Plasma autoantibodies against apolipoprotein B-100 peptide 210 in subclinical atherosclerosis

Atherosclerosis 232 (2014) 242e248

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Plasma autoantibodies against apolipoprotein B-100 peptide 210 in subclinical atherosclerosis Olga McLeod a, *, Angela Silveira a, Gunilla N. Fredrikson c, Karl Gertow a, Damiano Baldassarre d, Fabrizio Veglia d, Bengt Sennblad b, Rona J. Strawbridge a, Malin Larsson a, Karin Leander e, Bruna Gigante e, Jussi Kauhanen g, Rainer Rauramaa h, Andries J. Smit i, Elmo Mannarino j, Philippe Giral k, Steve E. Humphries f, Elena Tremoli d, Ulf de Faire e, John Öhrvik a, Jan Nilsson c, Anders Hamsten a a

Atherosclerosis Research Unit, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden Atherosclerosis Research Unit, Department of Medicine, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden c Department of Clinical Sciences, Skåne University Hospital, Lund University, Malmö, Sweden d Dipartimento di Scienze Farmacologiche e Biomolecolari, Università di Milano, Centro Cardiologico Monzino, IRCCS, Milan, Italy e Division of Cardiovascular Epidemiology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden f Centre for Cardiovascular Genetics, University College London, United Kingdom g Institute of Public Health and Clinical Nutrition, University of Eastern Finland, Kuopio, Finland h Foundation for Research in Health, Exercise and Nutrition, Kuopio Research Institute of Exercise Medicine, Kuopio, Finland i Department of Medicine, University Medical Center Groningen, The Netherlands j Internal Medicine, Angiology and Arteriosclerosis Diseases, Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy k Assistance Publique e Hopitaux de Paris, Service Endocrinologie-Metabolisme, Groupe Hôpitalier Pitie-Salpetriere, Unités de Prévention Cardiovasculaire, Paris, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 July 2013 Received in revised form 5 November 2013 Accepted 12 November 2013 Available online 23 November 2013

Objective: Experimental studies have suggested that autoimmunity is involved in atherosclerosis and provided evidence that both protective and pro-atherogenic immune responses exist. This concept has received support from small clinical studies implicating autoantibodies directed against apolipoprotein B-100 (apoB-100) in human atherosclerosis. We examined circulating autoantibodies directed against native and malondialdehyde (MDA)-modified epitope p210 of apoB-100 (IgG-p210nat and IgM-p210MDA) in relation to early atherosclerosis in a large, European longitudinal cohort study of healthy high-risk individuals. Approach and results: IgG-p210nat and IgM-p210MDA were quantified in baseline plasma samples of 3430 participants in the IMPROVE study and related to composite and segment-specific measures of severity and rate of progression of carotid intima-media thickness (cIMT) determined at baseline and after 30 months. IgM-p210MDA autoantibody levels were independently related to several cIMT measures both in the common carotid artery and in the carotid bulb, including measures of cIMT progression, higher levels being associated with lower cIMT or slower cIMT progression. Consistent inverse relationships were also found between plasma levels of IgG-p210nat and baseline composite measures of cIMT. These associations disappeared when adjusting for established and emerging risk factors, and there were no associations with rate of cIMT progression besides in certain secondary stratified analyses. Conclusions: The present study provides further evidence of involvement of autoantibodies against native and MDA-modified apoB-100 peptide 210 in cardiovascular disease in humans and demonstrates that these associations are present already at a subclinical stage of the disease. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Native apoB-100 peptide 210 Malondialdehyde-modified apoB-100 peptide 210 Carotid ultrasound Intima-media thickness Progression

1. Introduction * Corresponding author. Atherosclerosis Research Unit, Center for Molecular Medicine, Building L8:03, Karolinska University Hospital Solna, S-17176 Stockholm, Sweden. Tel.: þ46 8 51773201; fax: þ46 8 311298. E-mail addresses: [email protected], [email protected] (O. McLeod). 0021-9150/$ e see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2013.11.041

Atherosclerosis is a chronic inflammatory disease, in which adaptive immunity is considered to play an important role. In particular, immune responses directed against autoantigens in oxidized low-density lipoprotein (oxLDL) represent a key process in

