Passive immunization with monoclonal IgM antibodies against phosphorylcholine reduces accelerated vein graft atherosclerosis in apolipoprotein E-null mice

Atherosclerosis 189 (2006) 83–90

Passive immunization with monoclonal IgM antibodies against phosphorylcholine reduces accelerated vein graft atherosclerosis in apolipoprotein E-null mice Jose R. Faria-Neto 1 , Kuang-Yuh Chyu ∗,1 , Xiaojun Li, Paul C. Dimayuga, Carmel Ferreira, Juliana Yano, Bojan Cercek, Prediman K. Shah Atherosclerosis Research Center, Division of Cardiology, Department of Medicine and Burns and Allen Research Institute, Cedars-Sinai Medical Center and David Geffen School of Medicine at UCLA, Los Angeles, CA 90048, United States Received 13 July 2005; received in revised form 10 November 2005; accepted 20 November 2005 Available online 18 January 2006

Abstract Phosphorylcholine (PC) headgroup is one of the neoantigens exposed by LDL oxidation that can elicit an immune response. Active immunization with Streptococcus pneumoniae, which bears PC on its cell wall, reduced atherosclerosis in hypercholesterolemic mice and this effect was attributed to an immune response to PC. In this study we tested the hypothesis that passive immunization with a monoclonal anti-PC IgM antibody can be athero-protective in a murine model of native aortic and vein graft atherosclerosis. Inferior vena cava from 16-week-old donor male apoE-null mice was grafted into right carotid artery of age-matched male recipient apoE-null mice. Anti-PC IgM titers were evaluated before and 4 weeks after surgery. For the immunization protocol, a separate group of mice received weekly intraperitoneal injection of monoclonal anti-PC IgM (400 ␮g) for 4 weeks, starting the day of surgery. Controls received PBS or pooled polyclonal IgM. Anti-PC IgM titres significantly increased at 4 weeks following surgery. Passive immunization with anti-PC IgM reduced vein graft plaque size and neointimal thickness resulting in a larger luminal area; in addition immunization reduced the inflammatory cell content of the plaques. There was no significant effect on the established native aortic atherosclerotic lesions. Immunization did not affect circulating cholesterol levels. Taken together our data suggest that passive immunization with anti-PC IgM significantly reduces vein graft lesion size with less inflammatory phenotype without affecting cholesterol levels, indicating an athero-protective immune response to PC. Lack of effect on established native aortic lesions may have been due to short duration of therapy and/or reduced efficacy in established lesions as compared to evolving lesions of vein graft atherosclerosis. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Phosphorylcholine; Passive immunization

1. Introduction The role of innate and adaptive immunity in atherosclerosis has been increasingly recognized [1–3]. One of the components of innate immune response is natural IgM antibody [4], which recognizes pathogen-associated molecular patterns (PAMPs) to confer protection against offending ∗ 1

Corresponding author. Tel.: +1 310 423 4876; fax: +1 310 423 0245. E-mail address: [email protected] (K.-Y. Chyu). These authors contributed equally to this manuscript.

0021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2005.11.033

antigens. A monoclonal anti-phosphorylcholine IgM that bears the TEPC-15 isotype was cloned from a hypercholesterolemic mouse and found to bind to phosphorylcholine (PC) headgroups on oxidized LDL or phospholipids and block oxLDL uptake by macrophage [5,6]. Phosphorylcholine is the head group of many phospholipids and is also an immunodeterminant in the cell wall of Streptococcus pneumoniae. While active immunization with S. pneumoniae expands T15-IgM secreting splenocytes and appears to be associated with reduced atherosclerotic lesion formation [7], it is not clear that the effect is directly

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mediated by an adaptive humoral response. To clarify this issue, we injected anti-PC monoclonal IgM antibodies once a week for 4 weeks into atherosclerosis-prone apolipoprotein E (apoE)-deficient mice fed a Western-style diet that had first undergone surgical grafting of the inferior vena cava onto the right carotid artery. This model produces an opportunity to evaluate “native” atherosclerosis instigated by the genetically-induced hypercholesterolemia, as well as provide an evolving and accelerated form of atherosclerosis in the vein graft [8]. We thus tested the hypothesis that passive immunization via injection of anti-PC antibodies would reduce both accelerated vein graft atherosclerosis and native aortic atherosclerosis in hypercholesterolemic apoE-deficient mice. We report here that anti-PC antibody administration for 4 weeks did indeed inhibit accelerated atherosclerosis in the vein graft, but had no detectable effect on concurrent development of native atherosclerosis in the aorta.

