- Email: [email protected]
ROCK pathways prevents chronic rejection of rat cardiac allografts
Accepted Manuscript
Coinhibition of mTORC1/mTORC2 and RhoA /ROCK pathways prevents chronic rejection of rat cardiac allografts Wei Chen , Wenhao Chen , Xian C Li , Rafik M Ghobrial , Malgorzata Kloc PII: DOI: Reference:
S2451-9596(18)30010-6 https://doi.org/10.1016/j.tpr.2018.09.002 TPR 18
To appear in:
Transplantation Reports
Received date: Accepted date:
10 September 2018 19 September 2018
Please cite this article as: Wei Chen , Wenhao Chen , Xian C Li , Rafik M Ghobrial , Malgorzata Kloc , Coinhibition of mTORC1/mTORC2 and RhoA /ROCK pathways prevents chronic rejection of rat cardiac allografts , Transplantation Reports (2018), doi: https://doi.org/10.1016/j.tpr.2018.09.002
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ACCEPTED MANUSCRIPT Highlights
RhoA/ROCK inhibitor Y27632 in combination with everolimus inhibits chronic rejection of cardiac allografts in a rat transplantation model.
Everolimus alone or in combination with Y27632 drastically reduces macrophage infiltration into the graft, while it does not decrease the T cell infiltration
Because everolimus inhibits the mTORC2 pathway, this indicates that
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macrophage infiltration of the graft strongly depends on mTORC2/RhoA signaling.
The coinhibition of the mTORC2 and RhoA pathways seems to be a promising avenue for the development of anti-chronic rejection therapies in human
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transplantation.
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ACCEPTED MANUSCRIPT Coinhibition of mTORC1/mTORC2 and RhoA /ROCK pathways prevents chronic rejection of rat cardiac allografts Wei Chen1, 2, Wenhao Chen1,3, Xian C Li1,3, Rafik M Ghobrial*1,3, Malgorzata Kloc1,3,4* 1
The Houston Methodist Research Institute, Houston, Texas, USA; 2Department of
Nephrology, Second Xiangya Hospital, Central South University, Changsha 410011,
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China; 3Weill Cornell Medical College, New York, NY, USA; 4The University of Texas, M.D. Anderson Cancer Center, Department of Genetics, Houston, Texas, USA
Corresponding Authors:
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Malgorzata Kloc and Rafik Mark Ghobrial The Houston Methodist Hospital, Department of Surgery 6550 Fannin St., Houston, TX 77030 Tel.: 713.441.6875 Fax: 713.790.3755
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E-mail: [email protected]
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E-mail: [email protected]
Disclosure Statement
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None of the authors has any conflict of interests
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ACCEPTED MANUSCRIPT Authorship page Wei Chen- performed all experiments, transplantations follow up, pathology analysis, statistical analysis ang graphs email: [email protected]
Wenhao Chen- performed all transplantations
Xian C Li- data analysis e-mail: [email protected]
Rafik M Ghobrial- concept and data analysis
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Email: [email protected]
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e-mail: [email protected]
Malgorzata Kloc-concept, data analysis, manuscript writing and figures
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e-mail: [email protected]
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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
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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
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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
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abrogates chronic rejection of rat cardiac allografts, and will aid in the development of
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clinically applicable anti-chronic rejection therapies.
Key words: chronic rejection, transplantation, RhoA, ROCK, mTORC1/mTORC2,
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everolimus, Y27632, rat
Abbreviations Arg1- Arginase 1 HRP- horseradish peroxidase 4
ACCEPTED MANUSCRIPT 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.
mTORC2- rapamycin-insensitive mTOR complex 2 Rac1- Ras-Related C3 Botulinum Toxin Substrate 1 RhoA- Ras homolog gene family, member A
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mTORC1- mammalian target of rapamycin complex 1,
Rictor- Rapamycin-insensitive companion of mammalian target of rapamycin
ROCK [ROCK1 and ROCK 2]- p160ROCK, a Rho-associated, coiled-coil-containing
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protein kinase
VVG- Verhoeff-Van Gieson [VVG], a stain for elastic fibers
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Y27632 - Y-27632 2HCl, a selective inhibitor of ROCK1 [p160ROCK] kinase
Introduction The majority of transplanted organs fail within a few months to a few years posttransplantation due to chronic rejection (1-5). The main symptoms of chronic rejection are the occlusion of blood vessels (formation of neointima) and the deposition 5
ACCEPTED MANUSCRIPT of collagen (fibrosis), which starve the graft and destroy tissue integrity. Currently, there is no cure for chronic rejection. The development of chronic rejection depends on a massive accumulation of macrophages within the graft that induce the over-proliferation of blood vessel walls (formation of the neointima) and the deposition of collagen. We have been seeking a method that would inhibit macrophage infiltration into the graft, and thus, prevent chronic rejection. Macrophage movement into the graft depends on the actin cytoskeleton, which is regulated by small GTPase RhoA and its downstream
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effectors ROCK1 and ROCK2 kinases (6-10). We showed previously that when the early T cell response is blocked, the inhibition of ROCK1 kinase with Y27632 or
macrophage-specific deletion of RhoA prevents macrophage accumulation within the graft, abrogates blood vessel occlusion and fibrosis, and inhibits the chronic rejection of rat and mouse cardiac allografts (6, 7, 15,34). We also showed that in a rodent
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transplantation model, an inhibition of chronic rejection correlated with a downregulation of the mTORC2 subunit of the mechanistic target of rapamycin (mTOR) pathway, which is a reciprocal regulator of RhoA signaling (11-15, 35). The mTOR serine-threonine kinase is a component of two distinct complexes, which differ in function and substrate specificity: mTORC1, which regulates cell growth and proliferation; and mTORC2, which
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through RhoA/Rac1, regulates the actin cytoskeleton and cell movement. mTORC1 contains mTOR, regulatory-associated protein of mTOR (Raptor), mammalian lethal
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with Sec13 protein 8 or mLST8 (GβL), proline-rich AKT substrate 40 kDa (PRAS40) and DEP-domain-containing mTOR-interacting protein (Deptor) (16). mTORC2 is composed
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of mTOR; GβL; Deptor; rapamycin-insensitive companion of mTOR (Rictor), which dictates actin cytoskeleton substrate specificity; mammalian stress-activated protein kinase-interacting protein (mSIN1); and protein observed with Rictor-1 (Protor-1) (16,
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17). Everolimus (RAD001, Zortress, Certican, Afinitor, Votubia, Evertor), a 40-O-(2hydroxyethyl) derivative of rapamycin (sirolimus), is used as an immunosuppressant in