O. McLeod et al. / Atherosclerosis 232 (2014) 242e248

Abbreviations apoB-100 apolipoprotein B100 BIC Bayesian information criterion Bif-IMT bifurcation of the carotid artery CC-IMT common carotid intima-media thickness cIMT carotid intima-media thickness CVD cardiovascular disease HDL high-density lipoprotein ICA internal carotid artery IgG-p210nat immunoglobulin G directed against native p210 IgM-p210MDA immunoglobulin M directed against malondealdehyde-modified p210 IMPROVEstudy of carotid IMT and IMT progression as predictors of vascular events in a high risk population IMT intima-media thickness MDA malondealdehyde MDS multidimensional scaling oxLDL oxidized low-density lipoprotein SD standard deviation SNP single nucleotide polymorphism

atherogenesis [1e4]. Thus, T-cells cloned from human lesions respond to oxLDL in a major histocompatibility complex class IIdependent manner, atherosclerotic lesions contain high amounts of antibodies recognizing various epitopes of oxLDL and hypercholesterolaemic animals with genetic deficiencies introduced in the adaptive immune system develop less atherosclerosis [5e10]. However, anti-atherogenic effects of immune responses against oxLDL have also been reported from studies where experimental animals were immunized with oxLDL particles [11e13] or specific components of oxLDL such as modified phospholipids or fragments of apolipoprotein B-100 (apoB-100) [14e16]. Among different factors implicated in diverse inflammatory pathogenic mechanisms of atherosclerosis, certain patterns have been described [17]. Overproduction of antibodies against oxLDL has been associated with accelerated atherosclerosis and increased cardiovascular risk in inflammatory conditions such as systemic lupus erythematosus and primary antiphospholipid syndrome, but not in rheumatoid arthritis [17]. Circulating antibodies have been identified in human plasma against a large number of peptide sequences of apoB-100, both native and malondialdehyde (MDA)-modified, some of which (such as peptides 45 and 210) are related to atherosclerosis [6]. These findings prompted immunization studies in apoE knockout mice with some of these native and aldehyde-modified peptides, or the corresponding antibodies, which resulted in up to 70% reduction in atherosclerosis [18]. The corollary of these studies is that activation of adaptive immunity against specific peptides of apoB-100 may be atheroprotective. Accordingly, recent clinical cohort studies of circulating antibodies against native and MDA-modified peptides 45 and 210 have demonstrated associations with both severity of carotid and coronary atherosclerosis as well as with risk of clinical events [19e22]. Specifically, antibodies directed against the native peptide 210 of apoB-100 seem to be protective, and this peptide emerges as a potential target for immunization against atherosclerosis in humans. So far, clinical studies examining the significance of autoantibodies against apoB-100 for human atherosclerosis and its accompanying potentially fatal complications of myocardial infarction (MI) and stroke remain limited in number and restricted to small, diverse and specific populations. In the present study, we investigated the relationships of plasma levels of IgG antibodies

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directed against native peptide 210 (IgG-p210nat) respectively IgM antibodies against MDA-modified peptide 210 (IgM-p210MDA) of apoB-100 to severity and rate of progression of carotid intimamedia thickness (cIMT) in a large, multicentre, European longitudinal cohort study (acronym: IMPROVE) [23]. Participants were free of clinically overt cardiovascular disease (CVD) at enrollment and had at least 3 established cardiovascular risk factors. This setting allowed us to examine the role of apoB autoantibodies in relation to early atherosclerosis and arterial remodeling in asymptomatic individuals at increased risk of CVD distributed across the established north-to-south cardiovascular risk gradient in Europe [24]. 2. Materials and methods 2.1. Study population The biobank and databases of the IMPROVE study formed the basis of the present study. In brief, IMPROVE is a multicentre, longitudinal, observational cohort study of high-risk individuals, involving seven recruiting centers in five European countries: Finland, Sweden, the Netherlands, France and Italy [23]. A total of 3711 participants, men and women, were enrolled between March 2004 and April 2005, 3430 of whom were included in the present report. Reasons for exclusion or drop-out from the present investigation are provided in Fig. 1. Eligibility criteria included age from 55 to 79 years, presence of at least three cardiovascular risk factors and absence of symptoms of CVD as well as of conditions that might limit longevity or cIMT visualization. A total of 92 participants included in the present report did not complete the follow-up, leaving 3338 subjects for autoantibody analyses in relation to change over time in cIMT. Baseline characteristics of the entire IMPROVE cohort and methods for determination of established cardiovascular risk factors

Fig. 1. Flow chart illustrating reasons for exclusion of IMPROVE participants from the present study.

