TRANSLATIONAL BIOMEDICAL RESEARCH

Obesity Modulates the Immune Response to Oxidized LDLin Hypertensive Patients

Henrique Andrade R. Fonseca • Francisco A. Fonseca • Andrea M. Monteiro •

Henrique T. Bianco • Paulo Boschcov • Sergio A. Brandao • Luiz Juliano •

Magnus Gidlund • Maria C. Izar

� Springer Science+Business Media New York 2013

Abstract Obesity and hypertension have been recognized

as inflammatory diseases capable of activating the immune

system, thus contributing to an increased cardiovascular

risk. However, the link between adaptive immunity,

obesity, and hypertension is poorly understood. We

investigated the relationship of the body mass index (BMI)

on the inflammatory, vascular, and immune responses

in patients with hypertension naıve of anti-hypertensive

treatment. Hypertensive patients (N = 88) were divided

into three groups: normal weight (NW), overweight (OW),

and obese (OB) subjects. Anti-oxidized LDL autoantibod-

ies (anti-oxLDL Abs), anti-ApoB-D peptide (anti-ApoB-D)

Abs, interleukin (IL)-8 and IL-10, flow-mediated dilation

(FMD) of the brachial artery, and 24-h ambulatory blood

pressure monitoring (ABPM) were assessed. OB patients

presented lower levels of anti-oxLDL Abs and IL-10,

higher levels of IL-8, and impaired FMD, when compared

to NW and OW (P \ 0.05), without differences between

groups regarding anti-ApoB-D Abs. After adjusting for

age, systolic and diastolic blood pressure, anti-oxLDL Abs

were inversely correlated with BMI and waist circumfer-

ence (r = -0.24, P = 0.02 and r = -0.25, P = 0.02,

respectively), whereas ApoB-D correlated with 24-h

ABPM (r = 0.22, P = 0.05 for systolic, and r = 0.29,

P = 0.01 for diastolic blood pressure). Regression analyses

showed inverse associations of anti-oxLDL Abs with BMI

(b = -0.05, P = 0.01) and waist circumference (b =

-0.01, P = 0.02); anti-ApoB-D Abs were associated with

systolic and diastolic 24-h ABPM (b = 0.96, P = 0.04;

b = 1.02, P = 0.005, for systolic and diastolic 24-h

ABPM, respectively). Among hypertensive patients, obesity

modulates the immune and inflammatory milieu, determin-

ing an unfavorable balance of cytokines and reduction in

titers of anti-oxLDL Abs. Twenty-four hour ABPM is

associated with titers of anti-ApoB-D Abs.

Keywords Autoantibodies � Oxidized LDL �Inflammation � Body mass index � Hypertension

Introduction

Annually, raised blood pressure is responsible for 7.5

million deaths, while overweight and obesity accounts for

another 2.8 million deaths, worldwide [1]. Improvement in

the quality of care with better risk factors control has been

accompanied by reduction in coronary heart disease mor-

tality. However, the increase in body mass index attenuated

Electronic supplementary material The online version of thisarticle (doi:10.1007/s12013-013-9585-9) contains supplementarymaterial, which is available to authorized users.

H. A. R. Fonseca � F. A. Fonseca � H. T. Bianco �S. A. Brandao � M. C. Izar (&)

Cardiology Division, Department of Medicine, Federal

University of Sao Paulo, Rua Pedro de Toledo, 276,

Sao Paulo, SP 04039030, Brazil

e-mail: mcoizar@terra.com.br; mcoizar@cardiol.br

F. A. Fonseca � A. M. Monteiro � M. C. Izar

National Institute of Complex Fluids, Sao Paulo, SP, Brazil

A. M. Monteiro � M. Gidlund

Department of Immunology, Institute of Biomedical Science,

University of Sao Paulo, Sao Paulo, SP, Brazil

P. Boschcov � L. Juliano

Department of Biophysics, Federal University of Sao Paulo,

Sao Paulo, SP, Brazil

M. Gidlund

National Institute of Nanomaterials for Integrated Markers,

Recife, PE, Brazil

123

Cell Biochem Biophys

DOI 10.1007/s12013-013-9585-9

the full benefit of strategies aimed at preventing cardiovas-

cular disease [2].