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organ transplantation and cancer treatment (18-20). In contrast to rapamycin, which mainly affects mTORC1, everolimus antagonizes both mTORC1 and mTORC2. Everolimus inhibits mTORC1 by dissociating Raptor from mTOR, which in turn inhibits cell proliferation and migration and inhibits mTORC2 by dissociating Rictor and Sin1 from mTOR (21). Knowing that the inhibition of chronic rejection is correlated with a downregulation of mTORC2 and RhoA/ROCK, here, we studied the ability of the mTORC1/mTORC2 inhibitor everolimus in combination with the RhoA/ROCK inhibitor Y27632 to alleviate the chronic rejection of rat cardiac allografts. 6
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Material and Methods Animals All experiments were performed according to The Methodist Hospital Research Institute’s animal care and use NIH standards as set forth in the "Guide for the Care and Use of Laboratory Animals" [DHHS publication No. [NIH] 85-23 Revised 1985]. The Institute also mandates concordance with the PHS "Policy on Humane Care and Use of Animals Used in Testing, Research and Training.” Heart transplantations and treatments
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Laboratory Animals" and the NIH "Principles for the Utilization and Care of Vertebrate
Adult male inbred Wistar Furth (WF; RT1.Au) and ACI (RT1.Aa) rats were purchased
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from Envigo (USA). Heterotopic cardiac transplants were placed intra-abdominally as described previously (6, 22, 23). There were 12 transplantation groups: 1. Control group with a 3-day course of rapamycin alone (2 mg/kg body weight, gavage, day 0-3 posttransplantation) treatment; 2. Control group with a 3-day course of rapamycin (2 mg/kg body weight, gavage, day 0-3 posttransplantation) in conjunction with 1 dose of
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Y27632 (2 mg/kg, gavage, day 0); 3. Control group with a 3-day course of rapamycin (2 mg/kg body weight, gavage day 0-3 posttransplantation) in conjunction with 2 doses of
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Y27632 (2 mg/kg, gavage, day 0 and day 2); 4. Control group with a 3-day course of rapamycin (2 mg/kg body weight, gavage, day 0-3 posttransplantation) in conjunction
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with 3 doses of Y27632 (2 mg/kg, gavage, day 0, day 2, day 4); 5. everolimus alone group (0.5 mg/kg, gavage, day 0-28 posttransplantation); 6. everolimus (0.5 mg/kg, gavage, day 0-28 posttransplantation) in conjunction with 1 dose of Y27632 (2 mg/kg,
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gavage, day 0); 7. everolimus (0.5 mg/kg, gavage, day 0-28 posttransplantation) in conjunction with 2 doses of Y27632 (2 mg/kg, gavage, day 0, day 2); 8. everolimus (0.5
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mg/kg, gavage, day 0-28 posttransplantation) in conjunction with 3 doses of Y27632 (2 mg/kg, gavage, day 0, day 2, day 4); 9. everolimus (5 mg/kg, gavage, day 0-28 posttransplantation) alone; 10. everolimus (5 mg/kg, gavage, day 0-28 posttransplantation) in conjunction with 1 dose of Y27632 (2 mg/kg, gavage, day 0); 11. everolimus (5 mg/kg, gavage, day 0-28 posttransplantation) in conjunction with 2 doses of Y27632 (2 mg/kg, gavage, day 0, day 2); and 12. everolimus (5 mg/kg, gavage, day 0-28 posttransplantation) in conjunction with 3 doses of Y27632 (2 mg/kg, gavage, day
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ACCEPTED MANUSCRIPT 0, day 2, day 4). All rapamycin groups consisted of 3 animals each, and all everolimus groups consisted of 5 animals each.
Inhibitors Everolimus (RAD001, cat # S1120), rapamycin (cat #S103) and Y27632 (cat# S1049) were purchased from Selleck Chemicals (Houston TX, USA).
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Pathology
Transplanted hearts were fixed in 10% formalin, embedded in Paraplast, sectioned at 8 m, deparaffinized, and stained with Verhoeff-Van Gieson (VVG) or trichrome stain. VVG staining was used to analyze the neointima and is presented as the neointimal index [NI= [intimal area]/ [luminal area + intimal area] x100]. Collagen deposition was
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quantified from Trichrome stained slides using Image-Pro plus and is presented as % of stained area/total area. Five animals from each experimental group were analyzed. Standard error was calculated in Excel using formula STDEV [range] / SQRT[COUNT[range]] and plotted using Sigma plot.
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Immunostaining
Deparaffinized sections of transplanted hearts were immunostained as previously
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described (7) for macrophage markers: iNOS (ABCAM #ab153230), Arginase 1 (ABCAM #ab91279, CD68 (ABCAM #ab31630), and T cell markers: CD3 (Invitrogen # MA181580), CD4 (Invitrogen # MA181588), CD25 (Invitrogen # MA170019), and Foxp3
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(Invitrogen #14477482). The secondary antibodies were Rabbit Anti-Mouse IgG H&L (HRP, ABCAM #ab6728) and Goat Anti-Rabbit IgG H&L (HRP, ABCAM #ab6721). The
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infiltration levels of T cells and macrophages were assessed by analyzing areas of positive staining on three immunostained slides from each animal (3-5 animals per
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experimental group), using Image Pro Plus.
Results Treatment with everolimus alone or in conjunction with Y27632 prolongs graft survival. 8
ACCEPTED MANUSCRIPT Treatment with rapamycin (day 0-3 posttransplantation, 2 mg/kg body weight, gavage feed) alone resulted in the acute rejection of the graft at approximately 11 days posttransplantation (Fig. 1A). Administration of rapamycin in conjunction with 1, 2 or 3 doses of Y27632 (2 mg/kg, gavage, day 0, day 2, day 4) prolonged graft survival to 1317 days posttransplantation (Fig. 1A). All rapamycin groups consisted of 3 animals each. In striking contrast to these results, the everolimus (0.5 mg/kg or 5 mg/kg, gavage delivery, day 0-28 posttransplantation) alone or in conjunction with Y27632 (2 mg/kg,
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gavage delivery, day 0, day 2, day 4 posttransplantation) resulted in long-term (>100 days) graft survival (Fig. 1B, C). All everolimus groups consisted of 5 animals each. Our previous studies showed that, in the same rat transplantation model, a therapeutic dose of cyclosporin (CsA) alone (10 mg/kg, day 0 - 6, gavage delivery) or seven-day
treatment with Y-27632 alone (2 mg/kg, day 0 - 6, gavage delivery) resulted in long-term
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(>100 days) graft survival, but all these grafts had symptoms of chronic rejection
(6).These studies also showed that the pretransplantation gavage delivery of a single dose (2 mg/kg) of Y-27632 in conjunction with a subtherapeutic dose of CsA (10 mg/kg, day 0 - 2) resulted in long-term graft survival and inhibition of chronic rejection (Table 1;
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6).
Everolimus in combination with Y27632 inhibits chronic rejection
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Pathological analysis of vessel occlusion and tissue fibrosis in transplanted hearts showed that everolimus at the 5 mg/kg dose was more effective in inhibiting vessel
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occlusion than everolimus at the 0.5 mg /kg dose (NI= 24.6 ±18.5 versus NI =45.2 ± 23.8) (Fig. 2A, Table 1). The addition of Y27632 to the everolimus caused a statistically significant and dose-dependent decrease in vessel occlusion when compared to
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everolimus alone (Fig. 2A, B, Table 1 and Suppl. Fig. 1). The P values for the vessel occlusion graph shown in Fig. 2A are summarized in Suppl. Fig. 1.