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have been described [23]. Characteristics of participants included in the present report are provided in Supplementary Table 2. Single nucleotide polymorphism (SNP) genotypes generated by genotyping with the Illumina CardioMetabo 200K BeadArray were available in all participants. There were no differences between subjects who completed the study and those lost to follow-up with respect to age, sex, body mass index (BMI), diastolic blood pressure (DBP), established and emerging biochemical risk factors, and common carotid intima-media thickness (CC-IMT) at baseline. However, subjects who completed follow-up had a slightly lower baseline systolic blood pressure (SBP) (data not shown). 2.2. Autoantibody determinations Circulating IgG-p210nat and IgM-p210MDA antibodies (directed against peptide p210 containing amino acids 3136e3155 of apoB100) were quantified by an in-house ELISA in EDTA plasma samples, which had been prepared from blood samples drawn at baseline and stored at 80  C until analysis (Supplementary Table 1). Procedures for generation of the synthetic p210nat and p210MDA, and details of the ELISA protocol have been described [6] and are also provided as Supplementary Material. Antibody levels for a few samples, 6 for IgG-p210nat and 3 for IgM-p210MDA, were below the detection limit on repeated measurements and therefore replaced with a value confidently below the detection limit (1/100 of the lowest measured value) to allow inclusion of the samples. 2.3. Carotid ultrasonography The carotid ultrasound protocol and precision of the ultrasonographic measurements have been reported [23]. The ultrasonographic variables selected for the present study were the mean and maximum IMT of the common carotid arteries (CC-IMTmean and CCIMTmax), excluding the first centimeter closest to the bifurcation, the mean and maximum IMT of the bifurcations (Bif-IMTmean and Bif-IMTmax), the mean and maximum IMT of the internal carotid artery (ICA-IMTmean and ICA-IMTmax), the mean and maximum IMT of the whole carotid tree (IMTmean and IMTmax), and the average of maximum IMT values of the whole carotid tree (IMTmeanemax). The right and left carotid arteries were also evaluated separately in stratified analyses. To evaluate change of cIMT over time (the former designated cIMT progression), ultrasonographic measurements made at 0 and 30 months were compared. cIMT changes at 30 months, expressed in mm/year, were calculated by linear regression of IMT changes versus time. cIMT measurements were performed in a centralized laboratory (Department of Pharmacological Sciences e University of Milan, Italy) using a dedicated software (M’Ath, Metris SRL France) [25], that allows semiautomatic edge detection of the echogenic lines of the intimamedia complex. 2.4. Ethical considerations Ethics committee approvals for the study were obtained by the 7 recruiting centers. All patients gave separate written informed consents for general participation in the study and for genotyping. 2.5. Statistical analysis Variables are reported as number (percentages), mean  standard deviation (SD) or median (interquartile range). Variables with skewed distributions (e.g. ultrasonographic variables and plasma triglycerides) were log-transformed before they were used in linear regression analysis. Smoking habits were identified as described earlier [23]. Cumulative life-time smoking was expressed as a 5-

level categorical variable according to never-smoker status and quartiles of pack-years. Hypertension was defined as a diagnosis of hypertension and/or SBP of at least 140 mmHg and/or DBP of at least 90 mmHg and/or treatment with antihypertensive drugs. Among 3430 subjects included in the current study 900 subjects had type 2 diabetes mellitus. Diabetes was considered to be present in participants who had a diagnosis of diabetes, were on insulin and/or other hypoglycemic drugs, or had a fasting blood glucose 7 mmol/L at the baseline examination. Differences in autoantibody levels across recruitment centers were analyzed by KruskaleWallis non-parametric one-way analysis of variance. The JonckheereeTerpstra test for ordered alternatives was used to assess trends across centers defined by latitude. Associations between IgG-p210nat and IgM-p210MDA levels and established and emerging cardiovascular risk factors were assessed by Spearman rank correlation. Multivariable analysis of factors associated with autoantibody levels was performed by multiple linear regression analysis. Multiple robust regression, which is less sensitive to influence of outliers, was used to assess associations between autoantibody levels and cIMT measurements [26]. Age and sex were always forced into the regression models. To identify variables to be included in the multivariable analysis, a best subset analysis [27] based on the Bayesian information criterion (BIC) was performed. All of the following variables with a p-value <0.20 in a univariable regression were considered for inclusion in the multivariable analysis: latitude, weight, height, waist circumference, BMI, SBP and DBP, hypertension, low-density lipoprotein (LDL) cholesterol, highdensity lipoprotein (HDL) cholesterol, triglycerides, C-reactive protein, diabetes, blood glucose, smoking status, current cigarette consumption, cumulative smoking (pack-years), and physical activity. Partial correlation coefficients were calculated for the relationships between plasma autoantibody concentrations and baseline respectively progression measures of cIMT, using robust regression [28] and two sets of covariates. In the first model, age, sex and latitude were used as covariates (plus the IgM-p210MDA-sex interaction term as appropriate). In the second model, clinical and biochemical risk indicators selected by best subset analysis (see above) were added to the basic set of covariates. Stratification by gender or by side of the carotid circulation was made where appropriate. SNP genotypes generated with the Illumina CardioMetabo 200K BeadArray were used to control for population substructure. Multidimensional scaling (MDS) coordinates, reflecting genetic distances between individuals (population substructure), were calculated based on the genotype data using PLINK1.07 [29]. Outliers identified by visual inspection of plots of the first three MDS dimensions (MDS1-3) were excluded. Of note, MDS1 and latitude was strongly correlated (r ¼ 0.942, p < 0.001). No adjustment for multiple testing was performed since both baseline cIMT measurements and cIMT progression measurements were strongly correlated. A two-sided p-value <0.05 was considered significant for all analyses. STATA 11.1 (StataCorp LP) and SAS 9.2 for Windows were used for the statistical analyses. 3. Results 3.1. Autoantibody levels according to center and in relation to established and emerging risk factors Both IgG-p210nat and IgM-p210MDA levels differed significantly across centers (Fig. 2). The JonckheereeTerpstra (J) test for ordered alternatives showed a significant trend for lower IgG-p210nat levels in the north (J (corrected for ties) ¼ 2.96, p ¼ 0.003) whereas no trend in either direction was found for IgM-p210MDA levels.

