Abrogation Of Chronic Rejection Involves Modulation Of The MTORC1and MTORC2Pathways

[Objective]We seek to develop a clinically applicable pathway-targeted therapy to inhibit the chronic rejection of organ allografts.Although progress in immunopharmacology has markedly improved the outcome of clinical transplantation the current impressive immunosuppressive armamentarium has failed to avert chronic rejection, which is the major threat to long-term successful organ transplantation. Therefore, a critical goal in transplantation research is to prevent chronic rejection while avoiding the side effects of global immunosuppression.We established a unique rat cardiac allograft model to study the mechanisms involved in the inhibition of chronic rejection and showed that the inhibition of chronic rejection involved abrogation of T cell infiltration, a process that requires the actin cytoskeleton-propelled migration of T cells into the allografts (1-9). Our recent studies showed that the inhibition of chronic rejection depends on early (1-7days) post-transplantation events, which involve a dramatic change in actin cytoskeleton organization and down-regulation of RhoA GTPase pathway molecules participating in actin polymerization(4,6).This novel finding that inhibiting the chronic rejection of allografts involves disrupting the integrity of T cell actin cytoskeleton via interference with RhoA GTPase pathway establishes a new paradigm and opens new frontiers in the study of Rho GTPase pathway-targeted immunosuppression therapies.The most recent studies indicate that the mammalian target of rapamycin (mTOR) kinase signaling is involved in regulation of cell migration via regulation of actin cytoskeleton and RhoA signaling pathway (10-13). Thus, we expect that the targeting the molecular components of mTOR and its downstream target Rac-1pathway during first few days of post-transplantation will prevent chronic rejection of rat cardiac allografts and will serve as a model for the development of the mTOR pathway-targeted immunospupression in human clinical intervention.[Methods]1. Transplantation design:Adult male inbred Wistar Furth (WF; RT1.Au) and ACI (RT1.Aa) rats will be used. Heterotopic cardiac transplants will be placed intra-abdominally as described previously (1-8). There will be three experimental groups:1) transplantation control group without any treatment,2) transplantation in the presence of a subtherapeutic dose of CsA (acute rejection), and3) transplantation in the presence of subtherapeutic dose of CsA supplemented with the allochimeric MHC I molecule (no acute, no chronic rejection).The allochimeric peptide [alhl/u]-RT1.Aa (GenWay, San Diego CA;1mg/kg) will be delivered through the portal vein into ACI recipients of WF hearts at the time of transplantation (1-8).Each experimental group will consist of5rats2. T cell isolation:T cells will be isolated from spleens of ACI host rats at one, three and seven days posttransplantation, and purified via a positive T cell isolation kit using magnetic anti-T cell micro beads (Miltenyi Biotech)(4,6,8)3. Western blot analysis:This anlaysis will allow us to assess and compare the levels of protein expression in chronic rejection versus inhibition of chronic rejection state. The isolated splenic T cells will be homogenized on ice in RIPA buffer (0.15M NaCl,1%deoxycholate Na salt,1%Triton X-100,0.1%SDS,0.01M Tris HC1, pH7.2) in the presence of Complete proteinase inhibitor. The SDS-PAGE and western blotting with mTOR pathway antibodies:anti-mTOR,-Raptor,-mLST8/GpL,-PRAS40and DEPTOR (components of mTORC1complex) and anti-Rictor and-mSIN1(components of mTORC2), and with anti-RAC1antibodies. Bands will be visualized using Lumi-Light Western blotting substrate and band intensity for each experimental group will be determined using Quantity One4.6.1system (Biorad).4. Immunostaining and double co-immunostaining will be performed to identify the distribution and co-distribution and level of expression of proteins of interest within the T cells isolated from chronic rejection versus chronic rejection inhibition experimental groups. T cells will be fixed in4%formalin in PBS-0.05%Tween, blocked, immunostain with above antibodies and after extensive washing mounted in Antifade with Hoechst and observed with a fluorescent microscope (6).5. Heterotopic cardiac transplants were performed between donor Wistar Furth (WF) and ACI recipient rats. Controls were either untreated or received therapeutic dose of cyclosporine (CsA,10mg/kg) for7days. The experimental groups of ACI recipients received one preoperative dose of the Y-27632inhibitor (2mg/kg, gavage feed) in conjunction with the sub-therapeutic dose of CsA for3days, or inhibitor alone for7days. The cardiac grafts were harvested at100days of post-transplantation for histological and immunohistochemical assessment of chronic rejection. Rat hearts were fixed in formalin, embedded in paraffin and5μM sections were cut at the mid-heart region. Sections were stained with Hematoxylin&Eosin (H&E), Trichrome and Verhoeff-van Gieson (VVG) using standard protocols. to assess heart global architectural integrity and inflammation, collagen content and neointimal index respectively.6. Statistical analysis:The Western Blot results were analyzed using Sigma plot,collagen value was the ratio between the collagen area/(collagen area+muscle area)×100%, Neointimal Index(NI) was the neointimal area/(neointimal area+lumen area)×100%.All the results were expressed as average±standard deviation and analyzed by T test with P-value<0.05considered as being significant.[Results]1. We successfully developed the rat cardiac allograft model system to study molecular pathways involved in acute and chronic rejection of the allograft.2.Western blots analysis showed that certain components of mTOR complex1/2 and the downstream signal RAC1were down-regulated in rats treated with CsA+allochimeric peptide. The inhibition of mTOR pathway decreased proliferation and actin-dependent T cell motility and attenuated the T cell infiltration into the graft3.Immunostaining:Racl is involved in the re-localization of mTOR from the peri-nuclear endoplasmic reticulum (ER) and Golgi cisternae to the plasma membrane in T cells isolated from the experimental group.4.Immunohistochemical stain:Comprehensive analysis of the allograft tissue and an assessment of cardiac vasculopathy showed dramatic differences in the degree of chronic rejection between allografts from recipients that had received Y-27632inhibitor in con-junction with sub-therapeutic dose of CsA and those treated with inhibitor alone or CsA alone. There was a significant reduction of the neointimal hyperplasia (NI) in the allograft from the recipients that received Y-27632inhibitor in conjunction with sub-therapeutic dose of CsA in comparison to those treated with therapeutic dose of CsA or inhibitor alone. Similar trend persisted in the collagen deposition present in the allografts5.Tissue integrity and cellular infiltration of cardiac allografts assessed by Hematoxylin&Eosin (H&E) staining of transplanted hearts at100days post-transplantation showed well-preserved tissue integrity, low degree of fibrosis and low cellular infiltration in the grafts from the recipients treated with one preoperative dose of the Y-27632inhibitor in conjunction with the sub-therapeutic dose of CsA when compared to the controls[Conclusions]1.The heterotopic cardiac transplantation model is crucial for the study of molecules and pathways involved in acute and chronic rejection of the allograft.2. We showed that in rat cardiac allografts, the inhibition of chronic rejection involves not only downregulation and re-distribution of the RhoA pathway components but also a downregulation and re-distribution of some components of mTOR (mTORC1and mTORC2) pathway and the downstream target Racl, which similar to RhoA, is a regulator of the actin cytoskeleton. The changes in actin cytoskeleton affect proliferation, movement and formation of lamellipodia in T cells, leading to decreased infiltration of the graft and attenuation of its chronic rejection. These results indicate that a deregulation of two parallel (RhoA and Racl) actin pathways plays a role in the inhibition of chronic rejection.3. Our novel finding that a single pre-operative dose of the RhoA pathway inhibitor Y-27632attenuates chronic rejection in rat cardiac model system warrants its potential as a novel therapeutic agent specific for the inhibition of chronic rejection.