Heterozygous mutation of ataxia-telangiectasia mutated gene aggravates hypercholesterolemia in apoE-deficient mice

• DOI: 10.1194/jlr.m400430-jlr200
• Publication Date: 2005-05-02T00:12:57Z
• Authors: DongFang Wu; Hong Yang; Wei Xiang; LiChun Zhou; MingJian Shi; George Julies; Janice M. LaPlante; Billy R. Ballard; ZhongMao Guo
• Keywords: Cerebellar ataxia

Individuals with a heterozygous mutation at the ataxia-telangiectasia mutated gene (ATM) have been reported to be predisposed ischemic heart disease. This report examined for first time effect of ATM (ATM+/−) on plasma lipid levels and atherosclerosis intensity using ATM+/−, ATM+/+ (wild type), ATM+/+/LDLR−/− (low density lipoprotein receptor knockout), ATM+/−/LDLR−/−, ATM+/+/ApoE−/− (apolipoprotein E ATM+/−/ApoE−/− mice. Our data demonstrated that cholesterol triglyceride in ATM+/− ATM+/−/LDLR−/− mice were approximately same as those control mice, respectively. In contrast, level was significantly higher than addition, showed apoB-48 levels, slower clearance apoB-48-carrying lipoproteins, more advanced atherosclerotic lesions aorta compared mice.These novel results suggest product is involved an apoE-independent pathway catabolism remnants; therefore, superimposition onto ApoE deficiency background reduces lipoproteins from blood circulation promotes formation atherosclerosis. These ErrataJournal Lipid ResearchVol. 46Issue 11PreviewIn article "Heterozygous aggravates hypercholesterolemia apoE-deficient mice" by Wu et al., published July 2005 issue Journal Research (Volume 46, pages 1380–1387), correct spelling name one coauthors is: George Jules. Full-Text PDF Open Access The has nuclear protein several signaling pathways, including DNA damage recognition, cell cycle control, meiotic recombination (for review, see 1Rotman G. Shiloh Y. ATM: function.Hum. Mol. Genet. 1998; 7: 1555-1563Google Scholar). It now known fraction also present cytoplasm associated vesicular structures such peroxisomes (2Watters D. Kedar P. Spring K. Bjorkman J. Chen Gatei M. Birrell Garrone B. Srinivasa Crane D.I. al.Localization portion extranuclear peroxisomes.J. Biol. 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Zhou Campos-Torres Leder Pleiotropic protein-deficient mice.Proc. Natl. Acad. Sci. USA. 93: 13084-13089Google appear healthy. Similar subjects, greater sensitivity irradiation-induced cataracts (10Worgul B.V. Smilenov L. Brenner D.J. Junk W. Hall E.J. Atm radiation-induced their counterparts.Proc. 2002; 99: 9836-9839Google oncogenesis (11Smilenov L.B. Modest versus mammalian mice.Cancer 2001; 61: 5710-5713Google controls. this report, we effects By cross-breeding lacking apolipoprotein (ApoE−/−) low (LDLR−/−) alleles, following four inbred lines: 1) (ATM+/−/ApoE−/−); 2) intact (ATM+/+/ApoE−/−); 3) LDLR (ATM+/−/LDLR−/−); 4) (ATM+/+/LDLR−/−). ApoE−/− 4- 5-fold develop widespread vascular sites typically (12Piedrahita J.A. Zhang S.H. Hagaman J.R. Oliver P.M. Maeda N. Generation mutant inactivated targeting embryonic stem cells.Proc. 1992; 89: 4471-4475Google LDLR−/− ∼2-fold mice; however, they exhibit massive increases throughout response high-fat diet (13Ishibashi Goldstein J.L. Brown Herz Burns D.K. Massive xanthomatosis cholesterol-fed receptor-negative mice.J. Clin. Invest. 1994; 1885-1893Google study, observed among seen controls, littermates. Moreover, reduced lipoproteins. indicate that, deficiency, removal resulting severe kindly provided Dr. Anthony Wynshaw-Boris (University California, San Diego, CA). 