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Fig. 2. Plasma levels of IgG antibodies directed against native peptide 210 (IgGp210nat) respectively IgM antibodies against MDA-modified peptide 210 (IgMp210MDA) of apolipoprotein B-100 in subjects grouped according to recruitment center (with latitude indicated in parenthesis for each center). Box plots with darker shade are used for IgG-p210nat, whereas box plots with brighter shade are used for IgMp210MDA. Differences between centers were explored by using the KruskaleWallis test: p < 0.0001 for IgG-p210nat; p < 0.0001 for IgM-p210MDA.

Differences in established and emerging risk factors and medication across centers did not account for the between-center differences in autoantibody levels (data not shown). Between-individual differences in IgG-p210nat (SD 0.34 U) were greater than in IgMp210MDA (SD 0.19 U), whereas mean IgM-p210MDA (0.92 U) levels were higher than mean IgG-p210nat (0.88 U) levels (p < 0.001). IgMp210MDA levels were higher in women (0.93  0.004 U) than in men (0.91  0.005 U, p ¼ 0.013), but not IgG-p210nat (p ¼ 0.999). Subjects with diabetes had significantly lower IgG-p210nat levels (0.85  0.323 vs 0.90  0.346 U, p < 0.001) whereas IgM-p210MDA levels did not differ between those with and without diabetes. Significantly higher levels of IgM-p210MDA (0.92  0.186 vs 0.90  0.186 U, p ¼ 0.001) were noted in hypertensive patients. Patients on lipid-lowering medication had significantly lower titers of IgM-p210MDA (0.91  0.187 U) than those without

Table 1 Relationships of plasma levels of autoantibodies against native and MDA-modified peptide 210 of apolipoprotein B-100 to established and emerging risk factors. Risk factor

IgG-P210nat

IgM-P210MDA

Age Sex (male/female) Latitude BMI Waist circumference Systolic blood pressure Diastolic blood pressure Hypertension (no/yes) LDL-cholesterol HDL-cholesterol Triglycerides CRP Diabetes (no/yes) Blood glucose Smoking status (never/ever) Pack-years (categories) Cigarettes per day High physical activity (no/yes)

0.059y 0.000 0.051** 0.034* 0.020 0.035* 0.022 0.018 0.015 0.017 0.065y 0.024 0.074y 0.055** 0.083y 0.086y 0.075y 0.039*

0.016 0.043* 0.029 0.016 0.014 0.034* 0.013 0.053** 0.011 0.005 0.029 0.051** 0.007 0.003 0.041* 0.033 0.019 0.021

n ¼ 3430. Values are Spearman rank correlation coefficients. MDA, malondialdehyde; IgG-p210nat, IgG antibodies directed against native apoB peptide 210; IgM-p210MDA, IgM antibodies directed against MDA-modified apoB peptide p210; BMI, body mass index; LDL, low-density lipoprotein; HDL, highdensity lipoprotein; CRP, high-sensitivity C-reactive protein. *p < 0.05; **p < 0.01; yp < 0.001.

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(0.93  0.183 U, p ¼ 0.003) whereas IgG-p210nat levels did not differ with respect to lipid-lowering medication. Overall, the relationships of antibody concentrations to established and emerging risk factors were weak (Table 1). Significant inverse correlations were observed between IgG-p210nat and latitude, BMI, systolic blood pressure, triglycerides, presence of manifest diabetes, blood glucose and cigarette smoking, whereas positive correlations were noted for age and physical activity. In addition, IgG-p210nat levels were inversely correlated with the first multidimensional scaling dimension, MDS1 (r ¼ 0.053, p ¼ 0.002). For IgM-p210MDA, positive correlations were found with female sex, systolic blood pressure, presence of hypertension, Creactive protein, and smoking status. Contrary to IgG-p210nat, IgMp210MDA was not associated with MDS1 (r ¼ 0.013, p ¼ 0.455). Gender-specific correlations between autoantibodies and cardiovascular risk factors are provided in Supplementary Table 3. Table 2 reports the multivariable analyses of the relationships of established and emerging risk factors to autoantibody levels. In all, independently related variables accounted for a total of 1.9% and 1.5% of the variations in IgG-p210nat and IgM-p210MDA, respectively. Adjustment for medication showed that IgG-p210nat levels were completely independent of any type of medication (lipid-lowering, glucose-lowering, anti-hypertensive, platelet-inhibiting and antiinflammatory), which hence did not influence the risk factor relationships to IgG-p210nat, whereas lipid-lowering and antihypertensive medication was significantly related to IgM-p210MDA but added very little (<0.5%) to the variation in IgM-p210MDA accounted for by the model (data not shown). Multiple linear regression analyses stratified by gender are provided in Supplementary Table 4. Age and SBP respectively age and hypertension were common determinants in men and women for IgG-p210nat and IgMp210MDA, correspondingly. 3.2. Relationships of autoantibody levels to baseline cIMT Autoantibodies against apoB-100 peptides were further analyzed in relation to baseline cIMT (Table 3). Adjustments for age, sex and latitude (Table 3) revealed statistically significant inverse associations between the IgG-p210nat level and the composite cIMT Table 2 Multiple linear regression analyses of risk indicators related to levels of autoantibodies against native and MDA-modified peptide 210 of apolipoprotein B-100 in human plasma. Risk factor