2. Methods 2.1. Culture of hybridoma cell line A hybridoma cell line secreting monoclonal antiphosphorylcholine IgM with T15 idiotype (HPCM2 cells [9], a generous gift from Dr. Patricia Gearhart at NIH/NIA) was maintained in BD CellTM serum free Mab medium (BD Biosciences) in a membrane-based disposable cell cultivation system (CELLineTM Device, BD Biosciences) incubated in a CO2 incubator at 37 ◦ C using a 7/21 day harvest protocol recommended by the manufacturer. The device has a 15 ml cell compartment and a 1000 ml nutrient compartment. The antibody-containing medium from cell compartment was collected every 7 days. On day 21 the culture medium in the nutrient compartment was replaced with fresh medium to maintain cell growth. The procedure was followed until sufficient antibody was obtained.

PC IgM. The purity of IgM was confirmed by a single 85 kDa band on 8% reducing electrophoretic gel after staining with Coomassie blue (see Section 3). Western blot analysis confirmed the presence of IgM and no IgG (data not shown). 2.3. Determination of IgM concentration Ninety-six-well plates (MaxiSorp, Nalge Nunc) were coated with anti-mouse immunoglobulin (H + L chain, 5 ␮g/ml) overnight. Standard ELISA protocol was used. HRP-conjugated goat anti-mouse IgM (H chain) was used as detection antibody and ABTS was used for color development. Purified mouse IgM (Sigma) of known concentration was used to construct a standard curve to determine IgM concentration in the sample. 2.4. Anti-PC IgM ELISA Ninety-six-well microtitration plate (MaxiSorp, Nalge Nunc) was coated with 50 ␮l of PC-KLH or KLH (each 10 ␮g/ml) overnight. After washings and blocking with 1% BSA in PBS, medium was added into wells and incubated for 1 h at room temperature. After washing, biotinylated goat anti-mouse IgM (Pierce) was added for incubation at room temperature for 1 h. Avidin-conjugated alkaline phosphatase (DAKO) and pNPP system were used for color development and plate was read at O.D. 405 nm. O.D. readings from KLH and blank were subtracted from PC-KLH O.D. to determine anti-PC IgM titer. 2.5. Biotinylation of anti-PC IgM Purified anti-PC IgM was biotinylated using a commercially available kit (EZ-Link® Sulfo-NHS-LC-Biotinylation Kit, Pierce). This biotinylated anti-PC IgM was then used for direct immunohistochemical localization of the PC epitopes in the vein graft plaques using standard protocol.

2.2. Purification of anti-phosphorylcholine IgM

2.6. Vein graft bypass procedure

The collected antibody-containing medium from cell compartment were pooled and concentrated by centrifugation at 3000 × g for 30 min at 4 ◦ C using a Jumbosep device (PALL Corporation) with a 100 K molecular weight cutoff membrane. The concentrated medium were pooled again, centrifuged for a second time and then dialyzed against Tris buffer (Tris 20 mM, sodium chloride 1.25 M, pH 7.4) to remove phosphate ions. Antibodies in the dialyzed medium were subsequently purified by ImmunoPure IgM Purification Kit (Pierce) using manufacturer’s recommended protocol. The fractions with O.D. greater than 0.02 at 280 nm were pooled, concentrated and dialyzed against PBS and filtersterilized before injection. The final concentration ranged 2–3.5 mg/ml as determined by IgM ELISA. Approximately 1.5 l of culture medium could yield 20 mg purified anti-

Apolipoprotein E (apoE)-deficient mice (Jackson Laboratory) were fed a high-fat, high-cholesterol diet containing 21% (w/w) fat and 0.15% cholesterol from 6 weeks of age throughout the duration of the experiment. At 16 week of age, the vein graft surgery was performed as reported previously [8]. In each group there were several mice that died post-operatively. After sacrifice we excluded from analysis the vein grafts that were thrombotic and occluded. Therefore, for PBS group a total of 18 mice received vein graft surgery and 5 mice were excluded (2 deaths and 3 occlusions). For control IgM group, a total of 15 mice received vein graft surgery and 4 mice were excluded due to occlusion of vein graft. For anti-PC IgM group, a total of 21 mice received vein graft surgery and 8 mice were excluded (4 deaths, 4 occlusions).

