ApoE−/−Fas−/− C57BL/6 mice: a novel murine model simultaneously exhibits lupus nephritis, atherosclerosis, and osteopenia
2007; Elsevier BV; Volume: 48; Issue: 4 Linguagem: Inglês
10.1194/jlr.m600512-jlr200
ISSN1539-7262
AutoresXuebing Feng, Hongyun Li, Alexis A. Rumbin, Xuping Wang, Antonio La Cava, Katherine Brechtelsbauer, Lawrence W. Castellani, Joseph L. Witztum, Aldons J. Lusis, Betty P. Tsao,
Tópico(s)Caveolin-1 and cellular processes
ResumoTo establish a mouse model of accelerated atherosclerosis in lupus, we generated apolipoprotein E-deficient (apoE−/−) and Faslpr/lpr (Fas−/−) C57BL/6 mice. On a normal chow diet, 5 month old apoE−/−Fas−/− mice had enlarged glomerular tuft areas, severe proteinuria, increased circulating autoantibody levels, and increased apoptotic cells in renal and vascular lesions compared with either single knockout mice. Also, double knockout mice developed increased atherosclerotic lesions but decreased serum levels of total and non-HDL cholesterol compared with apoE−/−Fas+/+ littermates. Moreover, female apoE−/−Fas−/− mice had lower vertebral bone mineral density (BMD) and bone volume density (BV/TV) than age-matched female apoE−/−Fas+/+ mice. Compared with apoE−/−Fas+/+ and apoE+/+Fas−/− mice, apoE−/−Fas−/− mice had decreased circulating oxidized phospholipid (OxPL) content on apoB-100 containing lipoprotein particles and increased serum IgG antibodies to OxPL, which were significantly correlated with aortic lesion areas (r = 0.58), glomerular tuft areas (r = 0.87), BMD (r = −0.57), and BV/TV (r = −0.72). These results suggest that the apoE−/−Fas−/− mouse model might be used to study atherosclerosis and osteopenia in lupus. Correlations of IgG anti-OxPL with lupus-like disease, atherosclerosis, and bone loss suggested a shared pathway of these disease processes. To establish a mouse model of accelerated atherosclerosis in lupus, we generated apolipoprotein E-deficient (apoE−/−) and Faslpr/lpr (Fas−/−) C57BL/6 mice. On a normal chow diet, 5 month old apoE−/−Fas−/− mice had enlarged glomerular tuft areas, severe proteinuria, increased circulating autoantibody levels, and increased apoptotic cells in renal and vascular lesions compared with either single knockout mice. Also, double knockout mice developed increased atherosclerotic lesions but decreased serum levels of total and non-HDL cholesterol compared with apoE−/−Fas+/+ littermates. Moreover, female apoE−/−Fas−/− mice had lower vertebral bone mineral density (BMD) and bone volume density (BV/TV) than age-matched female apoE−/−Fas+/+ mice. Compared with apoE−/−Fas+/+ and apoE+/+Fas−/− mice, apoE−/−Fas−/− mice had decreased circulating oxidized phospholipid (OxPL) content on apoB-100 containing lipoprotein particles and increased serum IgG antibodies to OxPL, which were significantly correlated with aortic lesion areas (r = 0.58), glomerular tuft areas (r = 0.87), BMD (r = −0.57), and BV/TV (r = −0.72). These results suggest that the apoE−/−Fas−/− mouse model might be used to study atherosclerosis and osteopenia in lupus. Correlations of IgG anti-OxPL with lupus-like disease, atherosclerosis, and bone loss suggested a shared pathway of these disease processes. Cardiovascular complications related to atherosclerosis are common among patients with systemic lupus erythematosus (SLE) and contribute to their disability and death (1Bacon P.A. Stevens R.J. Carruthers D.M. Young S.P. Kitas G.D. Accelerated atherogenesis in autoimmune rheumatic diseases. Autoimmun. Rev. 2002; 1: 338-347Google Scholar, 2Borchers A.T. Keen C.L. Shoenfeld Y. Gershwin M.E. Surviving the butterfly and the wolf: mortality trends in systemic lupus erythematosus. Autoimmun. Rev. 2004; 3: 423-453Google Scholar, 3Roman M.J. Shanker B.A. Davis A. Lockshin M.D. Sammaritano