Rat Cardiac Allografts Research Articles

Assessment of Bax gene transfer influence on survival of heterotopic cardiac allograft in rats by high-frequency echocardiography

Objective To evaluate the value of high-frequency echocardiography in assessing Bax gene transfer influence on survival of heterotopic cardiac allograft in rats.Methods Thirty rat models of heterotopic heart transplantation were established.Group A received heart transplantation only; Group B received cyclosporin (CsA) after operatiom Group C received ultrasound targeted microbubble destruction (UTMD) combined with Bax-shRNA.All rats were tested by high-frequency echocardiography at day 1,3,6 after transplantation.The ultrasound parameters included left ventricular internal dimension diastole (LVIDd),left ventricular internal dimension systole(LVIDs),left ventricular ejection fraction(LVEF),left ventricular thickness (LVT),left ventricular thickening (LVTR) and so on.The pathologic examinations were carried out on five rats at day 6 after echocardiography exam.The other rats in each group were observed for the survival time of cardiac allograft.Results ①The left ventricular long axis view and the apical four chamber view could be displayed clearly by high-frequency echocardiography.②The survival time of allografts in group C was (16.21 ± 5.01)d,which was longer than that in group B [(11.14 ± 1.72)d,P ＜ 0.05] and group A [(7.26 ± 1.50)d,P ＜0.01].③LVT index got higher after transfection.At day 6,LVEF of group A was lower than group B and C markedly(P ＜0.05) while no significant difference was found between group B and C(P ＞0.05).At day 3 and 6,LVT of group A and B were higher than group C (P ＜0.05) while LVTR was lower(P ＜0.05).Conclusions The Bax gene transfer influence on survival of heterotopic cardiac allograft in rats could be evaluated accurately by high-frequency echocardiography which could assess cardiac structure and function.LVT and LVTR can be considered as early evaluation indexes for its high sensitivity over LVEF. Key words: Echocardiography; Heart transplantation; Ventricular function,left ; Transfection

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Combined intrathymic and intravenous injection of mesenchymal stem cells can prolong the survival of rat cardiac allograft associated with decrease in miR-155 expression

BackgroundMesenchymal stem cells (MSCs) have the potential to improve graft outcomes and promote allograft tolerance. In this study, we examined the effects and mechanism of combined intrathymic (IT) and intravenous (IV) injection of MSCs on the survival of transplanted hearts in a rat allograft model. MethodsRecipient Sprague-Dawley rats were transplanted with hearts from Wistar rats. Wistar rat MSCs were infused via IT or IV or combined IT and IV (IT/IV) injection at designated intervals. In vitro mixed lymphocyte reaction assays were performed to assess the immunosuppressive capacity of MSCs. Mesenchymal stem cell surface markers and CD4+, CD25+, and Foxp3+ T-cells in the peripheral blood were detected using flow cytometry analysis. The expression of microRNAs and cytokines in graft infiltrating lymphocytes was analyzed by real-time polymerase chain reaction. ResultsThe MSCs cultured in vitro had multipotential differentiation capacity. Mixed lymphocyte reaction assays showed that donor-derived MSCs could not stimulate a proliferative response of recipient lymphocytes and could markedly suppress T-cell responses. Survival of the allografts was significantly prolonged by administration of IT/IV injection of MSCs compared with controls, with a mean survival of 32.2 versus 6.5 d, respectively. Compared with the syngeneic groups posttransplant, miR-155 expression was significantly increased in the allogeneic group, and could be restored by injection of MSCs, especially IT/IV injection of MSCs. Moreover, IT/IV injection of MSCs decreased the level of interleukin (IL)-2 and interferon-gamma, but increased the levels of IL-4 and IL-10 in the allogeneic group. More important, IT/IV injection of MSCs was the best way to increase the percentage of CD4+, CD25+, and Foxp3+ T-cell peripheral blood. ConclusionsOur results indicated that IT/IV injection of MSCs can prolong the survival of rat cardiac allograft, which may be associated with down-regulating miR-155 expression, a shift in the Th1/Th2 balance, and up-regulation of Treg cells expression.