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Because graft recipients who were treated with rapamycin alone or in combination with Y2763 rejected their grafts within 11-17 days posttransplantation, the grafts did not yet have the vessel occlusion typical for chronic rejection. However, pathological analysis showed that, as expected in acute rejection, the integrity of the graft vessels and tissues was visibly compromised (Fig. 2B). The analysis of collagen deposition showed that there was a slight decrease in collagen deposition in recipients receiving everolimus at 5 mg/kg in comparison to everolimus at 0.5 mg/kg (Fig. 2C, Table 1). However, the addition of Y27632 to everolimus caused a statistically significant decrease in collagen 9
ACCEPTED MANUSCRIPT deposition (Fig. 2C, Table 1). The P values for the collagen content graph shown in Fig. 2C are summarized in Suppl. Fig. 2.
Everolimus inhibits macrophage but not T cell infiltration into the graft We used immunostaining with a macrophage marker, a transmembrane glycoprotein, cluster of differentiation 68 (CD68), to assess the extent of macrophage infiltration into the grafts. CD68 binds to lectins /selectins expressed by various tissues, allowing
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macrophages to crawl over selectin-bearing cells/tissue and home in on particular
targets. We also used immunostaining with inducible nitric oxide synthase (iNOS) and arginine (Arg1) markers to assess the degree of infiltration with M1 and M2
macrophages, respectively. These studies showed that everolimus alone or in
combination with Y27632 drastically reduced the number of CD68+, iNos+ and Arg1+
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macrophages when compared to rapamycin alone or in combination with Y27632
treatment (Fig. 3, 4). The P values of the differences in macrophage infiltration between different experimental groups are shown in Suppl. Fig. 3.
We also tested whether everolimus alone or in conjunction with Y27632 affected T cell infiltration of the grafts. Immunostaining was performed with the following T cell
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markers: the cluster of differentiation 3 (CD3) present on mature T cells, the cluster of differentiation 4 (CD4) present on T helper cells, the α-chain of the IL-2 receptor (CD25)
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expressed on activated T cells, and the forkhead box P3 transcription factor (Foxp3) expressed by natural T regulatory cells (nTregs) and adaptive/induced T regulatory cells
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(a/iTregs). This study showed that in contrast to the macrophage infiltration, the rapamycin alone or in combination with Y27632 was much more effective in decreasing T cell infiltration than everolimus alone or in combination with Y27632 (Fi.3, 4). The P
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values of the differences in T cell infiltration between different experimental groups are
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shown in Suppl. Fig. 4.
Discussion
It is well established that vessel occlusion during chronic rejection depends, to a high degree, on the function of graft infiltrating macrophages, which induce the overproliferation of smooth muscle in the vessel wall and the formation of the neointima (2, 26-33). Macrophages are also partially responsible for an increased deposition of collagen; they by themselves deposit collagen and stimulate fibroblasts to produce an excess of collagen, all of which leads to tissue fibrosis. (2, 26-33). There are two major 10
ACCEPTED MANUSCRIPT hypotheses concerning vascular remodeling during chronic rejection. The “inside–out” hypothesis argues that immune injury during transplantation is initiated at the endothelial layer of the intima, i.e., at the luminal (inside) surface of the vessel (Fig. 5A). The vascular endothelium produces cytokines and inflammatory molecules that recruit leukocytes (predominantly macrophages) and stimulate macrophages present on the surface of the intima. This immune cell infiltration results in endothelium injury and the initiation of an inflammatory process, which in turn, stimulate smooth muscle cells
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(SMCs) of the media to acquire a proliferative phenotype, which results in the thickening of the intima (formation of the neointima) and vessel occlusion (2). The more recent “outside-in” hypothesis assumes that the inflammatory response starts at the outer layer of the vessel, i.e., at the adventitia, and propagates inwards toward the intima (Fig. 5); then, macrophages stimulate a phenotypic switch of resident progenitor/stem cells
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residing in the adventitia into SMCs, which proliferate and migrate from the adventitia to form the neointima (2). No matter which of these two hypotheses (or both) is (are) correct, all these studies indicate that macrophage-targeted therapies (Fig. 5) should have beneficial effects on the long-term outcome of the grafts in clinical transplantation. We showed here that everolimus in conjunction with Y27632 improves the long-term
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outcome (inhibits chronic rejection) of cardiac allografts in a rat transplantation model. Everolimus and its analog rapamycin are mTOR inhibitors approved by the FDA for use
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in solid organ transplantation as immunosuppressants (24). Structurally and functionally, rapamycin and everolimus resemble tacrolimus (also known as fujimycin or
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FK506). They all bind to the intracellular tacrolimus (FK506)–binding proteins, such as FKBP12 and other proteins from the same family of proteins, which inhibit mTORC1, which regulates the cell cycle, proliferation and cell metabolism in response to
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interleukin 2 (IL-2) signaling. Recent studies indicate that everolimus, besides inhibiting mTORC1, is also very effective in the inhibition of mTORC2, which regulates, via the
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RhoA pathway, the actin cytoskeleton (16, 21, 24, 25). We have shown previously that inhibition of the RhoA pathway (by RhoA deletion or RhoA/ROCK inhibition by Y27632, Azaindole or Fasudil) in the recipients of cardiac allografts decreased macrophage infiltration, vessel occlusion and tissue fibrosis in rodent model systems (6, 7,15, 34). We also showed that the inhibition of chronic rejection was correlated with a downregulation of the mTORC2 pathway, which is an upstream regulator of RhoA signaling (35). This finding suggested that a coinhibition of RhoA and mTORC2 pathways should have beneficial effects on allografts in preventing chronic rejection. 11
ACCEPTED MANUSCRIPT Here, we showed that, indeed, the addition of the RhoA/ROCK inhibitor Y27632 in combination with everolimus improved the long-term outcome and inhibited chronic rejection of cardiac allografts in a rat transplantation model. We also showed that everolimus alone or in combination with Y27632 drastically reduced macrophage infiltration into the graft, while it did not decrease the T cell infiltration when compared to rapamycin alone or in combination with Y27632. Because everolimus is much more effective than rapamycin in inhibiting the mTORC2 pathway, this indicates that
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macrophage infiltration of the graft strongly depends on mTORC2/RhoA signaling.
Considering the prominent role of macrophages in the process of chronic rejection, the coinhibition of the mTORC2 and RhoA pathways seems to be a promising avenue for
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the development of anti-chronic rejection therapies in human transplantation.
Acknowledgements
We are grateful for the support from Novartis.
