Key points are not available for this paper at this time.
The present work investigates the occurrence and significance of aberrant DNA methylation patterns during early stages of atherosclerosis. To this end, we asked whether the genetically atherosclerosis-prone APOE-null mice show any changes in DNA methylation patterns before the appearance of histologically detectable vascular lesion. We exploited a combination of various techniques: DNA fingerprinting, in vitro methyl-accepting assay, 5-methylcytosine quantitation, histone post-translational modification analysis, Southern blotting, and PCR. Our results show that alterations in DNA methylation profiles, including both hyper- and hypomethylation, were present in aortas and PBMC of 4-week-old mutant mice with no detectable atherosclerotic lesion. Sequencing and expression analysis of 60 leukocytic polymorphisms revealed that epigenetic changes involve transcribed genic sequences, as well as repeated interspersed elements. Furthermore, we showed for the first time that atherogenic lipoproteins promote global DNA hypermethylation in a human monocyte cell line. Taken together, our results unequivocally show that alterations in DNA methylation profiles are early markers of atherosclerosis in a mouse model and may play a causative role in atherogenesis. The present work investigates the occurrence and significance of aberrant DNA methylation patterns during early stages of atherosclerosis. To this end, we asked whether the genetically atherosclerosis-prone APOE-null mice show any changes in DNA methylation patterns before the appearance of histologically detectable vascular lesion. We exploited a combination of various techniques: DNA fingerprinting, in vitro methyl-accepting assay, 5-methylcytosine quantitation, histone post-translational modification analysis, Southern blotting, and PCR. Our results show that alterations in DNA methylation profiles, including both hyper- and hypomethylation, were present in aortas and PBMC of 4-week-old mutant mice with no detectable atherosclerotic lesion. Sequencing and expression analysis of 60 leukocytic polymorphisms revealed that epigenetic changes involve transcribed genic sequences, as well as repeated interspersed elements. Furthermore, we showed for the first time that atherogenic lipoproteins promote global DNA hypermethylation in a human monocyte cell line. Taken together, our results unequivocally show that alterations in DNA methylation profiles are early markers of atherosclerosis in a mouse model and may play a causative role in atherogenesis. Atherosclerosis and its complications are a major cause of death and disability in the developed world. The disease is characterized by infiltration of lipid particles in the arterial wall, accompanied by the recruitment of inflammatory and immune cells, migration and proliferation of smooth muscle cells (SMC), 1The abbreviations used are: SMC, smooth muscle cells; mC, 5-methylcytosine; CG, CpG dinucleotide; DMP, DNA methylation polymorphism; HPCE, high performance capillary electrophoresis; MSAP, methylation-sensitive amplified polymorphism; SAM, S-adenosyl methionine; PBMC, peripheral blood mononuclear cell; WDMP, wild type DNA methylation polymorphism; MDMP, mutant DNA methylation polymorphism; UK, United Kingdom; EST, expressed sequence tag; WT, wild type; LDL, low density lipoprotein; VLDL, very low density lipoprotein; HDL, high density lipoprotein; HL, high VLDL + LDL mixture; WL, low VLDL + LDL mixture. and synthesis of extracellular matrix. These processes eventually result in the gradual development of an elevated lipid-rich, fibrocellular lesion (1Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19370) Google Scholar). In mammals, DNA methyltransferases use S-adenosyl methionine (SAM) as a methyl group donor to methylate the carbon in position 5 of cytosine residues in a CpG dinucleotide (CG) context (2Jeltsch A. Chem. Bio. Chem. 2002; 3: 274-293Crossref PubMed Google Scholar). DNA methylation regulates fundamental biological phenomena such as gene expression, genome stability, mutation rate, genomic imprinting, and X chromosome inactivation (3Chen R.Z. Pettersson U. Beard C. Jackson-Grusby L. Jaenisch R. Nature. 1998; 395: 89-93Crossref PubMed Scopus (795) Google Scholar, 4Li E. Nat. Rev. Genet. 2002; 3: 662-673Crossref PubMed Scopus (1584) Google Scholar, 5Jaenisch R. Bird A. Nat. Genet. 