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Accelerated Atherosclerosis in Low-Density LipoproteinReceptor–Deficient Mice Lacking the Membrane-Bound
Complement Regulator CD59Sheng Yun, Viola W.Y. Leung, Marina Botto, Joseph J. Boyle, Dorian O. Haskard
Objective—Whereas studies in humans and animal models have suggested a role for complement activation inatherosclerosis, there has been little analysis of the importance of complement regulators. We tested the hypothesis thatthe terminal pathway inhibitor CD59 plays an essential role in limiting the proinflammatory effects ofcomplement activation.
Methods and Results—CD59 gene targeted mice (CD59a�/�) mice were crossed with low-density lipoprotein receptor–deficient (Ldlr�/�) mice. CD59-deficient Ldlr�/� mice had significantly more extensive en face Sudan IV staining ofthoracoabdominal aorta than Ldlr�/� single knock-outs, both after a low-fat diet (6.51�0.36% versus 2.63�0.56%,P�0.001) or a high-fat diet (17.05�2.15% versus 7.69�1.17%, P�0.004). Accelerated lesion formation inCD59a�/�/Ldlr�/� mice on a high-fat diet was associated with increased lesional vascular smooth muscle cell (VSMC)number and fibrous cap formation.
Conclusion—Our data show that CD59 deficiency accelerates the development of lesions and increases plaque VSMCcomposition. Assuming that the main function of CD59 is to prevent the development of C5b-9 membrane attackcomplexes, our observations are consistent with the terminal complement pathway having proatherogenic potential inthe Ldlr�/� mouse model, and highlight the importance of complement regulation. (Arterioscler Thromb Vasc Biol.2008;28:000-000)
Although inflammatory mechanisms are recognized asplaying critical roles in atherosclerosis and its clinical
complications,1 the contribution of complement to atherogen-esis is still poorly defined. Previous experimental studiesinvestigating the role of complement in atherogenesis havefocused on the effects of deficiencies of individual comple-ment pathway components.2 Whereas there is evidence thatthe classical pathway has protective functions, the terminalpathway has been shown in rabbits to have proatherogeniceffects.3,4
Normally the complement system is controlled by thebalance between complement activators and a variety offluid-phase and membrane-bound regulatory proteins. Astransport of plasma-derived inhibitors into the arterial wallmay be limited, it is possible that complement regulation inatherosclerotic plaques may depend particularly on cell sur-face inhibitors, such as protectin (CD59), decay acceleratingfactor (DAF, CD55), membrane cofactor protein (MCP,CD46), and, in the mouse, complement receptor 1 (CR1)-related gene y (Crry),
CD59 is a glycophosphoinositol lipid-anchored glyocopro-tein that protects cells from complement-mediated injury byinhibiting the insertion of C9 into cell membranes andthereby preventing the development of C5b-9 membraneattack complexes.5,6 It is known to be expressed by macro-phages, T lymphocytes, endothelial cells, and vascularsmooth muscle cells (VSMCs) in human atherosclerosis.7 TheCD59 gene in mice is duplicated, with CD59a being widelyexpressed and CD59b restricted to testis. CD59a�/� miceappear healthy but show exacerbated inflammation in variousdisease models.8–10 We report herein the effect on atherogen-esis of deleting CD59a in Ldlr�/� mice.
Materials and MethodsReagentsOil Red O, dextrin, gelatin, Mayer Hematoxylin, L-glutamic acid,glycerol, sodium azide, calcium chloride, magnesium sulfate, andsodium phosphate were obtained from Merck/BDH. Buffered formalsaline (4% w/w formaldehyde solution) was from Pioneer ResearchChemicals. OCT compound was from CellPath. Other reagents werefrom Sigma-Aldrich.
Original received January 31, 2008; final version accepted June 26, 2008.From the Bywaters Centre for Vascular Inflammation, National Heart and Lung Institute (S.Y., V.W.Y.L., J.J.B., D.O.H.), the Division of Investigative
Sciences (V.W.Y.L., J.J.B.), and the Molecular Genetics and Rheumatology Section (M.B.), Division of Medicine, Imperial College, London, UK.S.Y. and V.W.Y.L. contributed equally to this study.Correspondence to Professor Dorian O. Haskard, NHLI Cardiovascular Sciences, Imperial College, Hammersmith Hospital, Du Cane Road, London,
Mice and DietsThe mice and diets used in the study are described in the supple-mental materials (please see http://atvb.ahajournals.org).
Lipoprotein, Cholesterol, andTriglyceride AnalysisAnalysis for lipoprotein profiles and serum total cholesterol andtriglycerides was as described.4
En Face Staining of AortaMethodology for en face staining of aortic lesions is in the supple-mental materials.