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Figure Legends
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ACCEPTED MANUSCRIPT
Fig.1. Allograft survival in everolimus and rapamycin treatment groups A) Survival curve of cardiac allografts in recipients treated with rapamycin alone, or in conjunction with different doses of Y27632. Grafts were acutely rejected within 11-17 days posttransplantation. Each experimental group consisted of 3 animals. B) Survival curve of cardiac allografts in recipients treated with 0.5 mg/kg everolimus alone or in 16
ACCEPTED MANUSCRIPT conjunction with different doses of Y27632 in comparison to rapamycin treatment alone. All everolimus groups had long-term (>100 days) graft survival. Each experimental group consisted of 5 animals. C) Survival curve of cardiac allografts in recipients treated with 5 mg/kg everolimus alone or in conjunction with different doses of Y27632 in comparison to rapamycin treatment alone. All everolimus groups had long-term (>100
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days) graft survival. Each experimental group consisted of 5 animals.
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Fig. 2. Everolimus inhibits vessel occlusion, fibrosis and chronic rejection A) The graph shows that addition of Y27632 to 0.5 mg/kg or 5 mg/kg everolimus results in a statistically significant decrease in blood vessel occlusion. The P values for the graph are summarized in Suppl. Fig. 1. B) The examples of blood vessels from the graft 18
ACCEPTED MANUSCRIPT of the recipient treated with rapamycin + 1 dose of Y27632, procured at 13 days posttransplantation (left panel), and from the graft of the recipient treated with everolimus 5 mg/kg + Y27632 (right panel), procured at 100 days posttransplantation, stained with VVG, show the compromised integrity of vessels and tissue in rapamycin treatment. The bar is equal to 100 μm. C) The graph shows that the addition of 1 dose of Y27632 to 0.5 mg/kg everolimus results in a statistically significant decrease in
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collagen content. The P values for the graph are summarized in Suppl. Fig. 2.
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Fig. 3. Everolimus alone or in combination with Y27632 inhibits macrophage but not T cell infiltration into the graft. A) The graph shows that treatment with everolimus at 0.5 mg/kg or 5 mg/kg dose alone or in combination with Y27632 significantly decreases CD68-positive macrophage infiltration (expressed in intensity of density (IOD) values) of the graft in comparison to the treatment with rapamycin alone or in combination with Y27632. The P values for the 20
ACCEPTED MANUSCRIPT graph are summarized in Suppl. Fig. 3. B) The graph shows that treatment with everolimus alone at 0.5 mg/kg or 5 mg/kg doses or in combination with Y27632 results in higher infiltration (expressed in intensity of density (IOD) values) with CD3-positive T cells than treatment with rapamycin alone or in combination with Y27632. The P values
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for the graph are summarized in Suppl. Fig. 4.
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Figure 4. Immunostaining of macrophage and T cell infiltration into the grafts Sections of cardiac allografts immunostained with macrophage markers: CD68, iNOS (M1 marker) and Arg-1 (M2 marker) or CD3 T cell marker. A-I) Sections of cardiac allografts from recipients treated with rapamycin in conjunction with Y27632 (day 0, day 2, day 4) immunostained with antibodies against CD68, iNos and Arg-1, and HRP22
ACCEPTED MANUSCRIPT conjugated secondary antibody show high infiltration with CD68+ and Arg1+ macrophages (represented by brown spots). Hearts were procured 14 days posttransplantation. D-F) Sections of cardiac allografts from recipients treated with everolimus alone at a 5 mg/kg dose immunostained with antibodies against CD68, iNos and Arg-1, and HRP-conjugated secondary antibody show low infiltration with CD68+, iNOS+, and Arg1+ macrophages. Hearts were procured 100 days posttransplantation. G-I) Sections of cardiac allografts from recipients treated with everolimus at a 5 mg/kg
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dose in combination with Y27632 (day 0, day 2, day 4) immunostained with antibodies against CD68, iNos and Arg-1, and HRP-conjugated secondary antibody show low
infiltration with CD68+, iNOS+, and Arg1+ macrophages. Hearts were procured at 100 days posttransplantation. J) A section of a cardiac allograft from a recipient treated with rapamycin in conjunction with Y27632 (day 0, day 2) immunostained with antibody
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against T cell marker CD3 shows very low infiltration with CD3+ T cells. The heart was procured at 13 days posttransplantation. K) A section of a cardiac allograft from a recipient treated with everolimus at 0.5 mg/kg in conjunction with Y27632 (day 0, day 2) immunostained with antibody against T cell marker CD3 shows higher infiltration with CD3+T cells than in rapamycin treatment. The heart was procured at 100 days
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posttransplantation. In all panels, the bar is equal to 100 μm.
Figure 5. Blood vessel composition, chronic rejection and the RhoA/mTOR pathway 23
ACCEPTED MANUSCRIPT A) The wall of large blood vessels (arteries and veins) contains three distinct layers: Tunica intima, which contains a single layer of endothelial cells; Tunica media, which is composed of extracellular matrix, smooth muscle cells (SMCs) and a thick elastic band called the external elastic lamina; and Tunica adventitia, which contains stem/progenitor cells and fibroblasts. The vascular remodeling that occurs during chronic rejection involves the thickening of the intima, which becomes the neointima through the recruitment and proliferation of smooth muscle cells (SMCs) and deposition
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of collagen from fibroblasts and macrophages. Vascular remodeling depends on the function of macrophages, which signal through the endothelial cells or adventitia or both (see “the inside-out” and “outside-in” hypothesis in the text). B) RhoA regulates the macrophage actin cytoskeleton via its downstream effector ROCK kinase. RhoA is
reciprocally regulated by mTORC2. In contrast, mTORC1 regulates cell proliferation,
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growth and metabolism. Everolimus inhibits both mTORC1 and mTORC2, while Y27632 inhibits ROCK kinase. The combination of everolimus and Y27632, by disorganizing the actin cytoskeleton, prevents macrophage infiltration into the graft and alleviates
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macrophage-driven vascular remodeling and chronic rejection.
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NI
Everolimus 0.5mg/kg
45.2 ± 23.8
Collagen Content 16.9%
Everolimus 0.5mg/kg + Y27632 day 0 Everolimus 0.5mg/kg + Y27632 day 0, day 2 Everolimus 0.5mg/kg + Y27632 day 0, day 2, day 4 Everolimus 5mg/kg
33.5 ± 19.7
10.7%
21.8 ± 8.7
15.3%
16.8 ± 2.7
11.2%
24.6 ± 18.5
14.9%
Everolimus 5mg/kg + Y27632 day 0 Everolimus 5mg/kg + Y27632 day 0, day 2 Everolimus 5mg/kg + Y27632 day 0, day 2, day 4
20.6 ± 4.4
15.2%
23.2 ± 4.5
11.9%
17.0 ± 3.48
11.5%
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Table 1 Treatment
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Coinhibition of mTORC1/mTORC2 and RhoA /ROCK pathways prevents chronic rejection of rat cardiac allografts Wei Chen , Wenhao Chen , Xian C Li , Rafik M Ghobrial , Malgorzata Kloc PII: DOI: Reference:
S2451-9596(18)30010-6 https://doi.org/10.1016/j.tpr.2018.09.002 TPR 18
To appear in:
Transplantation Reports
Received date: Accepted date:
10 September 2018 19 September 2018
Please cite this article as: Wei Chen , Wenhao Chen , Xian C Li , Rafik M Ghobrial , Malgorzata Kloc , Coinhibition of mTORC1/mTORC2 and RhoA /ROCK pathways prevents chronic rejection of rat cardiac allografts , Transplantation Reports (2018), doi: https://doi.org/10.1016/j.tpr.2018.09.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Highlights
RhoA/ROCK inhibitor Y27632 in combination with everolimus inhibits chronic rejection of cardiac allografts in a rat transplantation model.