IgG-p210nat Partial r

P-value

Partial r

P-value

Age Sex Latitude Weight HDL-cholesterol LDL-cholesterol Triglycerides Smoking status (never/ever) CRP Systolic blood pressure Diastolic blood pressure Hypertension (no/yes) High physical activity (no/yes) Diabetes (no/yes) Model multiple r2

0.051 0.010 0.041 e 0.023 e e 0.059 0.031 0.057 0.031 0.037 0.055 0.051 0.019

0.003 0.586 0.018 e 0.191 e e 0.001 0.078 0.001 0.069 0.034 0.009 0.003 <0.001

0.043 0.044 e 0.027 e 0.024 0.033 0.052 0.027 e e 0.069 0.024 e 0.015

0.014 0.011 e 0.126 e 0.158 0.059 0.003 0.114 e e <0.001 0.172 e <0.001

IgM-p210MDA

Variables included in the multiple linear regression analyses were selected by best subset analysis. Age and sex were forced into the best subset analysis. MDA, malondialdehyde; r, partial correlation coefficient; IgG-p210nat, IgG antibodies directed against native apoB peptide 210; IgM-p210MDA, IgM antibodies directed against MDA-modified apoB peptide p210; HDL, high-density lipoprotein; LDL, lowdensity lipoprotein; CRP, high-sensitivity C-reactive protein.

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Table 3 Relationships of levels of autoantibodies against native and MDA-modified peptide p210 of apolipoprotein B-100 to baseline dimension and change over time of carotid artery intima-media thickness. Regression model 1

Regression model 2

Baseline dimension

b  2SE IgG-p210nat

IgM-p210MDA

IMTmean IMTmax IMTmeanemax CC-IMTmean CC-IMTmax Bif-IMTmean Bif-IMTmax ICA-IMTmean ICA-IMTmax IMTmean IMTmax IMTmeanemax CC-IMTmean CC-IMTmax Bif-IMTmean Bif-IMTmax ICA-IMTmean ICA-IMTmax

0.0085 0.0229 0.0112 0.0022 0.0057 0.0129 0.0191 0.0056 0.0120 0.0145 0.0236 0.0110 0.0021 0.0108 0.0358 0.0424 0.0220 0.0178

                 

0.0084 0.0164 0.0090 0.0064 0.0092 0.0138 0.0168 0.0136 0.0188 0.0214 0.0420 0.0226 0.0164 0.0234 0.0352 0.0430 0.0342 0.0476

Change over time p

b  2SE

0.043 0.005 0.012 0.496 0.218 0.064 0.024 0.405 0.199 0.175 0.261 0.331 0.792 0.356 0.042 0.048 0.200 0.454

0.0026 0.0018 0.0024 0.0021 0.0038 0.0040 0.0086 0.0039 0.0045 0.0059 0.0031 0.0146 0.0070 0.0141 0.0031 0.0055 0.0010 0.0306

                 

0.0030 0.0126 0.0048 0.0024 0.0056 0.0064 0.0122 0.0044 0.0096 0.0080 0.0328 0.0126 0.0062 0.0148 0.0164 0.0316 0.0114 0.0250

Baseline dimension p

b  2SE

0.092 0.775 0.314 0.080 0.177 0.203 0.159 0.078 0.347 0.141 0.849 0.020 0.022 0.055 0.708 0.728 0.868 0.014

0.0038 0.0159 0.0064 0.0011 0.0017 0.0087 0.0143 0.0004 0.0025 0.0271 0.0318 0.0178 0.0025 0.0050 0.0423 0.0459 0.0273 0.0232

                 

0.0082 0.0164 0.0088 0.0064 0.0092 0.0138 0.0170 0.0136 0.0188 0.0204 0.0416 0.0224 0.0162 0.0234 0.0352 0.0410 0.0348 0.0476

