografts

Transplant ischemia-reperfusion injury (Tx-IRI) and allograft dysfunction remain as two of the major clinical challenges after heart transplantation. We investigated the role of angiopoietin-2 (Ang2) in Tx-IRI and rejection using fully MHC-mismatched rat cardiac allografts. We report that plasma levels of Ang2 were significantly enhanced in the human and rat recipients of cardiac allografts, but not in the rat recipients of syngrafts, during IRI. Ex vivo intracoronary treatment of rat cardiac allografts with anti-Ang2 antibody before 4-h cold preservation prevented microvascular dysfunction, endothelial cell (EC) adhesion molecule expression and leukocyte infiltration, myocardial injury and the development of cardiac fibrosis and allograft vasculopathy. Recipient preoperative and postoperative treatment with anti-Ang2 antibody produced otherwise similar effects without effecting microvascular dysfunction, and in additional experiments prolonged allograft survival. Recipient preoperative treatment alone failed to produce these effects. Moreover, ex vivo intracoronary treatment of allografts with recombinant Ang2 enhanced Tx-IRI and, in an add-back experiment, abolished the beneficial effect of the antibody. We demonstrate that neutralization of Ang2 prevents EC activation, leukocyte infiltration, Tx-IRI and the development of chronic rejection in rat cardiac allografts. Our results suggest that blocking Ang2 pathway is a novel, clinically feasible, T cell-independent strategy to protect cardiac allografts.

Ultrasound Molecular Imaging of Acute Cellular Cardiac Allograft Rejection in Rat With T-Cell–Specific Nanobubbles

Acute rejection (AR) is one of the main obstacles of cardiac transplantation; however, a noninvasive diagnostic method, which reflects its pathologic nature, has not been developed yet. In this study, we prepared a specific nanobubbles targeting to the activated T cells and applied it in the ultrasound molecular imaging of AR in heart transplantation by myocardial contrast echocardiography (MCE). Nanobubbles loading anti-CD25 antibody (NB(specific)) or isotype control antibody (NB(nonspecific)) were prepared and then applied in the ultrasound molecular imaging by MCE in a rat model. MCE was performed in 24 allografts and 18 isografts that were divided into three groups, including days 2, 4, and 6 after transplantation. Confocal laser scanning microscopy was used to evaluate the binding of nanobubbles and T cells in four allografts and four isografts. MCE with NB(specific) in allograft showed a "delayed enhancement," and the time-intensity curve presented a second peak. The intensity and time of second peak were both positively correlated with the transplant time (P<0.01) and the pathologic grade of AR (P<0.01). Confocal laser scanning microscopy demonstrated the binding of nanobubbles and lymphocytes in myocardium post-MCE with NB(specific). Ultrasound molecular imaging of AR after heart transplantation can be achieved by using MCE with the nanobubbles targeted to T cells. The appearance of delayed enhancement indicates the occurrence of AR, and the intensity and time of the second peak in time-intensity curve provide potential quantitative indications for diagnosis and severity of AR.

Systemic Inhibition of Angiopoietin-2 Prevents Endothelial Activation, and Acute Rejection in Rat Cardiac Allografts

Purpose Angiopoietin-2 (Ang2) is vascular growth factor promoting vascular instability and inflammation. In our previous study, heart-targeted Ang2-inhibition prevented ischemia-reperfusion injury (IRI). Ang2 production and release, however, continues after the acute phase of IRI, and activates endothelium to attract inflammatory cells. We investigated in allogeneic rat cardiac transplantation (Tx) model the effects of preop and postop systemic recipient treatment with anti-Ang2 antibody (Ab). Methods and Materials DA donor hearts were preserved in +4 °C PBS for 4h and then transplanted to fully MHC-mismatched WF recipient rats. The recipients received anti-Ang2 Ab or control-IgG i.p. 4h prior to the Tx and, in survival studies, consecutive doses on days 1, 3, and 5. We analyzed IRI-induced endothelial activation, myocardial damage and inflammation at 6h after the Tx. In survival experiments, the recipients received additional mild CyA (1mg/kg/d) and allograft survival was evaluated by daily palpation. Results The preop recipient anti-Ang2 Ab treatment reduced IRI-induced myocardial damage, microvascular VCAM1 and ICAM1 expression, and inflammatory cell influx at 6h after the Tx. Furthermore, when the anti-Ang2 Ab treatment was continued on days 1, 3, and 5 after the Tx, the allograft survival was significantly prolonged, while single preop treatment dose failed to prolong the survival. [ figure 1 ] Conclusions In conclusion, we show that systemic recipient treatment with anti-Ang2 Ab reduces endothelial activation, inflammatory cell influx, myocardial injury, and when continued after the Tx, prolongs allograft survival. These results suggest that Ang2 is important in IRI, but also in sustaining inflammation after IRI.

