Brief Report: Accelerated Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice Lacking the Membrane-Bound Complement Regulator CD59
Objective— Whereas studies in humans and animal models have suggested a role for complement activation in atherosclerosis, there has been little analysis of the importance of complement regulators. We tested the hypothesis that the terminal pathway inhibitor CD59 plays an essential role in limiting the proinflammatory effects of complement 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 of thoracoabdominal 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 in CD59a−/−/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 VSMC composition. Assuming that the main function of CD59 is to prevent the development of C5b-9 membrane attack complexes, our observations are consistent with the terminal complement pathway having proatherogenic potential in the Ldlr−/− mouse model, and highlight the importance of complement regulation.
Although inflammatory mechanisms are recognized as playing critical roles in atherosclerosis and its clinical complications,1 the contribution of complement to atherogenesis is still poorly defined. Previous experimental studies investigating the role of complement in atherogenesis have focused on the effects of deficiencies of individual complement pathway components.2 Whereas there is evidence that the classical pathway has protective functions, the terminal pathway has been shown in rabbits to have proatherogenic effects.3,4
Normally the complement system is controlled by the balance between complement activators and a variety of fluid-phase and membrane-bound regulatory proteins. As transport of plasma-derived inhibitors into the arterial wall may be limited, it is possible that complement regulation in atherosclerotic plaques may depend particularly on cell surface inhibitors, such as protectin (CD59), decay accelerating factor (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 glyocoprotein that protects cells from complement-mediated injury by inhibiting the insertion of C9 into cell membranes and thereby preventing the development of C5b-9 membrane attack complexes.5,6 It is known to be expressed by macrophages, T lymphocytes, endothelial cells, and vascular smooth muscle cells (VSMCs) in human atherosclerosis.7 The CD59 gene in mice is duplicated, with CD59a being widely expressed and CD59b restricted to testis. CD59a−/− mice appear healthy but show exacerbated inflammation in various disease models.8–10 We report herein the effect on atherogenesis of deleting CD59a in Ldlr−/− mice.
Materials and Methods
Oil Red O, dextrin, gelatin, Mayer Hematoxylin, L-glutamic acid, glycerol, sodium azide, calcium chloride, magnesium sulfate, and sodium phosphate were obtained from Merck/BDH. Buffered formal saline (4% w/w formaldehyde solution) was from Pioneer Research Chemicals. OCT compound was from CellPath. Other reagents were from Sigma-Aldrich.
Mice and Diets
The mice and diets used in the study are described in the supplemental materials (please see http://atvb.ahajournals.org).
Lipoprotein, Cholesterol, and Triglyceride Analysis
Analysis for lipoprotein profiles and serum total cholesterol and triglycerides was as described.4
En Face Staining of Aorta
Methodology for en face staining of aortic lesions is in the supplemental materials.
Aortic Root Histology and Quantification
Cryosections of the aortic root were stained with Oil Red O and Mayer hematoxylin and analyzed blind, as previously described.4
Immunohistochemistry and confocal microscopy techniques are described in the supplemental materials.
Data handling is described in the supplemental materials.
There was strong immunohistochemical staining of CD59 in the aortic root of Ldlr−/− but not in CD59a−/−/Ldlr−/− mice (supplemental Figures I and II). Lesions were barely detectable in the en face preparations of aortae of low-fat diet-fed Ldlr−/− mice but were significantly increased in the CD59a−/−/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 than 3-fold greater in CD59a−/−/ Ldlr−/− mice, either when expressed as absolute lesion area (P<0.006) or as an area fraction (P<0.001). High-fat diet feeding enhanced en face aortic 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 were not different between groups (Figure 1 and supplemental Figure III).
Lesions in low-fat-fed mice consisted almost exclusively of macrophages and extracellular debris. In contrast, aortic root lesions of high-fat-fed CD59a−/−/Ldlr−/− mice were more complex than those in Ldlr−/− mice, despite the similarity in size. Thus there was a reduction in the proportion of lesional cells staining with the macrophage marker and a 3-fold increased presence of alpha actin-positive VSMCs (47.7±3.7% versus 16.0±2.8% in Ldlr−/−, P<0.0001; Figure 2). Furthermore, fibrous caps covered all lesions in high-fat-fed CD59a−/−/Ldlr−/− mice, compared with ≈25% of lesions in Ldlr−/− mice. Further details of immunocytochemical staining, body weights and lipid profiles are given in the supplemental materials.
To our knowledge this is the first experimental study addressing the importance of an endogenous complement regulator in atherosclerosis. Our data show that CD59 deficiency accelerates the development of lesions and increases plaque VSMC composition. While we interpret this as evidence of accelerated plaque progression, the question arises as to whether the effects of CD59 deficiency might be to promote a relatively stable plaque phenotype characterized by a robust fibrous cap containing matrix and VSMC.11,12
The simplest explanation for our observations is that CD59 inhibits the development of MAC in the arterial wall, but this remains to be established. Whereas the assembly and insertion of C5b-9 into cell membranes may lyse nonnucleated cells, sublytic levels can activate proliferation or proinflammatory gene expression.13 It should be noted however that our data do not exclude the contribution of other mechanisms, such as an effect on the innate immune system of the mild hemolysis that has been reported in CD59a−/− mice.8
Our results need to be viewed alongside those showing an acceleration of atherosclerosis in Ldlr−/− mice that are deficient in the classical complement pathway activator C1q.4 Taken together with previous reports,14,15 a paradigm is emerging in which the controlled activation of the classical and possibly other upstream complement pathways is protective through facilitation of the clearance of apoptotic cells and probably also enzymatically-modified LDL and other debris, whereas complement regulators such as CD59 help prevent this upstream complement activation translating into the elaboration of downstream proinflammatory effects.
In summary, our data show that CD59 retards atherosclerosis. The relative roles of other fluid phase and membrane-bound complement regulators in atherosclerotic lesion development and in shaping plaque phenotype now deserve further investigation.
Sources of Funding
This study was funded by a Programme Grant from the British Heart Foundation.
S.Y. and V.W.Y.L. contributed equally to this study.
Original received January 31, 2008; final version accepted June 26, 2008.
Geertinger P, Sørensen H. On the reduced atherogenic effect of cholesterol feeding in rabbits with congenital complement (C6) deficiency. Artery. 1977; 1: 177–184.
Rollins SA, Sims PJ. The complement-inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9. J Immunol. 1990; 144: 3478–3483.
Holt DS, Botto M, Bygrave AE, Hanna SM, Walport MJ, Morgan BP. Targeted deletion of the CD59 gene causes spontaneous intravascular hemolysis and hemoglobinuria. Blood. 2001; 98: 442–449.
Turnberg D, Botto M, Warren J, Morgan BP, Walport MJ, Cook HT. CD59a deficiency exacerbates accelerated nephrotoxic nephritis in mice. J Am Soc Nephrol. 2003; 14: 2271–2279.
Flugelman MY, Virmani R, Correa R, Yu ZX, Farb A, Leon MB, Elami A, Fu YM, Casscells W, Epstein SE. Smooth muscle cell abundance and fibroblast growth factors in coronary lesions of patients with nonfatal unstable angina. A clue to the mechanism of transformation from the stable to the unstable clinical state. Circulation. 1993; 88: 2493–2500.
Gershov 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: 1353–1364.
Bhakdi S, Torzewski M, Paprotka K, Schmitt S, Barsoom H, Suriyaphol P, Han SR, Lackner KJ, Husmann M. Possible protective role for C-reactive protein in atherogenesis: complement activation by modified lipoproteins halts before detrimental terminal sequence. Circulation. 2004; 109: 1870–1876.