Complement C1q Reduces Early Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice
2007; Elsevier BV; Volume: 170; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.060406
ISSN1525-2191
AutoresVinay K. Bhatia, Sheng Yun, Viola Leung, David C. Grimsditch, G. Martin Benson, Marina Botto, Joseph J. Boyle, Dorian O. Haskard,
Tópico(s)Renal Diseases and Glomerulopathies
ResumoWe explored the role of the classic complement pathway in atherogenesis by intercrossing C1q-deficient mice (C1qa−/−) with low-density lipoprotein receptor knockout mice (Ldlr−/−). Mice were fed a normal rodent diet until 22 weeks of age. Aortic root lesions were threefold larger in C1qa−/−/Ldlr−/− mice compared with Ldlr−/− mice (3.72 ± 1.0% aortic root versus 1.1 ± 0.4%; mean ± SEM, P < 0.001). Furthermore, the cellular composition of lesions in C1qa−/−/Ldlr−/− was more complex, with an increase in vascular smooth muscle cells. The greater aortic root lesion size in C1qa−/−/Ldlr−/− mice occurred despite a significant reduction in C5b-9 deposition per lesion unit area, suggesting the critical importance of proximal pathway activity. Apoptotic cells were readily detectable by cleaved caspase-3 staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and electron microscopy in C1qa−/−/Ldlr−/−, whereas apoptotic cells were not detected in Ldlr−/− mice. This is the first direct demonstration of a role for the classic complement pathway in atherogenesis. The greater lesion size in C1qa−/−/Ldlr−/− mice is consistent with the emerging homeostatic role for C1q in the disposal of dying cells. This study suggests the importance of effective apoptotic cell removal for containing the size and complexity of early lesions in atherosclerosis. We explored the role of the classic complement pathway in atherogenesis by intercrossing C1q-deficient mice (C1qa−/−) with low-density lipoprotein receptor knockout mice (Ldlr−/−). Mice were fed a normal rodent diet until 22 weeks of age. Aortic root lesions were threefold larger in C1qa−/−/Ldlr−/− mice compared with Ldlr−/− mice (3.72 ± 1.0% aortic root versus 1.1 ± 0.4%; mean ± SEM, P < 0.001). Furthermore, the cellular composition of lesions in C1qa−/−/Ldlr−/− was more complex, with an increase in vascular smooth muscle cells. The greater aortic root lesion size in C1qa−/−/Ldlr−/− mice occurred despite a significant reduction in C5b-9 deposition per lesion unit area, suggesting the critical importance of proximal pathway activity. Apoptotic cells were readily detectable by cleaved caspase-3 staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and electron microscopy in C1qa−/−/Ldlr−/−, whereas apoptotic cells were not detected in Ldlr−/− mice. This is the first direct demonstration of a role for the classic complement pathway in atherogenesis. The greater lesion size in C1qa−/−/Ldlr−/− mice is consistent with the emerging homeostatic role for C1q in the disposal of dying cells. This study suggests the importance of effective apoptotic cell removal for containing the size and complexity of early lesions in atherosclerosis. The complement system contains a family of proteins that provides an important arm of the innate immune system.1Walport MJ Complement. 