L. Simantov R. Crow M.K. Schwartz J.E. Paget S.A. Devereux R.B. Salmon D.J. Prevalence and correlates of accelerated atherosclerosis in systemic lupus erythematosus. N. Engl. J. Med. 2003; 349: 2399-2406Google Scholar, 4Asanuma Y. Oeser A. Shintani A.K. Turner E. Olsen N. Fazio S. Linton M.F. Raggi P. Stein C.M. Premature coronary-artery atherosclerosis in systemic lupus erythematosus. N. Engl. J. Med. 2003; 349: 2407-2415Crossref PubMed Scopus (711) Google Scholar). Women with SLE between 35 and 44 years old have an estimated 50-fold increased risk of myocardial infarction compared with age- and gender-matched controls (5Manzi S. Meilahn E.N. Rairie J.E. Conte C.G. Medsger T.A. Jansen-McWilliams L. Agostino R.B. Kuller L.H. Age-specific incidence rates of myocardial infarction and angina in women with systemic lupus erythematosus: comparison with the Framingham Study. Am. J. Epidemiol. 1997; 145: 408-415Google Scholar). After controlling for traditional Framingham risk factors, the relative risk for early coronary heart disease attributed to SLE itself is 7.5-fold (6Esdaile J.M. Abrahamowicz M. Grodzicky T. Li Y. Panaritis C. du B.R. Cote R. Grover S.A. Fortin P.R. Clarke A.E. Senecal J.L. Traditional Framingham risk factors fail to fully account for accelerated atherosclerosis in systemic lupus erythematosus. Arthritis Rheum. 2001; 44: 2331-2337Google Scholar). An increased prevalence of subclinical atherosclerosis in SLE has also been established: 37% of SLE patients versus 15% of controls have plaques detectable by carotid artery ultrasound (3Roman M.J. Shanker B.A. Davis A. Lockshin M.D. Sammaritano L. Simantov R. Crow M.K. Schwartz J.E. Paget S.A. Devereux R.B. Salmon D.J. Prevalence and correlates of accelerated atherosclerosis in systemic lupus erythematosus. N. Engl. J. Med. 2003; 349: 2399-2406Google Scholar), and 31% of SLE patients versus 9% of controls exhibit coronary artery calcification (4Asanuma Y. Oeser A. Shintani A.K. Turner E. Olsen N. Fazio S. Linton M.F. Raggi P. Stein C.M. Premature coronary-artery atherosclerosis in systemic lupus erythematosus. N. Engl. J. Med. 2003; 349: 2407-2415Crossref PubMed Scopus (711) Google Scholar). The disease processes of SLE itself, including a longer duration of disease, a higher damage-index score, and less aggressive immunosuppressive therapy, are independent risk factors for atherosclerosis (3Roman M.J. Shanker B.A. Davis A. Lockshin M.D. Sammaritano L. Simantov R. Crow M.K. Schwartz J.E. Paget S.A. Devereux R.B. Salmon D.J. Prevalence and correlates of accelerated atherosclerosis in systemic lupus erythematosus. N. Engl. J. Med. 2003; 349: 2399-2406Google Scholar). Similar to accelerated atherosclerosis in SLE, bone loss in SLE involves both traditional osteoporosis risk factors and lupus-related factors. More than one-third of SLE patients were osteopenic by bone mineral density (BMD) scanning (7Bultink I.E. Lems W.F. Kostense P.J. Dijkmans B.A. Voskuyl A.E. Prevalence of and risk factors for low bone mineral density and vertebral fractures in patients with systemic lupus erythematosus. Arthritis Rheum. 2005; 52: 2044-2050Google Scholar, 8Redlich K. Ziegler S. Kiener H.P. Spitzauer S. Stohlawetz P. Bernecker P. Kainberger F. Grampp S. Kudlacek S. Woloszczuk W. Smolen J.S. Pietschmann P. Bone mineral density and biochemical parameters of bone metabolism in female patients with systemic lupus erythematosus. Ann. Rheum. Dis. 2000; 59: 308-310Google Scholar) More strikingly, at least 20% of SLE patients (mean age of 41 years) had osteoporotic vertebral fractures compared with a 12% bone facture frequency in a much older general population in Europe (65–69 years) (7Bultink I.E. Lems W.F. Kostense P.J. Dijkmans B.A. Voskuyl A.E. Prevalence of and risk factors for low bone mineral density and vertebral fractures in patients with systemic lupus erythematosus. Arthritis Rheum. 