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2.7. Antibody administration Immediately after the vein graft procedure, mice received intraperitoneal injection of purified 0.4 mg IgM antibody against PC followed by weekly injection for additional three times until euthanasia. The dose was chosen based on an observation that the mean level of IgM after injection of 0.4 mg IgM into Rag1(−/−) mouse was similar to that of normal mouse [10]. Mice in control groups received either pooled mouse IgM (Rockland, same dose and protocol as PC IgM) or PBS (same volume as IgM) administered at the same time-point. To establish that IgM injected intraperitoneally can be absorbed into systemic circulation, we demonstrated in a pilot study that plasma IgM concentration increased and reached plateau 24 h after intraperitoneal injection of 400 ␮g of antiPC IgM antibody and returned to near pre-injection level 150 h after injection (data not shown). 2.8. Tissue preparation and histomorphometry of vein graft lesions, aortic sinus lesions and aorta The preparation and histomorphometrical analysis of the vein graft and aortic sinus lesions was performed as reported previously [8]. The aortic tree was cleaned of adventitial fat and fixed in Histochoice (Amresco) overnight. The aorta was then carefully cut longitudinally and stained with oil-red-O. The stained aorta was placed with luminal side up on a slide freshly coated with egg albumin solution. After the albumin solution dried, images of the aorta were digitally acquired to assess the extent of atherosclerosis with computer-assisted histomorphometry. 2.9. Plasma cholesterol measurement Plasma cholesterol levels were measured with a commercially available kit (Sigma) following manufacturer’s instructions. Plasma samples were diluted 1:2 with PBS prior to assay. 2.10. Immunohistochemistry Sections were fixed in ice-cold acetone for 5 min. Immunohistochemical studies were performed using standard protocol with monocyte/macrophage antibody (MOMA-2, Serotec) or PCNA antibody (Santa Cruz Biotechnology). Negative controls included non-immune isotype antibody or omission of the primary antibody. 2.11. Oil-red-O staining Frozen sections were fixed in 4% paraformaldehyde at room temperature for 30 min. After washing in water for 2 min, slides were stained with 0.24% oil-red-O in 60% isopropanol for 20 min. After another wash in 60% isopropanol

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for 2 min, slides were counterstained with hematoxyline and fast-green. 2.12. Trichrome staining Collagen content in the plaque was assessed after staining with Trichrome using a standard method. Collagen appears blue under light microscopy. 2.13. Morphometric analysis Quantitative measurement of MOMA-2 positive area, the extent of oil-red-O or collagen stain area was determined by computer-assisted analysis using ImagePro Plus (Media Cybernetics). Positively stained area was presented as mean percentage of the total aortic sinus area or total area of aortic tree. The number of nuclei positively stained with PCNA in each section, using PCNA immunoreactivity in intestinal sections as positive control, was counted manually. The data are presented as PCNA density (number of PCNA positive nuclei/plaque size in mm2 ). The quantitative measurements of the vein graft lesions were presented as absolute size in mm2 , percent of luminal stenosis or averaged thickness. To measure the thickness of the vein graft lesion, each vein graft lesion was equally divided into 8 sections by 2 perpendicular crosshairs. The thickness of each section was summed and averaged to represent the average thickness of the lesion. 2.14. Statistical analysis Data are presented as mean ± S.D. Statistical method used is listed in text, table or figure legend. Normally distributed data were analyzed by parametric method, whereas nonnormally distributed data were analyzed by non-parametric method. p < 0.05 was considered as statistically significant.

