Chronic Cardiac Allograft Rejection Research Articles

MEK1/2-PKM2 Pathway Modulates the Immunometabolic Reprogramming of Proinflammatory Allograft-infiltrating Macrophages During Heart Transplant Rejection.

Emerging evidence has highlighted the role of macrophages in heart transplant rejection (HTR). However, the molecular signals modulating the immunometabolic phenotype of allograft-infiltrating macrophages (AIMs) during HTR remain unknown. We analyzed single-cell RNA sequencing data from cardiac graft-infiltrating immunocytes to characterize the activation patterns and metabolic features of AIMs. We used flow cytometry to determine iNOS and PKM2 expression and MEK/ERK signaling activation levels in AIMs. We then generated macrophage-specific Mek1/2 knockout mice to determine the role of the MEK1/2-PKM2 pathway in the proinflammatory phenotype and glycolytic capacity of AIMs during HTR. Single-cell RNA sequencing analysis showed that AIMs had a significantly elevated proinflammatory and glycolytic phenotype. Flow cytometry analysis verified that iNOS and PKM2 expressions were significantly upregulated in AIMs. Moreover, MEK/ERK signaling was activated in AIMs and positively correlated with proinflammatory and glycolytic signatures. Macrophage-specific Mek1/2 deletion significantly protected chronic cardiac allograft rejection and inhibited the proinflammatory phenotype and glycolytic capacity of AIMs. Mek1/2 ablation also reduced the proinflammatory phenotype and glycolytic capacity of lipopolysaccharides + interferon-γ-stimulated macrophages. Mek1/2 ablation impaired nuclear translocation and PKM2 expression in macrophages. PKM2 overexpression partially restored the proinflammatory phenotype and glycolytic capacity of Mek1/2 -deficient macrophages. Moreover, trametinib, an Food and Drug Administration-approved MEK1/2 inhibitor, ameliorated chronic cardiac allograft rejection. These findings suggest that the MEK1/2-PKM2 pathway is essential for immunometabolic reprogramming of proinflammatory AIMs, implying that it may be a promising therapeutic target in clinical heart transplantation.

Low-dose IL-2 prevents murine chronic cardiac allograft rejection: Role for IL-2-induced T regulatory cells and exosomes with PD-L1 and CD73

To determine the effects and immunological mechanisms of low-dose interleukin-2 (IL-2) in a murine model of chronic cardiac allograft rejection (BALB/c to C57BL/6) after costimulatory blockade consisting of MR1 (250 μg/ip day 0) and CTLA4-Ig (200 μg/ip day 2), we administered low-dose IL-2 (2000 IU/day) starting on posttransplant day 14 for 3 weeks. T regulatory (Treg) cell infiltration of the grafts was determined by immunohistochemistry; circulating exosomes by western blot and aldehyde bead flow cytometry; antibodies to donor MHC by immunofluorescent staining of donor cells; and antibodies to cardiac self-antigens (myosin, vimentin) by ELISA. We demonstrated that costimulation blockade after allogeneic heart transplantation induced circulating exosomes containing cardiac self-antigens and antibodies to both donor MHC and self-antigens, leading to chronic rejection by day 45. Treatment with low-dose IL-2 prolonged allograft survival (>100 days), prevented chronic rejection, and induced splenic and graft-infiltrating CD4+ CD25+ Foxp3 Treg cells by day 45 and circulating exosomes (Foxp3+) with PD-L1 and CD73. MicroRNA 142, associated with the TGFβ pathway, was significantly downregulated in exosomes from IL-2-treated mice. In conclusion, low-dose IL-2 delays rejection in a murine model of chronic cardiac allograft rejection and also induces graft-infiltrating Tregs and circulating exosomes with immunoregulatory molecules.

