Lack of Complement Factor C3, but Not Factor B, IncreasesHyperlipidemia and Atherosclerosis in Apolipoprotein E�/�

Low-Density Lipoprotein Receptor�/� MiceLinda Persson, Jan Boren, Anna-Karin L. Robertson, Ville Wallenius,

Goran K. Hansson, Marcela Pekna

Objective—To investigate the effect of complement deficiency on atherogenesis and lipidemia, we used mice deficient inthe third complement component (C3�/�) or factor B (FB�/�).

Methods and Results—Complement-deficient mice were crossed with mice deficient in both apolipoprotein E and thelow-density lipoprotein receptor (Apoe�/� LDLR�/�). The percent lesion area in the aorta at 16 weeks, determinedby en face analysis, was 84% higher in C3�/� mice than in controls (11.8%�0.4% versus 6.4%�0.8%, mean�SEM,P�0.00005). The C3�/� mice also had 58% higher serum triglyceride levels (P�0.05) and a more proatherogeniclipoprotein profile, with significantly more low-density lipoprotein cholesterol and very-low-density lipoproteintriglycerides than control mice. The C3�/� mice weighed 13% less (P�0.01) and had a lower body fat content(3.5%�1.0% versus 13.1%�3.0%, P�0.01). There were no differences between FB�/� mice and controls.

Conclusions—Complement activation by the classical or lectin pathway exerts atheroprotective effects, possibly throughthe regulation of lipid metabolism. (Arterioscler Thromb Vasc Biol. 2004;24:1062-1067.)

Key Words: atherosclerosis � complement � C3 � factor B � hyperlipidemia

The complement system plays an essential role in thehumoral immune response. Soluble complement compo-

nents are present in the blood in precursor forms and need to beactivated to fulfill their specific physiological roles. Activatedcomplement has diverse functions, including the initiation ofinflammation, recruitment of leukocytes, clearance of immunecomplexes, neutralization of pathogens, regulation of antibodyresponses, and disruption of cell membranes. The complementcascade can be activated by 3 pathways. The classical activationpathway depends on assembly of complement factors at sites ofantigen–antibody complexes. The lectin pathway is initiated bymannan-binding lectin bound to pathogen surfaces. Activationof the alternative pathway is triggered by a variety of pathogensurfaces and requires the interaction of third complement com-ponent (C3), factor B (FB), and factor D. Regardless of thepathway, activation leads to the cleavage of C3. This generatesthe smaller, proinflammatory C3a fragment and the larger C3bfragment. C3b can act as an opsonin and triggers the terminalpart of the cascade, which culminates in the assembly of theterminal complement complex (TCC) on the target surface.

C3-derived peptides have been implicated in the regulationof lipid metabolism. C3a and C3ades-Arg, a peptide formedwhen the C-terminal arginine is removed from C3a by

carboxypeptidase N, can act as acylation-stimulating protein(ASP).1 ASP has been implicated in adipose tissue functionand maintenance of metabolic homeostasis.2 ASP and C3aincrease fat storage in adipocytes through increased triglyc-eride synthesis3–5 and decreased intracellular lipolysis.5 Micethat are deficient in C3, and therefore unable to synthesizeASP, have delayed triglyceride clearance,6–8 which can benormalized by administration of ASP.7,9,10 However, Wetselet al did not detect any impaired ability of C3�/� mice toclear triglycerides and free fatty acids from the circulationafter an oral fat load.11 FB�/� mice are viable and fertile andhave no overall abnormalities of the immune system.12,13 Theeffect of FB deficiency on adipose tissue and blood lipidprofiles has thus far not been studied.

Atherosclerosis is characterized by an inflammatory re-sponse to the accumulation of subendothelial lipids, and thereis evidence that the activated complement system is involvedin atheroma formation.14 Complement components and acti-vation products have been identified in the walls of diseasedvessels,15–18 and mRNAs of complement components areexpressed in atherosclerotic plaques. However, the role ofcomplement inhibitors is more controversial.19,20 The degreeof TCC deposition correlates with the severity of arterial

Received December 22, 2003; revision accepted March 22, 2004.From the Department of Medical Biochemistry (L.P., M.P.), Wallenberg Laboratory for Cardiovascular Research (J.B.), and Research Center for

Endocrinology and Metabolism (V.W.), The Sahlgrenska Academy at Goteborg University, Goteborg; and the Center for Molecular Medicine (A.-K.L.R.,G.K.H.), Karolinska Institute, Stockholm, Sweden