Everolimus alone or in combination with Y27632 drastically reduces macrophage infiltration into the graft, while it does not decrease the T cell infiltration
Because everolimus inhibits the mTORC2 pathway, this indicates that
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macrophage infiltration of the graft strongly depends on mTORC2/RhoA signaling.
The coinhibition of the mTORC2 and RhoA pathways seems to be a promising avenue for the development of anti-chronic rejection therapies in human
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transplantation.
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ACCEPTED MANUSCRIPT Coinhibition of mTORC1/mTORC2 and RhoA /ROCK pathways prevents chronic rejection of rat cardiac allografts Wei Chen1, 2, Wenhao Chen1,3, Xian C Li1,3, Rafik M Ghobrial*1,3, Malgorzata Kloc1,3,4* 1
The Houston Methodist Research Institute, Houston, Texas, USA; 2Department of
Nephrology, Second Xiangya Hospital, Central South University, Changsha 410011,
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China; 3Weill Cornell Medical College, New York, NY, USA; 4The University of Texas, M.D. Anderson Cancer Center, Department of Genetics, Houston, Texas, USA
Corresponding Authors:
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Malgorzata Kloc and Rafik Mark Ghobrial The Houston Methodist Hospital, Department of Surgery 6550 Fannin St., Houston, TX 77030 Tel.: 713.441.6875 Fax: 713.790.3755
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E-mail: [email protected]
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E-mail: [email protected]
Disclosure Statement
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None of the authors has any conflict of interests
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ACCEPTED MANUSCRIPT Authorship page Wei Chen- performed all experiments, transplantations follow up, pathology analysis, statistical analysis ang graphs email: [email protected]
Wenhao Chen- performed all transplantations
Xian C Li- data analysis e-mail: [email protected]
Rafik M Ghobrial- concept and data analysis
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Email: [email protected]
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e-mail: [email protected]
Malgorzata Kloc-concept, data analysis, manuscript writing and figures
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e-mail: [email protected]
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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
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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
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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
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abrogates chronic rejection of rat cardiac allografts, and will aid in the development of
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clinically applicable anti-chronic rejection therapies.
Key words: chronic rejection, transplantation, RhoA, ROCK, mTORC1/mTORC2,
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everolimus, Y27632, rat
Abbreviations Arg1- Arginase 1 HRP- horseradish peroxidase 4
ACCEPTED MANUSCRIPT 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.
mTORC2- rapamycin-insensitive mTOR complex 2 Rac1- Ras-Related C3 Botulinum Toxin Substrate 1 RhoA- Ras homolog gene family, member A
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mTORC1- mammalian target of rapamycin complex 1,
Rictor- Rapamycin-insensitive companion of mammalian target of rapamycin
ROCK [ROCK1 and ROCK 2]- p160ROCK, a Rho-associated, coiled-coil-containing
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protein kinase
VVG- Verhoeff-Van Gieson [VVG], a stain for elastic fibers
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Y27632 - Y-27632 2HCl, a selective inhibitor of ROCK1 [p160ROCK] kinase
Introduction The majority of transplanted organs fail within a few months to a few years posttransplantation due to chronic rejection (1-5). The main symptoms of chronic rejection are the occlusion of blood vessels (formation of neointima) and the deposition 5
ACCEPTED MANUSCRIPT of collagen (fibrosis), which starve the graft and destroy tissue integrity. Currently, there is no cure for chronic rejection. The development of chronic rejection depends on a massive accumulation of macrophages within the graft that induce the over-proliferation of blood vessel walls (formation of the neointima) and the deposition of collagen. We have been seeking a method that would inhibit macrophage infiltration into the graft, and thus, prevent chronic rejection. Macrophage movement into the graft depends on the actin cytoskeleton, which is regulated by small GTPase RhoA and its downstream
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effectors ROCK1 and ROCK2 kinases (6-10). We showed previously that when the early T cell response is blocked, the inhibition of ROCK1 kinase with Y27632 or
macrophage-specific deletion of RhoA prevents macrophage accumulation within the graft, abrogates blood vessel occlusion and fibrosis, and inhibits the chronic rejection of rat and mouse cardiac allografts (6, 7, 15,34). We also showed that in a rodent
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transplantation model, an inhibition of chronic rejection correlated with a downregulation of the mTORC2 subunit of the mechanistic target of rapamycin (mTOR) pathway, which is a reciprocal regulator of RhoA signaling (11-15, 35). The mTOR serine-threonine kinase is a component of two distinct complexes, which differ in function and substrate specificity: mTORC1, which regulates cell growth and proliferation; and mTORC2, which
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through RhoA/Rac1, regulates the actin cytoskeleton and cell movement. mTORC1 contains mTOR, regulatory-associated protein of mTOR (Raptor), mammalian lethal
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with Sec13 protein 8 or mLST8 (GβL), proline-rich AKT substrate 40 kDa (PRAS40) and DEP-domain-containing mTOR-interacting protein (Deptor) (16). mTORC2 is composed
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of mTOR; GβL; Deptor; rapamycin-insensitive companion of mTOR (Rictor), which dictates actin cytoskeleton substrate specificity; mammalian stress-activated protein kinase-interacting protein (mSIN1); and protein observed with Rictor-1 (Protor-1) (16,
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17). Everolimus (RAD001, Zortress, Certican, Afinitor, Votubia, Evertor), a 40-O-(2hydroxyethyl) derivative of rapamycin (sirolimus), is used as an immunosuppressant in
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organ transplantation and cancer treatment (18-20). In contrast to rapamycin, which mainly affects mTORC1, everolimus antagonizes both mTORC1 and mTORC2. Everolimus inhibits mTORC1 by dissociating Raptor from mTOR, which in turn inhibits cell proliferation and migration and inhibits mTORC2 by dissociating Rictor and Sin1 from mTOR (21). Knowing that the inhibition of chronic rejection is correlated with a downregulation of mTORC2 and RhoA/ROCK, here, we studied the ability of the mTORC1/mTORC2 inhibitor everolimus in combination with the RhoA/ROCK inhibitor Y27632 to alleviate the chronic rejection of rat cardiac allografts. 6
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Material and Methods Animals All experiments were performed according to The Methodist Hospital Research Institute’s animal care and use NIH standards as set forth in the "Guide for the Care and Use of Laboratory Animals" [DHHS publication No. [NIH] 85-23 Revised 1985]. The Institute also mandates concordance with the PHS "Policy on Humane Care and Use of Animals Used in Testing, Research and Training.” Heart transplantations and treatments