Low Dose IL-2 Therapy Induces T Regulatory Cell Derived Circulatory Exosomes Containing PDL1 and CD73 and Abrogates Development of Chronic Cardiac Allograft Rejection

<h3>Purpose</h3> Aim of the study is to determine the effect of low dose interleukin-2 (IL-2) therapy on the kinetics of induction of circulating exosomes and their immunological properties resulting in prevention of chronic rejection following allogeneic murine cardiac transplantation (HTx). <h3>Methods</h3> BALB/c (H2^d) to C57BL/6 (H2^b) HTx was performed heterotopically in the abdominal cavity. Costimulatory blockade consisting of MR1 (250 μg/ip day 0) and CTLA4-Ig (200 μg/ip day 2) was administered to induce tolerance. IL-2 (2000IU/day) was administered from day 14 post-transplant for 3 weeks. Sera were collected on days 7-100 for analysis of exosome induction and characterization of cardiac self-antigens (SAgs) (Myosin, Vimentin) by western blot. Foxp3, PDL1, CD73 in exosomes were detected using aldehyde bead - based flow cytometry. Antibodies (Abs) to donor MHC and SAgs were detected using immunofluorescence and ELISA respectively. <h3>Results</h3> BALB/c to C57BL/6 HTx acutely rejected (AR) with median survival time of 8 days. Costimulatory blockade prevented AR but developed chronic rejection with mean of 42 days circulating exosomes contained significantly higher levels of Myosin and Vimentin in the exosomes day 15 (p<0.05) and day 30 (p<0.01). Abs to MHC (32±13.8% vs 2±1.1; p<0.001) was detected on day 30, whereas Abs to Myosin (1114.21±443.4 ng/ml; p<0.0001) and Vimentin (519±241 ng/ml; p<0.0001) were detected on day45 during chronic rejection with fibrosis (57.1±20.7 vs 2.3±1.7%). Low dose IL-2 significantly prolonged the survival, 115 vs 42 (p<0.001) and did not induce exosomes with cardiac SAgs or developed Abs to MHC or SAgs. Low dose IL-2 not only abrogated chronic rejection but also induced splenic CD4+CD25+ Foxp3 T regulatory (Treg) cells in the allograft by day 45. Flow cytometry of circulatory exosomes isolated in day 45 also demonstrated increased Foxp3+ PDL1+ (3.3 fold) and FoxP3+ CD73+ (3 fold) compared to exosomes isolated on day 45 from chronic rejecting animals. <h3>Conclusion</h3> Our results demonstrate that low dose IL-2 abrogated the development of chronic rejection following HTx and induced circulating Treg cells derived exosomes with PDL1 and CD73 along with splenic Tregs and graft infiltrating FoxP3 cells.

Sinomenine in Cardio-Cerebrovascular Diseases: Potential Therapeutic Effects and Pharmacological Evidences.

Cardio-cerebrovascular diseases, as a major cause of health loss all over the world, contribute to an important part of the global burden of disease. A large number of traditional Chinese medicines have been proved effective both clinically and in pharmacological investigations, with the acceleration of the modernization of Chinese medicine. Sinomenine is the main active constituent of sinomenium acutum and has been generally used in therapies of rheumatoid arthritis and neuralgia. Varieties of pharmacological effects of sinomenine in cardio-cerebrovascular system have been discovered recently, suggesting an inspiring application prospect of sinomenine in cardio-cerebrovascular diseases. Sinomenine may retard the progression of atherosclerosis by attenuating endothelial inflammation, regulating immune cells function, and inhibiting the proliferation of vascular smooth muscle cells. Sinomenine also alleviates chronic cardiac allograft rejection relying on its anti-inflammatory and anti-hyperplastic activities and suppresses autoimmune myocarditis by immunosuppression. Prevention of myocardial or cerebral ischemia-reperfusion injury by sinomenine is associated with its modulation of cardiomyocyte death, inflammation, calcium overload, and oxidative stress. The regulatory effects on vasodilation and electrophysiology make sinomenine a promising drug to treat hypertension and arrhythmia. Here, in this review, we will illustrate the pharmacological activities of sinomenine in cardio-cerebrovascular system and elaborate the underlying mechanisms, as well as give an overview of the potential therapeutic roles of sinomenine in cardio-cerebrovascular diseases, trying to provide clues and bases for its clinical usage.