Obesity, initially considered as a lipid storage disease, is

now recognized as a chronic inflammatory disorder, with

activation of innate and adaptive immunity, leading to

atherosclerosis formation [3, 4].

On the other hand, hypertension another important car-

diovascular risk factor, is also considered an inflammatory

disease associated with atherosclerosis, eliciting immune

responses and production of reactive oxygen species (ROS)

via enzymatic cellular complexes with NADPH oxidase [5, 6].

Retention of modified LDL in the artery wall, by

interactions with structural proteins of the sub-endothelial

space are a crucial event in atherosclerosis initiation and

progression [7]. LDL particle modification can occur by

oxidation, lipolysis, proteolysis, and also by ROS produc-

tion [8, 9], generating epitopes that are recognized by the

immune system by antibodies, forming immune complexes

with the LDL particle, with an important role in athero-

sclerosis [10]. After oxidation and proteolysis of the LDL

particle, peptide fragments derived from Apolipoprotein

B-100 (ApoB-100) [11, 12], can also trigger activation of

adaptive and innate immune responses. Further, there is

evidence for ApoB-derived peptides activating T cell

responses [13]. In spite of the current understanding of the

biological actions of modified LDL products in the ath-

erosclerosis process [14], the clinical significance of the

response of autoantibodies to different ApoB-100 frag-

ments is less reported.

Our group has recently reported that another peptide, a

danger-associated peptide from Apolipoprotein B-100

(ApoBDS-1), which is a non-oxidized peptide, not asso-

ciated with lipoperoxidation products, triggers innate

pro-atherogenic responses [11], and can be recognized by

circulating IgG autoantibodies.

The adaptive immune response to ApoB-D was showed

to be associated with markers of atherosclerotic process in

humans and in animal models [15, 16].

Therefore, this study aimed to examine the impact of

raised body mass index (BMI) in the inflammatory, vas-

cular, and immune environment in hypertensive subjects.

Methods and Procedures

Study Subjects

We prospectively included 88 subjects, aging 30–70 years,

of both genders with stage 1 hypertension, and naıve of

anti-hypertensive treatment at study initiation [17], with no

other risk factors, in primary prevention of CHD.

Hypertension was defined by office and/or 24-h ambu-

latory blood pressure monitoring (ABPM). For office blood

pressure (BP), the average of three measurements of sitting

BP C 140 mm Hg for systolic and/or C90 mm Hg for

diastolic BP, obtained with appropriate cuffs, after a 5-min

resting period and repeated at 5-min intervals was con-

sidered; for ABPM mean 24-h systolic blood pressure

(SBP) and/or diastolic blood pressure (DBP) above normal

range was considered as entry criteria [17]. Participants

were divided into three groups according to BMI: normal

weight (NW, BMI \ 25 kg/m2), overweight (OW, BMI C

25 \ 30 kg/m2), and obese (OB, BMI C 30 kg/m2). Waist

circumference was obtained following the recommenda-

tions of the National Cholesterol Education Program/Adult

Treatment Panel III [18]. These parameters were evaluated

in large prospective population studies that have shown

effects of obesity, measured by BMI and abdominal

obesity, on cardiovascular event risk [19, 20], and were

also associated with elevated blood pressure levels.

The study was conducted in accordance with the ethical

standards of our institutional committee on human exper-

imentation and the protocol was approved by the local

ethics committee of our university (#2137/11). Informed

consent was obtained from all participants prior to protocol

initiation.

Laboratory Parameters

Lipids, Biochemistry, and Apolipoproteins

Serum total cholesterol, high-density lipoprotein choles-

terol (HDL-C), and triglycerides were determined enzy-

matically (Opera Bayer, Leverkusen, Germany) with

low-density lipoprotein cholesterol (LDL-C) estimated by

the Friedewald equation when triglycerides were\400 mg/

dl [21]. Creatinine was measured automatically (Opera

Bayer, Leverkusen, Germany) and the glomerular filtration

rate (GFR) was estimated by the Cockcroft–Gault equation

[22]. Glucose was assessed by enzymatic method, and

glycated hemoglobin (HbA1c) was measured using high-

performance liquid chromatography. Concentrations of

apolipoproteins were determined by nephelometry (Array

360 Beckmann, Fullerton, CA).

Preparation of Oxidized LDL and Anti-oxLDL Abs

Blood was drawn from a single fasting normolipidemic

blood donor volunteer, and ethylenediamine tetraacetic

acid (EDTA)-plasma was obtained after centrifugation at

1000xg, 4 �C, for 15 min. Benzamidine (2 mmol/l),

gentamicin (0.5 %), chloramphenicol (0.25 %), phenyl-

methyl-sulfonylfluoride (0.5 mmol/l), and aprotinin (0.1 U/

ml) were added to the plasma. The LDL (1.006 \ d \1.063 g/ml) was isolated by sequential ultracentrifugation