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Laboratory Animals" and the NIH "Principles for the Utilization and Care of Vertebrate
Adult male inbred Wistar Furth (WF; RT1.Au) and ACI (RT1.Aa) rats were purchased
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from Envigo (USA). Heterotopic cardiac transplants were placed intra-abdominally as described previously (6, 22, 23). There were 12 transplantation groups: 1. Control group with a 3-day course of rapamycin alone (2 mg/kg body weight, gavage, day 0-3 posttransplantation) treatment; 2. Control group with a 3-day course of rapamycin (2 mg/kg body weight, gavage, day 0-3 posttransplantation) in conjunction with 1 dose of
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Y27632 (2 mg/kg, gavage, day 0); 3. Control group with a 3-day course of rapamycin (2 mg/kg body weight, gavage day 0-3 posttransplantation) in conjunction with 2 doses of
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Y27632 (2 mg/kg, gavage, day 0 and day 2); 4. Control group with a 3-day course of rapamycin (2 mg/kg body weight, gavage, day 0-3 posttransplantation) in conjunction
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with 3 doses of Y27632 (2 mg/kg, gavage, day 0, day 2, day 4); 5. everolimus alone group (0.5 mg/kg, gavage, day 0-28 posttransplantation); 6. everolimus (0.5 mg/kg, gavage, day 0-28 posttransplantation) in conjunction with 1 dose of Y27632 (2 mg/kg,
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gavage, day 0); 7. everolimus (0.5 mg/kg, gavage, day 0-28 posttransplantation) in conjunction with 2 doses of Y27632 (2 mg/kg, gavage, day 0, day 2); 8. everolimus (0.5
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mg/kg, gavage, day 0-28 posttransplantation) in conjunction with 3 doses of Y27632 (2 mg/kg, gavage, day 0, day 2, day 4); 9. everolimus (5 mg/kg, gavage, day 0-28 posttransplantation) alone; 10. everolimus (5 mg/kg, gavage, day 0-28 posttransplantation) in conjunction with 1 dose of Y27632 (2 mg/kg, gavage, day 0); 11. everolimus (5 mg/kg, gavage, day 0-28 posttransplantation) in conjunction with 2 doses of Y27632 (2 mg/kg, gavage, day 0, day 2); and 12. everolimus (5 mg/kg, gavage, day 0-28 posttransplantation) in conjunction with 3 doses of Y27632 (2 mg/kg, gavage, day
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ACCEPTED MANUSCRIPT 0, day 2, day 4). All rapamycin groups consisted of 3 animals each, and all everolimus groups consisted of 5 animals each.
Inhibitors Everolimus (RAD001, cat # S1120), rapamycin (cat #S103) and Y27632 (cat# S1049) were purchased from Selleck Chemicals (Houston TX, USA).
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Pathology
Transplanted hearts were fixed in 10% formalin, embedded in Paraplast, sectioned at 8 m, deparaffinized, and stained with Verhoeff-Van Gieson (VVG) or trichrome stain. VVG staining was used to analyze the neointima and is presented as the neointimal index [NI= [intimal area]/ [luminal area + intimal area] x100]. Collagen deposition was
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quantified from Trichrome stained slides using Image-Pro plus and is presented as % of stained area/total area. Five animals from each experimental group were analyzed. Standard error was calculated in Excel using formula STDEV [range] / SQRT[COUNT[range]] and plotted using Sigma plot.
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Immunostaining
Deparaffinized sections of transplanted hearts were immunostained as previously
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described (7) for macrophage markers: iNOS (ABCAM #ab153230), Arginase 1 (ABCAM #ab91279, CD68 (ABCAM #ab31630), and T cell markers: CD3 (Invitrogen # MA181580), CD4 (Invitrogen # MA181588), CD25 (Invitrogen # MA170019), and Foxp3
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(Invitrogen #14477482). The secondary antibodies were Rabbit Anti-Mouse IgG H&L (HRP, ABCAM #ab6728) and Goat Anti-Rabbit IgG H&L (HRP, ABCAM #ab6721). The
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infiltration levels of T cells and macrophages were assessed by analyzing areas of positive staining on three immunostained slides from each animal (3-5 animals per
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experimental group), using Image Pro Plus.
Results Treatment with everolimus alone or in conjunction with Y27632 prolongs graft survival. 8
ACCEPTED MANUSCRIPT Treatment with rapamycin (day 0-3 posttransplantation, 2 mg/kg body weight, gavage feed) alone resulted in the acute rejection of the graft at approximately 11 days posttransplantation (Fig. 1A). Administration of rapamycin in conjunction with 1, 2 or 3 doses of Y27632 (2 mg/kg, gavage, day 0, day 2, day 4) prolonged graft survival to 1317 days posttransplantation (Fig. 1A). All rapamycin groups consisted of 3 animals each. In striking contrast to these results, the everolimus (0.5 mg/kg or 5 mg/kg, gavage delivery, day 0-28 posttransplantation) alone or in conjunction with Y27632 (2 mg/kg,
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gavage delivery, day 0, day 2, day 4 posttransplantation) resulted in long-term (>100 days) graft survival (Fig. 1B, C). All everolimus groups consisted of 5 animals each. Our previous studies showed that, in the same rat transplantation model, a therapeutic dose of cyclosporin (CsA) alone (10 mg/kg, day 0 - 6, gavage delivery) or seven-day
treatment with Y-27632 alone (2 mg/kg, day 0 - 6, gavage delivery) resulted in long-term
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(>100 days) graft survival, but all these grafts had symptoms of chronic rejection
(6).These studies also showed that the pretransplantation gavage delivery of a single dose (2 mg/kg) of Y-27632 in conjunction with a subtherapeutic dose of CsA (10 mg/kg, day 0 - 2) resulted in long-term graft survival and inhibition of chronic rejection (Table 1;
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6).
Everolimus in combination with Y27632 inhibits chronic rejection
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Pathological analysis of vessel occlusion and tissue fibrosis in transplanted hearts showed that everolimus at the 5 mg/kg dose was more effective in inhibiting vessel
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occlusion than everolimus at the 0.5 mg /kg dose (NI= 24.6 ±18.5 versus NI =45.2 ± 23.8) (Fig. 2A, Table 1). The addition of Y27632 to the everolimus caused a statistically significant and dose-dependent decrease in vessel occlusion when compared to
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everolimus alone (Fig. 2A, B, Table 1 and Suppl. Fig. 1). The P values for the vessel occlusion graph shown in Fig. 2A are summarized in Suppl. Fig. 1.
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Because graft recipients who were treated with rapamycin alone or in combination with Y2763 rejected their grafts within 11-17 days posttransplantation, the grafts did not yet have the vessel occlusion typical for chronic rejection. However, pathological analysis showed that, as expected in acute rejection, the integrity of the graft vessels and tissues was visibly compromised (Fig. 2B). The analysis of collagen deposition showed that there was a slight decrease in collagen deposition in recipients receiving everolimus at 5 mg/kg in comparison to everolimus at 0.5 mg/kg (Fig. 2C, Table 1). However, the addition of Y27632 to everolimus caused a statistically significant decrease in collagen 9
ACCEPTED MANUSCRIPT deposition (Fig. 2C, Table 1). The P values for the collagen content graph shown in Fig. 2C are summarized in Suppl. Fig. 2.