Change over time p

b  2SE

0.352 0.053 0.146 0.728 0.708 0.210 0.092 0.948 0.793 0.008 0.127 0.113 0.758 0.671 0.016 0.033 0.116 0.330

0.0018 0.0025 0.0021 0.0017 0.0028 0.0035 0.0080 0.0036 0.0036 0.0052 0.0043 0.0132 0.0046 0.0097 0.0034 0.0057 0.0015 0.0343

p                  

0.0032 0.0128 0.0048 0.0024 0.0058 0.0064 0.0122 0.0044 0.0098 0.0082 0.0168 0.0063 0.0336 0.0150 0.0168 0.0316 0.0114 0.0252

0.240 0.696 0.399 0.154 0.327 0.275 0.189 0.107 0.463 0.199 0.798 0.037 0.157 0.195 0.691 0.718 0.796 0.007

Model 1: robust regression after adjustment for age, sex and latitude. Model 2: robust regression after adjustment for age, sex and latitude, and clinical and biochemical variables selected by best subset analysis. For IgM-p210MDA, adjustment was also made for IgM-p210MDA-sex interaction. b, regression coefficient; SE, standard error; IgG-p210nat, IgG antibodies directed against native apoB peptide 210; IgM-p210MDA, IgM antibodies directed against MDA-modified apoB peptide p210; IMTmean, mean IMT of the whole carotid tree; IMTmax, maximum IMT of the whole carotid tree; IMTmeanemax, average of maximum IMT values of the whole carotid tree; CC, common carotid artery; Bif, bifurcation (bulb); ICA, internal carotid artery. IMT measurements were log-transformed to make them more symmetrically distributed.

measures of IMTmean, IMTmax and IMTmeanemax. In addition, there was an inverse association between IgG-p210nat and Bif-IMTmax. IgM-p210MDA levels were only significantly inversely related to BifIMTmean and Bif-IMTmax. After inclusion of additional covariates (Table 3), the IgG-p210nat relationships to composite cIMT measures and Bif-IMTmax weakened and became statistically insignificant, whereas the IgM-p210MDA relationships to Bif-IMTmean and Bif-IMTmax were largely unaltered. Also, IgM-p210MDA now appeared as being significantly related to IMTmean. After introducing medication into the models, the significant relationship remained between IgM-p210MDA and Bif-IMTmean (Supplementary Table 5). Stratified analysis by gender is provided in Supplementary Table 6. Separate examinations of the right and left carotid arteries (Supplementary Table 7) indicated that effects appeared to be stronger on the right side for the composite and segment-specific measures that were associated with IgG-p210nat antibody levels in the whole group. However, specific inverse relationships were noted between IgM-p210MDA levels and the left ICA-IMTmean and ICA-IMTmax measures. 3.3. Relationships of autoantibody levels to cIMT progression Autoantibodies against apoB-100 peptides were also analyzed in relation to change in cIMT over time (Table 3). Statistically significant associations were sparse. Inverse relationships that were independent of cardiovascular risk factors were noted for IgMp210MDA in relation to the measures of IMTmeanemax and ICAIMTmax progression. These relationships remained statistically significant after additional adjustment for any type of medication (Supplementary Table 5). Stratification by gender showed significant inverse associations of IgM-p210MDA, which were independent of cardiovascular risk factors, with changes in ICA-IMTmax in men and IMTmeanemax in women (Supplementary Table 6). Further stratified analyses according to presence of at least one plaque (IMTmax > 1.5 mm) revealed significant inverse relationships of

IgG-p210nat levels to progression of ICA-IMTmean in subjects with plaques and of IgM-p210MDA levels to progression of IMTmeanemax in subject without plaques, after adjustment for age, gender and latitude (data not shown). Separate analyses of the right and left carotid arteries showed significant inverse associations between IgG-p210nat levels and progression of left IMTmean, IMTmeanemax and Bif-IMTmean, and a significant inverse association between IgMp210MDA levels and progression of right ICA-IMTmax. 4. Discussion This study is by far the largest clinical investigation of the role of autoantibodies directed against apoB-100 in CVD. Focus was placed on retrospectively assessing associations of baseline levels of specific autoantibodies with subclinical atherosclerosis as determined by ultrasound determination of baseline cIMT and cIMT progression. The choice of middle-aged to elderly high-risk individuals was considered optimal for exploring autoantibody associations with disease progression. By enrolling equal numbers of male and female participants from different parts of Europe, the study allows for data to be interpreted in a wider context than in previous studies. Some findings should be considered as particularly important. First, differences in both IgG-p210nat and IgM-p210MDA levels were found to exist between centers, which were not accounted for by differences in risk factor profiles or treatment, nor were they due to center-specific differences in laboratory performance since all assays were run by the same analyst in a central laboratory (at the Centre for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden). These observations suggest that IgG-p210nat and IgMp210MDA levels are largely regulated by mechanisms that are independent of established risk factors. One candidate mechanism for between-center difference in IgG-p210nat is genetic heterogeneity, as indicated by the association with the first MDS dimension. Interestingly, levels of the putatively protective IgG-p210nat were lower in the north. However, we could not demonstrate conclusive