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Activation of C3 generates leukocyte chemoattractants C3a and C5a and the assembly of terminal complement complexes (C5b-7, C5b-8, C5b-9), which insert into cell membranes and cause cell lysis or activation.3Niculescu F Rus H Mechanisms of signal transduction activated by sublytic assembly of terminal complement complexes on nucleated cells.Immunol Res. 2001; 24: 191-199Crossref PubMed Scopus (58) Google Scholar However, not all effects of complement activation are proinflammatory, since upstream complement components (C1q, MBL, C3bi) may facilitate the clearance of apoptotic cells and other debris.4Mevorach D Mascarenhas JO Gershov D Elkon KB Complement-dependent clearance of apoptotic cells by human macrophages.J Exp Med. 1998; 188: 2313-2320Crossref PubMed Scopus (571) Google Scholar, 5Gershov D Kim S Brot N Elkon KB C-Reactive protein binds to apoptotic cells, protects the cells from assembly of the terminal complement components, and sustains an antiinflammatory innate immune response: implications for systemic autoimmunity.J Exp Med. 2000; 192: 1353-1364Crossref PubMed Scopus (576) Google Scholar, 6Fishelson Z Attali G Mevorach D Complement and apoptosis.Mol Immunol. 2001; 38: 207-219Crossref PubMed Scopus (170) Google Scholar Programed cell death (apoptosis) is a feature of human atherosclerosis and is associated with development of the lesion necrotic core as well as instability of complex plaques.7Han DKM Hauderschild CC Hong MK Tinkle BT Leon MB Liau G Evidence for apoptosis in human atherogenesis and in a rat vascular injury model.Am J Pathol. 1995; 147: 267-277PubMed Google Scholar, 8Björkerud S Björkerud B Apoptosis is abundant in human atherosclerotic lesions, especially in inflammatory cells (macrophages and T cells), and may contribute to the accumulation of gruel and plaque instability.Am J Pathol. 1996; 149: 367-380PubMed Google Scholar, 9Mallat Z Ohan J Leseche G Tedgui A Colocalization of CPP-32 with apoptotic cells in human atherosclerotic plaques.Circulation. 1997; 96: 424-428Crossref PubMed Scopus (90) Google Scholar Stimuli that trigger macrophage apoptosis include ingestion of free cholesterol and oxidized low-density lipoprotein (LDL).10Yao PM Tabas I Free cholesterol loading of macrophages induces apoptosis involving the fas pathway.J Biol Chem. 2000; 275: 23807-23813Crossref PubMed Scopus (159) Google Scholar, 11Tabas I Consequences of cellular cholesterol accumulation: basic concepts and physiological implications.J Clin Invest. 2002; 110: 905-911Crossref PubMed Scopus (528) Google Scholar, 12Carpenter KL Challis IR Arends MJ Mildly oxidised LDL induces more macrophage death than moderately oxidised LDL: roles of peroxidation, lipoprotein-associated phospholipase A2 and PPARgamma.FEBS Lett. 2003; 553: 145-150Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar On the other hand, vascular smooth muscle cell (VSMC) apoptosis may be stimulated by modified LDL, tumor necrosis factor α, and other cytokines, or by surface contact interactions with activated macrophages.13Niemann-Jönsson A Ares MP Yan ZQ Bu DX Fredrikson GN Branen L Porn-Ares I Nilsson AH Nilsson J Increased rate of apoptosis in intimal arterial smooth muscle cells through endogenous activation of TNF receptors.Arterioscler Thromb Vasc Biol. 2001; 21: 1909-1914Crossref PubMed Scopus (40) Google Scholar, 14Boyle JJ Weissberg PL Bennett MR Human macrophage-induced vascular smooth muscle cell apoptosis requires NO enhancement of Fas/Fas-L interactions.Arterioscler Thromb Vasc Biol. 2002; 22: 1624-1630Crossref PubMed Scopus (91) Google Scholar, 15Loidl A Sevcsik E Riesenhuber G Deigner HP Hermetter A Oxidized phospholipids in minimally modified low density lipoprotein induce apoptotic signaling via activation of acid sphingomyelinase in arterial smooth muscle cells.J Biol Chem. 2003; 278: 32921-32928Crossref PubMed Scopus (78) Google Scholar Apoptotic cells are often detectable in late atherosclerotic lesions but are not usually identified in early lesions, in large part due to highly efficient removal of apoptotic cells by