sirolimus and Aortic-Diseases

Vascular calcification increases the risk of cardiovascular disease and death in patients with chronic kidney disease (CKD). Increased activity of mammalian target of rapamycin complex 1 (mTORC1) and endoplasmic reticulum (ER) stress-unfolded protein response (UPR) are independently reported to partake in the pathogenesis of vascular calcification in CKD. However, the association between mTORC1 activity and ER stress-UPR remains unknown. We report here that components of the uremic state [activation of the receptor for advanced glycation end products (RAGE) and hyperphosphatemia] potentiate vascular smooth muscle cell (VSMC) calcification by inducing persistent and exaggerated activity of mTORC1. This gives rise to prolonged and excessive ER stress-UPR as well as attenuated levels of sestrin 1 ( Sesn1) and Sesn3 feeding back to inhibit mTORC1 activity. Activating transcription factor 4 arising from the UPR mediates cell death via expression of CCAAT/enhancer-binding protein (c/EBP) homologous protein (CHOP), impairs the generation of pyrophosphate, a potent inhibitor of mineralization, and potentiates VSMC transdifferentiation to the osteochondrocytic phenotype. Short-term treatment of CKD mice with rapamycin, an inhibitor of mTORC1, or tauroursodeoxycholic acid, a bile acid that restores ER homeostasis, normalized mTORC1 activity, molecular markers of UPR, and calcium content of aortas. Collectively, these data highlight that increased and/or protracted mTORC1 activity arising from the uremic state leads to dysregulated ER stress-UPR and VSMC calcification. Manipulation of the mTORC1-ER stress-UPR pathway opens up new therapeutic strategies for the prevention and treatment of vascular calcification in CKD.

Topics: Activating Transcription Factor 4; Animals; Aorta; Aortic Diseases; Cell Death; Cell Proliferation; Cell Transdifferentiation; Disease Models, Animal; Endoplasmic Reticulum Stress; Extracellular Signal-Regulated MAP Kinases; HEK293 Cells; Humans; Mechanistic Target of Rapamycin Complex 1; Mice, Mutant Strains; Muscle, Smooth, Vascular; Osteogenesis; Phosphorylation; Receptor for Advanced Glycation End Products; S100 Proteins; Signal Transduction; Sirolimus; Taurochenodeoxycholic Acid; Unfolded Protein Response; Uremia; Vascular Calcification

Elastin deficiency because of heterozygous loss of an. Thoracic aortas of neonatal and juvenile mice with graded elastin deficiency exhibited increased signaling through both mTOR complex 1 and 2. Despite lower predicted wall stress, there was increased phosphorylation of focal adhesion kinase, suggestive of greater integrin activation, and increased transforming growth factor-β-signaling mediators, associated with increased collagen expression. Pharmacological blockade of mTOR by rapalogs did not improve luminal stenosis but reduced mechanosignaling (in delayed fashion after mTOR complex 1 inhibition), medial collagen accumulation, and stiffening of the aorta. Rapalog administration also retarded somatic growth, however, and precipitated neonatal deaths. Complementary, less-toxic strategies to inhibit mTOR via altered growth factor and nutrient responses were not effective.. In addition to previously demonstrated therapeutic benefits of rapalogs decreasing smooth muscle cell proliferation in the absence of elastin, we find that rapalogs also prevent aortic fibrosis and stiffening attributable to partial elastin deficiency. Our findings suggest that mTOR-sensitive perturbation of smooth muscle cell mechanosensing contributes to elastin aortopathy.

Topics: Animals; Aorta, Thoracic; Aortic Diseases; Cell Proliferation; Collagen; Elastin; Everolimus; Focal Adhesion Kinase 1; Genetic Predisposition to Disease; Humans; Imatinib Mesylate; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mechanotransduction, Cellular; Mice, Inbred C57BL; Mice, Knockout; Multiprotein Complexes; Muscle, Smooth, Vascular; Phenotype; Phosphorylation; Protein Kinase Inhibitors; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Vascular Stiffness; Williams Syndrome