3. Results 3.1. Characterization of anti-PC IgM Purified monoclonal anti-PC IgM with T15 idiotype from a hybridoma cell line was obtained as described in Methods. This IgM contained high titers against the PC epitope (Fig. 1A) and competitive ELISA indicated specific binding of this antibody to PC (Fig. 1B). A single 85 kDa band on 8% reducing SDS-PAGE gel after staining with Coomassie blue confirmed the purity of IgM (Fig. 1C). 3.2. Immune response against phosphorylcholine after vein graft surgery We have previously established an accelerated vein graft bypass atherosclerosis model in hypercholesterolemic apoE (−/−) mice [8]. The PC epitope was present in the vein

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taneously produced anti-PC IgM titer before and 4 weeks after surgery. Anti-PC IgM titer became significantly higher at 4 weeks after surgery, indicating an immune response against PC headgroups (Fig. 2B). 3.3. Effect of passive immunization with monoclonal anti-PC IgM antibody on vein graft atherosclerosis There was no significant difference of body weight (PBS: 30.9 ± 3.3 gm, control IgM: 30.3 ± 1.8 gm, anti-PC IgM: 28.9 ± 1.8 gm) or circulating cholesterol levels (PBS: 1234 ± 383 mg/dl, control IgM: 1322 ± 302 mg/dl, anti-PC IgM: 1151 ± 414 mg/dl) at sacrifice. The atherosclerotic plaques in the vein graft from mice receiving weekly injection of monoclonal anti-PC IgM for 4 weeks were smaller (0.455 ± 0.171 mm2 , n = 13) when compared to the plaques from mice that received polyclonal IgM (0.804 ± 0.566 mm2 , n = 11) or PBS (0.615 ± 0.254 mm2 , n = 13) (Fig. 3A, p < 0.05 by Kruskal–Wallis test). The mice that received anti-PC IgM also had the least luminal stenosis (Fig. 3B) and the smallest vein graft plaque thickness (Fig. 3C). PCNA immunoreactivity was also significantly less in the vein graft lesions from the mice receiving anti-PC IgM (Fig. 3D). The vein graft lesions from the mice receiving anti-PC IgM had the least macrophage immunoreactivity (Fig. 4A) and lipid content (Fig. 4B) as demonstrated by MOMA immunohistochemical and oil-red-O staining, respectively. 3.4. Effect of passive immunization with monoclonal anti-PC IgM antibody on established native atherosclerosis In contrast to the reduction of atherosclerotic lesions in the vein graft, weekly passive immunization with anti-PC IgM for 4 weeks did not significantly change the native atherosclerotic lesions in aortic sinus or aorta compared to PBS or polyclonal IgM treatment (Table 1).

4. Discussion

Fig. 1. (A) Anti-PC titer from the concentrated, purified conditioned medium of HPCM2 cells (black circle). Mouse IgM from pooled serum was used as control and showed minimal binding to PC-KLH (open circle). (B) Competition ELISA of anti-PC IgM from the condition medium of HPCM2 cells with increasing concentration of PC-KLH (␮g/ml). (C) Coomassie blue staining of an 8% reducing SDS-PAGE gel. Lane 1: purified anti- PC IgM from MBP column; lane 2: concentrated HPCM2 medium before purification by MBP column; lane 3: mouse IgM as positive control.

graft lesions as demonstrated by the PC immunoreactivity using biotinylated anti-PC IgM (Fig. 2A). To determine whether the presence of PC epitope and formation of accelerated atherosclerosis in the vein graft is accompanied with an immune response against PC epitope, we compared the spon-

Oxidation of low density lipoprotein (LDL) generates antigenic neoepitopes [11,12]. These antigenic sites activate host defenses: progression of atherosclerosis is accompanied by generation of autoantibodies against oxidized LDL (oxLDL) epitopes, and active immunization of atherosclerosis-prone hypercholesterolemic mice with LDL or oxidized LDL blunts plaque development [13–16]. Additionally immunization of atherosclerosis-prone mice against S. pneumoniae led to high circulating levels of IgM against the phorphorylcholine epitope and a reduction in the extent of atherosclerosis [7]. These studies show molecular mimicry between epitopes of oxLDL and S. pneumoniae and indicate that these immune responses can have athero-protective effects. Similar interest in PC as a potential immune target arises from data showing

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Fig. 2. (A) The PC epitope is present in the vein graft lesions (left) as demonstrated immunohistochemically using biotinylated anti-PC IgM. Its location is largely similar to that of lipid staining (right), as shown on consecutive sections. (B) Spontaneously produced anti-PC IgM titer at 4 weeks after vein graft bypass surgery was significantly higher when compared to pre-surgery level. * p < 0.05 by t-test.