Combined Donor Simvastatin and Methylprednisolone Treatment Prevents Ischemia-Reperfusion Injury in Rat Cardiac Allografts Through Vasculoprotection and Immunomodulation

Ischemia-reperfusion injury (IRI) and allograft dysfunction remain as two of the major clinical challenges after heart transplantation. Here, we investigated the effect of donor treatment with simvastatin and methylprednisolone on microvascular dysfunction and immunomodulation during IRI in rat cardiac allografts subjected to prolonged ischemia time. The DA rats received simvastatin, methylprednisolone, or both 2 hr before heart donation. The allografts were subjected to 4-hr hypothermic preservation and transplanted to the fully major histocompatibility complex-mismatched WF rat recipients. Six hours after reperfusion, donor treatment either with simvastatin alone or with high dose of methylprednisolone alone or in combination with simvastatin and methylprednisolone significantly reduced cardiac troponin T release and the number of allograft infiltrating ED1 macrophages MPO neutrophils. However, the combination donor treatment was superior in the prevention of IRI and significantly prolonged allograft survival. Donor simvastatin treatment inhibited allograft microvascular RhoA GTPase pathway activation, whereas methylprednisolone prevented activation of innate immune response and mRNA expression of hypoxia-inducible factor-1α and its multiple target genes. Our results show that donor treatment in combination with simvastatin and methylprednisolone prevents IRI and has beneficial effect on allograft survival in rat cardiac allografts. Minimizing microvascular injury and the activation of innate immunity may offer a novel therapeutic strategy to expand the donor pool and furthermore improve the function of the marginal donor organs.

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.

Intracoronary Treatment with VEGF-C/D Inhibitor Enhances Cardiac Allograft Survival and Prevents Chronic Rejection by Regulating Lymphatic Endothelial Cell Activation

Purpose VEGF-C/D/VEGFR-3 signaling may regulate lymphatic vessel activation and the initiation of alloimmunity after transplantation. We investigated the effect of perioperative intracoronary injection of synthetic VEGF-C/D inhibitor on lymphatic activation, antigen-presenting cell trafficking and subsequent development of alloimmune responses in rat cardiac allografts. Methods and Materials We perfused the coronaries of DA donor rat hearts ex vivo with VEGF-C/D inhibitor or PBS and preserved the grafts in +4°C PBS for 4h and then transplanted the grafts to fully MHC-mismatched WF recipient rats. We analyzed ischemia-reperfusion injury, and acute and chronic rejection responses. In acute and chronic rejection models, the recipients received CyA as immunosuppression. Results At 6h after the reperfusion, VEGF-C/D inhibitor did not affect the myocardial injury but reduced the number of LYVE-1+ lymphatic vessels expressing VEGFR-3, ICAM-1 and VCAM-1 in the allograft and reduced the number of dendritic cells migrating out of the allograft. VEGF-C/D inhibition enhanced allograft survival in acute rejection model. In chronic rejection model, VEGF-C/D inhibitor reduced allograft inflammation and prevented the development of cardiac fibrosis and vasculopathy. Conclusions Intracoronary treatment with VEGF-C/D inhibitor prevented early activation of lymphatic endothelial cells and trapped dendritic cells into the allograft. This interfered with allorecognition resulting in reduced acute and chronic rejection responses. Our results suggest donor single-dose intracoronary treatment with VEGF-C/D inhibitor as a novel clinically feasible lymphatic vessel targeted immunomodulatory approach. [ figure 1 ]

Differential Effects of Pharmacological HIF Preconditioning of Donors Versus Recipients in Rat Cardiac Allografts

Ischemia-reperfusion injury (IRI) induces hypoxia-inducible factor-1 (HIF-1) in the myocardium, but the consequences remain elusive. We investigated HIF-1 activation during cold and warm ischemia and IRI in rat hearts and cardiac syngrafts. We also tested the effect of HIF-α stabilizing prolyl hydroxylase inhibitor (FG-4497) on IRI or allograft survival. Ex vivo ischemia of the heart increased HIF-1α expression in a time- and temperature-dependent fashion. Immunohistochemistry localized HIF-1α to all cardiac cell types. After reperfusion, HIF-1α immunoreactivity persisted in smooth muscle cells and cardiomyocytes in the areas with IRI. This was accompanied with a transient induction of protective HIF-1 downstream genes. Donor FG-4497 pretreatment for 4 h enhanced IRI in cardiac allografts as evidenced by an increase in cardiac troponin T release, cardiomyocyte apoptosis, and activation of innate immunity. Recipient FG-4497 pretreatment for 4 h decreased infiltration of ED1(+) macrophages, and mildly improved the long-term allograft survival. In syngrafts donor FG-4497 pretreatment increased activation of innate immunity, but did not induce myocardial damage. We conclude that the HIF-1 pathway is activated in heart transplants. We suggest that pharmacological HIF-α preconditioning of cardiac allografts donors would not lead to clinical benefit, while in recipients it may result in antiinflammatory effects and prolonged allograft survival.

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.