Ang1 Reduces Ischemia-Reperfusion Injury-Induced Microvascular Dysfunction and Chronic Rejection in Rat Cardiac Allografts

Introduction: Ischemia-reperfusion injury (IRI) is an inevitable factor in current clinical solid organ transplantations resulting in microvascular dysfunction, innate immunity activation and correlating with the later development of chronic rejection. Angiopoietin-1 (Ang1) is endothelial cell-specific growth factor having essential role in vascular development and in endothelial cell maintenance. We investigated the effects of preoperative ex vivo intracoronary perfusion with recombinant variant of native Ang1 (COMP-Ang1) on IRI-associated vascular leakage, no-reflow phenomenon, cardiomyocyte damage, innate immune activation and subsequent development of cardiac fibrosis and allograft vasculopathy. Methods: We performed fully MHC-mismatched cardiac allograft transplantations between DA donor and WF recipient rats. We perfused the donor hearts intracoronarily with COMP-Ang1 or control-PBS perioperatively and preserved the graft was in +4 °C PBS for 4h before transplantation. We analyzed vascular permeability and perfusion at 30 mins, IRI induced myocardial damage and inflammation at 6 h and chronic rejection at 8 weeks after transplantation. Results: COMP-Ang1 reduced IRI-induced microvascular dysfunction by reducing vascular permeability measured by Evans Blue absorbance and increasing the number of perfused vessels 30 min after reperfusion. COMP-Ang1 also significantly reduced endothelial activation, measured by VCAM1 immunoreactivity, ED1+ macrophage and MPO+ neutrophil influx and innate immune activation 6h after reperfusion. Furthermore, periopreative COMP-Ang1 treatment reduced cardiac fibrosis and allograft vasculopathy analyzed 8 weeks after transplantation. (Figure 1)[Figure 1]Conclusion: Here, we show that COMP-Ang1 protects cardiac allografts from IRI-induced microvascular dysfunction and from subsequent chronic allograft rejection. Such results suggest COMP-Ang1 as clinically feasible method for early allograft protection.

331 COMP-Ang1 Reduces Ischemia-Reperfusion Injury-Induced Microvascular Dysfunction and Chronic Rejection in Rat Cardiac Allografts

331 COMP-Ang1 Reduces Ischemia-Reperfusion Injury-Induced Microvascular Dysfunction and Chronic Rejection in Rat Cardiac Allografts

Rock1 Inhibitor Abrogates Chronic Rejection in Rat Cardiac Model System

Background: We have shown previously that the abrogation of acute and chronic rejection of rat cardiac allografts occurs through the down-regulation of RhoA pathway and involves the changes in RhoA kinase (ROCK)-dependent actin cytoskeleton and T cell motility. Here we studied the ability of the Y-27632, a highly selective inhibitor of Rho-associated protein kinase p160ROCK (ROCK1), to abrogate chronic rejection of the allograft and influence T cell infiltration. Methods: Heterotopic cardiac transplants were performed between donor Wistar Furth (WF) and ACI recipient rats. Controls received sub-therapeutic dose of cyclosporine (CsA, 10 mg/kg) for 3 days or 7 days therapeutic dose of cyclosporine. The experimental groups of ACI recipient received one preoperative dose of the Y-27632 inhibitor (2 mg/kg, gavage feed) in conjunction with the sub-therapeutic dose of CsA for 3 days or inhibitor alone for 7 days. The cardiac grafts were harvested at 100 days of post-transplantation for histological and immunohistochemical assessment of chronic rejection, vascular sclerosis, and infiltration by different T cell subtypes. Results: Cardiac allografts from recipients exposed to Y-27632 inhibitor in conjunction with sub-therapeutic dose of CsA showed drastically reduced vascular sclerosis, minimal myocardial total cellular infiltration, and were selectively infiltrated with Foxp3+ T regulatory (Treg) cells. Conclusions: Our novel finding that a single dose of the ROCK1 inhibitor Y-27632 attenuates chronic rejection in rat cardiac model system by promoting development of Treg cells warrants its potential as a novel therapeutic agent specific for the inhibition of chronic rejection.