Everolimus inhibits macrophage but not T cell infiltration into the graft We used immunostaining with a macrophage marker, a transmembrane glycoprotein, cluster of differentiation 68 (CD68), to assess the extent of macrophage infiltration into the grafts. CD68 binds to lectins /selectins expressed by various tissues, allowing
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macrophages to crawl over selectin-bearing cells/tissue and home in on particular
targets. We also used immunostaining with inducible nitric oxide synthase (iNOS) and arginine (Arg1) markers to assess the degree of infiltration with M1 and M2
macrophages, respectively. These studies showed that everolimus alone or in
combination with Y27632 drastically reduced the number of CD68+, iNos+ and Arg1+
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macrophages when compared to rapamycin alone or in combination with Y27632
treatment (Fig. 3, 4). The P values of the differences in macrophage infiltration between different experimental groups are shown in Suppl. Fig. 3.
We also tested whether everolimus alone or in conjunction with Y27632 affected T cell infiltration of the grafts. Immunostaining was performed with the following T cell
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markers: the cluster of differentiation 3 (CD3) present on mature T cells, the cluster of differentiation 4 (CD4) present on T helper cells, the α-chain of the IL-2 receptor (CD25)
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expressed on activated T cells, and the forkhead box P3 transcription factor (Foxp3) expressed by natural T regulatory cells (nTregs) and adaptive/induced T regulatory cells
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(a/iTregs). This study showed that in contrast to the macrophage infiltration, the rapamycin alone or in combination with Y27632 was much more effective in decreasing T cell infiltration than everolimus alone or in combination with Y27632 (Fi.3, 4). The P
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shown in Suppl. Fig. 4.
Discussion
It is well established that vessel occlusion during chronic rejection depends, to a high degree, on the function of graft infiltrating macrophages, which induce the overproliferation of smooth muscle in the vessel wall and the formation of the neointima (2, 26-33). Macrophages are also partially responsible for an increased deposition of collagen; they by themselves deposit collagen and stimulate fibroblasts to produce an excess of collagen, all of which leads to tissue fibrosis. (2, 26-33). There are two major 10
ACCEPTED MANUSCRIPT hypotheses concerning vascular remodeling during chronic rejection. The “inside–out” hypothesis argues that immune injury during transplantation is initiated at the endothelial layer of the intima, i.e., at the luminal (inside) surface of the vessel (Fig. 5A). The vascular endothelium produces cytokines and inflammatory molecules that recruit leukocytes (predominantly macrophages) and stimulate macrophages present on the surface of the intima. This immune cell infiltration results in endothelium injury and the initiation of an inflammatory process, which in turn, stimulate smooth muscle cells
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(SMCs) of the media to acquire a proliferative phenotype, which results in the thickening of the intima (formation of the neointima) and vessel occlusion (2). The more recent “outside-in” hypothesis assumes that the inflammatory response starts at the outer layer of the vessel, i.e., at the adventitia, and propagates inwards toward the intima (Fig. 5); then, macrophages stimulate a phenotypic switch of resident progenitor/stem cells
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residing in the adventitia into SMCs, which proliferate and migrate from the adventitia to form the neointima (2). No matter which of these two hypotheses (or both) is (are) correct, all these studies indicate that macrophage-targeted therapies (Fig. 5) should have beneficial effects on the long-term outcome of the grafts in clinical transplantation. We showed here that everolimus in conjunction with Y27632 improves the long-term
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outcome (inhibits chronic rejection) of cardiac allografts in a rat transplantation model. Everolimus and its analog rapamycin are mTOR inhibitors approved by the FDA for use
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in solid organ transplantation as immunosuppressants (24). Structurally and functionally, rapamycin and everolimus resemble tacrolimus (also known as fujimycin or
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FK506). They all bind to the intracellular tacrolimus (FK506)–binding proteins, such as FKBP12 and other proteins from the same family of proteins, which inhibit mTORC1, which regulates the cell cycle, proliferation and cell metabolism in response to
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interleukin 2 (IL-2) signaling. Recent studies indicate that everolimus, besides inhibiting mTORC1, is also very effective in the inhibition of mTORC2, which regulates, via the
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RhoA pathway, the actin cytoskeleton (16, 21, 24, 25). We have shown previously that inhibition of the RhoA pathway (by RhoA deletion or RhoA/ROCK inhibition by Y27632, Azaindole or Fasudil) in the recipients of cardiac allografts decreased macrophage infiltration, vessel occlusion and tissue fibrosis in rodent model systems (6, 7,15, 34). We also showed that the inhibition of chronic rejection was correlated with a downregulation of the mTORC2 pathway, which is an upstream regulator of RhoA signaling (35). This finding suggested that a coinhibition of RhoA and mTORC2 pathways should have beneficial effects on allografts in preventing chronic rejection. 11
ACCEPTED MANUSCRIPT Here, we showed that, indeed, the addition of the RhoA/ROCK inhibitor Y27632 in combination with everolimus improved the long-term outcome and inhibited chronic rejection of cardiac allografts in a rat transplantation model. We also showed that everolimus alone or in combination with Y27632 drastically reduced macrophage infiltration into the graft, while it did not decrease the T cell infiltration when compared to rapamycin alone or in combination with Y27632. Because everolimus is much more effective than rapamycin in inhibiting the mTORC2 pathway, this indicates that
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macrophage infiltration of the graft strongly depends on mTORC2/RhoA signaling.
Considering the prominent role of macrophages in the process of chronic rejection, the coinhibition of the mTORC2 and RhoA pathways seems to be a promising avenue for
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the development of anti-chronic rejection therapies in human transplantation.
Acknowledgements
We are grateful for the support from Novartis.
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29. Libby P. Changing concepts in atherogenesis. J Int Med. 2000; 247:349-358. 30. Skaro AI, Liwski RS, Johnson P, Legare JF, Lee TD, Hirsch GM. Donor versus Recipient: Neointimal Cell Origin in Allograft Vascular Disease. Graft 2002; 5:390-398.
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ACCEPTED MANUSCRIPT 31. Li AC, Glass CK. The macrophage foam cell as a target for therapeutic intervention. Nature Medicine 2002;8:1235-1242. 32. Akyurek LM, Paul LC, Funa K, Larsson E, Fellstrom BC. Smooth muscle cell migration into intima and adventitia during development of transplant vasculopathy.Transplantation 1996; 62:1526-1529. 33. Murry CE, Gipaya CT, Bartosek T, Benditt EP, SchwartzSM. Monoclonality of smooth muscle cells in human atherosclerosis. Am J Pathol. 1997;151:697-705.