Cell Biochem Biophys

123

at 100,000xg, at 4 �C, using a 50Ti rotor (L-8 ultracentri-

fuge, Beckman Instruments, Palo Alto, CA), and thereafter

dialyzed at 4 �C against PBS, pH 7.4, with 0.01 % EDTA.

The LDL was sterilized via filtration through a 0.22-mm

filter (Milipore, Germany). To obtain oxidized LDL, LDL

was dialyzed overnight against PBS without EDTA, fol-

lowed by incubation with CuSO4, 2.5 mol/l/mg of LDL

protein, for 18 h at 37 �C. The oxidation was stopped by

the addition of 1 mmol/l EDTA [23]. This procedure is

standardized in our laboratory and results in a completely

oxidized LDL, as defined by the plateau phase using

TBARS assay, in addition to further increase in negative

charges [24].

To determine the Abs to copper-oxidized LDL, we used

an enzyme-linked immunosorbent assay (ELISA), devel-

oped in our laboratory, as previously described [15, 25]. To

increase the precision in quantifying anti-oxLDL, IgG

(10 mg/ml; purified human IgG, Pierce Protein Research

Products; Thermo Scientific, Rockford, IL), and a buffer

blank (PBS) were used as controls to compensate intra-

plate variation. Inter-plate imprecision in the ELISA was

minimized by processing all the samples in the same

period, at the end of the clinical protocol. To minimize

false positive results due to cross-reactivity with antigen

naıve epitopes, Abs titers were expressed as the reactivity

index (RI), calculated as RI = (ODsample - ODsample

blank)/(ODIgG - ODIgG blank), where IgG was used as a

control. Samples were run in triplicate and the variation

within the triplicates did not exceed 5 % of the mean.

Anti-ApoB-D Autoantibodies

Quantification of anti-ApoB-D Abs was assessed in total

plasma by ELISA [15], using a method modified from

Soares et al. [16]. Briefly, ApoB-D is a peptide with 22

amino acids in the hydrophobic region of ApoB, not

accessible to trypsin digestion. For analysis, a peroxidase-

conjugated secondary goat anti-human IgG antibody

(50 lL; 1:400) was used to detect anti-ApoB-D Abs. The

optical density was measured at 450 nm and Abs titers

were expressed as the reactivity index (RI), calculated as

RI = (ODsample - ODsample blank)/(ODIgG - ODIgG blank),

where IgG was used as control. Samples were run in trip-

licate and the variation within the triplicates did not exceed

5 % of the mean.

Interleukins, Markers of Plasma Lipoperoxidation

(TBARS) and High-sensitivity C-reactive Protein

(hsCRP)

The plasma was stored at -70 �C until analyzed. Interleukin

(IL)-8, and IL-10 concentrations were tested using commer-

cially available quantitative enzyme-linked immunosorbent

assay (ELISA) kits and according to the information provided

by the manufacturer (R&D Systems, Minneapolis, MN).

To quantify the degree of oxidation by the thiobarbituric

acid reactive substances (TBARS) assay, we incubated the

sample (50 ll) with the TBARS reagent (200 ml,1 %

thiobarbituric acid, 562.5 mM HCl, and 15 % trichloro-

acetic acid). The mixture was boiled for 15 min and cen-

trifuged (103 9 g for 10 min), and the absorbance of the

supernatant was measured at k = 540 nm using a spec-

trophotometer (GENiosTecan, Tecan Trading AG, Swit-

zerland) and compared to a malondialdehyde (MDA)

standard.

High-sensitivity C-reactive protein was measured by

nephelometry (R100 Analyser, Behringer, Germany).

Electrocardiogram

A 12-lead electrocardiogram was performed at entry. Patients

with left ventricular hypertrophy [26] were excluded.

