Rapamycin attenuates atherosclerosis induced by dietary cholesterol in apolipoprotein-deficient mice through a p27Kip1-independent pathway


      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 p27Kip1 in the antiproliferative and antimigratory activities of rapamycin in vitro. However, our studies with fat-fed mice doubly deficient for p27Kip1 and apoE disclosed an antiatherogenic effect of rapamycin comparable with that found in apoE-null mice with an intact p27Kip1 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 p27Kip1-independent pathway that involves reduced expression of positive cell cycle regulators and MCP-1 within the arterial wall.


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Atherosclerosis
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Ross R.
        Atherosclerosis: an inflammatory disease.
        N. Engl. J. Med. 1999; 340: 115-126
        • Lusis A.J.
        Nature. 2000; 407: 233-241
        • Dzau V.J.
        • Braun-Dullaeus R.C.
        • Sedding D.G.
        Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies.
        Nat. Med. 2002; 8: 1249-1256
        • Andrés V.
        • Castro C.
        Antiproliferative strategies for the treatment of vascular proliferative disease.
        Curr. Vasc. Pharmacol. 2003; 1: 85-98
        • Binder C.J.
        • Chang M.K.
        • Shaw P.X.
        • et al.
        Innate and acquired immunity in atherogenesis.
        Nat. Med. 2002; 8: 1218-1226
        • Greaves D.R.
        • Channon K.M.
        Inflammation and immune responses in atherosclerosis.
        Trends Immunol. 2002; 23: 535-541
        • Sehgal S.N.
        • Baker H.
        • Vezina C.
        Rapamycin (AY-22,989), a new antifungal antibiotic. II. Fermentation, isolation and characterization.
        J. Antibiot. (Tokyo). 1975; 28: 727-732
        • Sehgal S.N.
        Rapamune (RAPA, rapamycin, sirolimus): mechanism of action immunosuppressive effect results from blockade of signal transduction and inhibition of cell cycle progression.
        Clin. Biochem. 1998; 31: 335-340
        • Marx S.O.
        • Marks A.R.
        Bench to bedside: the development of rapamycin and its application to stent restenosis.
        Circulation. 2001; 104: 852-855
        • Morice W.G.
        • Brunn G.J.
        • Wiederrecht G.
        • Siekierka J.J.
        • Abraham R.T.
        Rapamycin-induced inhibition of p34cdc2 kinase activation is associated with G1/S-phase growth arrest in T lymphocytes.
        J. Biol. Chem. 1993; 268: 3734-3738
        • Sabatini D.M.
        • Erdjument-Bromage H.
        • Lui M.
        • Tempst P.
        • Snyder S.H.
        RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs.
        Cell. 1994; 78: 35-43
        • Nourse J.
        • Firpo E.
        • Flanagan W.M.
        • et al.
        Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin.
        Nature. 1994; 372: 570-573
        • Brown E.J.
        • Albers M.W.
        • Shin T.B.
        • et al.
        A mammalian protein targeted by G1-arresting rapamycin-receptor complex.
        Nature. 1994; 369: 756-758
        • Marx S.O.
        • Jayaraman T.
        • Go L.O.
        • Marks A.R.
        Rapamycin-FKBP inhibits cell cycle regulators of proliferation in vascular smooth muscle cells.
        Circ. Res. 1995; 76: 412-417
        • Dumont F.J.
        • Su Q.
        Mechanism of action of the immunosuppressant rapamycin.
        Life Sci. 1996; 58: 373-395
        • Hara K.
        • Yonezawa K.
        • Kozlowski M.T.
        • et al.
        Regulation of eIF-4E BP1 phosphorylation by mTOR.
        J. Biol. Chem. 1997; 272: 26457-26463
        • Kawamata S.
        • Sakaida H.
        • Hori T.
        • Maeda M.
        • Uchiyama T.
        The upregulation of p27Kip1 by rapamycin results in G1 arrest in exponentially growing T-cell lines.
        Blood. 1998; 91: 561-569
        • Gallo R.
        • Padurean A.
        • Jayaraman T.
        • et al.