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34. Chen W, Chen S, Chen W, Li XC, Ghobrial RM, Kloc M. Screening RhoA/ROCK inhibitors for the ability to prevent chronic rejection of mouse cardiac
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RM. (2013) Abrogation of chronic rejection in rat model system involves
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Figure Legends
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Fig.1. Allograft survival in everolimus and rapamycin treatment groups A) Survival curve of cardiac allografts in recipients treated with rapamycin alone, or in conjunction with different doses of Y27632. Grafts were acutely rejected within 11-17 days posttransplantation. Each experimental group consisted of 3 animals. B) Survival curve of cardiac allografts in recipients treated with 0.5 mg/kg everolimus alone or in 16
ACCEPTED MANUSCRIPT conjunction with different doses of Y27632 in comparison to rapamycin treatment alone. All everolimus groups had long-term (>100 days) graft survival. Each experimental group consisted of 5 animals. C) Survival curve of cardiac allografts in recipients treated with 5 mg/kg everolimus alone or in conjunction with different doses of Y27632 in comparison to rapamycin treatment alone. All everolimus groups had long-term (>100
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days) graft survival. Each experimental group consisted of 5 animals.
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Fig. 2. Everolimus inhibits vessel occlusion, fibrosis and chronic rejection A) The graph shows that addition of Y27632 to 0.5 mg/kg or 5 mg/kg everolimus results in a statistically significant decrease in blood vessel occlusion. The P values for the graph are summarized in Suppl. Fig. 1. B) The examples of blood vessels from the graft 18
ACCEPTED MANUSCRIPT of the recipient treated with rapamycin + 1 dose of Y27632, procured at 13 days posttransplantation (left panel), and from the graft of the recipient treated with everolimus 5 mg/kg + Y27632 (right panel), procured at 100 days posttransplantation, stained with VVG, show the compromised integrity of vessels and tissue in rapamycin treatment. The bar is equal to 100 μm. C) The graph shows that the addition of 1 dose of Y27632 to 0.5 mg/kg everolimus results in a statistically significant decrease in
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collagen content. The P values for the graph are summarized in Suppl. Fig. 2.
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Fig. 3. Everolimus alone or in combination with Y27632 inhibits macrophage but not T cell infiltration into the graft. A) The graph shows that treatment with everolimus at 0.5 mg/kg or 5 mg/kg dose alone or in combination with Y27632 significantly decreases CD68-positive macrophage infiltration (expressed in intensity of density (IOD) values) of the graft in comparison to the treatment with rapamycin alone or in combination with Y27632. The P values for the 20
ACCEPTED MANUSCRIPT graph are summarized in Suppl. Fig. 3. B) The graph shows that treatment with everolimus alone at 0.5 mg/kg or 5 mg/kg doses or in combination with Y27632 results in higher infiltration (expressed in intensity of density (IOD) values) with CD3-positive T cells than treatment with rapamycin alone or in combination with Y27632. The P values
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for the graph are summarized in Suppl. Fig. 4.
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Figure 4. Immunostaining of macrophage and T cell infiltration into the grafts Sections of cardiac allografts immunostained with macrophage markers: CD68, iNOS (M1 marker) and Arg-1 (M2 marker) or CD3 T cell marker. A-I) Sections of cardiac allografts from recipients treated with rapamycin in conjunction with Y27632 (day 0, day 2, day 4) immunostained with antibodies against CD68, iNos and Arg-1, and HRP22
ACCEPTED MANUSCRIPT conjugated secondary antibody show high infiltration with CD68+ and Arg1+ macrophages (represented by brown spots). Hearts were procured 14 days posttransplantation. D-F) Sections of cardiac allografts from recipients treated with everolimus alone at a 5 mg/kg dose immunostained with antibodies against CD68, iNos and Arg-1, and HRP-conjugated secondary antibody show low infiltration with CD68+, iNOS+, and Arg1+ macrophages. Hearts were procured 100 days posttransplantation. G-I) Sections of cardiac allografts from recipients treated with everolimus at a 5 mg/kg
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dose in combination with Y27632 (day 0, day 2, day 4) immunostained with antibodies against CD68, iNos and Arg-1, and HRP-conjugated secondary antibody show low
infiltration with CD68+, iNOS+, and Arg1+ macrophages. Hearts were procured at 100 days posttransplantation. J) A section of a cardiac allograft from a recipient treated with rapamycin in conjunction with Y27632 (day 0, day 2) immunostained with antibody
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against T cell marker CD3 shows very low infiltration with CD3+ T cells. The heart was procured at 13 days posttransplantation. K) A section of a cardiac allograft from a recipient treated with everolimus at 0.5 mg/kg in conjunction with Y27632 (day 0, day 2) immunostained with antibody against T cell marker CD3 shows higher infiltration with CD3+T cells than in rapamycin treatment. The heart was procured at 100 days
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posttransplantation. In all panels, the bar is equal to 100 μm.
Figure 5. Blood vessel composition, chronic rejection and the RhoA/mTOR pathway 23
ACCEPTED MANUSCRIPT A) The wall of large blood vessels (arteries and veins) contains three distinct layers: Tunica intima, which contains a single layer of endothelial cells; Tunica media, which is composed of extracellular matrix, smooth muscle cells (SMCs) and a thick elastic band called the external elastic lamina; and Tunica adventitia, which contains stem/progenitor cells and fibroblasts. The vascular remodeling that occurs during chronic rejection involves the thickening of the intima, which becomes the neointima through the recruitment and proliferation of smooth muscle cells (SMCs) and deposition
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of collagen from fibroblasts and macrophages. Vascular remodeling depends on the function of macrophages, which signal through the endothelial cells or adventitia or both (see “the inside-out” and “outside-in” hypothesis in the text). B) RhoA regulates the macrophage actin cytoskeleton via its downstream effector ROCK kinase. RhoA is
reciprocally regulated by mTORC2. In contrast, mTORC1 regulates cell proliferation,
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growth and metabolism. Everolimus inhibits both mTORC1 and mTORC2, while Y27632 inhibits ROCK kinase. The combination of everolimus and Y27632, by disorganizing the actin cytoskeleton, prevents macrophage infiltration into the graft and alleviates
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macrophage-driven vascular remodeling and chronic rejection.
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NI
Everolimus 0.5mg/kg
45.2 ± 23.8
Collagen Content 16.9%
Everolimus 0.5mg/kg + Y27632 day 0 Everolimus 0.5mg/kg + Y27632 day 0, day 2 Everolimus 0.5mg/kg + Y27632 day 0, day 2, day 4 Everolimus 5mg/kg
33.5 ± 19.7
10.7%
21.8 ± 8.7
15.3%
16.8 ± 2.7
11.2%
24.6 ± 18.5
14.9%
Everolimus 5mg/kg + Y27632 day 0 Everolimus 5mg/kg + Y27632 day 0, day 2 Everolimus 5mg/kg + Y27632 day 0, day 2, day 4
20.6 ± 4.4
15.2%
23.2 ± 4.5
11.9%
17.0 ± 3.48
11.5%
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Table 1 Treatment
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