Vascular calcification (VC) is a major risk factor for cardiovascular mortality in chronic renal failure (CRF) patients, but the pathogenesis remains partially unknown and effective therapeutic targets should be urgently explored. Here we pursued the therapeutic role of rapamycin in CRF-related VC. Mammalian target of rapamycin (mTOR) signal was activated in the aortic wall of CRF rats. As expected, oral rapamycin administration significantly reduced VC by inhibiting mTOR in rats with CRF. Further in vitro results showed that activation of mTOR by both pharmacological agent and genetic method promoted, while inhibition of mTOR reduced, inorganic phosphate-induced vascular smooth muscle cell (VSMC) calcification and chondrogenic/osteogenic gene expression, which were independent of autophagy and apoptosis. Interestingly, the expression of Klotho, an antiaging gene that suppresses VC, was reduced in calcified vasculature, whereas rapamycin reversed membrane and secreted Klotho decline through mTOR inhibition. When mTOR signaling was enhanced by either mTOR overexpression or deletion of tuberous sclerosis 1, Klotho mRNA was further decreased in phosphate-treated VSMCs, suggesting a vital association between mTOR signaling and Klotho expression. More importantly, rapamycin failed to reduce VC in the absence of Klotho by using either siRNA knockdown of Klotho or Klotho knockout mice. Thus, Klotho has a critical role in mediating the observed decrease in calcification by rapamycin in vitro and in vivo.

Topics: Animals; Aorta, Abdominal; Aorta, Thoracic; Aortic Diseases; Cells, Cultured; Disease Models, Animal; Gene Expression Regulation; Genetic Predisposition to Disease; Glucuronidase; Humans; Kidney Failure, Chronic; Klotho Proteins; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Osteogenesis; Phenotype; Protein Kinase Inhibitors; RNA Interference; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transfection; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins; Vascular Calcification

Medial arterial calcification involves chondrogenic/osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Zhao et al. report that phosphate activates the mammalian target of rapamycin (mTOR) cascade in VSMCs, leading to downregulation of Klotho. Furthermore, rapamycin was shown to halt medial calcification. This effect was blunted in the absence of Klotho. Given the concomitant anti-atherosclerotic effects of the mTOR inhibitor, this agent has clinical potential as an inhibitor of intimal atherosclerosis and medial calcification.

Topics: Animals; Aorta, Abdominal; Aorta, Thoracic; Aortic Diseases; Glucuronidase; Humans; Protein Kinase Inhibitors; Sirolimus; TOR Serine-Threonine Kinases; Vascular Calcification

To determine the efficacy of sirolimus-eluting bioabsorbable magnesium alloy stents (SEBMAS) in restenosis prevention.. A balloon-expandable bioabsorbable magnesium alloy stent (BMAS) was created and coated with biodegradable poly(lactic acid-co-trimethylene carbonate) that contained the antiproliferative drug sirolimus (140 ± 40 µg/cm²). Both the uncoated BMAS and the coated SEBMAS were deployed 2 cm apart in balloon-injured infrarenal abdominal aortas of 20 New Zealand white rabbits. The stented aortic segments were removed at 30, 60, 90, and 120 days (5 rabbits per interval) after implantation. The average stent strut sectional area of each group was measured to evaluate the degree of magnesium corrosion and to forecast the biodegradation time profile of the magnesium stent. Histology and histopathology of the sectioned stented aortic segments were performed to evaluate neointima formation, endothelialization, and inflammation.. The SEBMAS degraded gradually after being implanted into the rabbit aorta, and total biocorrosion occurred after ~120 days. In all groups, the lumen area was significantly greater, but the neointimal area was significantly smaller in SEBMAS segments compared with the uncoated BMAS segments (p < 0.05) at all time points. There was no significant difference in the injury or inflammation scores between the groups. Endothelialization was delayed at 30 days in the SEBMAS segments vs. the uncoated BMAS segments.. SEBMAS further reduces intimal hyperplasia and improves the lumen area when compared to uncoated BMAS; however, it delays vascular healing and endothelialization.