        Inhibition of intimal thickening after balloon angioplasty in porcine coronary arteries by targeting regulators of the cell cycle.
        Circulation. 1999; 99: 2164-2170
        • Isotani S.
        • Hara K.
        • Tokunaga C.
        • Inoue H.
        • Avruch J.
        • Yonezawa K.
        Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase alpha in vitro.
        J. Biol. Chem. 1999; 274: 34493-34498
        • Braun-Dullaeus R.C.
        • Mann M.J.
        • Seay U.
        • et al.
        Cell cycle protein expression in vascular smooth muscle cells in vitro and in vivo is regulated through phosphatidylinositol 3-kinase and mammalian target of rapamycin.
        Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1152-1158
        • Bruemmer D.
        • Yin F.
        • Liu J.
        • et al.
        Rapamycin inhibits E2F-dependent expression of minichromosome maintenance proteins in vascular smooth muscle cells.
        Biochem. Biophys. Res. Commun. 2003; 303: 251-258
        • Sacks S.H.
        Rapamycin on trial.
        Nephrol. Dial. Transplant. 1999; 14: 2087-2089
        • Kahan B.D.
        Efficacy of sirolimus compared with azathioprine for reduction of acute renal allograft rejection: a randomised multicentre study. The Rapamune US Study Group.
        Lancet. 2000; 356: 194-202
        • Shapiro A.M.
        • Lakey J.R.
        • Ryan E.A.
        • et al.
        Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.
        N. Engl. J. Med. 2000; 343: 230-238
        • Meiser B.M.
        • Billingham M.E.
        • Morris R.E.
        Effects of cyclosporin, FK506, and rapamycin on graft-vessel disease.
        Lancet. 1991; 338: 1297-1298
        • Gregory C.R.
        • Huie P.
        • Billingham M.E.
        • Morris R.E.
        Rapamycin inhibits arterial intimal thickening caused by both alloimmune and mechanical injury. Its effect on cellular, growth factor, and cytokine response in injured vessels.
        Transplantation. 1993; 55: 1409-1418
        • Morris R.E.
        • Cao W.
        • Huang X.
        • et al.
        Rapamycin (Sirolimus) inhibits vascular smooth muscle DNA synthesis in vitro and suppresses narrowing in arterial allografts and in balloon-injured carotid arteries: evidence that rapamycin antagonizes growth factor action on immune and nonimmune cells.
        Transplant. Proc. 1995; 27: 430-431
        • Poston R.S.
        • Billingham M.
        • Hoyt E.G.
        • et al.
        Rapamycin reverses chronic graft vascular disease in a novel cardiac allograft model.
        Circulation. 1999; 100: 67-74
        • Ikonen T.S.
        • Gummert J.F.
        • Hayase M.
        • et al.
        Sirolimus (rapamycin) halts and reverses progression of allograft vascular disease in non-human primates.
        Transplantation. 2000; 70: 969-975
        • Gregory C.R.
        • Huang X.
        • Pratt R.E.
        • et al.
        Treatment with rapamycin and mycophenolic acid reduces arterial intimal thickening produced by mechanical injury and allows endothelial replacement.
        Transplantation. 1995; 59: 655-661
        • Burke S.E.
        • Lubbers N.L.
        • Chen Y.W.
        • et al.
        Neointimal formation after balloon-induced vascular injury in Yucatan minipigs is reduced by oral rapamycin.
        J. Cardiovasc. Pharmacol. 1999; 33: 829-835
        • Suzuki T.
        • Kopia G.
        • Hayashi S.
        • et al.
        Stent-based delivery of sirolimus reduces neointimal formation in a porcine coronary model.
        Circulation. 2001; 104: 1188-1193
        • Roque M.
        • Reis E.D.
        • Cordon-Cardo C.
        • et al.
        Effect of p27 deficiency and rapamycin on intimal hyperplasia: in vivo and in vitro studies using a p27 knockout mouse model.
        Lab. Invest. 2001; 81: 895-903
        • Sousa J.E.
        • Costa M.A.
        • Abizaid A.C.
        • et al.