Consulting Editor for this article was Peter Libby, MD, Brigham and Women’s Hospital, Boston, Mass.Correspondence to Dr Marcela Pekna, Department of Medical Biochemistry, Goteborg University Box 440 S-405 30, Goteborg, Sweden. E-mail

Marcela.Pekna@medkem.gu.se© 2004 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000127302.24266.40

1062 by guest on February 9, 2016http://atvb.ahajournals.org/Downloaded from by guest on February 9, 2016http://atvb.ahajournals.org/Downloaded from by guest on February 9, 2016http://atvb.ahajournals.org/Downloaded from

damage.21,22 In rabbits, TCC and lipid colocalize in the aortic tunicaintima.23 Similarly, in human lesions, TCC colocalizes with enzy-matically modified lipoproteins.24 Lipoprotein particles in vitro aswell as modified lipoproteins are also potent activators of comple-ment.25,26 The severity of cholesterol-induced atherosclerosis isreduced in C6-deficient rabbits.27,28 In apolipoprotein E-deficient(Apoe�/�) mice, the absence of C5 does not affect atheromaformation,29 whereas aortic lipid-positive lesional size is reported tobe greater in C3�/� low-density lipoprotein receptor-deficient(LDLR�/�) mice than in controls.30

To assess the role of the early components of the comple-ment system in the pathogenesis of atherosclerosis and theregulation of plasma lipids, we crossed Apoe�/� LDLR�/�mice31 with C3�/�32 or FB�/� mice.12 C3�/� mice cannotactivate the complement cascade beyond the formation of theC3-convertase of the classical/lectin pathway, and FB�/�mice are unable to activate the alternative pathway. The useof these 2 complement-deficient strains enabled us to assessthe relative roles of these activation pathways.

Methods

MiceTo generate a complement-deficient, atherosclerosis-prone mousemodel, we crossed C3�/�32 and FB�/� mice12 with Apoe�/�LDLR�/� mice.31 Complement-deficient males from the mating ofC3�/� Apoe�/� LDLR�/� or FB�/�Apoe�/� LDLR�/� par-ents were compared with complement-sufficient Apoe�/�LDLR�/� siblings to minimize the effect of other genes on themixed genetic background (C57BL/6J/129Ola). The mice were fednormal chow and analyzed at 16 or 26 weeks of age. The study wasapproved by the ethics committee at Goteborg University.

Processing and Analysis of the AortaThe aortic root was processed and quantified as described.33 Sectionswere obtained 100, 200, 300, and 400 �m from the origin of theaortic valve cusps and stained with Oil Red O (Sigma-Aldrich, St.Louis, Mo). The aorta was prepared and analyzed en face asdescribed.34 In brief, the aortas were pinned on wax plates andstained with Sudan IV. The extent of atherosclerosis was expressedas the percentage of the aortic surface covered by lesions. The resultsfrom control Apoe�/� LDLR�/� mice fed normal chow werecomparable with data published for Apoe�/� and LDLR�/� micefed a high-cholesterol diet.30,33

Serum Lipid AnalysesBlood for lipid analyses was collected before perfusion of theanimals and allowed to clot, and the serum was stored at �20°C.Cholesterol and triglycerides were analyzed with a Konelab 30automated analyzer (Thermo Electron Clinical Chemistry & Auto-mation Systems) using the reagents triglycerides 981786 and cho-lesterol 981773, as recommended by the manufacturer. The lipiddistribution in plasma lipoprotein fractions was assessed by fast-per-formance liquid chromatography gel filtration with a Superose 6 HR10/30 column (Pharmacia, Uppsala, Sweden).35

Postprandial Triglyceride ClearancePostprandial triglyceride clearance was measured as described.7

Blood samples were collected before and 1, 2, 3, 4, and 6 hours afteroral administration of olive oil.

Body Fat MeasurementBody fat content was measured in vivo by dual-energy x-rayabsorptiometry (DXA).36

Serum Anti-LDL Antibody MeasurementsSpecific antibodies to malondialdehyde (MDA)-modified low-density lipoprotein (LDL) were quantified by enzyme-linked immu-nosorbent assay.37

Histological Evaluation and ImmunohistochemistryAortic root sections were stained with hematoxylin and erythrosineor Masson trichrome (Bio-Optica, Milan, Italy) to identify foamcells, fibrosis, cholesterol clefts, acellular areas, and fibrous cap inatherosclerotic plaques. For immunohistochemistry, sections werefixed in acetone, incubated with 1% bovine serum albumin inphosphate-buffered saline–Tween, and stained with MOMA-2 (Se-rotec, Oxford, UK) and �-smooth muscle actin (EPOS; Dako A/S,Glostrup, Denmark) antibodies. The MOMA-2 antibody was de-tected by biotin-conjugated rabbit antiserum against rat immuno-globulins (Dako A/S) followed by ABC Vectastain Elite kit (VectorLaboratories, Burlingame, Calif) and visualized with 3, 3�-diaminobenzidine tetrahydrochloride (Sigma).