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evidence that this contributes to the previously demonstrated north-to-south gradient in cIMT [23] and risk of CVD [24]. Secondly, consistent, albeit weak inverse associations were found between IgG-p210nat and baseline composite measures of cIMT in the entire cohort. These associations disappeared when adjusting for established and emerging risk factors, and there were no associations with rate of cIMT progression. This would speak against a direct causal protective role of IgG-p210nat in early atherosclerosis and either suggests that these antibodies are innocent bystanders or that they exert their effect through conventional risk factor mechanisms. In the former scenario, some components of the metabolic syndrome, which by themselves promote atherosclerosis, would potentially depress the adaptive immune system leading to lower levels of antibodies against native p210. Conversely, a lower risk factor burden would be associated with less atherosclerosis and a more active adaptive immune system. In the latter scenario, higher levels of IgG-p210nat, reflecting a stronger activation of adaptive immunity against certain epitopes, would counteract some facets of the metabolic syndrome whereas lower levels would lead to a poorer risk factor control. However, in secondary analyses with stratification for the right and left carotid circulation, significant inverse associations were observed between IgG-p210nat levels and progression of left IMTmean, IMTmeanemax and Bif-IMTmean. Of note, when interpreting these results, it should be remembered that the baseline measures of cIMT reflect processes that have occurred in the intima-media layers of the artery wall over decades prior to the autoantibody determinations whereas the measures of cIMT progression relate to changes taking place within a couple of years after plasma was drawn for autoantibody determination. In contrast, the cIMT results for IgM-p210MDA in the entire cohort showed independent inverse associations to bifurcation IMT measured at baseline, but not to the corresponding progression measures, whereas it was independently related to progression of IMTmeanemax and ICA-IMTmax, a composite measure of lesion severity respectively a measure of maximum lesion size in the right and left internal carotid artery segments. Thus, contrary to IgGp210nat, IgM-p210MDA autoantibody levels were independently related to some cIMT measures, including measures of cIMT progression, higher levels being associated with lower cIMT or slower cIMT progression. In addition, the pattern of independent associations for IgM-p210MDA was further reinforced in secondary analyses, particularly with stratification for gender. Studies of autoantibodies against apoB-100 performed so far in human subjects have examined diverse antibody species, patient groups and clinical phenotypes using different ultrasound protocols, rendering comparisons across studies complicated. Of two prior cIMT studies [19,20], one focused on IgG- and IgM-p210MDA relationships to baseline cIMT and cIMT progression in symptomless subjects who, however, had a demonstrable carotid plaque [20] whereas the other investigated relationships between the IgG- and IgM-p45MDA antibody species, cIMT and incident myocardial infarction (MI) or sudden cardiac death [19]. Comparisons between the two studies measuring IgM-p210MDA reveal similar findings of an inverse relationship with respect to baseline measurements of the bifurcation (Bif-IMT) but discrepant results for the common carotid (CC-IMT). High levels of IgG-p210nat autoantibodies have previously been found to be associated with less coronary artery calcification in persons with type-2 diabetes, but no clinical signs of coronary artery disease [22], as well as with less severe coronary lesions in subjects who had suffered MI before the age of 60 [21], suggesting a potential protective role of these autoantibodies in atherosclerosis. In the present study, inverse associations were also seen between IgGp210nat levels and severity of subclinical atherosclerosis, as reflected