2003; 33: 245-254Crossref PubMed Scopus (4739) Google Scholar, 6Hashimshony T. Zhang J. Keshet I. Bustin M. Cedar H. Nat. Genet. 2003; 34: 187-192Crossref PubMed Scopus (289) Google Scholar). Both global and gene-specific alterations in DNA methylation are associated with abnormal phenotypes in disease (7Issa J.-P. J. Nutr. 2002; 132: 2388S-2392SCrossref PubMed Google Scholar, 8Feinberg A.P. Tycko B. Nat. Rev. Cancer. 2004; 4: 143-153Crossref PubMed Scopus (1816) Google Scholar). For example, cancer cells show global genomic hypomethylation and dense hypermethylation of CpG islands, which are normally unmethylated (9Esteller M. Herman J.G. J. Pathol. 2002; 196: 1-7Crossref PubMed Scopus (596) Google Scholar). The identification of cancer type- and stage-specific changes in DNA methylation has justified hopes for novel diagnostic and therapeutic avenues (10Laird P.W. Nat. Rev. Cancer. 2003; 3: 253-266Crossref PubMed Scopus (1283) Google Scholar). Two general observations suggest that alterations in DNA methylation patterns are involved in atherogenesis (11Newman P.E. Med. Hypotheses. 1999; 53: 421-424Crossref PubMed Scopus (52) Google Scholar, 12Dong C. Yoon W. Goldschmidt-Clermont P.J. J. Nutr. 2002; 132: 2406S-2409SCrossref PubMed Google Scholar, 13Hiltunen M.O. Ylä-Herttuala S. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1750-1753Crossref PubMed Scopus (98) Google Scholar). First, global hypomethylation and dense hypermethylation of certain CpG islands are associated with aging, a major risk factor for atherosclerosis (14Issa J.-P. Crit. Rev. Oncol. Hematol. 1999; 32: 31-43Crossref PubMed Scopus (169) Google Scholar). Second, hyperhomocysteinemia and the subsequent decreased production or bioavailability of SAM is associated with an increased risk of cardiovascular disease (15Brattström L. Wilcken D.E.L. Am. J. Clin. Nutr. 2000; 72: 315-323Crossref PubMed Scopus (375) Google Scholar). Accordingly, mice with genetically reduced levels of methylenetetrahydrofolate reductase, a key enzyme in the pathway generating SAM, show hyperhomocysteinemia, DNA hypomethylation, and aortic lipid infiltrations (16Chen Z. Karaplis A.C. Ackerman S.L. Pogribny I.P. Melnyk S. Lussier-Cacan S. Chen M.F. Pai A. John S.W.M. Smith R.S. Bottiglieri T. Bagley P. Selhub J. Rudnicki M.A. James S.J. Rozen R. Hum. Mol. Gen. 2001; 10: 433-443Crossref PubMed Scopus (509) Google Scholar). Furthermore, a global DNA hypomethylation has been observed in vascular lesions and leukocytes of atherosclerosis patients and proliferating SMC in animal models (17Laukkanen M.O. Mannermaa S. Hiltunen M.O. Aittomäki S. Airenne K. Jänne J. Ylä-Herttuala S. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2171-2178Crossref PubMed Scopus (142) Google Scholar, 18Hiltunen M.O. Turunen M.P. Hakkinen T.P. Rutanen J. Hedman M. Makinen K. Turunen A.M. Aalto-Setala K. Ylä-Herttuala S. Vasc. Med. 2002; 7: 5-11Crossref PubMed Scopus (205) Google Scholar, 19Castro R. Rivera I. Struys E.A. Jansen E.E.W. Ravasco P. Camilo M.E. Blom H.J. Jakobs C. Tavares de Almeida I. Clin. Chem. 2003; 49: 1292-1296Crossref PubMed Scopus (350) Google Scholar). One unresolved issue is whether DNA methylation patterns are altered at early stages of atherosclerosis and are associated with susceptibility to the disease. Answers to these questions may have important implications for prevention and therapy of atherosclerosis. We therefore asked whether changes in DNA methylation patterns occur prior to the appearance of any vascular lesions in PBMC and aorta of mice lacking APOE, compared with matched WT animals (20Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Science. 1992; 258: 468-471Crossref PubMed Scopus (1849) Google Scholar). Moreover, in an attempt to provide a mechanism for the changes in DNA methylation patterns observed in vivo, we asked whether atherogenic lipoprotein profiles could affect DNA methylation and histone post-translational modifications in the human monocyte-macrophage cell line THP-1 (21Auverx J. Experientia (Basel). 1991; 47: 22-31Crossref PubMed Scopus (666) Google Scholar). The present study is the first analysis of DNA methylation at early stages of atherosclerosis and the first description of genomic DNA sequences undergoing epigenetic changes in a mouse model. The implications of our findings for understanding human atherosclerosis are discussed. Animal Work and Tissue Manipulation—All procedures used in this study were approved by the local ethical committee (Malmö/Lunds Djurförsöksetiska Nämnd, license M89-01). Apoe nullizygous mice (Apoe–/–) of the strain created in the laboratory of N. Maeda (20Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Science. 