Statistical AnalysisThe results are presented as mean�SEM. The data were analyzed byunpaired t test. Repeated-measures analysis of variance was used toanalyze the postprandial triglyceride clearance data. Differenceswere considered statistically significant at P�0.05.

ResultsC3 Deficiency Increases Lesion Size at 16 Weeksof AgeLesion size increased with age in all groups examined. All micehad advanced lesions composed of foam cells and abundantextracellular lipid at 16 weeks and fully developed atheromaswith lipid-rich core regions covered by fibrous caps at 26 weeks.En face analysis of aortas at 16 weeks (Figure 1A and 1C)showed that the lesions were 84% larger in C3�/� than incomplement-sufficient mice (11.8%�0.4% versus 6.4%�0.8%of surface area, P�0.00005). At 26 weeks, however, the lesionswere of similar size in the 2 groups (Figure 2A). There were nodifferences in lesion size between FB�/� mice and controls atany time point (Figure 2B). In serial sections of the aortic root,the extent of atherosclerosis did not significantly differ betweencomplement-deficient mice and control Apoe�/� LDLR�/�mice (not shown).

Atherosclerotic Lesions Are Qualitatively Similarin C3�/� and Control MiceMorphological examination of aortic root sections at 16weeks revealed lesions in all mice, ranging from fatty streakscomposed of foam cells to advanced fibrofatty lesions cov-ered by fibrous cap. At 26 weeks, the lesions often coveredthe whole inner circumference of the vessel, and all wereexclusively of the fibroatheromatous type, the majority withlarge acellular areas, a fibrous cap of varying thickness, andextensive necrosis. No difference in plaque progression wasnoted between groups. As shown by MOMA-2 staining,macrophages were the predominant cell type in all lesions,although lesions in the older mice were less cellular. Theextent and distribution of MOMA-2 staining were similar incomplement-deficient mice and controls. Fibrous caps, visu-alized by �-smooth muscle actin immunostaining, wereobserved in all sections analyzed, with no difference betweenthe experimental groups (Figure I, available online athttp://atvb.ahajournals.org).

Persson et al Complement Activation and Atherosclerosis 1063

by guest on February 9, 2016http://atvb.ahajournals.org/Downloaded from

C3 Deficiency Increases Serum Triglycerides andAlters Plasma Lipoprotein ProfilesIn C3�/� mice, serum triglyceride levels were 58% higherthan in controls at 16 weeks (6.8�0.9 versus4.3�0.4 mmol/L, P�0.05) and 79% higher at 26 weeks(8.4�1.3 versus 4.4�0.6 mmol/L, P�0.05) (Figure 3A). C3

deficiency did not affect serum cholesterol levels at any age(Figure 3B). Serum cholesterol and triglyceride levels weresimilar in FB�/� and control mice (Figure 3C and 3D).Superose 6 chromatography of pooled plasma showed a moreproatherogenic lipoprotein profile in C3�/� mice, withsignificantly more LDL cholesterol and very-low-densitylipoprotein (VLDL) triglycerides than controls at both 16 and26 weeks (Figure 4).

C3�/� Mice Have Normal PostprandialTriglyceride ClearanceNext, we determined if the increased serum lipid levels of theC3�/� mice could be explained by an aberrant postprandial

Figure 1. Sudan IV staining of aortasfrom 16-week-old C3�/� (A) and C3�/�Apoe�/� LDLR�/� (C) mice. Scale bar,2.5 mm. Oil Red O staining of aorticroots of 26-week-old C3�/� (B) andC3�/� Apoe�/� LDLR�/� (D) mice.Scale bar, 100 �m.

Figure 2. Percent lesion area in en face preparations of aortasfrom C3�/� and C3�/� (A) and FB�/� and FB�/� Apoe�/�LDLR�/� (B) mice. ***P�0.00005.

Figure 3. Serum lipid concentrations in 16- and 26-week-oldApoe�/� LDLR�/� mice (A through D). *P�0.05.