129vEv genetic backcrossed into C57BL/6 12 generations. ApoE−/−, LDLR−/−, ApoB48/48/ApoE−/− Jackson Laboratory (Bar Harbor, ME). Piedrahita 10 Ishibashi five crossbreeding ApoB48/48 Farese (14Farese Jr., R.V. Veniant M.M. Cham C.M. Flynn L.M. Pierotti V. Loring J.F. Traber Ruland Stokowski Huszar al.Phenotypic analysis expressing exclusively B48 B100.Proc. 6393-6398Google expressed only apoB-100. deficient genes ATM+/−/ApoE−/−, used 14 weeks age. appeared healthy ATM+/+, ATM+/+/ApoE−/−, controls fed chow containing ∼5% fat 19% weight (Harlan Teklad, Madison, WI) after weaning. From 6 littermates either 15% 1.25% Teklad) 8 weeks. All procedures handling animals approved Institutional Animal Care Use Committees Meharry Medical College performed accordance guidelines American Association Accreditation National Institutes Health. Approximately 0.5 ml collected posterior vena cava anesthetized analgesic cocktail described previously (15Guo Z.M. Mitchell-Raymundo Yang H. Ikeno Nelson Diaz Richardson Reddick Dietary restriction oxidative stress E-deficient mice.Mech. Ageing Dev. 123: 1121-1131Google 100 μl sample individual fractionated fast-performance liquid chromatography (FPLC) (Äkta FPLC 900; Amersham Biosciences, Piscataway, NJ) buffer 0.15 M NaCl, 0.01 Na2HPO4, 0.1 mM EDTA, pH 7.5, flow rate ml/min. Forty fractions (0.5 ml/fraction) collected. triglycerides, phospholipids, total cholesterol, free measured spectrophotometric quantification reagents Sigma Chemical Co. (St. Louis, MO) Wako Chemicals USA (Richmond, VA). Briefly, aliquoted mixed triglyceride, phophospholipid, reaction glass microplate. mixtures incubated 37°C min. absorbance spectra read Dynex microplate reader (Thermo Labsystems, Franklin, MA) wavelength manufacturer. concentrations determined based incubation phospholipid, standards Wako. contents various calculated Hasty (16Hasty A.H. Linton M.F. L.L. Fazio Determination lower threshold remnant clearance.J. 40: 1529-1538Google Fractions 14–17 contained VLDL chylomicrons, 18–25 LDL, 26–40 HDL. 0.4 overlaid 0.6 KBr gradient solution (d = 1.215). Total isolated samples Sorvall Discovery M150 ultracentrifuge (Kendro Products, Asheville, NC) 120,000 rpm 2 h. Lipoproteins centrifuge tubes pooled delipidated ethyl ether Mindham Mayes (17Mindham M.A. P.A. simple rapid method preparation apolipoproteins.Electrophoresis. 16: 998-1001Google extracts (50 μg) boiled 3 min sampling 125 Tris-HCl, 20% glycerol, 4% SDS, 0.05% bromophenol blue, 2-mercaptoethanol. electrophoresed SDS-5%/15% polyacrylamide gel. Proteins stained Coomassie blue. gel images captured AlphaImager system (Alpha Innotech Co., Leandro, CA), relative band quantified software. ApoB-48-carrying prepared Plasma < 1.006) centrifuged h ultracentrifuge. collected, dialyzed PBS (pH 7.4) EDTA 48 4°C, filtered through 0.45 μm filter. One milliliter concentration 1 mg/ml 0.2 glycine 0.25 NaOH 10) then 7 iodine monochloride, μCi/μl 125I, 25 10). mixture room temperature applied DG column (Bio-Rad Laboratories, Hercules, CA) remove (18Sobal Resch U. Sinzinger Modification low-density different radioiodination methods.Nucl. Med. 2004; 31: 381-388Google 125I-labeled eluted extensively against 7.4). radiolabeled (1.5 μg protein/4 μl/g body weight) injected tail vein Blood (25 μl) drawn retro-orbital venous plexus puncture indicated points injection. Aliquots analyzed radioactivity universal γ counter (1282 Compugamma; Perkin-Elmer Life Analytic Science, Shelton, CT). counts assuming represents (v/w) weight. After last procedure, flush-perfused