IRF4 ablation in B cells abrogates allogeneic B cell responses and prevents chronic transplant rejection

B cells contribute to chronic transplant rejection by producing donor-specific antibodies and promoting T cell response, but how these processes are regulated at the transcriptional level remains unclear. Herein, we investigate the role of transcription factor interferon regulatory factor 4 (IRF4) in controlling B cell response during chronic transplant rejection. We generated the Irf4gfp reporter mice to determine IRF4 expression in B cell lineage. We then used mice with B cell-specific IRF4 deletion to define the role of IRF4 in B cell response after NP-KLH immunization or allogeneic heart transplantation. In particular, graft survival and histology, as well as B and T cell responses, were evaluated after transplantation. IRF4 is dynamically expressed at different stages of B cell development and is absent in germinal center (GC) B cells. However, IRF4 ablation in the B cell lineage primarily eliminates GC B cells in both naïve and NP-KLH immunized mice. In the transplantation setting, IRF4 functions intrinsically in B cells and governs allogeneic B cell responses at multiple levels, including GC B cell generation, plasma cell differentiation, donor-specific antibody production, and support of T cell response. B cell-specific IRF4 deletion combined with transient CTLA4-Ig treatment abrogates acute and chronic cardiac allograft rejection in naïve recipient mice but not in donor skin-sensitized recipients. B cells require IRF4 to mediate chronic transplant rejection. IRF4 ablation in B cells abrogates allogeneic B cell responses and may also inhibit the ability of B cells to prime allogenic T cells. Targeting IRF4 in B cells represents a potential therapeutic strategy for eliminating chronic transplant rejection.

Low Dose Interleukin-2 Induces Exosomes with Tolerance Markers (PDL1, CD73) and Significantly Delays Development of Chronic Rejection Following Murine Heterotopic Cardiac Transplantation

Purpose Aim of the study is to determine the effect of low dose interleukin-2 (IL-2) therapy on the kinetics of induction of circulating exosomes with tolerance markers (PDL1, CD73) and their role in abrogating chronic rejection following murine heterotopic cardiac transplant (HTx). Methods Allogenic (BALB/c to C57BL/6) HTx was performed heterotopically in abdominal cavity. Costimulatory blockade consisting of MR1 (250 μg/ip day 0) and CTLA4-Ig (200 μg/ip day 2) was performed. Low dose IL-2 (2000IU/day) was administered from day 15 post-transplant for 3 weeks. Sera was collected on days 7-100 for kinetic analysis of exosome induction and characterization of PDL1, CD73, cardiac self-antigens (SAgs) (Myosin, Vimentin), class II MHC trans-activator (CIITA), and 20S proteasome by western blot using specific antibodies (Abs) and Abs to SAgs. Results BALB/c to C57BL/6 HTx rejected transplanted heart with median survival time of 8 (7-9) days. HTx done following costimulatory blockade developed chronic rejection with mean survival time 26 (7-47) days. Exosome and Ab development demonstrated induction of exosomes with Vimentin (2.2± 1.0 vs 0.6±0.1; p Conclusion Circulating exosomes containing cardiac SAgs and development of Abs to SAgs lead to chronic rejection in a murine model occurring after costimulation blockade following allogenic HTx. Low dose IL-2 abrogated chronic rejection and induced circulating exosome with tolerance markers. This result supports an important role for exosomes, inducing tolerance and significantly delaying development of chronic rejection in murine model of chronic cardiac allograft rejection.

Allograft or Recipient ST2 Deficiency Oppositely Affected Cardiac Allograft Vasculopathy via Differentially Altering Immune Cells Infiltration.

The role of IL-33/ST2 signaling in cardiac allograft vasculopathy (CAV) is not fully addressed. Here, we investigated the role of IL-33/ST2 signaling in allograft or recipient in CAV respectively using MHC-mismatch murine chronic cardiac allograft rejection model. We found that recipients ST2 deficiency significantly exacerbated allograft vascular occlusion and fibrosis, accompanied by increased F4/80+ macrophages and CD3+ T cells infiltration in allografts. In contrast, allografts ST2 deficiency resulted in decreased infiltration of F4/80+ macrophages, CD3+ T cells and CD20+ B cells and thus alleviated vascular occlusion and fibrosis of allografts. These findings indicated that allografts or recipients ST2 deficiency oppositely affected cardiac allograft vasculopathy/fibrosis via differentially altering immune cells infiltration, which suggest that interrupting IL-33/ST2 signaling locally or systematically after heart transplantation leads different outcome.