Tranilast, an Analogue of Tryptophan Catabolites, Induces Allograft Tolerance by CD161+ Cells

Background: Indoleamine 2, 3-dioxygenase (IDO) converts tryptophan in various catabolites and has been shown to induce immune tolerance in different immune-mediated diseases, including organ transplantation. One of these tolerogenic metabolites is anthranilic acid. Tranilast is a clinically approved, structural and functional analogue of anthranilic acid that has been recently shown to be effective in murine models of multiple sclerosis and rheumatoid arthritis. We examined the effect of tranilast in a rat cardiac allograft model. Materials/methods: Lewis 1W rat hearts were grafted in MHC-mismatched Lewis 1A rats. The receiver is orally treated with 650mg/kg of tranilast daily for 30 days. Total splenocytes and purified spleen cell subtypes sorted by FACS Aria were transferred to sublethaly irradiated rats by i.v injection the day before transplantation. Results: Graft survival in recipients treated with tranilast were significantly prolonged (64, 6±42.7 days, n=15, p< 0, 0001) when compared to control group (8.3±2.3 days, n=6) and in 50% of recipients (n=15) tranilast induced tolerance (>100 days survival). Adoptive transfer of total splenocytes from tolerant tranilast-treated rats to naïve rats resulted in tolerance in all animals (n=5). Moreover, splenocytes from these adoptively transferred tolerant recipients were again capable of transferring tolerance to all naïve recipients (n=5). Tolerant splenocytes depleted of T and B cells (n=6) or depleted of T, B and DCs (n=5) transferred tolerance. Importantly, depletion of CD161+ cells from T, B and DCs-depleted splenocytes abrogated tolerance transfer (9.3 ± 0.6 n=3). To confirm these results, we adoptively transferred CD161+TCR- cells from tolerant rats which resulted in tolerance (130±91.6 days, n=5, 3/5 recipients >100 days) whereas CD161-TCR- cells from the same animals did not (12.7±4.6 days, n=3, p< 0,005). Among CD161+TCR- cells, we could distinguish 2 cell populations, one CD161high (NK cells) and another CD161low (myeloid-derived suppressor cells). When we transferred CD161high and CD161 low from the same animals, we only obtained tolerance to cardiac allografts with CD161low cells (59 ±64days, n=5, 2/5 recipients >100 days vs 18±14 days, n=3, respectively) Discussion: This is the first demonstration that tranilast mediates transplantation tolerance. Tolerance was active and transferable by CD161lowTCR− cells, these cells in the rat have been described in the rat as of myeloid-derived suppressor cells. Experiments are under way to better define this cell population that mediate tranilast-induced tolerance and by which downstream mechanisms.

An Oral Sphingosine 1-Phosphate Receptor 1 (S1P1) Antagonist Prodrug with Efficacy in Vivo: Discovery, Synthesis, and Evaluation

A prodrug approach to optimize the oral exposure of a series of sphingosine 1-phosphate receptor 1 (S1P(1)) antagonists for chronic efficacy studies led to the discovery of (S)-2-{[3'-(4-chloro-2,5-dimethylphenylsulfonylamino)-3,5-dimethylbiphenyl-4-carbonyl]methylamino}-4-dimethylaminobutyric acid methyl ester 14. Methyl ester prodrug 14 is hydrolyzed in vivo to the corresponding carboxylic acid 15, a potent and selective S1P(1) antagonist. Oral administration of the prodrug 14 induces sustained peripheral blood lymphocyte reduction in rats. In a rat cardiac transplantation model coadministration of a nonefficacious dose of prodrug 14 with a nonefficacious dose of sotrastaurin (19), a protein kinase C inhibitor, or everolimus (20), an mTOR inhibitor, effectively prolonged the survival time of rat cardiac allografts. This demonstrates that clinically useful immunomodulation mediated by the S1P(1) receptor can be achieved with an S1P(1) antagonist generated in vivo after oral administration of its prodrug.

Immunoregulatory Function of IL-27 and TGF-β1 in Cardiac Allograft Transplantation