1064 Arterioscler Thromb Vasc Biol. June 2004

by guest on February 9, 2016http://atvb.ahajournals.org/Downloaded from

lipid clearance. No differences in serum triglyceride levelswere observed after an oral fat load in 21-week-old C3�/�(n�4) and control mice (n�5) (not shown).

C3 Deficiency Reduces Body Weight and Body FatCompared with controls, C3�/� mice weighed 13% less at16 weeks (34.2�0.96 versus 38.8�1.29 g, n�17/group,P�0.01) and 22% less at 26 weeks (34.7�1.12 g, n�10,versus 42.3�1.93 g, n�11, P�0.005). There were no differ-ences in body weight between the FB�/� and control miceat either time point. As shown by DXA analysis of bodycomposition, C3�/� mice had approximately two-thirds lessbody fat than controls at 16 weeks (3.5%�1.0% versus13.1%�3.0%, P�0.01) and 26 weeks (2.8%�0.7% versus9.3�1.9%, P�0.01) (Figure 5).

C3 Deficiency Does Not AlterAnti-MDA–Modified-LDL Antibody TitersTo determine if the effect of complement on atherogenesiscould result from changes in B-cell–associated protectiveimmunity,38 we assessed the immunoglobulin M (IgM) andimmunoglobulin G (IgG) isotypes of autoantibodies againstMDA-modified LDL. There were no differences in anti-MDA–LDL IgM or IgG titers between C3�/� mice andcontrols (not shown).

DiscussionComplement activation has been suggested to contribute tothe progression of atherosclerotic lesions, and complement-derived ASP has been implicated in the regulation of lipidmetabolism. To address the relative roles of the classical and

Figure 4. Cholesterol and triglyceridedistribution as assessed by Superose 6chromatography of pooled plasma fromC3�/� and C3�/� mice on theApoe�/� LDLR�/� background at 16weeks (left) or 26 weeks (right) (n�5 pergenotype). VLDL corresponds to frac-tions 2 to 6, intermediate-densitylipoproteins/LDL to fractions 7 to 15, andhigh-density lipoproteins to fractions 16to 20.

Figure 5. A, Representative DXA imagesof 16-week-old male C3�/� and C3�/�mice. White represents areas with �50%fat. B, Quantification of DXA measure-ments. **P�0.01.

Persson et al Complement Activation and Atherosclerosis 1065

by guest on February 9, 2016http://atvb.ahajournals.org/Downloaded from

lectin pathways versus the alternative pathway of complementactivation in these processes, we studied C3�/� and FB�/�mice. Remarkably, the aortic lesions were 84% larger in C3�/�mice than in complement-sufficient mice at 16 weeks of age.This finding is consistent with a recent report showing that C3deficiency increased lipid-positive lesional size and impairedlesion maturation beyond the foam cell stage in 15-week-oldLDLR�/� mice.30 Because serum lipoprotein/cholesterol pro-files were not changed, the authors concluded that local effectsof complement lead to lesion differences.

In our study, histopathological and immunohistochemicalanalyses of the aortic root showed no qualitative differencesin lesion morphology between C3�/� and control mice.However, we cannot exclude that the C3 deficiency may haveresulted in morphological differences such as reduced colla-gen and smooth muscle cell content in the lesions distal to theaortic root as reported for the C3�/� LDLR�/� mice.30 Inaddition, the strong atherogenic drive in the Apoe�/�LDLR�/� mice might have masked a milder anti-atherogenic effect of the complement system in the vascularwall and might also explain why C3�/� and control micehad lesions of similar size at 26 weeks. Given the possiblerole of antibodies in atheroprotective immunity,33,38–40 weanalyzed sera from C3�/� and control mice for antibodiesagainst MDA–LDL. No differences in antibody levels weredetected, indicating that the increased atherosclerosis inC3�/� Apoe�/� LDLR�/� mice is not likely a result ofimpairments in B-cell–associated protective immunity orprotective antibody production.

C3�/� mice also had a more atherogenic plasma lipopro-tein profile than controls, with elevated serum triglyceridelevels, increased VLDL triglycerides, and increased LDLcholesterol. In addition, the C3�/� mice weighed less andhad a lower body fat mass. The latter findings are in line withrecent reports showing that C3�/� mice had reduced bodyweight and fat mass and were resistant to weight gain on ahigh-fat diet, despite increased food intake.8,41 These pheno-typic abnormalities may reflect reduced triglyceride synthesisby adipocytes because of the absence of C3-derived ASP, asproposed by Sniderman et al.42 Thus, the absence of ASPleads to a more atherogenic lipoprotein profile and increasedatherosclerosis. Cianflone et al2 suggested that discrepanciesin plasma lipid levels and triglyceride clearance in C3�/�mice in different reports6–8,11 could be explained by theinfluence of genetic background, resulting in differences ininsulin sensitivity, lipoprotein lipase activity, or triglyceridesynthesis by adipose tissue.