Everolimus-eluting stents versus sirolimus-eluting stents in patients with cardiac allograft vasculopathy.

IntroductionCardiac allograft vasculopathy remains one of the most important factors leading to chronic cardiac allograft rejection. When revascularization is needed percutaneous coronary interventions are the method of choice.AimTo compare the short- and long-term outcomes of cardiac allograft vasculopathy patients treated with everolimus- (EES) or sirolimus-eluting stents (SES).Material and methodsBetween December 2012 and December 2020, 319 patients after heart transplantation undergoing coronary angiography at our institution were analysed. Subsequently 39 patients underwent de novo angioplasty with second-generation EES. The primary study endpoint was angiographic restenosis as evaluated by quantitative coronary angiography. Secondary outcomes included binary restenosis, target lesion revascularization and cardiac death during the follow-up period (6 months).ResultsTwenty-four patients were treated with EES and 15 treated with SES. No significant differences were observed regarding the rate of risk factors of cardiovascular diseases and comorbidities. The patients treated with EES were younger (55.8 ±11.8 vs. 60.1 ±12.2) and less frequently male (79% vs. 93%). The majority of patients were diagnosed with single vessel disease with LAD involvement (62% and 86% in the EES group, and 47% and 56% in the SES group). In 6 months follow-up, late lumen loss was comparable in both groups, 0.19 ±0.15 vs. 0.14 ±0.15, and binary restenosis was 4% and 0% for EES and SES groups, respectively.ConclusionsSecond generation drug-eluting stents eluting rapamycin analogues are associated with high direct efficacy of procedures and low incidence of restenosis in a 6-month follow-up.

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.

P6345Chronic cardiac allograft rejection after heterotopic rat heart transplantation: ed-a but not ed-b domain containing fibronectin as novel serum biomarker of cardiac allograft vasculopathy

Abstract Background and aims Diagnosis of Cardiac Allograft Vasculopathy (CAV) following heart transplantation (HTX) is still challenging but of prognostic relevance. Thus, the identification of biomarkers non-invasively detecting the disease is an hitherto unmet clinical need. Fetal variants of the cell adhesion modulating protein Fibronectin (Fn) could be proven to re-occur in the context of chronic rejection in rat and human cardiac tissue after HTX in spatial association to CAV and fibrosis. This study was aimed to determine serum levels of fetal Fn variants in a heterotopic rat HTX model of chronic rejection. Methods Chronic cardiac allograft rejection was induced by heterotopic rat HTX using inbred male Lewis (LEW) rats as donors and Fisher (F344) rats as recipients (n=24). After microsurgical modifications, the allograft was implanted into the abdomen of the recipient animal. Rats were sacrificed after 70 days. Untreated F344 rats were used as controls (n=6). Cardiac tissue was subjected to histological analysis according to a recently established sum-score system between 0 and 10 (0 = no detectable rejection; 1 to 3 = weak rejection; 4 to 7 = moderate rejection; and 8 to 10 = severe rejection). The score includes all relevant parameters of chronic rejection (CAV, lymphocyte infiltration, cardiomyocyte degeneration, fibrosis). Brain natriuretic peptide (BNP) was determined by a commercially available ELISA. Serum levels of both, ED-A domain containing Fn (ED-A+ Fn) and ED-B domain containing Fn (ED-B+ Fn) were quantified using ELISA protocols established in our group. Results Final experiments were performed on day 70, mortality rate was 0%. Histological analysis revealed a chronic cardiac allograft rejection in all transplanted animals (mean sum-score 6.92±1.94) while controls did not exhibit relevant alterations (mean sum-score 0.1±0.2; p&lt;0.001). There were significantly elevated BNP levels in HTX rats (76.16±23.22 pg/ml) compared to controls (352.23±132.09 pg/ml; p&lt;0.001). BNP showed a strong positive correlation to the histological sum-score (r=0.653; p&lt;0.001). Serum levels of ED-A+ Fn (HTX: 34.42±11.4 μg/ml, controls: 17.3±6.13 μg/ml; p=0.009) but not ED-B+ Fn (HTX: 8.58±4.12 μg/ml, controls: 9.4±2.61 μg/ml; p=0.804) were significantly increased in HTX rats qualifying ED-A+ Fn for further analysis with respect to histological signs of chronic rejection. Interestingly, there is a noticeable association to the occurrence of CAV representing 1 out of 4 parameters included in the histological sum-score (r=0.51; p=0.006). Conclusions Chronic cardiac allograft rejection and, in particular, CAV as major long-term prognosis-limiting factor after HTX are accompanied by both, a re-expression and vessel associated tissue deposition of ED-A+ Fn and also a relevant liberation into blood. These results underline the potential impact of ED-A+ Fn as biomarker or potential therapeutic target patients that underwent HTX.

