sirolimus and Arteriosclerosis

The proliferation of vascular smooth muscle cells has an important role in the pathogenesis of atherosclerosis. The results of recent studies on drugs interfering on this process are briefly summarized.

Topics: Antineoplastic Agents; Arteriosclerosis; Cell Division; Humans; Muscle, Smooth, Vascular; Paclitaxel; Sirolimus

To investigate the effect and related mechanism of sirolimus (SRL) in arteriosclerosis(AS) induced by advanced glycation end products (AGEs) in kidney transplantation recipients (KTRs). Human kidney tissues from KTRs before and after treatment with SRL were assessed by hematoxylin-eosin and immunohistochemical staining. Rat vascular smooth muscle cells (VSMCs) were treated with AGEs and/or SRL. The expressions of α-smooth muscle actin (α-SMA), osteopontin (OPN), actinin-associated LIM protein (ALP), proliferating cell nuclear antigen (PCNA), integrin-linked kinase (ILK) and the mTOR signaling pathway proteins were examined using western blot assay. Cytosolic calcium present in VSMCs was also measured by the calcium assay kit and von Kossa staining assay. The expression of α-SMA was remarkably higher while OPN expression was significantly lower in recipients with AS after they were administered SRL. Rat VSMCs treated with AGEs exhibited significantly lower expression of α-SMA and overexpression of OPN, ALP and PCNA than the other groups. In contrast, the expression of α-SMA was significantly higher while the expression of OPN, ALP and PCNA was significantly lower in VSMCs treated with both AGEs and SRL. Moreover, the ILK/mTOR signaling pathway was activated in rat VSMCs treated with AGEs, while treatment with AGEs and SRL led to significant inhibition of the ILK/mTOR signaling pathway. AGEs play a critical role in the development and progression of AS after kidney transplantation, but SRL can reverse these effects and therefore slow down the development of AS through inhibition of the ILK/mTOR signaling pathway.

Topics: Adult; Animals; Arteriosclerosis; Calcium; Cell Differentiation; Cell Proliferation; Cytosol; Female; Glycation End Products, Advanced; Humans; Kidney Transplantation; Male; Muscle, Smooth, Vascular; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

It was previously shown that the combination of clopidogrel and everolimus reduced the development of transplant arteriosclerosis. The aim of this study was to investigate whether delayed onset of treatment, similar to the clinical situation after heart transplantation, inhibits progression of transplant arteriosclerosis.. Fully allogeneic C57BL/6 (H2-b) donor aortas were transplanted into CBA.J (H2-k) recipients treated with clopidogrel and everolimus alone or in combination starting on Days 1, 7 or 14. Grafts were analysed by histology and alloantibodies were detected by fluorescence activated cell sorting after transplantation.. Delayed platelet inhibition with clopidogrel reduced the development of transplant arteriosclerosis [neointima formation (Day 14): 50±4 vs 84±9% (control)]. The combination of clopidogrel and everolimus almost abolished formation of transplant arteriosclerosis when therapy was started on Day 1 [neointima formation (Day 1): 14±5 vs 84±9% (control)] and also showed a remarkable reduction in both delayed treatment groups [neointima formation (Day 7): 24±7 vs 84±9% (control); neointima formation (Day 14): 28±11 vs 84±9% (control)]. Platelet inhibition alone and in combination with everolimus resulted in reduced alloantibody production.. These results demonstrate that delayed treatment with clopidogrel and everolimus-representative of a clinical setting-prevents the progression of transplant arteriosclerosis and impairs humoral immunity in this experimental model.

Topics: Allografts; Animals; Aorta; Arteriosclerosis; Clopidogrel; Disease Progression; Everolimus; Immunity, Humoral; Isoantibodies; Mice; Mice, Inbred C57BL; Sirolimus; Ticlopidine