macrophages and also, possibly, a more proapoptotic environment in advanced lesions.16Kockx MM Apoptosis in the atherosclerotic plaque: quantitative and qualitative aspects.Arterioscler Thromb Vasc Biol. 1998; 18: 1519-1522Crossref PubMed Scopus (211) Google Scholar, 17Tabas I Consequences and therapeutic implications of macrophage apoptosis in atherosclerosis: the importance of lesion stage and phagocytic efficiency.Arterioscler Thromb Vasc Biol. 2005; 25: 2255-2264Crossref PubMed Scopus (541) Google Scholar Complement C1q initiates activation of the classic pathway, typically through binding immunoglobulin Fc in immune complexes. However, C1q also binds apoptotic cells and plays an important role in their disposal.18Korb LC Ahearn JM C1q binds directly and specifically to surface blebs of apoptotic human keratinocytes: complement deficiency and systemic lupus erythematosus revisited.J Immunol. 1997; 158: 4525-4528PubMed Google Scholar, 19Navratil JS Watkins SC Wisnieski JJ Ahearn JM The globular heads of C1q specifically recognize surface blebs of apoptotic vascular endothelial cells.J Immunol. 2001; 166: 3231-3239PubMed Google Scholar, 20Taylor PR Carugati A Fadok VA Cook HT Andrews M Carroll MC Savill JS Henson PM Botto M Walport MJ A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo.J Exp Med. 2000; 192: 359-366Crossref PubMed Scopus (623) Google Scholar, 21Nauta AJ Trouw LA Daha MR Tijsma O Nieuwland R Schwaeble WJ Gingras AR Mantovani A Hack EC Roos A Direct binding of C1q to apoptotic cells and cell blebs induces complement activation.Eur J Immunol. 2002; 32: 1726-1736Crossref PubMed Scopus (254) Google Scholar The mechanism underlying C1q-mediated clearance of apoptotic cells is unclear but is likely to involve calreticulin and CD91-dependent docking of C1q on phagocytes.22Ogden CA deCathelineau A Hoffmann PR Bratton D Ghebrehiwet B Fadok VA Henson PM C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells.J Exp Med. 2001; 194: 781-795Crossref PubMed Scopus (949) Google Scholar In this study, we have used C1q gene-targeted mice to test the hypothesis that the classic complement pathway plays a role in apoptotic cell clearance in atherosclerosis and that defective apoptotic cell clearance increases lesion development. Oil Red O, dextrin, gelatin, Mayer's hematoxylin, l-glutamic acid, glycerol, sodium azide, calcium chloride, magnesium sulfate, and sodium phosphate were obtained from Merck/BDH, Poole, UK. Buffered formal saline (2% formaldehyde in phosphate-buffered saline) was from Pioneer Research Chemicals, Colchester, Essex, UK. OCT compound was from CellPath, Newtown, Powys, UK. Other reagents were from Sigma, St. Louis, MO. C1q gene-targeted mice (C1qa−/−) were generated in-house.23Botto M Dell'Agnola C Bygrave AE Thompson EM Cook HT Petry F Loos M Pandolfi P Walport MJ Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies.Nat Genet. 1998; 19: 56-59Crossref PubMed Scopus (1252) Google Scholar Ldlr−/− mice were obtained from Jackson Laboratories (Bar Harbor, ME). Both C1qa−/− and Ldlr−/− mice were backcrossed for 10 generations onto the C57BL/6 background before intercrossing to form C1qa−/−/Ldlr−/− double knockout mice. Mice genotypes were determined by polymerase chain reaction. All mice in the study were female. We conducted two separate experiments using mice fed a normal laboratory diet. In the first, 14 Ldlr−/− and 19 C1qa−/−/Ldlr−/− mice were analyzed for serum lipids, lipoprotein profiles, serum autoantibodies, and aortic root lesion area. In the second, 19 Ldlr−/− and eight C1qa−/−/Ldlr−/− mice were assessed for aortic root lesion area and cellular composition of lesions. A further