2005; 52: 2044-2050Google Scholar). In addition to age, diabetes, a proinflammatory state, increased cholesterol and estrogen levels, and treatment with corticosteroids also contribute to bone mineral loss in SLE patients (9Carlsten H. Immune responses and bone loss: the estrogen connection. Immunol. Rev. 2005; 208: 194-206Google Scholar). Of interest, a recent cross-sectional study revealed an association between disease damage and lower BMD in women with SLE independent of prior use of corticosteroid (10Lee C. Almagor O. Dunlop D.D. Manzi S. Spies S. Chadha A.B. Ramsey-Goldman R. Disease damage and low bone mineral density: an analysis of women with systemic lupus erythematosus ever and never receiving corticosteroids. Rheumatology (Oxford). 2006; 45: 53-60Google Scholar), a finding similar to the described nontraditional risk factor for accelerated atherosclerosis in SLE. Inflammatory responses to oxidized phospholipids (OxPLs) seem to be shared by atherosclerosis, osteoporosis, and lupus (11Demer L.L. Vascular calcification and osteoporosis: inflammatory responses to oxidized lipids. Int. J. Epidemiol. 2002; 31: 737-741Google Scholar, 12Frostegard J. Autoimmunity, oxidized LDL and cardiovascular disease. Autoimmun. Rev. 2002; 1: 233-237Google Scholar). Oxidized low density lipoprotein (OxLDL) has been recognized as a key component in modulating atherosclerosis (13Binder C.J. Chang M.K. Shaw P.X. Miller Y.I. Hartvigsen K. Dewan A. Witztum J.L. Innate and acquired immunity in atherogenesis. Nat. Med. 2002; 8: 1218-1226Google Scholar, 14Frostegard J. SLE, atherosclerosis and cardiovascular disease. J. Intern. Med. 2005; 257: 485-495Google Scholar, 15Berliner J.A. Watson A.D. A role for oxidized phospholipids in atherosclerosis. N. Engl. J. Med. 2005; 353: 9-11Crossref PubMed Scopus (218) Google Scholar). Several OxPLs present in mildly modified/OxLDL, such as 1-palmitoyl-2(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), can activate genes necessary for the cellular responses observed in the development of fatty streaks (16Navab M. Hama S.Y. Reddy S.T. Ng C.J. Van Lenten B.J. Laks H. Fogelman A.M. Oxidized lipids as mediators of coronary heart disease. Curr. Opin. Lipidol. 2002; 13: 363-372Crossref PubMed Scopus (91) Google Scholar). Increased OxLDL (17Frostegard J. Svenungsson E. Wu R.H. Gunnarsson I. Lundberg I.E. Klareskog L. Horkko S. Witztum J.L. Lipid peroxidation is enhanced in patients with systemic lupus erythematosus and is associated with arterial and renal disease manifestations. Arthritis Rheum. 2005; 52: 192-200Google Scholar) and IgG antibodies to OxLDL (17Frostegard J. Svenungsson E. Wu R.H. Gunnarsson I. Lundberg I.E. Klareskog L. Horkko S. Witztum J.L. Lipid peroxidation is enhanced in patients with systemic lupus erythematosus and is associated with arterial and renal disease manifestations. Arthritis Rheum. 2005; 52: 192-200Google Scholar, 18Soep J.B. Mietus-Snyder M. Malloy M.J. Witztum J.L. von Scheven E. Assessment of atherosclerotic risk factors and endothelial function in children and young adults with pediatric-onset systemic lupus erythematosus. Arthritis Rheum. 2004; 51: 451-457Google Scholar) have been observed in SLE patients compared with normal controls, implying a potential contribution to accelerated atherosclerosis in SLE. Additionally, oxidized lipids can inhibit osteoblastic differentiation and calcium uptake of bone-derived preosteoblasts in vitro (19Parhami F. Morrow A.D. Balucan J. Leitinger N. Watson A.D. Tintut Y. Berliner J.A. Demer L.L. Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation. A possible explanation for the paradox of arterial calcification in osteoporotic patients. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 680-687Google Scholar). Osteoporosis has been observed more frequently in SLE patients with cardiovascular disease, which is also associated with increased OxLDL and autoantibodies to OxLDL (12Frostegard J. Autoimmunity, oxidized LDL and cardiovascular disease. Autoimmun. Rev. 2002; 1: 233-237Google Scholar). To date, progress in understanding the underlying pathogenesis of atherosclerosis and osteoporosis in SLE has been impaired by the lack of appropriate animal models. Recently, apolipoprotein E-deficient (apoE−/−) gld/gld (Fas ligand-deficient; FasL−/−) C57BL/6 (B6) mice and LDLr.Sle mice (radiation chimeras of LDL receptor null B6 mice transplanted with B6.Sle1.2.3.) were reported as models of accelerated atherosclerosis (20Aprahamian T. Rifkin I. Bonegio A. Hugel B. Freyssinet J.M. Sato K. Castellot J.J. Walsh K. Impaired clearance of apoptotic cells promotes synergy between atherogenesis and autoimmune disease. J. Exp. Med. 2004; 199: 1121-1131Google Scholar, 21Stanic A.K. Stein C.M. Morgan A.C. Fazio S. Linton M.F. Wakeland E.K. Olsen N.J. Major A.S. Immune dysregulation accelerates atherosclerosis and modulates plaque composition in systemic lupus erythematosus. Proc. Natl. Acad. Sci. USA. 2006; 103: 7018-7023Google Scholar). These mice develop atherosclerotic lesions, lymphadenopathy, splenomegaly, and autoantibodies on a Western diet. Meanwhile, MRL/lpr mice, which spontaneously develop decreased bone formation and bone turnover compared with age-matched MRL/n mice, are a promising model of osteoporosis in murine lupus (22Schapira D. Kabala A. Raz B. Israeli E. Osteoporosis in murine systemic lupus erythematosus—a laboratory model. Lupus. 2001; 10: 431-438Google Scholar). To better understand the link between atherosclerosis, osteopenia, and lupus, we have established a new mouse model, double knockout apoE−/−Fas−/− B6 mice, which spontaneously develop lupus-like disease, increased atherosclerotic lesions, and decreased BMD by 5 months of age on a normal chow diet. This mouse model is characterized by increased apoptotic cells in renal and heart vessels, decreased levels of OxPL on apoB-100-containing particles, and increased levels of autoantibodies to OxPL in the circulation. Correlations of IgG anti-OxPL levels with atherosclerotic lesions, glomerular tuft areas, and BMD suggest that shared pathways may promote synergy leading to the development of accelerated atherosclerosis, glomerulonephritis, and osteopenia. ApoE−/− and Fas−/− (lpr/lpr) mice on the B6 background were purchased from the Jackson Laboratories (Bar Harbor, ME). Single knockout mice were intercrossed and backcrossed to the apoE−/− parents to produce three groups of mice with the following genotypes: apoE−/−Fas+/+, apoE−/−Fas+/−, and apoE−/−Fas−/−. DNA was extracted from ear tissue using the Qiagen DNeasy Tissue Kit. Genotyping of the wild-type versus the apoE knockout allele (23Piedrahita J.A. Zhang S.H. Hagaman J.R. Oliver P.M. Maeda N. Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc. Natl. Acad. Sci. USA. 1992; 89: 4471-4475Google Scholar) and the lpr allele (24Croker B.P. Gilkeson G. Morel L. Genetic interactions between susceptibility loci reveal epistatic pathogenic networks in murine lupus. Genes Immun. 