In contrast to the results in C3�/� mice, the extent ofatherosclerosis, serum triglyceride levels, and body weightwere not significantly different in FB�/� Apoe�/�LDLR�/� mice and controls. Because C3, FB, and factor Dare produced in adipose tissue, ASP is believed to begenerated by the alternative pathway of complement activa-tion. In support of this possibility, there is no evidence thatadipocytes produce C2, an essential component of the clas-sical and lectin pathways.43,44 Although FB�/� mice havenormal C3 levels, they cannot generate ASP through proteo-lytic cleavage mediated by FB and factor D. Our findingssuggest that the alternative pathway is not required for

C3-mediated control of energy storage and energy use andthat ASP is generated by other mechanisms, such as activa-tion of the classical and/or lectin pathway locally in adiposetissue or in the circulation. Interestingly, genes encodingproteins of the classical pathway are expressed in humanadipose tissue.45

An alternative explanation for the phenotype of the C3�/�mice is the absence of uncleaved C3. One possibility worthexploring is the capacity of C3(H2O) to bind to the C3a/ASPreceptor C5L246 and to exhibit the functions ascribed to ASP.C3(H2O)—the product of spontaneous hydrolysis of theinternal thioester bond of the native C3 molecule—is confor-mationally and functionally similar to C3b. It can bind FBand form the C3 convertase of the alternative pathway.However, it still contains the C3a fragment47–49 and thereforecould, theoretically, act like ASP.

En face assessment of the whole aorta revealed a signifi-cant difference in percent lesion area between C3�/� andcontrol mice at 16 weeks of age that were not seen by analysisof aortic root sections. Babaev et al also reported a loss ofcorrelation between lesion areas determined by these twomethods when lesions in the proximal aorta became compli-cated.50 Thus, for detecting effects on disease development,en face analysis of the percent lesion area appears to be amore sensitive method than measuring plaque area in sectionsfrom the aortic root, at least in the Apoe�/� LDLR�/�model.

ConclusionIn conclusion, deficiency in C3, but not FB, leads to increasedhyperlipidemia, a more atherogenic lipoprotein profile, moreextensive atherosclerotic lesions, and reduced body weightand body fat in Apoe�/� LDLR�/� mice. These resultssuggest that complement activation via the classical or lectinpathway exerts atheroprotective effects, possibly through theregulation of lipid metabolism. However, the exact mecha-nisms remain unclear and further investigation is warranted.

AcknowledgmentsThe authors thank Kristina Skålen, Maria Garcia, Anna Berndtsson,Lisbeth Lindgren, and Ingrid Tornberg for technical help. The workwas supported by grants from The Swedish Medical ResearchCouncil (Projects 13470 and 6816), King Gustaf V 80th AnniversaryFoundation, The Swedish Heart-Lung Foundation, The SwedishSociety of Medicine, The Swedish Society for Medical Research,and Sigurd and Elsa Golje’s Foundation.

References1. Baldo A, Sniderman AD, St-Luce S, Avramoglu RK, Maslowska M,

Hoang B, Monge JC, Bell A, Mulay S, Cianflone K. The adipsin-acylation stimulating protein system and regulation of intracellular tri-glyceride synthesis. J Clin Invest. 1993;92:1543–1547.

2. Cianflone K, Xia Z, Chen LY. Critical review of acylation-stimulatingprotein physiology in humans and rodents. Biochim Biophys Acta. 2003;1609:127–143.

3. Murray I, Parker RA, Kirchgessner TG, Tran J, Zhang ZJ, Westerlund J,Cianflone K. Functional bioactive recombinant acylation stimulating protein isdistinct from C3a anaphylatoxin. J Lipid Res. 1997;38:2492–2501.

4. Cianflone K, Roncari DA, Maslowska M, Baldo A, Forden J, Sniderman AD.Adipsin/acylation stimulating protein system in human adipocytes: regulation oftriacylglycerol synthesis. Biochemistry. 1994;33:9489–9495.