        Sustained suppression of neointimal proliferation by sirolimus-eluting stents: one-year angiographic and intravascular ultrasound follow-up.
        Circulation. 2001; 104: 2007-2011
        • Serruys P.W.
        • Degertekin M.
        • Tanabe K.
        • et al.
        Intravascular ultrasound findings in the multicenter, randomized, double-blind RAVEL (randomized study with the sirolimus-eluting velocity balloon-expandable stent in the treatment of patients with de novo native coronary artery lesions) trial.
        Circulation. 2002; 106: 798-803
        • Regar E.
        • Serruys P.W.
        • Bode C.
        • et al.
        Angiographic findings of the multicenter randomized study with the sirolimus-eluting Bx velocity balloon-expandable stent (RAVEL): sirolimus-eluting stents inhibit restenosis irrespective of the vessel size.
        Circulation. 2002; 106: 1949-1956
        • Morice M.C.
        • Serruys P.W.
        • Sousa J.E.
        • et al.
        A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization.
        N. Engl. J. Med. 2002; 346: 1773-1780
        • Nurse P.
        Ordering S phase and M phase in the cell cycle.
        Cell. 1994; 79: 547-550
        • Wei G.L.
        • Krasinski K.
        • Kearney M.
        • Isner J.M.
        • Walsh K.
        • Andrés V.
        Temporally and spatially coordinated expression of cell cycle regulatory factors after angioplasty.
        Circ. Res. 1997; 80: 418-426
        • Abe J.
        • Zhou W.
        • Taguchi J.
        • et al.
        Suppression of neointimal smooth muscle cell accumulation in vivo by antisense cdc2 and cdk2 oligonucleotides in rat carotid artery.
        Biochem. Biophys. Res. Commun. 1994; 198: 16-24
        • Kearney M.
        • Pieczek A.
        • Haley L.
        • et al.
        Histopathology of in-stent restenosis in patients with peripheral artery disease.
        Circulation. 1997; 95: 1998-2002
        • Ihling C.
        • Technau K.
        • Gross V.
        • Schulte-Monting J.
        • Zeiher A.M.
        • Schaefer H.E.
        Concordant upregulation of type II-TGF-beta-receptor, the cyclin-dependent kinases inhibitor p27Kip1 and cyclin E in human atherosclerotic tissue: implications for lesion cellularity.
        Atherosclerosis. 1999; 144: 7-14
        • Vidal A.
        • Koff A.
        Cell-cycle inhibitors: three families united by a common cause.
        Gene. 2000; 247: 1-15
        • Chen D.
        • Krasinski K.
        • Sylvester A.
        • Chen J.
        • Nisen P.D.
        • Andrés V.
        Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27Kip1, an inhibitor of neointima formation in the rat carotid artery.
        J. Clin. Invest. 1997; 99: 2334-2341
        • Tanner F.C.
        • Yang Z.-Y.
        • Duckers E.
        • Gordon D.
        • Nabel G.J.
        • Nabel E.G.
        Expression of cyclin-dependent kinase inhibitors in vascular disease.
        Circ. Res. 1998; 82: 396-403
        • Roque M.
        • Cordon-Cardo C.
        • Fuster V.
        • Reis E.D.
        • Drobnjak M.
        • Badimon J.J.
        Modulation of apoptosis, proliferation, and p27 expression in a porcine coronary angioplasty model.
        Atherosclerosis. 2000; 153: 315-322
        • Diez-Juan A.
        • Andres V.
        The growth suppressor p27Kip1 protects against diet-induced atherosclerosis.
        FASEB J. 2001; 15: 1989-1995
        • Dı́ez-Juan A.
        • Castro C.
        • Edo M.D.
        • Andrés V.
        Role of the growth suppressor p27Kip1 during vascular remodeling.
        Curr. Vasc. Pharmacol. 2003; 1: 99-106
        • Diez-Juan A.
        • Andres V.
        Coordinate control of proliferation and migration by the p27Kip1/cyclin-dependent kinase/retinoblastoma pathway in vascular smooth muscle cells and fibroblasts.
        Circ. Res. 2003; 92: 402-410
        • Poon M.