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by the baseline composite measures of cIMT. However, these associations were not independent of established and emerging risk factors. One of the possible mechanisms underlying the proatherogenic effects of IgG-p210nat could involve apolipoprotein H and, thus, facilitated binding of antiphospholipid antibodies to lipids of the endothelial cell membrane, eventually resulting in apoptosis of endothelial cells [17]. Based on the findings listed above, it could be speculated that IgG-p210nat autoantibodies may have a more important protective role in advanced as opposed to early subclinical atherosclerosis. Alternatively, autoantibodies directed against apoB100 may exert different effects in the coronary as opposed to the carotid circulation. It should also be emphasized in this context that neither previous clinical studies nor the present one have demonstrated associations between high IgG-p210nat and slower disease progression or lowered risk of future clinical events. This fact can be interpreted to further question the causeeeffect relationship between IgG-p210nat antibody levels and various cardiovascular phenotypes. The level of IgG-210nat was found to be lower in subjects with type 2 diabetes. The reason for this remains to be clarified. However, it has previously been shown that diabetic subjects with a poor metabolic control have lower levels of IgG-210nat levels [22], which may have contributed to the findings in the present study. It is also interesting to note that IgG-210nat levels have been reported to be lower in type 2 diabetes patients with a high degree of coronary calcification (>400 Agatston units) than in those with low to moderate calcification [22]. The strengths of the present study include the sample size and the uniquely detailed ultrasound protocol. Whereas the European setting allows for data to be interpreted in a wider context than in previous studies and thus would add to the strengths of the study, it also potentially introduced heterogeneity in terms of population substructure. Of note, the first MDS dimension, a measure of genetic distance between individuals, was significantly correlated with IgGp210nat autoantibody levels. Thus, studies of the genetic regulation of IgG-p210nat are warranted. The inter-individual variation in IgGp210nat levels was greater than that for IgM-p210MDA. This contributed to a higher inter-assay variation of the IgG-p210nat ELISA. The focused autoantibody panel, which did not include the p45 epitope, although based on findings made by our group in relation to MI, constitutes a limitation. An additional limitation of the present study is that antibody levels were only determined at baseline; hence, we lack data regarding changes in antibody levels during follow-up. However, previous studies analyzing changes in autoantibodies against p210 over a 12-month period suggest that these remain relatively stable [20]. In conclusion, the present study provides further evidence of an involvement of autoantibodies against the native and MDA-modified peptide 210 in atherosclerosis. Furthermore, the results obtained here provide stronger indications of a direct causal protective role for IgM-p210MDA than for IgG-p210nat. However, whereas mainly IgM-p210MDA autoantibody levels were inversely and independently related to some cIMT measures, including measures of cIMT progression, higher IgG-p210nat levels were consistently related to lower values of baseline global measures of cIMT, but these associations disappeared when controlled for the effects of cardiovascular risk factors. Further large-scale prospective cohort studies of autoantibodies directed towards apoB-100 are thus warranted that focus on both the p45 and the p210 epitopes. Such studies should combine a comprehensive analysis of clinical and biochemical risk factors and detailed cardiovascular imaging with adequate numbers of incident cardiovascular events and sequential blood sampling. Due to the existing evidence of accelerated progression of atherosclerosis in some rheumatic diseases characterized by inflammation [17], it is also worth examining to what extent antibodies against native and MDAmodified peptide 210 may be involved in inflammation-induced

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atherosclerosis. Until such studies have been completed, the mechanistic role and biomarker status of antibodies directed against the native and MDA-modified peptide epitopes on apoB-100 remain unresolved. However, these restrictions notwithstanding, the present study provides further support for exploring apoB-100 peptide 210 as a target for immunization against atherosclerosis in humans. 5. Sources of funding The IMPROVE study was supported by the European Commission (Contract number: QLG1-CT-2002-00896), the Swedish HeartLung Foundation, the Swedish Research Council (projects 8691, 0593, 0125 and 8311), the Knut and Alice Wallenberg Foundation, the Foundation for Strategic Research, the Stockholm County Council (project 592229), the Strategic Cardiovascular and Diabetes Programmes of Karolinska Institutet and Stockholm County Council, the European Union Framework Programme 7 (FP7/2007e2013) for the Innovative Medicine Initiative under grant agreement n IMI/115006 (the SUMMIT consortium), the Magnus Bergvall Foundation, the Academy of Finland (Grant #110413) the British Heart Foundation (RG2008/08, RG2008/014) and the Italian Ministry of Health (Ricerca Corrente). Acknowledgments None. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2013.11.041. References [1] Nilsson J, Hansson GK. Autoimmunity in atherosclerosis: a protective response losing control? J Intern Med 2008;263:464e78. [2] Hansson GK, Hermansson A. The immune system in atherosclerosis. Nat Immunol 2011;12:204e12. [3] Ketelhuth DF, Tonini GC, Carvalho MD, Ramos RF, Boschcov P, Gidlund M. Autoantibody response to chromatographic fractions from oxidized LDL in unstable angina patients and healthy controls. Scand J Immunol 2008;68: 456e62. [4] Binder CJ. Natural IgM antibodies against oxidation-specific epitopes. J Clin Immunol 2010;30(Suppl. 1):S56e60. [5] Stemme S, Faber B, Holm J, Wiklund O, Witztum JL, Hansson GK. T lymphocytes from human atherosclerotic plaques recognize oxidized low density lipoprotein. Proc Natl Acad Sci U S A 1995;92:3893e7. [6] Fredrikson GN, Hedblad B, Berglund G, et al. Identification of immune responses against aldehyde-modified peptide sequences in apoB associated with cardiovascular disease. Arterioscler Thromb Vasc Biol 2003;23:872e8. [7] Nilsson J, Hansson GK, Shah PK. Immunomodulation of atherosclerosis: implications for vaccine development. Arterioscler Thromb Vasc Biol 2005;25: 18e28. [8] Hansson GK, Libby P. The immune response in atherosclerosis: a doubleedged sword. Nat Rev Immunol 2006;6:508e19.

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