Regulatory T cells (T(reg)) are currently being tested in clinical trials as a potential therapy in cell and solid organ transplantation. The immunosuppressive drug rapamycin has been shown to preferentially promote T(reg) expansion. Here, we hypothesized that adjunctive rapamycin therapy might potentiate the ability of ex vivo expanded human T(reg) to inhibit vascular allograft rejection in a humanized mouse model of arterial transplantation. We studied the influence of combined treatment with low-dose rapamycin and subtherapeutic T(reg) numbers on the development of transplant arteriosclerosis (TA) in human arterial grafts transplanted into immunodeficient BALB/cRag2(-/-) Il2rg(-/-) mice reconstituted with allogeneic human peripheral blood mononuclear cell. In addition, we assessed the effects of the treatment on the proliferation and apoptosis of naïve/effector T cells. The combined therapy efficiently suppressed T-cell proliferation in vivo and in vitro. Neointima formation in the human arterial allografts was potently inhibited compared with each treatment alone. Interestingly, CD4(+) but not CD8(+) T lymphocytes were sensitive to T(reg) and rapamycin-induced apoptosis in vitro. Our data support the concept that rapamycin can be used as an adjunctive therapy to improve efficacy of T(reg)-based immunosuppressive protocols in clinical practice. By inhibiting TA, T(reg) and rapamycin may prevent chronic transplant dysfunction and improve long-term allograft survival.

Topics: Animals; Apoptosis; Arteries; Arteriosclerosis; Cell Proliferation; Dose-Response Relationship, Drug; Humans; Mice; Mice, Inbred BALB C; Sirolimus; T-Lymphocytes, Regulatory; Transplantation

Our group has shown that platelet inhibition with clopidogrel, an antagonist of the P2Y12 adenosine diphosphate receptor on platelets, reduced the formation of transplant arteriosclerosis. The aim of this study was to investigate whether a combination of cyclosporin or everolimus with clopidogrel has a beneficial effect on the development of transplant arteriosclerosis. Fully MHC mismatched C57Bl/6 (H2(b)) donor aortas were transplanted into CBA.J (H2(k)) recipients and mice received either clopidogrel alone (1 mg/kg/day) or in combination with cyclosporin (2 mg/kg/day) or everolimus (0.05 mg/kg/day). Grafts were analysed by histology and morphometry on day 30 after transplantation. In mice treated with clopidogrel alone, transplant arteriosclerosis was significantly reduced [intima proliferation 56 +/- 11% vs. 81 +/- 7% (control)/n = 7]. Daily application of everolimus reduced the development of transplant arteriosclerosis compared with untreated controls [intima proliferation of 29 +/- 9% vs. 81 +/- 7% (control)/n = 7]. Strikingly, combination of clopidogrel and everolimus almost abolished the formation of transplant arteriosclerosis [intima proliferation: 11 +/- 8% vs. 81 +/- 7% (control)/n = 7]. By contrast, combination of cyclosporin and clopidogrel compared with clopidogrel alone showed no additive effect. These results demonstrate that combination of platelet- and mammalian target of Rapamycin-inhibition can dramatically reduce the development of transplant arteriosclerosis.

Topics: Animals; Aorta, Abdominal; Arteriosclerosis; Clopidogrel; Disease Models, Animal; Drug Therapy, Combination; Everolimus; Graft Rejection; Immunosuppressive Agents; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Platelet Aggregation; Platelet Aggregation Inhibitors; Sirolimus; Ticlopidine; Transplantation, Homologous; Treatment Outcome

Cytomegalovirus infection after heart transplantation is considered as risk factor for the development of transplant arteriosclerosis. Therefore, the aim of this study was to investigate the effect of murine cytomegalovirus (MCMV) as a single risk factor on transplant arteriosclerosis in an experimental aortic allograft model.. Major histocompatibility complex class I-mismatched aortas of C.B10-H2(b)/LilMcdJ donor were transplanted into BALB/c recipients, which were either mock-infected or infected with MCMV (strain Smith) on day 7 and harvested 37 days after transplantation. In one experimental group animals received a daily dose of everolimus to increase the viral load of recipients. Grafts were analyzed by histology, morphometry, and immunofluorescence. Intragraft cytokine mRNA production was analyzed by real-time polymerase chain reaction (PCR), and persistence of cytomegalovirus infection was confirmed by TaqMan PCR.. After infection with MCMV, there was significantly more intimal proliferation when compared with uninfected controls (intimal proliferation 83.5%+/-9.6% [MCMV] vs. 43.9%+/-5.1% [MCMV]), indicating that MCMV plays a role in the development of transplant arteriosclerosis. Even after treatment with everolimus, MCMV infection pronounced significantly more intimal proliferation (intimal proliferation 52.5%+/-7.3% [MCMV] vs. 20.2%+/-1.7% [MCMV]). Intragraft mRNA expression showed significantly higher production of CD62E, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 after infection with MCMV. Cellular infiltration revealed significantly more CD4, CD8, and dendritic cells. We could also confirm the presence of MCMV for the duration of the experimental protocol by PCR within the spleen, liver, salivary glands, lung, and the aortic transplant.. These data suggest that MCMV infection plays an important role in the development of transplant arteriosclerosis.