5. Van Harmelen V, Reynisdottir S, Cianflone K, Degerman E, Hoffstedt J,Nilsell K, Sniderman A, Arner P. Mechanisms involved in the regulation

1066 Arterioscler Thromb Vasc Biol. June 2004

by guest on February 9, 2016http://atvb.ahajournals.org/Downloaded from

of free fatty acid release from isolated human fat cells by acylation-stimulating protein and insulin. J Biol Chem. 1999;274:18243–18251.

6. Murray I, Sniderman AD, Havel PJ, Cianflone K. Acylation stimulatingprotein (ASP) deficiency alters postprandial and adipose tissue metabo-lism in male mice. J Biol Chem. 1999;274:36219–36225.

7. Murray I, Sniderman AD, Cianflone K. Mice lacking acylation stimu-lating protein (ASP) have delayed postprandial triglyceride clearance. JLipid Res. 1999;40:1671–1676.

8. Xia Z, Sniderman AD, Cianflone K. Acylation-stimulating protein (ASP)deficiency induces obesity resistance and increased energy expenditure inob/ob mice. J Biol Chem. 2002;277:45874–45879.

9. Murray I, Sniderman AD, Cianflone K. Enhanced triglyceride clearancewith intraperitoneal human acetylation stimulating protein (ASP) inC57BL/6 mice. Am J Physiol Endocrinol Metab. 1999;277:E474–E480.

10. Saleh J, Blevins JE, Havel PJ, Barrett JA, Gietzen DW, Cianflone K. Acylationstimulating protein (ASP) acute effects on postprandial lipemia and food intakein rodents. Int J Obes Relat Metab Disord. 2001;25:705–713.

11. Wetsel RA, Kildsgaard J, Zsigmond E, Liao W, Chan L. Genetic defi-ciency of acylation stimulating protein (ASP(C3ades-Arg)) does notcause hyperapobetalipoproteinemia in mice. J Biol Chem. 1999;274:19429–19433.

12. Pekna M, Hietala MA, Landin A, Nilsson A-K, Lagerberg C, Betsholtz C,Pekny M. Mice deficient for complement factor B develop and reproducenormally. Scand J Immunol. 1998;47:375–380.

13. Matsumoto M, Fukuda W, Circolo A, Goellner J, Strauss-Schoenberger J,Wang X, Fujita S, Hidvegi T, Chaplin DD, Colten HR. Abrogation of thealternative complement pathway by targeted deletion of murine factor B.Proc Natl Acad Sci U S A. 1997;94:8720–8725.

14. Niculescu F, Rus H. Complement activation and atherosclerosis. MolImmunol. 1999;36:949–955.

15. Pang ASD, Katz A, Minta JO. C3 deposition in cholesterol-inducedatherosclerosis in rabbits: A possible etiologic role for complement inatherogenesis. J Immunol. 1979;123:1117–1122.

16. Hansson GK, Holm J, Kral IG. Accumulation of IgG and complementfactor C3 in human arterial endothelium and atherosclerotic lesions. ActaPathol Microbiol Immunol Scand Sect A. 1984;92:429–435.

17. Vlaicu R, Rus HG, Niculescu F, Cristea A. Immunoglobulins and complementcomponents in human aortic intima. Atherosclerosis. 1985;55:35–50.

18. Seifert PS, Messner M, Roth I, Bhakdi S. Analysis of complement C3activation products in human atherosclerotic lesions. Atherosclerosis.1991;91:155–162.

19. Yasojima K, Schwab C, McGeer EG, McGeer PL. Complement com-ponents, but not complement inhibitors, are upregulated in atheroscleroticplaques. Arterioscler Thromb Vasc Biol. 2001;21:1214–1219.

20. Seifert PS, Hansson GK. Complement receptors and regulatory proteinsin human atherosclerotic lesions. Arteriosclerosis. 1989;9:802–811.

21. Niculescu F, Rus HG, Vlaicu R. Immunohistochemical localization ofC5b-9, S-protein, C3d and apolipoprotein B in human arterial tissues withatherosclerosis. Atherosclerosis. 1987;65:1–11.

22. Seifert PS, Kazatchkine MD. The complement system in atherosclerosis.Atherosclerosis. 1988;73:91–104.

23. Seifert PS, Hugo F, Hansson GK, Bhakdi S. Prelesional complementactivation in experimental atherosclerosis. Terminal C5b-9 complementdeposition coincides with cholesterol accumulation in the aortic intima ofhypercholesterolemic rabbits. Lab Invest. 1989;60:747–754.