Ambulatory Blood Pressure Monitoring (ABPM)

Twenty-four hour (24 h) ABPM was recorded (Spacelabs

90207, Seattle, WA), as previously described [17].

Vascular Reactivity Measurements

The evaluation of vascular reactivity was performed in the

morning after an overnight fast, by an experienced

sonographer in accordance with the Guidelines for the

Ultrasound Assessment of Endothelial-dependent Flow-

mediated Dilation (FMD) of the brachial artery [27]. All

studies were performed in temperature-controlled rooms

(24–26 �C). An ultrasound system (Sonos 5500; Hewlett-

Packard-Phillips, Palo Alto, CA), equipped with vascular

software for two-dimensional imaging, color and spectral

doppler ultrasound modes, internal electrocardiogram

monitor, and linear-array transducer (with a frequency

range from 7.5 to 12.0 MHz), was used. Image acquisition,

endothelial-dependent FMD, and endothelium-independent

dilation (NID) by isosorbidedinitrate (5 mg; sublingual)

were assessed. The percent change in vessel diameter from

the baseline value was calculated to determine FMD or

NID. The intra- and inter-sonographer variability values

were \1 and 2 %, respectively.

Statistical Analyses

Categorical variables are expressed as n (%); and compared

by Pearson’s Chi square test. Numerical variables are

medians and interquartile ranges (IQR). Distribution of

normality was assessed by Kolmogorov–Smirnov test.

Glucose, glomerular filtration rate, 24-h ABPM did not

Cell Biochem Biophys

123

present Gaussian distribution and were log10-transformed

prior to analyses. Numerical variables were compared

between BMI strata by using analyses of variance (ANOVA)

with Tukey’s post-test. Interaction between Abs titers and

other variables was tested for partial correlation analyses

(adjusted for SBP, DBP, and age). Variables identified to

have significant interaction were tested with multiple linear

regression analyses, with anti-oxLDL and anti-ApoB-D Abs

as dependent variables. All tests were performed using the

SPSS 17.0 software package (Statistical Package for Social

Science, SPSS Inc., Chicago, IL, USA). A two-sided

P value \ 0.05 was considered statistically significant.

Results

Demographic Characteristics

Eighty-eight subjects with hypertension were recruited; of

them, 19 presented normal body weight (NW), 35 were

overweight (OW), and 34 were obese (OB). Gender dis-

tribution along BMI categories was similar (P = 0.98), as

well as the values for systolic blood pressure. However,

diastolic blood pressure was higher in obese compared with

overweight patients (P = 0.01) (Table 1). As expected,

waist circumference differed along BMI groups

(OB [ OW, OB [ NW; OW [ NW, P \ 0.05). Obese

subjects had lower HDL-C values compared to NW

(P = 0.02), whereas LDL-C values in OW subjects were

lower than in those with NW (P = 0.04).

Increase in BMI was accompanied by elevation in

fasting glucose (NW vs. OW, P = 0.04; NW vs. OB,

P = 0.01), without differences on HbA1c among groups.

GFR showed higher values for OB than OW and NW

(P \ 0.05).

Anti-oxLDL and Anti-ApoB-D Autoantibodies Titers

Obese patients had lower median titers (IQR) of anti-

oxLDL Abs when compared with NW [1.36(1.15–1.73)

vs. 1.71(1.51–2.88), P = 0.01], and to OW patients [1.79

(1.29–2.30); P = 0.01] (Fig. 1a).

On the other hand, we found that IL-8 distribution increased

with increasing BMI, with OB patients showing the highest

median concentration (IQR) of this inflammatory biomarker

[50.3(32.1–115.9)] compared to NW [36.7(15.1–57.7),

P = 0.02] and OW subjects [39.8(22.3–53.8), P = 0.04]

(Fig. 1b). Conversely, the IL-10 median (IQR) levels were

lower in OB patients [4.60(1.57–5.90), when compared with

NW [6.43(3.11–6.95); P = 0.01], and OW individuals

[5.90(3.14–6.87); P = 0.04] (Fig. 1c).

Titers of anti-ApoB-D Abs were similar among groups,

according to BMI, categorized as obesity, overweight, and

normal weight. Median titers (IQR) were 0.41 (0.25–0.65)

in OB, 0.43 (0.32–0.65) in OW, and 0.45 (0.21–0.57) in

NW, suggesting that in the present study, body mass did

not influence or modulate immune responses to ApoB-D

peptide in hypertensive patients (Fig. 1d).