experiment was conducted in which 10 Ldlr−/− and 10 C1qa−/−/Ldlr−/− mice were fed a cholate-free high-fat diet (Diet W; Hope Farms, Woerden, The Netherlands) consisting of (w/w) cocoa butter (15%), cholesterol (0.25%), sucrose (40.5%), cornstarch (10%), corn oil (1%), cellulose (5.95%), casein (20%), 50% choline chloride (2%), methionine (0.2%), and mineral mixture (5.1%) These mice were gradually transferred onto the high-fat diet at 10 weeks of age and sacrificed at 22 weeks of age. Animals were housed in a specific pathogen-free environment and studied according to UK Home Office regulations. Urinary protein was determined using Hema-combistick (Bayler plc, Newbury, Berks, UK). Terminal blood was collected in Microvette CB-300 blood tubes (Sarstedt, Nümbrecht, Germany) and allowed to clot at 4°C. Serum was pooled from all mice in the same group and kept at 4°C for up to 24 hours before analysis. Lipoprotein profiles were analyzed on pooled sera by size-exclusion chromatography using a SMART micro-FPLC system (Pharmacia, Stockholm, Sweden).24Benson GM Schiffelers R Nicols C Latcham J Vidgeon-Hart M Toseland CD Suckling KE Groot PH Effect of probucol on serum lipids, atherosclerosis and toxicology in fat-fed LDL receptor deficient mice.Atherosclerosis. 1998; 141: 237-247Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar Total cholesterol was measured enzymatically on each individual mouse using Kit CII (no. 270-54399/54499) from Wako Chemicals, GmbH, Neuss, Germany. Serum triglycerides were measured enzymatically using a kit purchased from ABX Diagnostics, Montpellier, France. Antibodies to single-stranded DNA (ssDNA) were measured by enzyme-linked immunosorbent assay.25Burlingame RW Rubin RL Subnucleosome structures as substrates in enzyme-linked immunosorbent assays.J Immunol Methods. 1990; 134: 187-199Crossref PubMed Scopus (111) Google Scholar The levels of IgM and IgG autoantibody titers to malondialdehyde-LDL and copper-oxidized LDL were determined in serum from individual mice in the laboratory of Dr. Joseph Witztum at the University of California, San Diego, in La Jolla, CA, using previously described methods.26Tsimikas S Palinski W Witztum JL Circulating autoantibodies to oxidized LDL correlate with arterial accumulation and depletion of oxidized LDL in LDL receptor-deficient mice.Arterioscler Thromb Vasc Biol. 2001; 21: 95-100Crossref PubMed Scopus (133) Google Scholar Hearts and aortae were perfused in situ with oxygenated Krebs-Henseleit buffer at 37°C under a pressure of ∼110 cm water via a cannula inserted in the left ventricle and an outlet created by incision of the right atrium. After 30 minutes, the buffer was replaced with 2% buffered formal saline at 37°C for a further 30 minutes. The heart, aortae, liver, and kidneys were then removed and stored in 2% formal saline. Cryosections of the aortic root were taken as previously described.27Paigen B Morrow A Holmes PA Mitchell D Williams RA Quantitative assessment of atherosclerotic lesions in mice.Atherosclerosis. 1987; 68: 231-240Abstract Full Text PDF PubMed Scopus (789) Google Scholar, 28Groot PH van Vlijmen BJ Benson GM Hofker MH Schiffelers R Vidgeon-Hart M Havekes LM Quantitative assessment of aortic atherosclerosis in APOE*3 Leiden transgenic mice and its relationship to serum cholesterol exposure.Arterioscler Thromb Vasc Biol. 1996; 16: 926-933Crossref PubMed Scopus (93) Google Scholar For each mouse, the entire aortic root from where the three valve leaflets first appeared was serially sectioned into 5-μm sections, and every 10th section (50 μm) was stained with Oil Red O and counterstained with Mayer's hematoxylin. Aortic root sections