2003; 4: 575-585Google Scholar) was performed as described. Mice were fasted for 16 h before bleeding. Serum samples of apoE−/−Fas+/+, apoE−/−Fas+/−, and apoE−/−Fas−/− littermates at 6 weeks and 5 months of age, and serum samples from apoE+/+Fas−/− mice at 5 months old, were collected. Mice at 5 or 9 months old were euthanized, with kidneys and heart harvested and spleen weighed. Lumbar vertebrae and limbs were excised. All mice were treated in conformity with Public Health Service policy. They were fed with a regular chow diet and maintained in a temperature-controlled room with a 12 h light/dark cycle according to the approved protocol by the University of California, Los Angeles Animal Research Committee. Total IgG level was measured with an ELISA kit (Bethyl Laboratories). Serum samples were diluted 1:50 to measure levels of IgG anti-double-stranded DNA (dsDNA) using a streptavidin-biotin method of ELISA (25Haywood M.E.K. Rogers N.J. Rose S.J. Boyle J. McDermott A. Rankin J.M. Thiruudaian V. Lewis M.R. Fossati-Jimack L. Izui S. Walport M.J. Morley B.J. Dissection of BXSB lupus phenotype using mice congenic for chromosome 1 demonstrates that separate intervals direct different aspects of disease. J. Immunol. 2004; 173: 4277-4285Google Scholar) and IgG anti-cardiolipin ELISA (26Edwards M.H. Pierangeli S. Liu X. Barker J.H. Anderson G. Harris E.N. Hydroxychloroquine reverses thrombogenic properties of antiphospholipid antibodies in mice. Circulation. 1997; 96: 4380-4384Google Scholar). Serially diluted, pooled serum samples from 43 week old NZB/NZW F1 mice were used to generate standard curves for IgG antibodies to dsDNA or cardiolipin. Serum IgG and IgM antibodies to POVPC and PGPC were measured with anti-cardiolipin ELISA. Briefly, POVPC or PGPC (Avanti Polar Lipids) at 50 μg/ml in 100% ethanol was coated onto an Immunon 2 flat plate overnight at 4°C and blocked with 0.25% gelatin/PBS for 1 h at room temperature. After the addition of diluted mouse serum (1:50) samples in duplicate, the plate was incubated for 2 h and developed with the addition of HRP-conjugated goat anti-mouse total IgG or IgM (1:3,000) and tetramethylbenzidine plus hydrogen peroxide as substrate. Serum from one of the apoE−/−Fas−/− mice with the highest anti-OxPL level was serially diluted to generate standard curves. Peripheral blood samples from 5 month old apoE−/− and apoE−/−Fas−/− B6 mice were collected to analyze the composition of circulating leukocytes after red-cell lysis and multi-color flow cytometry on a FACSCalibur (BD Biosciences, San Jose, CA) with CellQuest software (BD Biosciences). For the identification of leukocyte subsets, the following markers were used: CD4 and CD8 for T-cells, CD19 for B-cells, NK1.1 for NK-cells, DEC205 for dendritic cells, CD11b+CD16− for neutrophils, and CD11b for monocytes/macrophages. Flurochrome-conjugated monoclonal antibodies were all purchased from BD PharMingen (San Diego, CA). Serum lipid levels (total cholesterol, unesterified cholesterol, triglycerides, HDL, and free fatty acids) were measured from 5 month old mice after 16 h of fasting using enzymatic colorimetric assays as described previously (27Shi W. Wang X. Wang N.J. McBride W.H. Lusis A.J. Effect of macrophage-derived apolipoprotein E on established atherosclerosis in apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2261-2266Crossref PubMed Scopus (32) Google Scholar). The OxPL content of mouse apoB-100-containing lipoprotein particles was assessed using a sandwich chemiluminescence immunoassay in which EO6 was used to detect OxPL epitopes on the captured particles and LF5 was used to capture serum apoB-100 (28Binder C.J. Hartvigsen K. Chang M.K. Miller M. Broide D. Palinski W. Curtiss L.K. Corr M. Witztum J.L. IL-5 links adaptive and natural immunity specific for epitopes of oxidized LDL and protects from atherosclerosis. J. Clin. Invest. 