Topics: Animals; Aorta, Abdominal; Arteriosclerosis; Cytomegalovirus Infections; DNA Primers; DNA Probes; E-Selectin; Everolimus; Genes, MHC Class I; Immunosuppressive Agents; Intercellular Adhesion Molecule-1; Interferon-gamma; Mice; Mice, Inbred BALB C; Mice, Inbred Strains; RNA, Messenger; Sirolimus; Transplantation, Homologous; Vascular Cell Adhesion Molecule-1

Accelerated arteriosclerosis remains a major limitation to therapeutic interventions such as angioplasty, stent deployment, and solid organ transplantation. Rapamycin, a powerful new immunosuppressant set to replace calcineurin inhibitors in the transplant setting, and imatinib mesylate, a receptor tyrosine kinase inhibitor, are both angioprotective. Here, we explored the pharmacological and therapeutic interactions of these two agents in a rat model of neointimal hyperplasia.. Wistar rats, subjected to balloon catheter-induced aortic injury, received daily drug treatment until postoperative day 14 and were subsequently sacrificed or followed up to day 40 without further treatment. Development of neointimal lesions was assessed histologically and immunohistochemically. Steady-state rapamycin levels in whole blood were determined by HPLC-UV.. Rapamycin and imatinib, administered individually or in combination, produced no signs of overt toxicity. Continuous postoperative therapy with either rapamycin (0.5-1.5 mg/kg/day) or imatinib (2- 50 mg/kg/day) dose-dependently suppressed neointimal hyperplasia on day 14. Combined treatment (0.5 or 1 + 10 mg/kg/day, respectively) showed a trend towards synergistic action on day 14. Withdrawal of medication on day 14 nullified the early therapeutic effect of either agent by day 40. In contrast, early combination therapy (1 + 10 mg/kg/day) achieved long-term suppression of neointimal hyperplasia by approximately 81%. Notably, coadministration of imatinib appeared to reduce exposure to rapamycin, although this finding did not reach statistical significance.. Short-term combination therapy with rapamycin and imatinib is well tolerated and produces synergistic, sustained suppression of neointimal hyperplasia in rats. Subject to clinical evaluation, this new drug regimen may afford definitive prophylaxis against accelerated arteriosclerosis.

Topics: Angioplasty, Balloon; Animals; Aorta, Abdominal; Arteriosclerosis; Benzamides; Drug Synergism; Hyperplasia; Imatinib Mesylate; Male; Piperazines; Protein-Tyrosine Kinases; Pyrimidines; Rats; Rats, Wistar; Sirolimus

Because of its antiproliferative properties and its known effects on plasma lipids, we evaluated the mechanisms underlying the effect of rapamycin (RPM) on endothelial nitric oxide synthase (eNOS) and matrix metalloproteinases in Apo-E knockout mice. Apo-E-/- mice fed a high-cholesterol diet were given RPM (3 mg/kg per day intraperitoneally) or no treatment for 10 weeks (n = 8 each). Blood was drawn for serum lipid analysis. Protein was extracted from the abdominal aortas for Western immunoblotting and zymography. Cellular localization was assessed by histology and immunohistochemistry. The data, expressed as mean +/- SEM, were compared by Student's t test or analysis of variance (ANOVA). Lipid levels at 10 weeks were similar in both groups except for higher triglyceride levels in RPM-treated animals. RPM-treated mice expressed greater amounts of eNOS and p-eNOS compared with controls (P < .05). Akt, p-Akt, Caveolin-1, and p-Caveolin-1 were not significantly affected by RPM treatment. RPM treatment was associated with increased activation of pro-MMP-9, a significant decrease in MMP-2 tissue levels, and corresponding increases in TIMP-2 and TIMP-3 expression. The increased expression and phosphorylation of eNOS with RPM appears to be regulated by mechanisms other than Akt or Caveolin-1. Alterations in eNOS expression, in addition to changes in MMP/TIMP ratios and MMP-2 and MMP-9 activation, may partially explain the changes observed in the aorta of treated Apo-E-/- mice induced by RPM.