IL-3 Triggers Chronic Rejection of Cardiac Allografts by Activation of Infiltrating Basophils.

Chronic rejection is a major problem in transplantation medicine, largely resistant to therapy, and poorly understood. We have shown previously that basophil-derived IL-4 contributes to fibrosis and vasculopathy in a model of heart transplantation with depletion of CD4+ T cells. However, it is unknown how basophils are activated in the allografts and whether they play a role when cyclosporin A (CsA) immunosuppression is applied. BALB/c donor hearts were heterotopically transplanted into fully MHC-mismatched C57BL/6 recipients and acute rejection was prevented by depletion of CD4+ T cells or treatment with CsA. We found that IL-3 is significantly upregulated in chronically rejecting allografts and is the major activator of basophils in allografts. Using IL-3-deficient mice and depletion of basophils, we show that IL-3 contributes to allograft fibrosis and organ failure in a basophil-dependent manner. Also, in the model of chronic rejection involving CsA, IL-3 and basophils substantially contribute to organ remodeling, despite the almost complete suppression of IL-4 by CsA. In this study, basophil-derived IL-6 that is resistant to suppression by CsA, was largely responsible for allograft fibrosis and limited transplant survival. Our data show that IL-3 induces allograft fibrosis and chronic rejection of heart transplants, and exerts its profibrotic effects by activation of infiltrating basophils. Blockade of IL-3 or basophil-derived cytokines may provide new strategies to prevent or delay the development of chronic allograft rejection.

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.

Screening RhoA/ROCK inhibitors for the ability to prevent chronic rejection of mouse cardiac allografts

BackgroundChronic rejection of transplanted organs is a major obstacle in organ transplantation. The main symptoms of chronic rejection are vessel occlusion and tissue fibrosis. Macrophages play a crucial role in chronic rejection. We showed previously that RhoA deletion or RhoA/Rock inhibition using Y27632 inhibitor reorganizes macrophage actin cytoskeleton, prevents macrophage movement to the cardiac allografts, and abrogates chronic rejection in rodent models. Although besides Y27632 there are other RhoA/ROCK inhibitors available commercially, their efficacy in inhibition of chronic rejection remains unknown. MethodsWe screened four RhoA/Rock inhibitors for their ability to inhibit chronic rejection of BALB/c [H-2d] mouse cardiac allografts heterotopically transplanted into C57BL/6 [H-2b] recipients. We also tested the effect of inhibitors on macrophages in vitro. ResultsWe found that out of four tested compounds, the Fasudil and Azaindole, inhibited vessel occlusion, tissue fibrosis, decreased M2 macrophage infiltration and abrogated chronic rejection of mouse cardiac allografts. The remaining inhibitors, SAR-407899 and SLX-2119, decreased only tissue fibrosis, and were ineffective or only slightly effective in inhibiting vessel occlusion. We also found that Azaindole and Fasudil affected actin cytoskeleton and protein expression in mouse peritoneal macrophages ConclusionResults of these studies might help in development of anti-chronic rejection therapy for clinical use.

Necroptosis Is Involved in CD4+ T Cell-Mediated Microvascular Endothelial Cell Death and Chronic Cardiac Allograft Rejection.