2004; 114: 427-437Google Scholar, 29Schneider M. Witztum J.L. Young S.G. Ludwig E.H. Miller E.R. Tsimikas S. Curtiss L.K. Marcovina S.M. Taylor J.M. Lawn R.M. Innerarity T.L. Pitas R.E. High-level lipoprotein [a] expression in transgenic mice: evidence for oxidized phospholipids in lipoprotein [a] but not in low density lipoproteins. J. Lipid Res. 2005; 46: 769-778Google Scholar). Murine apoB-100-specific monoclonal antibodies LF3 and LF5 (30Zlot C.H. Flynn L.M. Veniant M.M. Kim E. Raabe M. McCormick S.P.A. Ambroziak P. McEvoy L.M. Young S.G. Generation of monoclonal antibodies specific for mouse apolipoprotein B-100 in apolipoprotein B-48-only mice. J. Lipid Res. 1999; 40: 76-84Google Scholar) were purified from the ascites (kindly provided by S. G. Young) using the HiTrap Protein G column (Amersham Biosciences). The protein concentration of the IgG fraction was determined using the Bio-Rad Protein Assay (Bio-Rad). The monoclonal LF5 IgG was biotinylated with EZ-link Sulfo-NHS-Biotin (Pierce) and dialyzed in PBS using Slide-A-Lyzer Dialysis Cassettes (10K MWCO; Pierce). The apoB-100-specific monoclonal antibody LF3 was coated on FluoroNunc 96-well MaxiSorp plates (Fisher Scientific) at 5 μg/ml in PBS overnight at 4°C. After blocking with 0.25% gelatin-PBS, the plates were incubated with serum (1:80 dilution in PBS) for 1 h at room temperature. Then, biotin-labeled EO6 (2 μg/ml in PBS) was added with biotin-labeled LF5 (2 μg/ml in PBS) in parallel wells to detect the relative amount of apoB captured in each sample for 1 h. The plates were incubated with alkaline phosphatase streptavidin (Vector Laboratories) for 1 h, then incubated with 50% Lumi-Phos (Lumigen, Inc.) in distilled water for 30 min in the dark. The chemiluminescence was read on an Orion Microplate Luminometer (Dynex Technologies), and data were expressed in relative light units (RLU) measured over 100 ms. Each serum sample was measured in duplicate for both EO6 and LF5. The relative number of EO6 epitopes (OxPL) bound per apoB-100 particle was then determined by dividing the bound EO6 RLU by the LF5 RLU. Five month old mice were euthanized, and one half of a kidney was fixed in 10% formaldehyde and embedded into paraffin. Three micrometer sections of the kidney tissue were cut and observed for various morphologic lesions after periodic acid Schiff staining. Stained sections were coded and digitally photographed using a microscope fitted with a digital camera (Nikon Eclipse CFI60). Images were analyzed using Image ProPlus software (Media Cybernetics) by a blinded observer to measure the size of the glomerular tuft area as described (31Muhlfeld A.S. Segerer S. Hudkins K. Carling M.D. Wen M. Farr A.G. Ravetch J.V. Alpers C.E. Deletion of the fcgamma receptor IIb in thymic stromal lymphopoietin transgenic mice aggravates membranoproliferative glomerulonephritis. Am. J. Pathol. 2003; 163: 1127-1136Google Scholar). At least 25 glomeruli per sample were observed in five photographs from different fields of duplicate slides to calculate the mean glomerular tuft area for each mouse. The other half of the kidney was embedded in Tissue-Tec OCT medium, frozen in liquid nitrogen, and stored at −70°C until sectioning. Five micrometer frozen sections were fixed with 4% paraformaldehyde and blocked with 2% BSA. Subsequently, slides were stained with fluorescein-conjugated goat anti-mouse IgG (1:150 dilution; Sigma) or fluorescein-conjugated goat IgG fraction to mouse complement C3 (1:150 dilution; Cappel Laboratories), mounted with Gel/Mount (Biomeda), and analyzed by fluorescence microscopy. At least 25 glomeruli were observed for each sample. Mice were monitored for proteinuria at ∼1–2 months and 5 months of age. Proteinuria was estimated by examination of fresh urine using Albustix (Bayer, Elkhart, IN). Grades of proteinuria were expressed as follows: 0 = none, 1 = trace, 2 = ∼30 mg/dl, 3 = ∼100 mg/dl, 4 = ∼300 mg/ml, and 5 = >2,000 mg/dl. The basal portion of the heart and the proximal aorta were harvested from 5 month old mice, washed in PBS to remove blood, embedded in Tissue-Tec OCT medium, frozen in liquid nitrogen, and stored at −70°C until sectioning. Serial 10 μm thick cryosections were collected, stained with Oil Red O and hematoxylin and counterstained with fast green, and then examined by light microscopy for the identification of atheromatous lesions (32Qiao J.H. Xie P.Z. Fishbein M.C. Kreuzer J. Drake T.A. Demer L.L. Lusis A.J. Pathology of atheromatous lesions in inbred and genetically-engineered mice—genetic determination of arterial calcification. Arterioscler. Thromb. 