1992; 258: 468-471Crossref PubMed Scopus (1849) Google Scholar) were purchased from M PubMed Scopus Google Scholar). PBMC were by according to the from the United muscle tissue dissected from the DNA or were by the or according to The levels of and were as S. Pettersson L. B. L. M. R. J. J. J. Biol. Chem. 2002; PubMed Scopus Google Scholar). DNA methylation profiles were by the as J. J. Mol. Gen. Genet. PubMed Scopus Google Scholar) with the following of DNA and of used in the were by of genomic DNA were for with of and in a the and were for an in a of The of of of DNA 5 and The were for at and a performed for with 5 of and by The sequences were as and and performed with to the of the sequences and the sequence of and The sequences of the and the were and Two of the were to the of of of and of The were as for for by as for for for A performed at for sequence of the to the included the of a of at the The of the were as and The and of in a of of and of The by of with of of and 5 of The for at by inactivation for at The a amplified P. R. M. M. de T. M. A. J. J. M. M. 23: PubMed Scopus Google Scholar). were with and and on for at were and to for at For or to from or were the results were used for of patterns were on of performed as J. Biol. Chem. PubMed Scopus Google with the that with and DNA on a and Sequencing of were from in and for at One of the used in a with the The were in the and were were The CpG by the of observed by with the of of of Southern and of of genomic DNA were overnight with the and with by the according to from the for which sequences could from were by methylation-sensitive S. A. R. The 1999; PubMed Scopus Google Scholar). Two of DNA amplified with the following of and to of sequence with of and to of sequence with of and to of sequence with were as for by at and for and a at for were and by analysis of leukocytic to or genomic sequences by amplified as an control and are Southern of a sequence in mouse Mol. Biol. 2000; PubMed Google Scholar) by mouse genomic DNA to with the and were as for by at and for and a at for The and used in Southern as in the and THP-1 cells were in with and were used as or to by with for with of For both and were for in with lipoprotein as were by of from M. L. C. A.M. A. Nutr. 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Nutr. 2002; 132: PubMed Google Scholar). the of mutant mice were any detectable or fibrocellular lesions at the ascending aorta or at the aortic arch A and lesions of a fibrocellular and a were detectable in the portion of the aortic in 6-month-old Apoe–/– mice as and (20Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Science. 1992; 258: 468-471Crossref PubMed Scopus (1849) Google Scholar). elevated in Apoe–/– mice of both to WT A and with and (20Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Science. 1992; 258: 468-471Crossref PubMed Scopus (1849) Google Scholar). To PBMC for DNA methylation polymorphisms Apoe–/– mice and matched we exploited the is a modification of amplified a that is on of by of genomic DNA with the and P. R. M. M. de T. M. A. J. J. M. M. 23: PubMed Scopus Google Scholar). In MSAP, is by the methylation-sensitive enzyme by methylation at cytosine in the J. J. Mol. Gen. Genet. 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Accordingly, in the present of the an and any that hypermethylation of at the of genome methylation by Two and of the polymorphisms observed at and polymorphisms that are to both changes in A and a in methylation or a mutation The of in 4-week-old PBMC or of A analysis on 6-month-old mice revealed that the of increased by to or of compared with the group that were present in both a of at of or were a at analysis on aortic a of showed a in with PBMC The of showed or no with and and of at and Furthermore, of were in both The of PBMC and aortic revealed that were to the PBMC and the aorta of aortic at and polymorphisms that are to both analysis on and of 6-month-old mice by In these the of in matched PBMC and aorta of in that the of observed in Apoe–/– mice from the of on and or epigenetic that are to the Apoe for example, S. U. S. 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Lund et al. (Thu,) studied this question.