were coded and analyzed blind. Sections were imaged using an Olympus BH-2 microscope (Tokyo, Japan) equipped with ×4 objective (total magnification ×40), three neutral density filters (2× ND-6 and 1× ND-25), and a video camera (HV-C10; Hitachi, Yokohama, Japan). Twenty-four-bit color images were acquired and analyzed using a PC (Datacell Pentium P5-133; Datacell, Berks, UK) fitted with a framegrabbing board (IC-PCI; Imaging Technologies, Bedford, MA) and Optimas software (version 6.1; Optimas Corp., Bothell, WA). The images were captured under identical lighting, microscope, camera, and PC conditions. Quantification was performed by drawing around the lesions and the aortic wall using the Image ProPlus software (version 4.5; Media Cybernetics, Silver Spring, MD). Absolute values for cross-sectional area were obtained by calibrating the software using an image of a micrometer slide taken at the same magnification. The individual lesion areas per aortic root section of each mouse were averaged to obtain the mean lesion area per mouse. The lesion area fraction was calculated by dividing the mean lesion area by the mean area of the aortic wall and expressed as a percentage, as previously described.29Robertson AK Rudling M Zhou X Gorelik L Flavell RA Hansson GK Disruption of TGF-beta signaling in T cells accelerates atherosclerosis.J Clin Invest. 2003; 112: 1342-1350Crossref PubMed Scopus (388) Google Scholar Immunohistochemistry was performed by standard procedures on residual sections not required for analysis of lesion size. We phenotyped lesions for macrophages (Moma-2; Serotec, Oxford, UK) and smooth muscle cell α-actin (clone α-1-A4; Sigma-Aldrich, Poole, UK), detecting positive cells with alkaline phosphatase and Vector Blue. Other primary antibodies used were against B lymphocytes (CD19; Pharmingen, Oxford, UK), T lymphocytes (CD3; Pharmingen), IgM (Abcam, Cambridge, UK), IgG (biotinylated anti-mouse IgG Vector), C3 (anti-mouse C3; MP Biomedicals, London, UK), and rabbit anti-C5b-9 (Calbiochem, Merck Biosciences, Darmstadt, Germany) and were detected with avidin-biotin peroxidase (Dako, Ely, UK) and diaminobenzidine (DAB) substrate (brown). Anti-C5b-9 was titrated in doubling dilutions on atherosclerotic sections, using a standard avidin-biotin-peroxidase-DAB detection layer. Then, anti-C5b-9 was diluted to working concentration in normal human serum or in C3-deficient human serum (Sigma). C5b-9-coated beads for adsorption of anti-C5b-9 antibodies were prepared by mixing polystyrene beads (no. 17136; Polysciences, Warrington, PA) 1:1 with 1 mg/ml IgM solution (The Binding Site, Birmingham, UK) at 4°C for 24 hours. The beads were washed once in phosphate-buffered saline (PBS) by centrifugation and resuspension and then added to the anti-C5b-9 antibodies in sera. As a complement-fixing immunoglobulin, IgM activates C5b-9 generation in normal serum but not C3-deficient serum. The beads were incubated for 120 minutes at 37°C and then removed by centrifugation. The supernatants were added to tissue sections, and immunostaining was performed as above. Apoptotic cells were detected by immunostaining with a rabbit antibody to an epitope on caspase-3 (1 μg/ml Clone CM1; Becton, Dickinson and Company, Oxford, UK) that is revealed in cells undergoing apoptosis and detected with biotinylated swine anti-rabbit (Dako), avidin-biotin peroxidase (Dako), and DAB substrate. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was performed using each of two kits (Roche, Welwyn Garden City, UK; and Oncogene Sciences, Gaithersburg, MD) according to the manufacturer's instructions. These enzymatically incorporate fluoresceinated nucleotides into (broken) DNA ends. Peroxidase conversion with peroxidase-labeled anti-fluorescein antibodies and DAB were used to give a stable brown product. For each kit, the TUNEL incubation time was carefully titrated on positive control sections to optimize staining intensity. The TUNEL indices obtained with each kit were closely correlated (Spearman's correlation coefficient = 0.95). Results of immunocytochemistry are presented as a percentage area fraction of the aortic root or as the percentage of lesional cells. Confocal immunostaining and microscopy was by modification of routine immunohistochemistry, primarily substituting fluorescent antibodies for the peroxidase second layers and TOPRO-3 for hematoxylin. In brief, cryostat sections were blocked in 10% normal goat serum (Dako) and incubated with antibodies to Moma-2 and/or smooth muscle actin (as above). Sections were washed briefly in PBS and then incubated in 1:200 goat-anti-rat-AlexaFluor 488 (Molecular Probes, Eugene, OR). Anti-activated caspase-3 immunostaining was performed with anti-cleaved caspase-3 epitope, as above, but detected with goat-anti-rabbit-AlexaFluor 488 (Molecular Probes). TUNEL was performed with the Roche kit (as above) under the same conditions but was stopped after incorporation of the fluoresceinated thymidine. For use with TUNEL and anti-cleaved caspase-3, Moma-2 staining was visualized with goat anti-rat AlexaFluor 568 or AlexaFluor 546 (which are spectrally similar). Throughout, after the second layer, sections were washed briefly in PBS and then incubated 10 minutes with TOPRO-3 (Molecular Probes), rinsed in PBS, and mounted in PBS/glycerol. Staining and storage were in the dark as much as practicable. Sections were examined with a Zeiss LSM 510 Meta inverted confocal microscope (Carl Zeiss GmbH, Jena, Germany), illuminated using Argon 488, HeNe 543, and HeNe 633 lines. Pinhole and tunable filter settings were at defaults for the objectives and the fluorescein isothiocyanate (FITC)/Cy3/Cy5MT wavelengths. Scan and photomultiplier settings were set to optimize signal/noise ratio for each emission wavelength. Processing was conducted with a Zeiss LSM Image Browser and comprised the addition of scale bars and adjustment of brightness and contrast before import into Microsoft Powerpoint (Redmond, WA) for assembly of montage figures. One parallel cryostat section was selected from each of four C1qa−/−/Ldlr−/− mice to correspond closely to those used for the confocal analysis of double immunostaining of Moma-2 and cleaved caspase-3 or TUNEL. These were postfixed in 4% glutaraldehyde at room temperature for 30 minutes. An area of interest was defined from the cleaved caspase-3 staining of the adjacent section. The relevant part of the section was then captured into a resin block. Semithin sections were used to confirm the local topography, after which the resin was ultramicrotomed and ultrathin sections were stained with lead and imaged on a Hitachi 7650 transmission electron microscope (Hitachi Software Engineering, Yokohama, Japan) with digital capture. Because the cryostat-reprocessed tissue had low contrast staining, contrast was adjusted after acquisition in Adobe Photoshop (Adobe Systems, Mountain View, CA) in line with digital image guidelines. Data were expressed as mean ± SE unless otherwise stated and tested by one-tailed Student's t-test [Excel (Microsoft) and SigmaStat (Systat Software, Inc., Point Richmond, CA)], with significance assumed at P < 0.05. Mice were fed a normal laboratory diet or a high-fat diet from weaning until sacrifice at 22 weeks of age. As shown in Table 1, no differences were observed between