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

Abstract

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. Keywords: Chronic rejection, Transplantation, RhoA, ROCK, mTORC1/mTORC2, Everolimus, Y27632, Rat, : Abbreviations: Arg1, Arginase 1, HRP, horseradish peroxidase, iNos, inducible nitric oxide synthase, M1, pro-inflammatory macrophage subtype, M2, anti-inflammatory macrophage subtype, mTOR, mechanistic target of rapamycin, also known as mammalian target of rapamycin or FK506-binding protein 12-rapamycin-associated protein 1 [FRAP1], a kinase that is a member of the phosphatidylinositol 3-kinase-related protein kinase family., mTORC1, mammalian target of rapamycin complex 1, mTORC2, rapamycin-insensitive mTOR complex 2, Rac1, Ras-Related C3 Botulinum Toxin Substrate 1, RhoA, Ras homolog gene family, member A, Rictor, Rapamycin-insensitive companion of mammalian target of rapamycin, ROCK [ROCK1 and ROCK 2], p160ROCK, a Rho-associated, coiled-coil-containing protein kinase, VVG, Verhoeff-Van Gieson [VVG], a stain for elastic fibers, Y27632, Y-27632 2HCl, a selective inhibitor of ROCK1 [p160ROCK] kinase