Vascular Function and Twenty-four Hour ABPM

Vascular function parameters as well as 24-h ABPM are

presented in Table 2. OB patients had lower FMD when

compared with NW (P = 0.04) and OW counterparts

(P \ 0.001), without differences in NID. Resting brachial

artery diameter (RBAD) was also similar among groups.

Twenty-four hour ABPM revealed lower DBP in OB

patients compared with NW (P = 0.02), without differ-

ences in 24-h SBP.

Correlations of Anti-oxLDL and Anti-ApoB-D Abs

Partial correlations between titers of anti-oxLDL Abs (as

dependent variable) and clinical and laboratory variables

(as independent variables) are shown in Supplementary

Table 1. Titers of anti-oxLDL Abs had inverse correlation

with BMI (r = -0.24, P = 0.02) and with waist circum-

ference (r = -0.25, P = 0.02) independently of blood

pressure and age. Positive correlation of anti-oxLDL Abs

was observed for triglycerides (r = 0.30, P = 0.006), and

inverse correlation was seen for ApoB/ApoA1 (r = -0.26,

P = 0.01). We also observed inverse and independent

associations between FMD (%) and waist circumference

(r = -0.34, P = 0.001) or with BMI (r = -0.27,

P = 0.01); positive associations between glucose and waist

circumference (r = 0.31, P = 0.004) or with BMI

(r = 0.27, P = 0.01) (Fig. 2).

Partial correlations showed that titers of anti-ApoB-D

Abs were associated with mean 24-h systolic and diastolic

blood pressure (r = 0.22; P = 0.05, and r = 0.29;

P = 0.01, respectively), when adjusted for other covariates

(Fig. 3). In addition, titers of anti-ApoB-D Abs were

associated with ApoB/ApoA1 ratio (r = 0.26; P = 0.01)

and triglycerides (r = 0.21; P = 0.05) (Supplementary

Table 1).

Regression Analyses

Multiple linear regression analyses revealed that anti-

oxLDL titers were associated with markers of obesity

(BMI: b = -0.053, P = 0.01; waist circumference:

b = 0.018, P = 0.02). An inverse association between the

anti-oxLDL Abs titers and glomerular filtration rate

(b = -1.377, P = 0.04) was observed (Table 3).

Cell Biochem Biophys

123

Titers of anti-ApoB-D Abs showed a positive associa-

tion with mean 24-h systolic (b = 0.96, P = 0.03) and

diastolic (b = 1.02, P = 0.005) blood pressure (Table 3).

Discussion

Our study has shown that obesity, in hypertensive patients,

is a condition associated with decreased concentrations of

both IL-10 and titers of anti-oxLDL Abs, as well as with

increased concentrations of the inflammatory cytokine

IL-8, when compared with OW or NW. In addition, we

observed that reduction of titers of anti-oxLDL Abs was

inversely correlated with BMI and waist circumference,

independently of blood pressure values. FMD was reduced

among hypertensive subjects with obesity, when compared

with other groups.

Otherwise, we observed that titers of anti-ApoB-D Abs

were associated with mean 24-h systolic and diastolic

blood pressure (BP), thus suggesting a possible modulation

of BP in the humoral response to ApoB-D peptide, espe-

cially when high BP values are present. These findings are

in accordance with previous results published by our group,

where individuals with hypertension, under drug treatment

and not at BP goals, had higher titers of anti-ApoB-D Abs

[15].

We have previously showed that treatment of hyper-

tension improved endothelial function and augmented titers

of anti-oxLDL Abs, regardless of the type of drugs used

[25]. In obese hypertensive patients, naıve of anti-hyper-

tensive therapy, the decreased titers of anti-oxLDL Abs

suggest increased oxidized LDL formation and/or deficient

humoral responses to these particles. Previous studies have

demonstrated that patients with hypertension have reduced

titers of anti-oxLDL Abs [28, 29], and unstable clinical

conditions are also associated with lower titers of these Abs

[30, 31]. This phenomenon can be explained by the greater

formation of immune complexes with oxLDL (IC-oxLDL),

leading to consumption of free, circulating antibodies,

showing an individual pattern of immune reactivity [32].