Topics: Analysis of Variance; Animals; Aorta; Apolipoproteins E; Arteries; Arteriosclerosis; Cholesterol; Enzyme Activation; Immunosuppressive Agents; Inflammation; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Sirolimus; Tissue Inhibitor of Metalloproteinases

Rapamycin has been shown to reduce neointimal thickening in the setting of balloon angioplasty and chronic graft vessel disease. This study was designed to test the effect of oral rapamycin on atherosclerotic plaque progression and the possible mechanism involved. Apolipoprotein E (apoE) knockout mice were fed either a diet supplemented with cholesterol or with cholesterol and rapamycin. At 4 and 8 weeks, quantitative analyses of plaque area and macrophage numbers were determined. Plasma cholesterol, triglyceride, and whole-blood rapamycin levels were measured. Rapamycin could be detected in the blood of mice (117+/-7 pg/mL). In mice fed with rapamycin, atherosclerotic lesions covered 22% of the aortic arch as compared with 41% in cholesterol-fed mice. The macrophage count was significantly lower in the rapamycin-fed mice as compared with cholesterol-fed mice. Rapamycin, in a dose-dependent manner, inhibited monocyte chemotaxis elicited by stromal cell-derived factor-1. Lesions in the cholesterol-fed mice had complex atherosclerotic plaque with acellular core, cholesterol clefts, and an abundant collection of monocytes/macrophages. Lesions in the rapamycin-fed mice were mainly composed of monocytes/macrophages. Oral rapamycin is effective in slowing the progression of atherosclerosis. Along with its multitude actions, attenuation of monocyte chemotaxis may be one more way by which rapamycin attenuates plaque progression.

Topics: Animals; Aorta; Apolipoproteins E; Arteriosclerosis; Body Weight; Chemokine CXCL12; Chemokines, CXC; Chemotaxis, Leukocyte; Dietary Supplements; Disease Progression; Dose-Response Relationship, Drug; Immunosuppressive Agents; Mice; Mice, Knockout; Monocytes; Sirolimus; Triglycerides

Activation of immune cells and dysregulated growth and motility of vascular smooth muscle cells contribute to neointimal lesion development during the pathogenesis of vascular obstructive disease. Inhibition of these processes by the immunosuppressant rapamycin is associated with reduced neointimal thickening in the setting of balloon angioplasty and chronic graft vessel disease (CGVD). In this study, we show that rapamycin elicits a marked reduction of aortic atherosclerosis in apolipoprotein E (apoE)-null mice fed a high-fat diet despite sustained hypercholesterolemia. This inhibitory effect of rapamycin coincided with diminished aortic expression of the positive cell cycle regulatory proteins proliferating cell nuclear antigen and cyclin-dependent kinase 2. Moreover, rapamycin prevented the normal upregulation of the proatherogenic monocyte chemoattractant protein-1 (MCP-1, CCL2) seen in the aorta of fat-fed mice. Previous studies have implicated the growth suppressor p27(Kip1) in the antiproliferative and antimigratory activities of rapamycin in vitro. However, our studies with fat-fed mice doubly deficient for p27(Kip1) and apoE disclosed an antiatherogenic effect of rapamycin comparable with that found in apoE-null mice with an intact p27(Kip1) gene. Taken together, these findings extend the therapeutic application of rapamycin from the restenosis and CGVD models to the setting of diet-induced atherosclerosis. Our results suggest that rapamycin-dependent atheroprotection occurs through a p27(Kip1)-independent pathway that involves reduced expression of positive cell cycle regulators and MCP-1 within the arterial wall.

Topics: Animals; Aortic Diseases; Apolipoproteins; Arteriosclerosis; Blotting, Western; Cell Cycle Proteins; Chemokine CCL2; Cholesterol, Dietary; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Immunosuppressive Agents; Mice; Mice, Inbred C57BL; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus; Tumor Suppressor Proteins; Tunica Intima; Up-Regulation