24. Torzewski M, Klouche M, Hock J, Messner M, Dorweiler B, TorzewskiJ, Gabbert HE, Bhakdi S. Immunohistochemical demonstration of enzy-matically modified human LDL and its colocalization with the terminalcomplement complex in the early atherosclerotic lesion. ArteriosclerThromb Vasc Biol. 1998;18:369–378.

25. Bhakdi S, Torzewski M, Klouche M, Hemmes M. Complement and athero-genesis: binding of CRP to degraded, nonoxidized LDL enhances complementactivation. Arterioscler Thromb Vasc Biol. 1999;19:2348–2354.

26. Seifert PS, Hugo F, Tranum-Jensen J, Zahringer U, Muhly M, Bhakdi S.Isolation and characterization of a complement-activating lipid extractedfrom human atherosclerotic lesions. J Exp Med. 1990;172:547–557.

27. Geertinger P, Sorensen H. On the reduced atherogenic effect of choles-terol feeding in rabbits with congenital complement (C6) deficiency.Artery. 1977;1:177–184.

28. Schmiedt W, Kinscherf R, Deigner H-P, Kamencic H, Nauen O, Kilo J,Oeler H, Metz J, Bhakdi S. Complement C6 deficiency protects againstdiet-induced atherosclerosis in rabbits. Arterioscler Thromb Vasc Biol.1998;18:1790–1795.

29. Patel S, Thelander EM, Hernandez M, Montenegro J, Hassing H, BurtonC, Mundt S, Hermanowski-Vosatka A, Wright SD, Chao Y-S, Detmers P.

ApoE�/� mice develop atherosclerosis in the absence of complementcomponent C5. Biochem Biophys Res Commun. 2001;286:164–170.

30. Buono C, Come CE, Witztum JL, Maguire GF, Conelly PW, Carroll MC,Lichtman AH. Influence of C3 deficiency on atherosclerosis. Circulation.2002;105:3025–3031.

31. Witting PK, Pettersson K, Ostlund-Lindqvist AM, Westerlund C,Eriksson AW, Stocker R. Inhibition by a coantioxidant of aorticlipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E andlow density lipoprotein receptor gene double knockout mice. FASEB J.1999;13:667–675.

32. Pekna M, Hietala MA, Rosklint T, Betsholtz C, Pekny M. Targeteddisruption of the murine gene coding for the third complement component(C3). Scand. J Immunol. 1998;47:25–29.

33. Nicoletti A, Kaveri S, Caligiuri G, Bariety J, Hansson G. Immunoglobulintreatment reduces atherosclerosis in apo E knockout mice. J Clin Invest.1998;102:910–918.

34. Tangirala RK, Rubin EM, Palinski W. Quantitation of atherosclerosis inmurine models: correlation between lesions in the aortic origin and in theentire aorta, and differences in the extent of lesions between sexes in LDLreceptor-deficient and apolipoprotein E-deficient mice. J Lipid Res. 1995;36:2320–2328.

35. Purcell-Huynh DA, Farese RV, Jr., Johnson DF, Flynn LM, Pierotti V, NewlandDL, Linton MF, Sanan DA, Young SG. Transgenic mice expressing high levelsof human apolipoprotein B develop severe atherosclerotic lesions in response toa high-fat diet. J Clin Invest. 1995;95:2246–2257.

36. Sjogren K, Wallenius K, Liu JL, Bohlooly YM, Pacini G, Svensson L,Tornell J, Isaksson OG, Ahren B, Jansson JO, Ohlsson C. Liver-derivedIGF-I is of importance for normal carbohydrate and lipid metabolism.Diabetes. 2001;50:1539–1545.

37. Robertson 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;112:1342–1350.

38. Caligiuri G, Nicoletti A, Poirier B, Hansson GK. Protective immunityagainst atherosclerosis carried by B cells of hypercholesterolemic mice.J Clin Invest. 2002;109:721–724.

39. Palinski W, Miller EJ, Witztum JL. Immunization of low densitylipoprotein (LDL) receptor-deficient rabbits with homologousmalondialdehyde-modified LDL reduces atherogenesis. Proc Natl AcadSci U S A. 1995;92:821–825.

40. Zhou X, Caligiuri G, Hamsten A, Lefvert AK, Hansson G. LDL immunizationinduces T-cell-dependent antibody formation and protection against atheroscle-rosis. Arterioscler Thromb Vasc Biol. 2001;21:108–114.

41. Murray I, Havel P, Sniderman AD, Cianflone K. Reduced body weight, adiposetissue, and leptin levels despite increased energy intake in female mice lackingacetylation-stimulating protein. Endocrinology. 2000;141:1041–1049.