Eicosapentenoic Acid Attenuates Allograft Rejection in an HLA-B27/EGFP Transgenic Rat Cardiac Transplantation Model.

The development of an animal model bearing definite antigens is important to facilitate the evaluation and modulation of specific allo-antigen responses after transplantation. In the present study, heterotopic cardiac transplantation was performed from F344/EGFPTg and F344/HLA-B27Tg rats to F344 rats. The F344 recipients accepted the F344/EGFPTg transplants, whereas they rejected the cardiac tissue from the F344/HLA-B27Tg rats by 39.4 ± 6.5 days, due to high production of anti-HLA-B27 IgM- and IgG-specific antibodies. In addition, immunization of F344 rats with skin grafts from F344/HLA-B27Tg rats resulted in robust production of anti- HLA-B27 IgM and IgG antibodies and accelerated the rejection of a secondary cardiac allograft (7.4 ± 1.9 days). Of interest, the F344 recipients rejected cardiac grafts from double transgenic F344/HLA-B27&EGFPTg rats within 9.0 ± 3.2 days, and this was associated with a significant increase in the infiltration of lymphocytes by day 7, suggesting a role for cellular immune rejection. Eicosapentenoic acid (EPA), one of the ω-3 polyunsaturated fatty acids in fish oil, could attenuate the production of anti-HLA IgG antibodies and B-cell proliferation, significantly prolonging double transgenic F344HLA-B27&EGFPTg to F344 rat cardiac allograft survival (36.1 ± 13.6 days). Moreover, the mRNA expression in the grafts was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), revealing an increase in the expression of the HO-1, IL-10, TGF-β, IDO, and Foxp3 genes in the EPA-treated group. Hence, our data indicate that HLA-B27 and/or GFP transgenic proteins are useful for establishing a unique animal transplantation model to clarify the mechanism underlying the allogeneic cellular and humoral immune response, in which the transplant antigens are specifically presented. Furthermore, we also demonstrated that EPA was effective in the treatment of rat cardiac allograft rejection and may allow the development of novel immunomodulatory strategies for organ transplantation.

The Effects of AEB071 (Sotrastaurin) with Tacrolimus on Rat Heterotopic Cardiac Allograft Rejection and Survival

AEB071 (sotrastaurin) is a specific inhibitor of protein kinase C that prevents T-cell activation. Our previous study demonstrated that AEB071 monotherapy could prevent acute cardiac allograft rejection in rats. Herein, we investigated the effects of AEB071 combined with various doses of tacrolimus (Tac) on the allograft rejection and survival in a rat heart transplantation model. Heterotopic cardiac transplantation from Brown-Norway to Lewis rats was performed. Cardiac allograft survival was assessed by monitoring heartbeats in six recipients of each experimental group. Another four recipient rats were selectively sacrificed in each group at d 7 post-transplantation for histologic examination. Serum transaminases, blood urea nitrogen, and creatinine concentrations were measured. AEB071 monotherapy prolonged allograft mean survival time (MST) compared with the untreated control group. Also a combination of AEB071 and Tac prolonged MST compared with monotherapy groups with higher dose of Tac. In the cardiac graft histology, AEB071 combined with Tac 0.6 mg/kg/d significantly decreased the rejection grade as indicative of decreased inflammatory cell infiltration into the graft. No experimental group was found with any abnormal histologic or serologic evidence of liver and kidney toxicity. AEB071 combined with a smaller dosage of Tac may be clinically possible to establish calcineurin inhibitor (CNI) minimization protocol in solid organ transplantation.

Allograft inflammatory factor-1 is overexpressed and induces fibroblast chemotaxis in the skin of sclerodermatous GVHD in a murine model

Allograft inflammatory factor (AIF)-1 has been identified in chronic rejection of rat cardiac allografts and is thought to be involved in the immune response. We previously showed that AIF-1 was strongly expressed in synovial tissues in rheumatoid arthritis and that rAIF-1 increased the IL-6 production of synoviocytes and peripheral blood mononuclear cells. Recently, the expression of AIF-1 has been reported in systemic sclerosis (SSc) tissues, whose clinical features and histopathology are similar to those of chronic graft-vs-host disease (GVHD). To clarify the pathogenic mechanism of fibrosis, we examined the expression and function of AIF in sclerodermatous (Scl) GVHD mice. We demonstrated that immunoreactive AIF-1 and IL-6 were significantly expressed in infiltrating mononuclear cells and fibroblasts in thickened skin of Scl GVHD mice compared with control. The immunohistochemical findings were confirmed by Western blot analysis. Wound healing assay also revealed that rAIF-1 increased the migration of normal human dermal fibroblasts (NHDF) directly, but cell growth assay did not show that rAIF-1 increased the proliferation of them. These findings suggest that AIF-1, which can induce the migration of fibroblasts and the production of IL-6 in affected skin tissues, is an important molecule promoting fibrosis in GVHD. Although the biological function of AIF-1 has not been completely elucidated, AIF-1 can induce IL-6 secretion on mononuclear cells and fibroblast chemotaxis. AIF-1 may accordingly provide an attractive new target for antifibrotic therapy in SSc as well as Scl GVHD.