However, the maintenance of circulating antibody iso-

types is dependent on the formation of ‘‘neoepitopes’’ that

are products generated by lipoprotein oxidation/modifica-

tion or ApoB peroxidation and proteolysis [33, 34]. In this

Table 1 Clinical and laboratory characteristics of study groups, according to body mass index

Variable NW OW OB P value

(n = 19) (n = 35) (n = 34)

Age (years) 58 (56–63) 59 (48–66) 56 (49–62) 0.569

Gender (F/M) 12/7 17/18 15/19 0.126

BMI (kg/m2)# 23.9 (22.8–24.5) 26.4 (25.9–27.4) 32.2 (31.5–34.8) \0.001

Waist circumference (cm)£ 85 (80–88) 90 (87–97) 106 (99–111) \0.001

SBP (mm Hg) 158 (140–166) 153 (145–159) 144 (140–165) 0.516

DBP (mm Hg)w 90 (90–96) 90 (88–91) 91 (90–100) 0.018

Total cholesterol (mg/dl) 210 (194–233) 189 (168–209) 188 (169–215) 0.048

HDL-C (mg/dl)@ 50 (44–63) 45 (37–59) 44 (39–55) 0.035

LDL-C (mg/dl)€ 130 (122–151) 111 (93–136) 116 (97–135) 0.058

Triglyceridesa (mg/dl) 93 (75–138) 112 (75–185) 123 (90–185) 0.432

Glucosea (mg/dl)b 81 (72–88) 87 (81–96) 92 (83–104) 0.020

Hb A1c (%) 5.4 (5.1–5.5) 5.3 (5.1–5.8) 5.5 (5.2–5.7) 0.746

apoB/apoA1 0.72 (0.67–1.06) 0.71 (0.57–0.92) 0.84 (0.57–0.96) 0.699

Creatinine (mg/ml) 1.0 (0.9–1.0) 0.9 (0.9–1.2) 1.0 (0.8–1.1) 0.120

GFRa (ml/min)§ 65.6 (55.1–76.5) 76.3 (62.9–91.4) 98.4 (76.8–125.5) \0.001

hsCRP (mg/dl) 0.43 (0.29–1.00) 0.42 (0.36–0.99) 0.46 (0.28–0.80) 0.837

TBARS (lmol/ml) 1.57 (0.90–1.70) 1.47 (0.87–1.92) 1.70 (0.90–2.10) 0.845

Categorical data are expressed as n(%); numerical data are medians and interquartile ranges. NW normal weight, OW overweight, OB obesity,

BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, HDL-C high-density lipoprotein cholesterol, LDL-C low-

density lipoprotein cholesterol, HbA1c hemoglobin A1C, ApoB/ApoA1 apolipoprotein B/apolipoprotein A1 ratio, GFR glomerular filtration rate,

hsCRP high-sensitivity C-reactive protein, TBARS thiobarbituric acid reactive substances

P \ 0.05; ANOVA-Tukey; # NW versus OW, P \ 0.001; OW versus OB, P \ 0.001 and NW versus OB, P \ 0.001; w OW versus OB,

P = 0.01; £ NW versus OW, P = 0.02 and OW versus OB, P \ 0.001 and NW versus OB, P \ 0.001; @ NW versus OB, P = 0.02; € NW

versus OW, P = 0.04; b NW versus OW, P = 0.04 and NW versus OB, P = 0.01; § OW versus OB, P = 0.001 and NW versus OB, P \ 0,001a Log-transformed variable

Cell Biochem Biophys

123

scenario, it is possible that transient changes on titers of

antibodies reflect the clinical condition of the patient, since

this phenomenon comprises a greater concentration of

some immunoglobulin isotypes to the most prevalent epi-

tope [35]. Humoral response to oxidized LDL and ApoB-

derived peptides seems to be dependent of the maturation

of B cells, thus reflecting how the antibodies produced will

contribute to atherogenesis [36].

In experimental models, immunization with oxLDL con-

fers atheroprotection, with high titers of circulating antibodies

[37]; however, in humans, available data showed positive or

negative associations with atherosclerosis, depending on the

Fig. 1 Box-plots showing medians, 25–75th percentiles, and extreme

values for IL-8 (a), IL-10 (b), anti-oxLDL Abs (c), and anti-ApoB-D

Abs (d) in patients with hypertension stratified by body mass index.