42. Sniderman AD, Cianflone K, Arner P, Summers LK, Frayn KN. Theadipocyte, fatty acid trapping, and atherogenesis. Arterioscler ThrombVasc Biol. 1998;18:147–151.

43. Choy LN, Rosen BS, Spiegelman BM. Adipsin and an endogenouspathway of complement from adipose cells. J Biol Chem. 1992;267:12736–12741.

44. Choy LN, Spiegelman BM. Regulation of alternative pathway activationand C3a production by adipose cells. Obes Res. 1996;4:521–532.

45. Gabrielsson BG, Johansson JM, Lonn M, Jernas M, Olbers T, PeltonenM, Larsson I, Lonn L, Sjostrom L, Carlsson B, Carlsson LM. Highexpression of complement components in omental adipose tissue in obesemen. Obes Res. 2003;11:699–708.

46. Kalant D, Cain SA, Maslowska M, Sniderman AD, Cianflone K, Monk PN.The chemoattractant receptor-like protein C5L2 binds the C3a des-Arg77/acylation-stimulating protein. J Biol Chem. 2003;278:11123–11129.

47. Pangburn MK, Muller-Eberhard HJ. Relation of a putative thiolester bondin C3 to activation of the alternative pathway and the binding of C3b tobiological targets of complement. J Exp Med. 1980;152:1102–1114.

48. Pangburn MK, Schreiber RD, Muller-Eberhard HJ. Formation of theinitial C3 convertase of the alternative complement pathway. Acquisitionof C3b-like activities by spontaneous hydrolysis of the putative thiolesterin native C3. J Exp Med. 1981;154:856–867.

49. Isenman DE, Kells DIC, Cooper NR, Muller-Eberhard HJ, Pangburn MK.Nucleophilic modification of human complement protein C3: correlationof conformational changes with acquisition of C3b-like functional prop-erties. Biochemistry. 1981;20:4458–4467.

50. Babaev VR, Patel MB, Semenkovich CF, Fazio S, Linton MF. Macro-phage lipoprotein lipase promotes foam cell formation and atherosclerosisin low density lipoprotein receptor-deficient mice. J Biol Chem. 2000;275:26293–26299.

Persson et al Complement Activation and Atherosclerosis 1067

by guest on February 9, 2016http://atvb.ahajournals.org/Downloaded from

Marcela PeknaLinda Persson, Jan Borén, Anna-Karin L. Robertson, Ville Wallenius, Göran K. Hansson and

Mice−/− Low-Density Lipoprotein Receptor−/−Atherosclerosis in Apolipoprotein E Lack of Complement Factor C3, but Not Factor B, Increases Hyperlipidemia and

Print ISSN: 1079-5642. Online ISSN: 1524-4636 Copyright © 2004 American Heart Association, Inc. All rights reserved.

Greenville Avenue, Dallas, TX 75231is published by the American Heart Association, 7272Arteriosclerosis, Thrombosis, and Vascular Biology

doi: 10.1161/01.ATV.0000127302.24266.402004;24:1062-1067; originally published online April 1, 2004;Arterioscler Thromb Vasc Biol. 

http://atvb.ahajournals.org/content/24/6/1062World Wide Web at:

The online version of this article, along with updated information and services, is located on the

http://atvb.ahajournals.org/content/suppl/2004/06/23/01.ATV.0000127302.24266.40.DC1.htmlData Supplement (unedited) at:

  http://atvb.ahajournals.org//subscriptions/

at: is onlineArteriosclerosis, Thrombosis, and Vascular Biology Information about subscribing to Subscriptions:

  http://www.lww.com/reprints

Information about reprints can be found online at: Reprints: 

document. Question and AnswerPermissions and Rightspage under Services. Further information about this process is available in the

which permission is being requested is located, click Request Permissions in the middle column of the WebCopyright Clearance Center, not the Editorial Office. Once the online version of the published article for

can be obtained via RightsLink, a service of theArteriosclerosis, Thrombosis, and Vascular Biologyin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions:

by guest on February 9, 2016http://atvb.ahajournals.org/Downloaded from

Figure I. Noqualitative differences

in atherosclerotic

lesions between C3-/-

mice and Apoe-/-LDLR-/-

controls at 16 or 26weeks of age were

demonstrated by

Masson trichrome (MT)staining, MOMA-2

staining, or α-smooth

muscle actin (ASMA)

immunostaining.Representative images

are shown. Scale bar,100 µm.