38: Preservation Enhances Rat Cardiac Allograft Vasculopathy by Advancing Innate Immune and Dendritic Cell Activation, and Inducing IL-17 Production

We investigated sequential effects of hypothermic preservation on ischemia-reperfusion injury activated innate and adaptive immunity responses, and their effect on chronic rejection in rat cardiac allografts.

A new approach for noninvasive assessment of chronic cardiac allograft rejection in rat by cellular cardiac MRI

Introduction Long-term cardiac transplant survival rates can be improved only if patients undergo proper monitoring and treatment after transplantation. Repetitive endomyocardial biopsy, the gold standard for monitoring rejection status, is not only invasive but is also prone to sampling errors because of the limited size and location of graft tissue available. We have developed cellular and functional MRI techniques as a non-invasive method to detect and stage acute cardiac allograft rejection. Our recent studies indicate that macrophages can be detected by MRI in our rat model of CCAR. Therefore, we seek to develop CCMRI for non-invasive evaluation of CCAR.

Cyclic AMP prolongs graft survival by suppressing apoptosis and inflammatory gene expression in acute cardiac allograft rejection

This study was designed to investigate the effects of cAMP on immune regulation and apoptosis during acute rat cardiac allograft rejection. We found that the production of immune markers such as inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha), iNOS expression, and nitric oxide (NO) production, was significantly increased in the blood and transplanted hearts of allograft recipients, but not of isograft controls. These increases were effectively suppressed by the administration of the membrane permeable cAMP analog dibutyryl cAMP (db-cAMP). Administration of db-cAMP reduced allograft-induced elevation of several biochemical markers, such as adhesion molecule expression, iron-nitrosyl complex formation, caspase-3 activation, and apoptotic DNA fragmentation in an animal model. Furthermore, treatment of allograft recipients with db-cAMP prolonged median graft survival to 11 days compared with a median graft survival time of 8 days in saline-treated allograft recipients. These results suggest that db-cAMP exerts a beneficial effect on murine cardiac allograft survival by modulating allogeneic immune response and cytotoxicity.

Effect of the inosine 5′-monophosphate dehydrogenase inhibitor BMS-566419 on rat cardiac allograft rejection

Inosine 5′-monophosphate dehydrogenase (IMPDH) inhibition is a critical target in solid organ transplantation, and the development of mycophenolate mofetil (MMF) represents a major advance in transplant medicine. In this study, the in vitro and in vivo pharmacological effects of BMS-566419, a novel chemically synthesized IMPDH inhibitor, were compared to those of mycophenolic acid (MPA) and MMF based on results from several immunological experiments. The in vitro inhibitory activity of BMS-566419 on IMPDH type I/II, immune cell proliferation and antibody production from lipopolysaccharide (LPS)-stimulated B cells was similar, albeit slightly less potent than that of MPA. In a rat heterotopic cardiac transplant model, monotherapy using orally administered BMS-566419 60 mg/kg or MMF 40 mg/kg prolonged the median survival time (MST) of transplanted grafts in the vehicle group from 5 to 18 and 18.5 days, respectively. In the presence of a sub-therapeutic dose of FK506, BMS-566419 30 mg/kg and MMF 20 mg/kg showed identical efficacy with an MST of 21.5 days. In dinitrophenol-LPS-stimulated rats in which calcineurin inhibitors failed to inhibit antibody production, in vivo oral administration of BMS-566419 resulted in antibody production suppression with similar efficacy to MMF. The in vivo antibody production against alloantigen was also suppressed by MMF or BMS-566419 treatment. In addition, gastrointestinal toxicity, considered a dose-limiting factor of MMF, was reduced in BMS-566419 treatment. These results suggest that BMS-566419 and other chemically synthesized IMPDH inhibitors have beneficial pharmacological effects similar to those of MMF, and are potential pharmaceutical candidates in transplant indications.