Aortic Root Histology and QuantificationCryosections of the aortic root were stained with Oil Red O andMayer hematoxylin and analyzed blind, as previously described.4
ImmunohistochemistryImmunohistochemistry and confocal microscopy techniques aredescribed in the supplemental materials.
StatisticsData handling is described in the supplemental materials.
ResultsThere was strong immunohistochemical staining of CD59 inthe aortic root of Ldlr�/� but not in CD59a�/�/Ldlr�/� mice(supplemental Figures I and II). Lesions were barely detect-able in the en face preparations of aortae of low-fat diet–fed Ldlr�/� mice but were significantly increased in theCD59a�/�/Ldlr�/� mice on this diet (CD59a�/�/Ldlr�/�
6.51�0.36% versus Ldlr�/� 2.63�0.56%, mean�SEM, P�0.001). Similarly, lesions in the aortic root were more than3-fold greater in CD59a�/�/ Ldlr�/� mice, either when ex-pressed as absolute lesion area (P�0.006) or as an areafraction (P�0.001). High-fat diet feeding enhanced en faceaortic lesion area in Ldlr�/� mice, and again CD59a�/�/Ldlr�/� mice had significantly larger lesions (CD59a�/�/Ldlr�/� 17.05�2.15% versus Ldlr�/� 7.69�1.17%, P�0.004). Aortic root lesion areas in high-fat–fed mice werenot different between groups (Figure 1 and supplementalFigure III).
Lesions in low-fat–fed mice consisted almost exclusivelyof macrophages and extracellular debris. In contrast, aorticroot lesions of high-fat–fed CD59a�/�/Ldlr�/� mice weremore complex than those in Ldlr�/� mice, despite the simi-larity in size. Thus there was a reduction in the proportion oflesional cells staining with the macrophage marker and a3-fold increased presence of alpha actin–positive VSMCs(47.7�3.7% versus 16.0�2.8% in Ldlr�/�, P�0.0001; Figure2). Furthermore, fibrous caps covered all lesions in high-fat–fed CD59a�/�/Ldlr�/� mice, compared with �25% of lesionsin Ldlr�/� mice. Further details of immunocytochemicalstaining, body weights and lipid profiles are given in thesupplemental materials.
DiscussionTo our knowledge this is the first experimental study address-ing the importance of an endogenous complement regulatorin atherosclerosis. Our data show that CD59 deficiencyaccelerates the development of lesions and increases plaque
VSMC composition. While we interpret this as evidence ofaccelerated plaque progression, the question arises as towhether the effects of CD59 deficiency might be to promotea relatively stable plaque phenotype characterized by a robustfibrous cap containing matrix and VSMC.11,12
The simplest explanation for our observations is that CD59inhibits the development of MAC in the arterial wall, but thisremains to be established. Whereas the assembly and inser-tion of C5b-9 into cell membranes may lyse nonnucleatedcells, sublytic levels can activate proliferation or proinflam-matory gene expression.13 It should be noted however that ourdata do not exclude the contribution of other mechanisms,such as an effect on the innate immune system of the mildhemolysis that has been reported in CD59a�/� mice.8
Our results need to be viewed alongside those showing anacceleration of atherosclerosis in Ldlr�/� mice that are defi-
Figure 1. CD59 deficiency accelerates aortic lipid depositionand atherosclerosis in the aortic root: Comparison betweenLdlr�/� and CD59a�/�/Ldlr�/�mice of (A) aortic en face lesionareas and (B) aortic root lesion area expressed as % fraction ofthe aortic root area.
2 Arterioscler Thromb Vasc Biol October 2008
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cient in the classical complement pathway activator C1q.4
Taken together with previous reports,14,15 a paradigm isemerging in which the controlled activation of the classicaland possibly other upstream complement pathways is protec-tive through facilitation of the clearance of apoptotic cells andprobably also enzymatically-modified LDL and other debris,whereas complement regulators such as CD59 help preventthis upstream complement activation translating into theelaboration of downstream proinflammatory effects.
In summary, our data show that CD59 retards atheroscle-rosis. The relative roles of other fluid phase and membrane-bound complement regulators in atherosclerotic lesion devel-opment and in shaping plaque phenotype now deserve furtherinvestigation.
Sources of FundingThis study was funded by a Programme Grant from the British HeartFoundation.
DisclosuresNone.
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Figure 2. CD59 deficiency increases lesion complexity in high-fat–fed mice: Aortic root VSMCs (red) in (A) Ldlr�/� and (B)CD59a�/�/Ldlr�/� mice. L indicates lumen. Nuclei are stainedpurple with TOPRO-3. Green arrowheads illustrate increasedfibrous cap formation in CD59a�/�/Ldlr�/� mice. C, Quantifica-tion of VSMC.
Yun et al CD59 Protects From Atherosclerosis 3
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