Deciphering the mechanisms of tolerance represents a crucial aim of research in transplantation. We previously identified by DNA chip interleukin (IL)-27 p28 and transforming growth factor (TGF)-β1 as overexpressed in a model of rat cardiac allograft tolerance mediated by regulatory CD4CD25 T cells. The role of these two molecules on the control of the inflammatory response remains controversial. However, both are involved in the regulation of the T helper 17/Treg axis, suggesting their involvement in tolerance. We analyzed regulation of IL-27 and TGF-β1 expression in allograft response and their role in tolerance by using blocking anti-TGF-β antibody and by generating an adeno-associated virus encoding IL-27. Here, we confirmed the overexpression of IL-27 and TGF-β1 in tolerated cardiac allografts in two different rodent models. We observed that their expression correlates with inhibition of T helper 17 differentiation and with expansion of regulatory CD4CD25 T cells. We showed in a rat model that anti-TGF-β treatment abrogates infectious tolerance mediated by the transfer of regulatory CD4CD25 T cells. Moreover, overexpression of IL-27 by adeno-associated virus administration in combination with a short-term immunosuppression allows prolongation of cardiac allograft survival and one tolerant recipient. We found that IL-27 overexpression did not induce Foxp3CD4CD25 T-cell expansion but rather IL-10-expressing CD4 T cells in the tolerant recipient. Taken together, these data suggest that both TGF-β1 and IL-27 play a role in the mechanisms of tolerance. However, in contrast to TGF-β1, IL-27 seems not to be involved in regulatory CD4CD25 T-cell expansion but rather in their mode of action.

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

630 Donor Pharmacologically Induced Hypothermia with DADLE Abolishes Rat Cardiac Allograft Ischemia/Reperfusion Injury through Akt, ERK1/2 Signaling

630 Donor Pharmacologically Induced Hypothermia with DADLE Abolishes Rat Cardiac Allograft Ischemia/Reperfusion Injury through Akt, ERK1/2 Signaling

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.

A selective and potent CXCR3 antagonist SCH 546738 attenuates the development of autoimmune diseases and delays graft rejection

BackgroundThe CXCR3 receptor and its three interferon-inducible ligands (CXCL9, CXCL10 and CXCL11) have been implicated as playing a central role in directing a Th1 inflammatory response. Recent studies strongly support that the CXCR3 receptor is a very attractive therapeutic target for treating autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis and psoriasis, and to prevent transplant rejection. We describe here the in vitro and in vivo pharmacological characterizations of a novel and potent small molecule CXCR3 antagonist, SCH 546738.ResultsIn this study, we evaluated in vitro pharmacological properties of SCH 546738 by radioligand receptor binding and human activated T cell chemotaxis assays. In vivo efficacy of SCH 546738 was determined by mouse collagen-induced arthritis, rat and mouse experimental autoimmune encephalomyelitis, and rat cardiac transplantation models. We show that SCH 546738 binds to human CXCR3 with a high affinity of 0.4 nM. In addition, SCH 546738 displaces radiolabeled CXCL10 and CXCL11 from human CXCR3 with IC50 ranging from 0.8 to 2.2 nM in a non-competitive manner. SCH 546738 potently and specifically inhibits CXCR3-mediated chemotaxis in human activated T cells with IC90 about 10 nM. SCH 546738 attenuates the disease development in mouse collagen-induced arthritis model. SCH 546738 also significantly reduces disease severity in rat and mouse experimental autoimmune encephalomyelitis models. Furthermore, SCH 546738 alone achieves dose-dependent prolongation of rat cardiac allograft survival. Most significantly, SCH 546738 in combination with CsA supports permanent engraftment.ConclusionsSCH 546738 is a novel, potent and non-competitive small molecule CXCR3 antagonist. It is efficacious in multiple preclinical disease models. These results demonstrate that therapy with CXCR3 antagonists may serve as a new strategy for treatment of autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, and to prevent transplant rejection.

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.

A selective and potent CXCR3 antagonist SCH 546738 attenuates the development of autoimmune diseases and delays graft rejection

The CXCR3 receptor and its three interferon-inducible ligands (CXCL9, CXCL10 and CXCL11) have been implicated as playing a central role in directing a Th1 inflammatory response. Recent studies strongly support that the CXCR3 receptor is a very attractive therapeutic target for treating autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis and psoriasis, and to prevent transplant rejection. We describe here the in vitro and in vivo pharmacological characterizations of a novel and potent small molecule CXCR3 antagonist, SCH 546738. In this study, we evaluated in vitro pharmacological properties of SCH 546738 by radioligand receptor binding and human activated T cell chemotaxis assays. In vivo efficacy of SCH 546738 was determined by mouse collagen-induced arthritis, rat and mouse experimental autoimmune encephalomyelitis, and rat cardiac transplantation models. We show that SCH 546738 binds to human CXCR3 with a high affinity of 0.4 nM. In addition, SCH 546738 displaces radiolabeled CXCL10 and CXCL11 from human CXCR3 with IC50 ranging from 0.8 to 2.2 nM in a non-competitive manner. SCH 546738 potently and specifically inhibits CXCR3-mediated chemotaxis in human activated T cells with IC90 about 10 nM. SCH 546738 attenuates the disease development in mouse collagen-induced arthritis model. SCH 546738 also significantly reduces disease severity in rat and mouse experimental autoimmune encephalomyelitis models. Furthermore, SCH 546738 alone achieves dose-dependent prolongation of rat cardiac allograft survival. Most significantly, SCH 546738 in combination with CsA supports permanent engraftment. SCH 546738 is a novel, potent and non-competitive small molecule CXCR3 antagonist. It is efficacious in multiple preclinical disease models. These results demonstrate that therapy with CXCR3 antagonists may serve as a new strategy for treatment of autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, and to prevent transplant rejection.