1994; 14: 1480-1497Google Scholar). Methods for IgG and C3 immunofluorescence staining of heart cryosections were the same as those described for kidney immunofluorescence. TUNEL staining of kidney and heart samples was performed according to the manufacturer's protocol (Roche Diagnostics Corp.) with a slight modification. Briefly, cryosections were fixed with 4% paraformaldehyde, permeabilized with freshly prepared 0.2% Triton X-100 in 0.1% sodium citrate on ice, added with the label solution in the presence or absence (negative control) of terminal deoxynucleotidyl transferase, and incubated for 1 h at 37°C. Subsequently, sections were counterstained with 4′,6-diamino-phenylindole (1:4,700 dilution; Sigma), and stained images were photographed by fluorescence microscopy. Total cells (stained by 4′,6-diamino-phenylindole) and apoptotic cells were counted using Image ProPlus software by a blinded observer. Excised limbs and vertebrae (including L2–L6 lumbar vertebrae) from 5 or 9 month old female mice were measured for BMD by dual-energy X-ray absorptiometry using a Lunar PIXImus2 instrument (GE Medical Systems, Madison, WI). Limb BMD was the average BMD of both arms and legs. The average value of the left and right femur was calculated as femoral BMD. Three-dimensional morphometric evaluation of the L5 lumbar vertebra was performed with a microtomographic imaging system (μCT 40; Scanco Medical, Bassersdorf, Switzerland). Parameters determined in the vertebral body included bone volume density (BV/TV), trabecular thickness, trabecular separation, trabecular number, and connectivity density. The results are presented as means ± SEM. Unpaired Student's t-test was conducted for comparisons between two groups if the variance was normally distributed, whereas the Mann-Whitney U test was used if the variance was not normally distributed. Comparisons among three or more groups were conducted using one-way ANOVA. Correlation between groups was evaluated using the Pearson rank test. Data were calculated using the Prism 3.0 program (GraphPad), and P < 0.05 was considered significant. Five month old apoE−/−Fas−/− B6 mice had significantly enlarged spleens (294 ± 33 mg; n = 16) compared with apoE−/−Fas+/− (98 ± 9 mg; n = 11) and apoE−/−Fas+/+ (72 ± 6 mg; n = 10) littermates as well as age-matched apoE+/+Fas−/− mice (132 ± 32 mg; n = 3) (P < 0.0001 by ANOVA). Similarly, apoE−/−Fas−/− mice had enlarged thymus and lymph nodes. The double knockout mice had significantly reduced levels of circulating CD4+ and CD8+ T-cells compared with age-matched apoE−/−Fas+/+ mice [18 ± 4.5% vs. 31 ± 1.3% (P = 0.036) and 17 ± 1.8% vs. 22 ± 0.4% (P = 0.024), respectively]. No significant differences in other leukocyte subsets (B-cells, NK-cells, monocytes/macrophages, and neutrophils) were found between 5 month old apoE−/−Fas−/− and apoE−/−Fas+/+ mice (data not shown). Serum levels of total IgG and IgG anti-dsDNA were significantly higher in apoE−/−Fas−/− mice than in apoE−/−Fas+/+ and apoE−/−Fas+/− littermates at both 6 weeks and 5 months of age (overall P < 0.0001 by ANOVA) (Fig. 1 ). Compared with 5 month old apoE+/+Fas−/− mice, age-matched apoE−/−Fas−/− mice had increased IgG antibodies to dsDNA (P < 0.05). IgG anti-cardiolipin levels were also increased