PBS. Whole livers liver determined. Fasted placed fat-free food via 200 10% Triton WR1339 (Sigma). (40 before injection times. triglycerides above. presence quantity apoB-100 Western blots. separated 6% SDS-PAGE gel, proteins transferred nitrocellulose membranes electrophoretically semidry transfer Laboratories). membrane immunoblotted sequentially apoB antibody (Santa Cruz Biotechnology, Santa Cruz, horseradish peroxidase-conjugated secondary antibody. ECL plus blotting detection (Amersham NJ), fluorescence detected laser scanner (Typhoon 9410; Biosciences), image-analysis (ImageQuant; Biosciences). Under anesthesia, perfusion-fixed paraformaldehyde μM butylated hydroxytoluene constant, near-physiological pressure (80 mm Hg) fixation, tree removed body. cut heart. proximal attached prepare microscope sections transversely sectioned immediately below parallel plane formed drawing line between atrial leaflets. section discarded, metal stub optimal cutting compound (Sakura Finetek USA, Torrance, sectioning followed toward root where aortic valves attached. Sections (8 μm) valve cups emerge root. Every other set slides Oil Red O viewed (E600; Nikon Instruments, Inc., Melville, NY) equipped color digital camera (CoolSnaps; Instruments) computer image-acquisition (MetaMorph image system; Instruments). average area (μm2) morphological features (foam deposition, clefts, acellular areas, fibrous caps) 16 each distal (2 iliac bifurcation) opened longitudinally microscissors pinned flat black wax surface dissecting pan (SMZ1000; en face fixed overnight Sudan IV photo-image CoolSnaps (Nikon mounted SMZ1000 microscope. lesion MetaMorph Data percentages covered lesions. measurements conducted double-blind manner (i.e., person taking aware tissue sources, identified when all tissues analyzed). means ± SEM. differences multiple-factor ANOVA Shapiro-Wilk test. Differences considered significant P 0.05. statistical analyses STASTIX software (Analytical Software, Tallahassee, FL). Table shows lipids diet. total, free, esterified ratio indistinguishable ∼60% being esterified. To investigate distribution system. As shown Fig. 1, arose mainly LDL fractions. mean (Table 1). HDL comparable phospholipid slightly, significantly, mice.TABLE 1Lipid lipoproteinsSourceTotal CholesterolFree CholesterolEsterified CholesterolPhospholipidTriglycerideATM+/+/ApoE−/−Plasma486 29196 15314 324 18 146 18VLDL304 23140 8189 13 159 11 95 12LDL139 1648 789 97 22 6HDL47 613 437 5 72 9 5ATM+/−/ApoE−/−Plasma652 33aSignificantly (P 0.05).276 17aSignificantly 0.05).405 25aSignificantly 0.05). 395 22aSignificantly 162 32VLDL416 18aSignificantly 0.05).197 13aSignificantly 0.05).237 10aSignificantly 201 14aSignificantly 115 18LDL177 0.05).72 6aSignificantly 0.05).113 8aSignificantly 31 11HDL57 714 444 78 7Plasma Materials Methods. VLDL, Esterified difference cholesterol. Values mg/dl represent SEM 15 mice.a Significantly table new tab We SDS-PAGE. Whereas barely detectable greatest change 2, ∼30% contrast changes apoB-48, (Fig. 2). result compares Feeding content did triglycerides. respectively, under selectively severity ApoE-deficient 2Cholesterol plasmaNormal ChowHigh-Fat DietMouseTotal CholesterolTriglycerideTotal CholesterolTriglycerideATM+/+ 105 60 9174 16aSignificantly genotype 57 5ATM+/− 109 8192 63 5ATM+/+/LDLR−/− 263 158 17922 81aSignificantly 342 61aSignificantly 0.05).ATM+/−/LDLR−/− 274 143 12905 58aSignificantly 355 30aSignificantly 0.05).ATM+/− fasting. whether resulted proteins, circulation. Figure 3Ashows lipop