Despite advances in immunosuppressive therapies, the rate of chronic transplant loss remains substantial. Organ injury involves various forms of cell death including apoptosis and necrosis. We now recognize that early injury of cardiac transplants involves a newly described form of programmed necrotic cell death, termed necroptosis. Because this involves receptor-interacting protein (RIP) kinase 1/3, this study aimed to establish the role of RIP3 in chronic cardiac allograft rejection. We used major histocompatibility complex class II mismatched C57BL/6N (H-2; B6) or B6.RIP3 (H-2; RIP3) mice to B6.C-H-2 (H2-Ab1; bm12) mouse cardiac transplantation. Microvascular endothelial cells (MVEC) were developed from B6 and RIP3 cardiac grafts. CD4 T cell-mediated cardiac graft rejection is inhibited using RIP3 deficient donor grafts, with reduced cellular infiltration and vasculopathy compared with wild type cardiac grafts. Alloreactive CD4 T cell-mediated MVEC death involves TNFα, Fas ligand (FasL) and granzyme B. Although necroptosis and release of danger molecule high-mobility group box 1 are eliminated by the absence of RIP3, CD4 T cells had attenuated MVEC death through granzyme B and FasL. CD4 T cell-mediated MVEC death involves in TNFα, FasL and granzyme B. Necroptotic cell death and release of the danger molecule may promote inflammatory responses and transplant rejection. Although loss of RIP3 does not eliminate alloimmune responses, chronic graft injury is reduced. RIP3 is an important therapeutic target but additional granzyme and caspases inhibition is required for sufficiently improving long-term graft survival.

Chronic Rejection of Cardiac Allografts Is Associated With Increased Lymphatic Flow and Cellular Trafficking.

Cardiac transplantation is an excellent treatment for end-stage heart disease. However, rejection of the donor graft, in particular, by chronic rejection leading to cardiac allograft vasculopathy, remains a major cause of graft loss. The lymphatic system plays a crucial role in the alloimmune response, facilitating trafficking of antigen-presenting cells to draining lymph nodes. The encounter of antigen-presenting cells with T lymphocytes in secondary lymphoid organs is essential for the initiation of alloimmunity. Donor lymphatic vessels are not anastomosed to that of the recipient during transplantation. The pathophysiology of lymphatic disruption is unknown, and whether this disruption enhances or hinders the alloimmune responses is unclear. Although histological analysis of lymphatic vessels in donor grafts can yield information on the structure of the lymphatics, the function following cardiac transplantation is poorly understood. Using single-photon emission computed tomography/computed tomography lymphoscintigraphy, we quantified the lymphatic flow index following heterotrophic cardiac transplantation in a murine model of chronic rejection. Ten weeks following transplantation of a minor antigen (HY) sex-mismatched heart graft, the lymphatic flow index was significantly increased in comparison with sex-matched controls. Furthermore, the enhanced lymphatic flow index correlated with an increase in donor cells in the mediastinal draining lymph nodes; increased lymphatic vessel area; and graft infiltration of CD4+, CD8+ T cells, and CD68+ macrophages. Chronic rejection results in increased lymphatic flow from the donor graft to draining lymph nodes, which may be a factor in promoting cellular trafficking, alloimmunity, and cardiac allograft vasculopathy.

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.

What's Hot, What's New From the 2016 American Transplant Congress.

From June 11-15, 2016 the American Transplant Congress, the joint meeting of the American Society of Transplantation and the American Society of Transplant Surgeons, held its annual meeting in Boston, MA. The meeting, attended by 5200 registrants, included pre-meeting conferences, focused topic sessions, and hundreds of high-quality presentations from the transplant field. This meeting report highlights key findings from specific basic science, translational, and clinical research presentations deemed to have notable impact in thematic areas. In particular, there were a number of transformative studies indicating important advances in the understanding of alloimmunity, chronic rejection, tolerance, and organ-specific outcomes. Many of these results are discussed in the context of the published literature to showcase rapid advances in the transplant field.

Macrophage/monocyte-specific deletion of Ras homolog gene family member A (RhoA) downregulates fractalkine receptor and inhibits chronic rejection of mouse cardiac allografts