significantly in apoE−/−Fas−/− mice at 5 months of age compared with those in apoE+/+Fas−/−, apoE−/−Fas+/+, and apoE−/−Fas+/− mice (overall P < 0.0001 by ANOVA). ApoE−/−Fas−/− mice exhibited renal damage similar to that found in lupus nephritis patients. Immunofluorescence staining of glomeruli showed that both IgG and C3 were present in the mesangium and peripheral capillary loops of 5 month old apoE−/−Fas−/− mice (Fig. 2A ). Kidneys from apoE−/−Fas−/− mice exhibited proliferation of glomerular cells and lobule formation, and mean glomeruli tuft areas of apoE−/−Fas−/− mice were enlarged compared with those of apoE−/−Fas+/− and apoE−/−Fas+/+ littermates and age-matched apoE+/+Fas−/− mice (5,778 ± 394 μm2 vs. 2,988 ± 82 μm2 and 3,409 ± 206 μm2, respectively; P = 0.004 and P = 0.024) (Fig. 2B, C). There were no significant differences in glomeruli tuft areas between male and female mice in each of the four groups. Consistent with the histological findings, 5 month old apoE−/−Fas−/− mice had increased proteinuria compared with age-matched apoE+/+Fas−/− mice (Fig. 2D), which was not evident in younger (∼1–2 month old) mice. The extent of atherosclerotic lesions was measured at the level of aortic valves and in the proximal aorta of each mouse. Measured lesion areas in apoE−/−Fas+/+ mice were in agreement with values reported previously (27Shi W. Wang X. Wang N.J. McBride W.H. Lusis A.J. Effect of macrophage-derived apolipoprotein E on established atherosclerosis in apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2261-2266Crossref PubMed Scopus (32) Google Scholar). Neither apoE−/−Fas−/− nor apoE−/−Fas+/+ mice showed gender differences in their lesion areas. Significantly, increased lesion area was noted at aortic valves in the apoE−/−Fas−/− mice (n = 11) on normal chow compared with apoE−/−Fas+/+ littermates (n = 16) (0.15 ± 0.012 mm2 vs. 0.088 ± 0.007 mm2, respectively; P = 0.0003) (Fig. 3A ). In contrast, 5 month old apoE+/+Fas−/− mice exhibited no evidence for aortic lesions either on a normal chow diet or after a 5 week high-fat diet (data not shown). Serum total cholesterol and unesterified cholesterol levels of apoE−/−Fas−/− mice were lower than those of apoE−/−Fas+/+ littermates (P < 0.05). No differences in serum triglycerides, HDL cholesterol, or free fatty acids were observed among mice of the three groups (Fig. 3B). The concentrations of HDL and non-HDL cholesterol could not explain the accelerated atherosclerosis in apoE−/−Fas−/− mice compared with apoE−/−Fas+/+ mice. Immunofluorescence staining showed IgG deposition in thickened aortic intimas of 5 month old apoE−/−Fas−/− mice compared with apoE−/−Fas+/+ littermates and age-matched apoE+/+Fas−/− mice (Fig. 3C). Negligible or trace staining of complement C3 deposition was observed in apoE−/−Fas−/− mice using immunofluorescence. Increased IgG deposition in the aortic intima and glomerulus support the hypothesis that IgG antibodies and their immune complexes were involved in the development of both atherosclerosis and lupus-like diseases in apoE−/−Fas−/− mice. Dual-energy X-ray absorptiometry analyses showed that BMD of all four limbs from each 5 month old female apoE−/−Fas −/− mouse was significantly lower than that from male apoE−/−Fas−/− mice (41.9 ± 0.44 mg/cm2vs. 47.1 ± 1.0 mg/cm2; P = 0.01). Compared with age-matched female apoE−/−Fas+/+ littermates, 5 month old female apoE−/−Fas−/− mice had decreased femoral BMD (49.6 ± 1.9 mg/cm2vs. 55.5 ± 1.5 mg/cm2; P = 0.040) (Fig. 3D). Vertebrae BMD in apoE−/−Fas−/− mice also showed a trend of lower levels than in apoE−/−Fas+/+ littermates (52.1 ± 1.7 mg/cm2vs. 57.0 ± 1.8 mg/cm2; P = 0.081). There were no significant differences in femoral and vertebral BMD
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