that monoclonal IgM against PC also exists in vivo, appears to be an important neo-antigen exposed by LDL oxidation, and the immune response to PC is linked to inhibition of atherogenesis [17]. These studies involved stimulation of host defenses, but raise the question as to whether antibodies that might protect against atherosclerosis can themselves be used as a therapeutic agent. We therefore tested whether an antibody against a specific PC epitope prevents or slows the rate on accelerated vein graft and native aortic atherosclerosis. We chose this model because it provides an opportunity to study the effect of intervention on vein graft atherosclerosis as well as native aortic atherosclerosis. The vein graft lesions

in this model display the classical morphological and cellular features in matured atherosclerotic plaques, consisting of a fibrous cap, inflammatory cells infiltrate, foam cells, cholesterol crystal, necrotic core and calcification [18]. Here we report that an immune response to PC paralleled the development of accelerated vein graft atherosclerosis. Moreover, passive immunization using an anti-PC IgM reduced the extent of accelerated vein graft atherosclerotic lesion and plaque macrophage immunoreactivity without changing circulating cholesterol levels. We reported previously that the exacerbated intimal thickening in hypercholesterolemic mice exposed to cigarette smoke was associated

Table 1 Effect of passive immunization with monoclonal anti-PC IgM antibody on established native atherosclerosis Aortic sinus Size PBS group (n = 13) Control IgM group (n = 10) PC IgM group (n = 13)

(mm2 )

0.505 ± 0.145 0.466 ± 0.117 0.471 ± 0.090

Aortic flat prep MOMA (%)

Lipid content (%)

Collagen content (%)

Plaque size (% aortic size)

11.0 ± 4.6 16.1 ± 3.7 13.5 ± 2.1

31.3 ± 4.5 34.1 ± 11.0 33.6 ± 9.5

21.3 ± 4.5 21.3 ± 7.2 20.8 ± 9.2

11.8 ± 5.4 13.3 ± 6.9 (n = 11) 10.2 ± 4.5 (n = 12)

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Fig. 3. Atherosclerotic lesions in the bypassed vein graft are smaller in mice receiving anti-PC IgM when compared to that from PBS group or from mice receiving polyclonal IgM (A). Luminal stenosis (B), plaque thickness (C) and PCNA density (D) were also significantly smaller in the vein graft lesions from the mice receiving anti-PC IgM when compared to the other 2 groups. * p < 0.05 by ANOVA.

with anti-PC IgM [19]. These findings collectively suggest that an immune response to PC develops in hypercholesterolemic mice that modulates development of accelerated vein graft atherosclerosis without affecting circulating cholesterol levels. Nevertheless, treatment with PC did not affect development of native aortic atherosclerosis. Studies comparing histopathologic features of vein graft atherosclerosis with native plaques have generally shown these two pathologies to be remarkably similar. The effectiveness of therapeutic interventions and the risk factor determinants appear to be similar as well [20–22]. For this reason, vein graft atherosclerosis is thought to be essentially the same as native arterial atherosclerosis, except that the former develops considerably more rapidly, for reasons that remain unclear [23]. Our experimental mice were treated with monoclonal PC IgM antibodies for only 4 weeks. This was done because our previous studies had demonstrated that this was sufficient time

for marked development of vein graft atherosclerosis, and longer treatment duration would have incurred increasing risk of total occlusion. The likely explanation for the lack of effect of anti-PC antibodies on development of native aortic atherosclerosis was that the 4-week duration of treatment was insufficient to produce detectable differences between treated and control mice or the treatment was started too late to have an effect. Consistent with this interpretation, we have previously observed a similar differential effect of treatments with apolipoprotein A-I-mimetic peptides in this same model of accelerated vein graft atherosclerosis in apoE (−/−) mice. We speculate that longer duration of treatment (and to be started earlier in life) would probably have revealed that anti-PC antibody treatment also slowed progression of native aortic atherosclerosis, but we cannot completely exclude the possibility that anti-PC antibodies somehow uniquely affect plaque development only in vein grafts but not in native arteries. Further studies in apoE (−/−) mice that have not been