Topics: Alloys; Angioplasty; Animals; Aorta, Abdominal; Aortic Diseases; Arterial Occlusive Diseases; Cardiovascular Agents; Cell Proliferation; Coated Materials, Biocompatible; Constriction, Pathologic; Dioxanes; Disease Models, Animal; Drug-Eluting Stents; Endothelial Cells; Hyperplasia; Lactic Acid; Magnesium; Male; Polyesters; Polymers; Prosthesis Design; Rabbits; Secondary Prevention; Sirolimus; Time Factors; Wound Healing

Topics: Alloys; Angioplasty; Animals; Aorta, Abdominal; Aortic Diseases; Arterial Occlusive Diseases; Cardiovascular Agents; Coated Materials, Biocompatible; Drug-Eluting Stents; Magnesium; Male; Sirolimus

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

Rapamycin is a specific inhibitor of the mammalian target of rapamycin (mTOR). We recently reported that administration of rapamycin before exposure to ascending aortic constriction significantly attenuated the load-induced increase in heart weight by approximately 70%.. To examine whether rapamycin can regress established cardiac hypertrophy, mice were subjected to pressure overload (ascending aortic constriction) for 1 week, echocardiography was performed to verify an increase in ventricular wall thickness, and mice were given rapamycin (2 mg x kg(-1) x d(-1)) for 1 week. After 1 week of pressure overload (before treatment), 2 distinct groups of animals became apparent: (1) mice with compensated cardiac hypertrophy (normal function) and (2) mice with decompensated hypertrophy (dilated with depressed function). Rapamycin regressed the pressure overload-induced increase in heart weight/body weight (HW/BW) ratio by 68% in mice with compensated hypertrophy and 41% in mice with decompensated hypertrophy. Rapamycin improved left ventricular end-systolic dimensions, fractional shortening, and ejection fraction in mice with decompensated cardiac hypertrophy. Rapamycin also altered the expression of some fetal genes, reversing, in part, changes in alpha-myosin heavy chain and sarcoplasmic reticulum Ca2+ ATPase.. Rapamycin may be a therapeutic tool to regress established cardiac hypertrophy and improve cardiac function.

Topics: Adaptation, Physiological; Animals; Aorta; Aortic Diseases; Cardiomegaly; Cell Size; Constriction, Pathologic; Drug Evaluation, Preclinical; Gene Expression Regulation; Male; Mice; Myocytes, Cardiac; Organ Size; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; Ribosomal Protein S6; Sirolimus; Stroke Volume; TOR Serine-Threonine Kinases

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

Chronic allograft dysfunction, the leading cause of solid-organ transplant failure, is characterised by histological evidence of extracellular matrix (ECM) accumulation (fibrosis). The aim of this study was to compare the effect of combined rapamycin and cyclosporine therapy on fibrosis-associated gene expression and ECM turnover during the development of allograft vasculopathy, compared with either agent alone. Lewis recipients of F344 rat thoracic-to-abdominal aorta transplants were administered rapamycin, cyclosporine, combined rapamycin and cyclosporine or no treatment. F344-to-F344 isografts served as controls. Six grafts in each group were harvested at 16 weeks. Vascular remodelling and ECM accumulation (Sirius red) were measured by computerised histomorphometry of aortic sections. Messenger RNA was extracted from frozen tissue, and expression of fibrosis-associated genes was studied by means of semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Rapamycin (0.5 mg/kg per day) or cyclosporine (5 mg/kg per day) inhibited intimal hyperplasia, medial ECM accumulation and expansive vascular remodelling (increasing vessel circumference) in rat aortic allografts. This was associated with attenuation of the graft inflammatory infiltrate and a reduction in intra-graft gelatinase, collagen III and tissue inhibitor of metalloproteinase (TIMP)-1 mRNA levels. Combined rapamycin and cyclosporine inhibited intimal hyperplasia; however, there was a lesser effect on vascular remodelling and medial ECM accumulation. Combined-treatment aortic allografts were also seen to have a more-severe inflammatory infiltrate and larger amounts of intra-graft matrix metalloproteinase (MMP)-9, transforming growth factor (TGF)-beta and TIMP-1 mRNA than those treated with monotherapy. Rapamycin and cyclosporine act synergistically to inhibit intimal hyperplasia but not the inflammatory infiltrate, allograft fibrosis or vessel remodelling. In the high-responder F344-to-Lewis rat model, effective immunosuppression is required to reduce graft fibrosis.

Topics: Animals; Aorta; Aortic Diseases; Azo Compounds; Cyclosporine; Drug Therapy, Combination; Fibrosis; Gene Expression; Graft Rejection; Immunosuppressive Agents; Matrix Metalloproteinase 9; Rats; Rats, Inbred F344; Rats, Inbred Lew; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus; Transforming Growth Factor beta; Transplantation, Homologous