Ldlr−/− and C1qa−/−/Ldlr−/− strains for final body weight or for total serum cholesterol and triglyceride levels. Furthermore, there was no difference in the lipoprotein profile of Ldlr−/− and C1qa−/−/Ldlr−/− mice, as determined by fast-performance liquid chromatography (FPLC) (not shown). No mouse of either strain had evidence of autoimmunity related to C1q deficiency, as shown by lack of proteinuria, the absence of glomerulonephritis, and the absence of antibodies to anti-ssDNA (not shown). Furthermore, there were no differences between Ldlr−/− and C1qa−/−/Ldlr−/− in the titers of antibodies to oxidatively modified lipoproteins (not shown).Table 1Body Weights and Total Serum Cholesterol and Triglycerides in 22-Week-Old C57BL/6 Ldlr−/− and C1qa−/−/Ldlr−/−Mouse strainDietnFinal body weight (g) (mean ± SD)Total cholesterol (mmol/L) (mean ± SD)Triglyceride (mmol/L) (mean ± SD)Ldlr−/−Normal1424 ± 28.56 ± 1.031.89 ± 0.56C1qa−/−/Ldlr−/−Normal1925 ± 28.97 ± 1.632.10 ± 0.54Ldlr−/−High fat1025 ± 325.60 ± 5.843.02 ± 1.00C1qa−/−/Ldlr−/−High fat1025 ± 330.66 ± 6.423.74 ± 1.02 Open table in a new tab In the first experiment, we analyzed lesion areas in aortic roots of Ldlr−/− and C1qa−/−/Ldlr−/− mice fed a normal laboratory diet by standard methods and found that aortic root lesion areas were significantly greater in the C1qa−/−/Ldlr−/− mice (P < 0.006, data not shown). We therefore performed a confirmatory experiment, in which we found that C1qa−/−/Ldlr−/− mice had approximately threefold larger absolute lesion areas in the aortic root than Ldlr−/− animals (C1qa−/−/Ldlr−/− 116 ± 30 × 103 μm2 versus Ldlr−/− 28.8 ± 9.0 × 103 μm2; mean ± SEM, P < 0.001) (Figure 1A). Furthermore, the lesion areas were also significantly increased in C1qa−/−/Ldlr−/− mice when expressed as a fraction of the aortic root (C1qa−/−/Ldlr−/− 3.72 ± 1.0% versus LDLR−/− 1.1 ± 0.4%; mean ± SEM, P < 0.001) (Figure 1B). Lesions in Ldlr−/− mice fed a high-fat diet from 10 to 22 weeks of age were approximately 10-fold greater than those fed a normal laboratory diet, but, following a high-fat diet, there were no differences between C1qa−/−/Ldlr−/− (n = 10) and Ldlr−/− (n = 10) mice in aortic root lesion area or fraction of the aortic occupied by lesions (lesion areas, C1qa−/−/Ldlr−/− 443.34 ± 190.28 × 103 μm2 versus Ldlr−/− 254.04 ± 75.28 × 103 μm2; aortic root fraction, C1qa−/−/Ldlr−/− 23.71 ± 7.61% versus Ldlr−/− 18.00 ± 5.63%). Subsequent analysis of lesions was therefore focused on mice fed a normal laboratory diet. Staining of aortic root lesions with the macrophage marker Moma-2 highlighted cells with the features of foamy macrophages. The area of Moma-2 immunoreactive cells was significantly greater in C1qa−/−/Ldlr−/− mice than from Ldlr−/− controls (area fraction of Moma-2-positive cells in C1qa−/−/Ldlr−/− 3.3 ± 0.6% versus Ldlr−/− 1.1 ± 0.3%; mean ± SEM, P = 0.01) (Figure 2A). Whereas the cells in lesions of Ldlr−/− mice consisted exclusively of macrophages, lesions in C1qa−/−/Ldlr−/− mice had ∼12% of cells negative for Moma-2 (Figure 2, B and D) and positive for smooth muscle cell α-actin (Figure 2, C and D). We failed to detect B or T lymphocytes in lesions in either strain. There were no significant differences between strains in the proportion of lesions occupied by collagen, as detected using the Picrosirius Red method, or in immunohistochemical staining for IgG, IgM, or C3 (not shown). Reactivity of anti-C5b-9 antibody, which was raised against human material, was established by showing that staining of aortic roots was abolished by prior absorption with IgM-coated polystyrene beads opsonized with normal human serum but not by IgM-coated beads