Atherosclerosis is a chronic inflammatory disease that develops in response to injury to the vessel wall, and is augmented by hypercholesterolemia. To further delineate the role of the immune system and local factors in this process, we assessed the effects of the immunosuppressant sirolimus (Rapamycin, RAPAMUNE, Wyeth, Collegeville, PA) on atherosclerosis in the apoE-deficient (apoE KO) mouse, a well-accepted model of cardiovascular disease. ApoE KO mice were fed a high fat diet and sirolimus was administered. After 12 weeks, atherosclerotic lesions and plasma lipoproteins were measured. The expression of cytokines associated with atherosclerosis was also examined. All groups demonstrated plasma total cholesterol (TC) >1100 mg/dL. Sirolimus treatment was associated with a 30% increase in LDL-cholesterol (LDLc) and a dose-dependent elevation in HDL-cholesterol (HDLc). Despite increased LDLc, aortic atherosclerosis was markedly reduced in all sirolimus-treated groups. Sirolimus treatment resulted in decreased expression of IL-12p40, IFN-gamma and IL-10 mRNA. In contrast, TGF-beta1 was elevated. Sirolimus significantly reduced atherosclerosis in apo E-KO mice; this effect is independent of, and obviates, elevated plasma TC and LDLc. Sirolimus might therefore be of benefit on atherosclerosis in patients undergoing therapy, independent of any impact on circulating lipids.

Topics: Animals; Aorta; Aortic Diseases; Apolipoproteins E; Arteriosclerosis; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Immunosuppressive Agents; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; RNA; Sirolimus; Th1 Cells; Th2 Cells; Transcription, Genetic

Topics: Animals; Arteriosclerosis; Cell Cycle; Cell Cycle Proteins; Cell Movement; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Graft Occlusion, Vascular; Growth Substances; Humans; Microtubule-Associated Proteins; Muscle, Smooth, Vascular; Paclitaxel; Sirolimus; Tumor Suppressor Proteins; Tunica Media

The aim of this study was to compare the effects of cyclosporine, tacrolimus, mycophenolate mofetil and SDZ RAD on an animal model of transplant arteriosclerosis involving alloantigen dependent and independent mechanisms.

Topics: Animals; Aorta, Abdominal; Arteriosclerosis; Chronic Disease; Cyclosporine; Disease Models, Animal; Everolimus; Graft Rejection; Immunosuppressive Agents; Isoantigens; Male; Mycophenolic Acid; Rats; Rats, Inbred F344; Rats, Inbred Lew; Sirolimus; Tacrolimus

Topics: Animals; Aorta, Thoracic; Arteriosclerosis; Cyclosporine; Enzyme Induction; Immunosuppressive Agents; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Polyenes; Postoperative Complications; Rats; Rats, Inbred ACI; Rats, Inbred WF; Sirolimus; Transplantation, Homologous; Transplantation, Isogeneic

Topics: Animals; Aorta, Abdominal; Arteriosclerosis; Everolimus; Immunosuppressive Agents; Ischemia; Male; Polyenes; Postoperative Complications; Rats; Rats, Inbred F344; Sirolimus; Transplantation, Isogeneic; Tunica Intima

Rat aortic allografts develop arteriosclerotic alterations in the vascular wall that are histologically similar to those observed in chronic rejecting vascular allografts. We used cyclosporine and rapamycin (RAPA) in two different rat strain aorta transplantation models to investigate the effect of immunosuppression on posttransplant graft arteriosclerosis. High dose CsA (25 mg/kg, 3 times/week) treatment significantly inhibited intimal proliferation in the "strong" WAG-BN model (P < 0.01) as well as in the "weak" BN-WAG combination (P < 0.001), compared with untreated allogeneic controls. In the latter combination, even fewer intimal lesions were present than in WAG autotransplants, suggesting that CsA may also inhibit the arteriosclerotic response to mechanical injury. RAPA (3 mg/kg, 3 times/week) was as effective as CsA in reducing intimal lesions (P < 0.01 and P < 0.001 in the BN-WAG and WAG-BN models, respectively). Low dose CsA (5 mg/kg, 3 times/week) was only partially effective in preventing intimal lesions. Histology of the nontreated allografts showed ongoing acute rejection in the adventitial layer. The degree of cellular infiltration correlated with the severity of arteriosclerotic lesions. High dose CsA and RAPA treatment prevented adventitial infiltration in both models, while low dose CsA was only moderately effective. In conclusion, in the present study, the degree of arteriosclerotic involvement after allogeneic aorta transplantation was related to the severity of cellular adventitial infiltration. The myointimal thickening was inhibited by effective immunosuppressive treatment. These observations may imply that chronic rejection develops after ineffective immunosuppression.

Topics: Animals; Aorta; Arteriosclerosis; Cyclosporine; Graft Rejection; Immunosuppressive Agents; Male; Polyenes; Rats; Rats, Inbred BN; Rats, Inbred Strains; Sirolimus; Transplantation, Autologous; Transplantation, Homologous