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Fig. 4. Atherosclerotic lesions in the vein graft from mice receiving anti-PC IgM had the least macrophage immunoreactivity (A) and lipid content (B) as demonstrated by MOMA staining and oil-red-O staining, respectively, when compared to the other 2 groups. * p < 0.05 by ANOVA.

subjected to vein grafting involving longer treatment conditions are needed and being planned to directly address this issue. How anti-PC IgM reduces vein graft atherosclerosis remains unclear. Similar to previous findings with antibodies against oxidized phospholipids or oxidized phospholipidprotein adducts [5], passive immunization with anti-PC IgM reduced lipid content and macrophage immunoreactivity in vein graft lesions (but not aortic plaques). Although rich in inflammatory cells, the accelerated lesion formation that occurs in the vein graft model is also characterized by significant presence of proliferating smooth muscle cells [24]. The reduced PCNA density in mice treated with anti-PC IgM suggests that such treatment may have reduced cell proliferation in the plaque, contributing to the reduced plaque size. This reduced cell proliferation could be due to concomitant reduction in macrophage and lipid content in the plaques since inflammation and a lipid-rich milieu potentially create a local environment with high oxidative stress and all these factors have been linked to atherogenesis. Other mechanisms of action may be operative as well, but must await the results of further studies. It is also not known whether the IgG isotype antibody against the phosphorylcholine head-group produces simi-

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lar athero-protective effects. Active immunization with S. pneumoniae induced predominantly IgM antibody and weak IgG3 antibody response against oxLDL with concomitant reduction of aortic atherosclerosis without inducing oxLDL specific IgG1 and IgG2a antibody responses [7]. Whether exogenously administered IgG has similar athero-protective effect when compared to anti-PC IgM remains to be determined. Our data may have certain clinical implications. The concept of using immunoglobulin as a therapeutic agent has been widely applied in clinical settings. The immunoglobulin used is either pooled immunoglobulin against no specific epitope or monoclonal antibody with epitope specificity. In the field of cardiovascular disease, abciximab is a well-known monoclonal antibody used in percutaneous coronary intervention. Although the specific epitopes in atherogenesis have not been well defined, intravenous administration of immunoglobulin has been shown to reduce atherogenesis in animal models [25]. Recently Schiopu et al. reported that monoclonal IgG antibodies against specific peptide sequence of human apo B-100 protein reduced atherosclerotic lesion formation [26]. To prevent atherosclerosis and its subsequent complication, an active immunization program likely is a cost-effective strategy. Although this approach has been effective in experimental animals, its human application has been hindered by the lack of knowledge such as specific epitopes, duration, interval and timing of immunization [16], route or appropriate adjuvant for immunization. In the meantime, a suitable monoclonal antibody against a specific antigenic epitope could be an alternative for treating atherosclerotic vascular disease. Our results support this view.

Acknowledgements The authors thank Ang Ji for his technical assistance. The authors gratefully acknowledge the support from The Eisner Foundation, and the Heart Foundation, United Hostesses Charities and Sam Spaulding Fund at Cedars Sinai Medical Center. Dr. Faria-Neto received a fellowship grant from FUNCOR of the Brazilian Society of Cardiology.

References [1] Binder CJ, Chang MK, Shaw PX, et al. Innate and acquired immunity in atherogenesis. Nat Med 2002;8:1218–26. [2] Hansson GK, Berne GP. Atherosclerosis and the immune system. Acta Paediatr Suppl 2004;93:63–9. [3] Nilsson J, Hansson GK, Shah PK. Immunomodulation of atherosclerosis: implications for vaccine development. Arterioscler Thromb Vasc Biol 2005;25:18–28. [4] Boes M. Role of natural and immune IgM antibodies in immune responses. Mol Immunol 2000;37:1141–9. [5] Horkko S, Bird DA, Miller E, et al. Monoclonal autoantibodies specific for oxidized phospholipids or oxidized phospholipid-protein adducts inhibit macrophage uptake of oxidized low-density lipoproteins. J Clin Invest 1999;103:117–28.