        • Marx S.O.
        • Gallo R.
        • Badimon J.J.
        • Taubman M.B.
        • Marks A.R.
        Rapamycin inhibits vascular smooth muscle cell migration.
        J. Clin. Invest. 1996; 98: 2277-2283
        • Luo Y.
        • Marx S.O.
        • Kiyokawa H.
        • Koff A.
        • Massague J.
        • Marks A.R.
        Rapamycin resistance tied to defective regulation of p27Kip1.
        Mol. Cell. Biol. 1996; 16: 6744-6751
        • Sun J.
        • Marx S.O.
        • Chen H.J.
        • Poon M.
        • Marks A.R.
        • Rabbani L.E.
        Role for p27Kip1 in vascular smooth muscle cell migration.
        Circulation. 2001; 103: 2967-2972
        • Plump A.S.
        • Smith J.D.
        • Hayek T.
        • et al.
        Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells.
        Cell. 1992; 71: 343-353
        • Zhang S.H.
        • Reddick R.L.
        • Piedrahita J.A.
        • Maeda N.
        Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E.
        Science. 1992; 258: 468-471
        • Gu L.
        • Okada Y.
        • Clinton S.K.
        • et al.
        Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice.
        Mol. Cell. 1998; 2: 275-281
        • Boring L.
        • Gosling J.
        • Cleary M.
        • Charo I.F.
        Decreased lesion formation in CCR2−/− mice reveals a role for chemokines in the initiation of atherosclerosis.
        Nature. 1998; 394: 894-897
        • Gosling J.
        • Slaymaker S.
        • Gu L.
        • et al.
        MCP-1 deficiency reduces susceptibility to atherosclerosis in mice that overexpress human apolipoprotein B.
        J. Clin. Invest. 1999; 103: 773-778
        • Peters W.
        • Charo I.F.
        Involvement of chemokine receptor 2 and its ligand, monocyte chemoattractant protein-1, in the development of atherosclerosis: lessons from knockout mice.
        Curr. Opin. Lipidol. 2001; 12: 175-180
        • Inoue S.
        • Egashira K.
        • Ni W.
        • et al.
        Anti-monocyte chemoattractant protein-1 gene therapy limits progression and destabilization of established atherosclerosis in apolipoprotein E-knockout mice.
        Circulation. 2002; 106: 2700-2706
        • van Royen N.
        • Hoefer I.
        • Bottinger M.
        • et al.
        Local monocyte chemoattractant protein-1 therapy increases collateral artery formation in apolipoprotein E-deficient mice but induces systemic monocytic CD11b expression, neointimal formation, and plaque progression.
        Circ. Res. 2003; 92: 218-225
        • Wasowska B.A.
        • Zheng X.X.
        • Strom T.B.
        • Kupieck-Weglinski J.W.
        Adjunctive rapamycin and CsA treatment inhibits monocyte/macrophage associated cytokines/chemokines in sensitized cardiac graft recipients.
        Transplantation. 2001; 71: 1179-1183
        • Oliveira J.G.
        • Xavier P.
        • Sampaio S.M.
        • et al.
        Compared to mycophenolate mofetil, rapamycin induces significant changes on growth factors and growth factor receptors in the early days post-kidney transplantation.
        Transplantation. 2002; 73: 915-920
        • Nelken N.A.
        • Coughlin S.R.
        • Gordon D.
        • Wilcox J.N.
        Monocyte chemoattractant protein-1 in human atheromatous plaques.
        J. Clin. Invest. 1991; 88: 1121-1127
        • Yla-Herttuala S.
        • Lipton B.A.
        • Rosenfeld M.E.
        • Sarkioja T.
        • Yoshimura T.
        • Leonard E.J.
        Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions.
        Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 5252-5256
        • Takeya M.
        • Yoshimura T.
        • Leonard E.J.
        • Takahashi K.
        Detection of monocyte chemoattractant protein-1 in human atherosclerotic lesions by an anti-monocyte chemoattractant protein-1 monoclonal antibody.
        Hum. Pathol. 1993; 24: 534-539