IL-8 was higher in obesity than in overweight (P = 0.04) and normal

weight (P = 0.02). IL-10 was lower in obesity than in overweight

(P = 0.04) and in normal weight (P = 0.01). Anti-oxLDL Abs titers

were lower in subjects with obesity compared to those with

overweight (P = 0.01) and normal weight (P = 0.01). No differences

were observed in anti-ApoB-D Abs titers among groups. P \ 0.05;

ANOVA-Tukey. Anti-oxLDL Abs and anti-ApoB-D Abs expressed

as RI; IL-8 and IL-10 expressed as pg/mL. Anti-oxLDL Abs anti-

oxidized LDL autoantibodies, anti-ApoB-DAbs autoantibodies anti-

ApoB-D, IL interleukin, RI reactivity index, ns not significant

Table 2 Vascular reactivity parameters and 24-h ABPM in subjects with hypertension, by group

Variables NW OW OB P value

(n = 19) (n = 35) (n = 34)

NID (%) 10.7 (8.2–12.6) 11.6 (9.0–13.0) 10.6 (8.3–15.0) 0.831

FMD (%)} 7.5 (4.3–10.2) 8.5 (6.1–10.5) 5.5 (3.6–7.48) 0.001

RBAD (mm) 0.36 (0.32–0.41) 0.40 (0.40–0.47) 0.43 (0.34–0.48) 0.251

24-h SBPa (mmHg) 130 (117–137) 128 (117–138) 117 (118–132) 0.118

24-h DBPa (mmHg)h 75 (71–84) 81 (74–88) 71 (62–80) 0.027

NW normal weight, OW overweight, OB obesity, ABPM ambulatory blood pressure monitoring, NID nitrate-induced dilation, FMD flow-

mediated dilation, RBAD resting brachial artery diameter, SBP systolic blood pressure, DBP diastolic blood pressure

P \ 0.05; ANOVA-Tukey; } OW versus OB, P \ 0.001 and NW versus OB, P = 0.042; h OW versus OB, P = 0.027a Log-transformed variables

Cell Biochem Biophys

123

Fig. 2 Dot plots representing partial correlations between anti-

oxLDL Abs and BMI (a) or waist circumference (b). An inverse

correlation was observed between anti-oxLDL Abs and BMI (r =

-0.24, P = 0.02) and waist circumference (r = -0.25, P = 0.02).

anti-oxLDL Abs expressed as RI; waist circumference expressed as

cm. anti-oxLDL Abs anti-oxidized LDL autoantibodies; BMI body

mass index, RI reactivity index, SBP systolic blood pressure, DBPdiastolic blood pressure. P \ 0.05; partial correlation test adjusted for

SBP, DBP, and age

Fig. 3 Dot plots representing partial correlations between anti-

ApoB-D Abs titers and mean 24-h SBP (a) or 24-h DBP (b).

Significant correlation was observed between anti-ApoB-D Abs titers

and mean 24-h SBP (r = 0.22; P = 0.05) and 24-h DBP (r = 0.29;

P = 0.01). anti-ApoB-D Abs expressed as RI. Partial correlation test

adjusted for age. Anti-ApoB-D Abs autoantibodies anti-ApoB-D, SBPsystolic blood pressure, DBP diastolic blood pressure, RI reactivity

index

Table 3 Multiple linear regression analyses for oxLDL Abs and ApoB-D Abs in hypertensive patients

Anti-oxLDL Abs Anti-ApoB-D Abs

b SE 95 % CI P b SE 95 % CI P

BMI -0.053 0.022 -0.097 to -0.009 0.018 -0.001 0.005 -0.012–0.009 0.818

Waist circumference -0.018 0.008 -0.315 to -0.002 0.029 000.1 0.003 -0.005–0.003 0.784