Tranilast, an analogue of tryptophan catabolites, induces allograft tolerance by CD161+ cells

Background Indoleamine 2, 3-dioxygenase (IDO) converts tryptophan in various catabolites and has been shown to induce immune tolerance in different immune-mediated diseases, including organ transplantation. One of these tolerogenic metabolites is anthranilic acid. Tranilast is a clinically approved, structural and functional analogue of anthranilic acid that has been recently shown to be effective in murine models of multiple sclerosis and rheumatoid arthritis. We examined the effect of tranilast in a rat cardiac allograft model. Materials and methods Lewis 1W rat hearts were grafted in MHC-mismatched Lewis 1A rats. The receiver is orally treated with 650mg/kg of tranilast daily for 30 days. Total splenocytes and purified spleen cell subtypes sorted by FACS Aria were transferred to sublethaly irradiated rats by i.v injection the day before transplantation. Results

Multiple roles of angiopoietins in atherogenesis

The roles of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) during vascular development have been extensively investigated, as has been their role in controlling the responsiveness of the endothelium to exogenous cytokines. However, very little is known about the role of these vascular morphogenic molecules in the pathogenesis of atherosclerosis. Here, we summarize the recent research into angiopoietins in atherosclerosis. Angiopoietin-2 is a context-dependent agonist that protects against the development of arteriosclerosis in rat cardiac allograft. A recent study showed, contrary to expectations, that a single systemic administration of adenoviral Ang-2 to apoE mice, fed a Western diet, reduced atherosclerotic lesion size and LDL oxidation in a nitric oxide synthase dependent manner. In contrast, overexpression of Ang-1 fails to protect from rat cardiac allograft due to smooth muscle cell activation. The potential proatherogenic effect of Ang-1 is further supported by the induction of chemotaxis of monocytes by Ang-1 in a manner that is independent of Tie-2 and integrin binding. These studies highlight the need for extensive research to better understand the role of angiopoietins in the cardiovascular setting. Ang-2 inhibits atherosclerosis by limiting LDL oxidation via stimulation of nitric oxide production. In contrast, Ang-1 can promote monocyte and neutrophil migration. The angiopoietin-Tie-2 system provides an important new target for modulating vascular function.

Donor Simvastatin Treatment Abolishes Rat Cardiac Allograft Ischemia/Reperfusion Injury and Chronic Rejection Through Microvascular Protection

Ischemia/reperfusion injury may have deleterious short- and long-term consequences for cardiac allografts. The underlying mechanisms involve microvascular dysfunction that may culminate in primary graft failure or untreatable chronic rejection. Here, we report that rat cardiac allograft ischemia/reperfusion injury resulted in profound microvascular dysfunction that was prevented by donor treatment with peroral single-dose simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase and Rho GTPase inhibitor, 2 hours before graft procurement. During allograft preservation, donor simvastatin treatment inhibited microvascular endothelial cell and pericyte RhoA/Rho-associated protein kinase activation and endothelial cell-endothelial cell gap formation; decreased intragraft mRNA levels of hypoxia-inducible factor-1α, inducible nitric oxide synthase, and endothelin-1; and increased heme oxygenase-1. Donor, but not recipient, simvastatin treatment prevented ischemia/reperfusion injury-induced vascular leakage, leukocyte infiltration, the no-reflow phenomenon, and myocardial injury. The beneficial effects of simvastatin on vascular stability and the no-reflow phenomenon were abolished by concomitant nitric oxide synthase inhibition with N-nitro-l-arginine methyl ester and RhoA activation by geranylgeranyl pyrophosphate supplementation, respectively. In the chronic rejection model, donor simvastatin treatment inhibited cardiac allograft inflammation, transforming growth factor-β1 signaling, and myocardial fibrosis. In vitro, simvastatin inhibited transforming growth factor-β1-induced microvascular endothelial-to-mesenchymal transition. Our results demonstrate that donor simvastatin treatment prevents microvascular endothelial cell and pericyte dysfunction, ischemia/reperfusion injury, and chronic rejection and suggest a novel, clinically feasible strategy to protect cardiac allografts.