The cellular and molecular mechanisms of chronic rejection of transplanted organs remain obscure; however, macrophages are known to play a critical role in the injury and repair of allografts. Among multiple factors influencing macrophage infiltration to allografts, the fractalkine chemokine (C-X3-C motif) ligand 1(CX3CL1)/chemokine (C-X3-C motif) receptor 1 (CX3CR1) signaling pathway and actin cytoskeleton, which is regulated by a small guanosine-5׳-triphosphatase Ras homolog gene family member A (RhoA), are of the utmost importance. To define the role of macrophage/RhoA pathway involvement in chronic rejection, we generated mice with monocyte/macrophage-specific deletion of RhoA. Hearts from BALB/c (H-2d) donors were transplanted into RhoAflox/flox (no Cre) and heterozygous Lyz2Cre+/-RhoAflox/flox recipients treated with cytotoxic T-lymphocyte-associated protein 4 immunoglobulin to inhibit early T-cell response. Allografts were assessed for chronic rejection and monocyte/macrophage functions. The deletion of RhoA inhibited macrophage infiltration, neointimal hyperplasia of vasculature, and abrogated chronic rejection of the allografts. The RhoA deletion downregulated G protein-coupled fractalkine receptor CX3CR1, which activates the RhoA pathway and controls monocyte/macrophage trafficking into the vascular endothelium. This in turn promotes, through overproliferation and differentiation of smooth muscle cells in the arterial walls, neointimal hyperplasia. Our finding of codependence of chronic rejection on monocyte/macrophage CX3CR1/CX3CL1 and RhoA signaling pathways may lead to the development of novel anti-chronic rejection therapies.

Basophils Trigger Fibroblast Activation in Cardiac Allograft Fibrosis Development.

Fibrosis is a major component of chronic cardiac allograft rejection. Although several cell types are able to produce collagen, resident (donor-derived) fibroblasts are mainly responsible for excessive production of extracellular matrix proteins. It is currently unclear which cells regulate production of connective tissue elements in allograft fibrosis and how basophils, as potential producers of profibrotic cytokines, are involved this process. We studied this question in a fully MHC-mismatched model of heart transplantation with transient depletion of CD4(+) T cells to largely prevent acute rejection. The model is characterized by myocardial infiltration of leukocytes and development of interstitial fibrosis and allograft vasculopathy. Using depletion of basophils, IL-4-deficient recipients and IL-4 receptor-deficient grafts, we showed that basophils and IL-4 play crucial roles in activation of fibroblasts and development of fibrotic organ remodeling. In the absence of CD4(+) T cells, basophils are the predominant source of IL-4 in the graft and contribute to expansion of myofibroblasts, interstitial deposition of collagen and development of allograft vasculopathy. Our results indicated that basophils trigger the production of various connective tissue elements by myofibroblasts. Basophil-derived IL-4 may be an attractive target for treatment of chronic allograft rejection.

Platelet-derived Growth Factor-B Protects Rat Cardiac Allografts From Ischemia-reperfusion Injury.

Microvascular dysfunction and cardiomyocyte injury are hallmarks of ischemia-reperfusion injury (IRI) after heart transplantation. Platelet-derived growth factors (PDGF) have an ambiguous role in this deleterious cascade. On one hand, PDGF may exert vascular stabilizing and antiapoptotic actions through endothelial-pericyte and endothelial-cardiomyocyte crosstalk in the heart; and on the other hand, PDGF signaling mediates neointimal formation and exacerbates chronic rejection in cardiac allografts. The balance between these potentially harmful and beneficial actions determines the final outcome of cardiac allografts. We transplanted cardiac allografts from Dark Agouti rat and Balb mouse donors to fully major histocompatibility complex-mismatched Wistar Furth rat or C57 mouse recipients with a clinically relevant 2-hour cold ischemia and 1-hour warm ischemia. Ex vivo intracoronary delivery of adenovirus-mediated gene transfer of recombinant human PDGF-BB upregulated messenger RNA expression of anti-mesenchymal transition and survival factors BMP-7 and Bcl-2 and preserved capillary density in rat cardiac allografts at day 10. In mouse cardiac allografts PDGF receptor-β, but not -α intragraft messenger RNA levels were reduced and capillary protein localization was lost during IRI. The PDGF receptor tyrosine kinase inhibitor imatinib mesylate and a monoclonal antibody against PDGF receptor-α enhanced myocardial damage evidenced by serum cardiac troponin T release in the rat and mouse cardiac allografts 6 hours after reperfusion, respectively. Moreover, imatinib mesylate enhanced rat cardiac allograft vasculopathy, cardiac fibrosis, and late allograft loss at day 56. Our results suggest that PDGF-B signaling may play a role in endothelial and cardiomyocyte recovery from IRI after heart transplantation.