Triglyceridesa 0.003 0.001 0.001 to 0.006 0.010 0.001 0.001 0.000–0.001 0.171

GFRa -1.377 0.666 -2.701 to -0.054 0.040 0.041 0.150 -0.273–0.355 0.790

Glucosea 1.058 1.383 -1.712 to 3.828 0.45 0.420 0.320 -0.187–1.039 0.173

ApoB/ApoA 0.697 0.292 0.117 to 1.277 0.019 0.042 0.073 -0.104–0.189 0.571

24-h SBPa 0.006 0.006 -0.007 to 0.019 0.38 0.960 0.443 0.077–1.844 0.037

24-h DBPa 0.075 1.599 -3.107 to 3.257 0.96 1.022 0.350 0.324–1.715 0.005

BMI body mass index, GFR glomerular filtration rate, ApoB/ApoA1 apolipoprotein B/apolipoprotein A1 ratio, SBP systolic blood pressure, DBPdiastolic blood pressure, b association coefficient, SE standard error, CI confidence intervala Log-transformed variables. Anti-oxLDL Abs and anti-ApoB-D Abs were analyzed as dependent variables, with other parameters as inde-

pendent variables

Cell Biochem Biophys

123

isotype of immunoglobulin used [38]. In our study, decreased

anti-oxLDL Abs titers in obese hypertensive subjects

appeared to be related to high consumption of these antibodies

in response to raised concentrations of oxidized LDL

particles.

Yet, the generation of antibodies to specific ApoB epi-

topes, can be expressed by different clinical settings, rel-

evant to progression of atherosclerosis [33], or also, that

these peptides can activate the inflammatory cells, and

accelerate the atherogenic process [11]. Some authors have

studied different ApoB-derived peptides and their respec-

tive autoantibodies, in association with clinical presenta-

tions, or using them as tools for production of vaccines for

prevention of atherosclerotic disease [39, 40]. Determina-

tion of autoantibodies to ApoB100 peptides can be a tool

for standardization of antigens for Abs analysis compared

to oxidized LDL, which can easily be modified ex vivo

[11]. The fragments of native ApoB100 or associated with

products of lipoperoxidation can be more stable in different

conditions. In this context, our group has demonstrated that

elevated anti-ApoB-D native Abs titers are associated with

the atherosclerotic process, and can be a potentially new

marker for autoimmune response in atherosclerosis [15, 16].

Studies addressing quantification of anti-ApoB-D auto-

antibodies titers have demonstrated immune reactivity of

ApoB-D antigens in various clinical scenarios [15, 16,

33, 39]. These autoantibodies to ApoB-100-derived pep-

tides can be markers of various stages of atherosclerosis

process, as well as fragments of the ApoB-100 can be new

approaches for therapeutic vaccines. Molecular studies of

ApoB-D in atherosclerosis and development of monoclonal

antibodies to detect ApoB-D peptide in serum are under-

way. In fact, there are few studies addressing ApoB-

derived peptides in relation to atherosclerosis. An elegant

paper by Ketelhuth et al. screened a peptide library of

ApoB-100, and identified ApoBDS-1, a previously unrec-

ognized peptide with robust proinflammatory activity,

contributing to the disease-promoting effects of low-den-

sity lipoprotein in the pathogenesis of atherosclerosis [11].

The ApoB-derived peptides may also modulate the

expression of cytokines, among them IL-8 [11] that was

increased in OB subjects in the present study. However,

because no differences were observed in the anti-ApoB-D

Abs titers in OB, OW, and NW groups, we could not

demonstrate a causal relationship for such findings.

Another important finding was that obese patients had

lower concentrations of the IL-10, an anti-atherogenic

cytokine, and a marker of adverse prognosis, related to

endothelial dysfunction and coronary artery disease [41].

Conversely, these subjects have increased titers of IL-8,

a pro-atherogenic cytokine, related to obesity and cardio-

vascular complications [42].

Study Limitations

In our study, we did not include normotensive-eutrophic or

normotensive-obese subjects. However, all patients in the

present study were naıve of anti-hypertensive therapy,

ruling out any drug effect and allowing us to report that

overweight and obesity can modulate the adaptive immune

responses to oxLDL and ApoB-D peptide among hyper-

tensive subjects.

Conclusion

Obesity is related to decreased titers of anti-oxLDL Abs,

endothelial dysfunction, and with an imbalance between

protective and inflammatory cytokines among hypertensive

subjects. The observed reduction of titers of anti-oxLDL

Abs was inversely correlated with BMI and waist cir-

cumference, independently of age and blood pressure val-

ues. Twenty-four hour systolic and diastolic blood pressure

was associated with titers of anti-ApoB-D Abs.

Acknowledgments This study was supported by a research Grant

from the National Institute of Complex Fluids, Sao Paulo, SP, Brazil.

Conflict of interest The authors have no conflicts of interest to

disclose.

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