opsonized with C3-deficient serum (not shown). C5b-9 staining in aortic root lesions of Ldlr−/− mice was identified particularly around lesional foam cells (Figure 3A). The percentage of aortic root lesions with positive C5b-9 staining was significantly less in C1qa−/−/Ldlr−/− mice compared with Ldlr−/− mice (2.78 versus 24.6%; P < 0.0001) (Figure 3, B and C). To quantify apoptotic cells, we performed immunohistochemical staining with an antibody against cleaved caspase-3. Lesions in C1qa−/−/Ldlr−/− mice contained 15.6 ± 1.8% cells (mean ± SEM) staining positively for activated caspase-3, whereas positive cells were barely detectable in the lesions of Ldlr−/− mice (P < 0.001) (Figure 4A). TUNEL staining confirmed the increase in apoptotic cells in C1qa−/−/Ldlr−/− mice (4.7 ± 1.2 versus 0 ± 0 mean ± SEM % lesional cells) (Figure 4B). The number of TUNEL-positive cells was less than the number of cells stained by anti-caspase-3, at least in part due to anti-caspase-3 identifying cells at more stages of apoptosis than TUNEL.30Belloc F Belaud-Rotureau MA Lavignolle V Bascans E Braz-Pereira E Durrieu F Lacombe F Flow cytometry detection of caspase 3 activation in preapoptotic leukemic cells.Cytometry. 2000; 40: 151-160Crossref PubMed Scopus (100) Google Scholar, 31Duan WR Garner DS Williams SD Funckes-Shippy CL Spath IS Blomme EA Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts.J Pathol. 2003; 199: 221-228Crossref PubMed Scopus (291) Google Scholar It is also possible that our careful titration of the TUNEL reaction to avoid nonspecific labeling led to an underestimation of apoptotic cell number with this technique. No cells outside lesions stained positively in C1qa−/−/Ldlr−/− for cleaved caspase-3 or by TUNEL. Double staining of lesions of C1qa−/−/Ldlr−/− revealed the apoptotic cells to be macrophages (ie, Moma-2-positive and α-actin-negative) (Figure 5). The presence of apoptotic cells in aortic root lesions of C1qa−/−/Ldlr−/− was confirmed by electron microscopy (Figure 6).Figure 5Double staining of apoptotic cells assessed by confocal microscopy. A: Low-power composite of cleaved caspase-3 (Alexa488, green), Moma-2 (Alexa 568, red), and TOPRO-3 nuclear dye (purple, pseudocolored; far red, original); (B) single channels of boxed area in A, with composite at bottom right. White arrows show cells triple-positive for TOPRO-3, Moma-2, and cleaved caspase-3; (C) equivalent of B in parallel section stained with Moma-2 (Alex 568, red) and TUNEL (FITC, green), with composite at bottom right. White arrows show cells triple-positive for TOPRO-3, TUNEL, and Moma-2. No such colocalization was seen on actin double staining (not shown, the resolution was less because the actin-positive cells were distant from the cleaved caspase-3-positive cells).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Apoptotic cells in EM aortic root lesions of C1qa−/−/Ldlr−/− mice. A: Low magnification for orientation. L, lumen. White arrowhead points to endothelial layer. Fine arrows point to apoptotic cells, identified by characteristic electron dense peripheral chromatin condensation. B and C are higher magnifications. Gray-filled block arrows marked with F, foam cell(s). In B, the white block arrow points to a shrunken cell with characteristic electron dense peripheral chromatin condensation and cytoplasmic collapse, characteristic of apoptosis; in C, the white block arrow points to an early apoptotic cell with early electron dense peripheral chromatin condensation and characteristic blebbing, containing electron dense material (Bl, white script within filled black block arrow). Gray
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