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[6] Shaw PX, Horkko S, Chang MK, et al. Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity. J Clin Invest 2000;105:1731–40. [7] Binder CJ, Horkko S, Dewan A, et al. Pneumococcal vaccination decreases atherosclerotic lesion formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL. Nat Med 2003;9:736–43. [8] Li X, Chyu KY, Neto JR, et al. Differential effects of apolipoprotein A-I-mimetic peptide on evolving and established atherosclerosis in apolipoprotein E-null mice. Circulation 2004;110:1701–5. [9] Gearhart PJ, Johnson ND, Douglas R, Hood L. IgG antibodies to phosphorylcholine exhibit more diversity than their IgM counterparts. Nature 1981;291:29–34. [10] Williams JP, Pechet TT, Weiser MR, et al. Intestinal reperfusion injury is mediated by IgM and complement. J Appl Physiol 1999;86:938–42. [11] Palinski W, Horkko S, Miller E, et al. Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein Edeficient mice. Demonstration of epitopes of oxidized low density lipoprotein in human plasma. J Clin Invest 1996;98:800–14. [12] Palinski W, Yla-Herttuala S, Rosenfeld ME, et al. Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein. Arteriosclerosis 1990;10:325–35. [13] Ameli S, Hultgardh-Nilsson A, Regnstrom J, et al. Effect of immunization with homologous LDL and oxidized LDL on early atherosclerosis in hypercholesterolemic rabbits. Arterioscler Thromb Vasc Biol 1996;16:1074–9. [14] Freigang S, Horkko S, Miller E, Witztum JL, Palinski W. Immunization of LDL receptor-deficient mice with homologous malondialdehydemodified and native LDL reduces progression of atherosclerosis by mechanisms other than induction of high titers of antibodies to oxidative neoepitopes. Arterioscler Thromb Vasc Biol 1998;18:1972–82. [15] Zhou X, Caligiuri G, Hamsten A, Lefvert AK, Hansson GK. LDL immunization induces T-cell-dependent antibody formation and protection against atherosclerosis. Arterioscler Thromb Vasc Biol 2001;21:108–14.

[16] Chyu KY, Reyes OS, Zhao X, et al. Timing affects the efficacy of LDL immunization on atherosclerotic lesions in apo E (−/−) mice. Atherosclerosis 2004;176:27–35. [17] Shaw PX, Horkko S, Chang MK, et al. Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity. J Clin Invest 2000;105:1731–40. [18] Reddick RL, Zhang SH, Maeda N. Atherosclerosis in mice lacking apo E. Evaluation of lesional development and progression. Arterioscler Thromb 1994;14:141–7. [19] Tani S, Dimayuga PC, Anazawa T, et al. Aberrant antibody responses to oxidized LDL and increased intimal thickening in apoE−/− mice exposed to cigarette smoke. Atherosclerosis 2004;175:7–14. [20] Mautner SL, Mautner GC, Hunsberger SA, Roberts WC. Comparison of composition of atherosclerotic plaques in saphenous veins used as aortocoronary bypass conduits with plaques in native coronary arteries in the same men. Am J Cardiol 1992;70:1380–7. [21] Sarjeant JM, Rabinovitch M. Understanding and treating vein graft atherosclerosis. Cardiovasc Pathol 2002;11:263–71. [22] Silva JA, White CJ, Collins TJ, Ramee SR. Morphologic comparison of atherosclerotic lesions in native coronary arteries and saphenous vein graphs with intracoronary angioscopy in patients with unstable angina. Am Heart J 1998;136:156–63. [23] Motwani JG, Topol EJ. Aortocoronary saphenous vein graft disease: pathogenesis, predisposition, and prevention. Circulation 1998; 97:916–31. [24] Dietrich H, Hu Y, Zou Y, et al. Rapid development of vein graft atheroma in ApoE-deficient mice. Am J Pathol 2000;157:659– 69. [25] Nicoletti A, Kaveri S, Caligiuri G, Bariety J, Hansson K. Immunoglobulin treatment reduces atherosclerosis in apo E knockout mice. J Clin Invest 1998;102:910–8. [26] Schiopu A, Bengtsson J, Soderberg I, et al. Recombinant Human antibodies against aldehyde-modified apolipoprotein B-100 peptide sequences inhibit atherosclerosis. Circulation 2004;110:2047– 52.