Autores: Viviane Aparecida Rodrigues Sant’Anna, Maria Cristina Oliveira Izar, Magnus Gidlund, Henrique Andrade Rodrigues da Fonseca e Vaccine Heart Group (Esteferson Fernandes Rodrigues, Henrique Tria Bianco e Francisco Antonio Helfenstein Fonseca)
European Journal of Internal Medicine, 2023 | https://doi.org/10.1016/j.ejim.2023.07.018
Dear Editor,
Patients with diabetes mellitus (DM) have accelerated atherosclerosis progression and increased cardiovascular morbidity and mortality. Therefore, patients with type 2 DM (T2DM) are increasing in the general population, and they have a higher risk of cardiovascular diseases (CVDs) than patients without T2DM [1]. However, the mechanisms underlying the increased CVD risk of patients with T2DM and atherosclerosis progression are not completely understood. One possible mechanism is the increased production of advanced glycation and lipoperoxidation end products, such as malondialdehyde (MDA) and lysophosphatidylcholine, implicated in the oxidation of low-density lipoprotein (LDL) particles, one of the main atherogenic components associated with CVD risk [2,3].
In DM, hormonal modulations promote LDL particle changes, and there is a possible association between the immune response and apolipoprotein B (apoB) regions associated with CVD risk [4,5]. Here, we aimed to evaluate the presence of antibody subclasses against oxidized LDL particles (oxLDL) and apoB peptides associated with long-term major adverse cardiovascular events (MACE) in patients with T2DM.
We conducted a cohort, prospective, multicenter study including patients age over 30 years of both sexes, with T2DM, as defined by the American Diabetes Association [6]. T2DM patients were recruited from 10 research centers in nine cities of Brazil.
Patients with stable coronary artery diseases were included. However, patients with type 1 diabetes, previous stroke, metastasis or under anti-cancer therapy, autoimmune diseases and CVD history less than 60 days and unstable heart failure or unstable angina prior study admission were excluded.
The data collected at baseline were used to determine the predictors associated to humoral immune response related to the outcome. The study protocol was approved by the central and local ethics committees and the signed informed consent form was obtained from all participants.
The primary outcome was defined as MACE, composited by CV death, myocardial infarction (MI), or stroke. All study outcomes were adjudicated by an independent clinical events committee.
Briefly, the ApoB-D is a synthetic apoB peptide based on the human apo B conservative immunodominant sequence of 22 amino acids [5]. The Abs anti-ApoB-D were performed in ELISA test [5]. All description and methods of the laboratory analyzes are described in detail in the supplementary material.
Data were presented in median and mean, interquartile interval and error mean, respectively. Categorical variables were compared using Fisher's exact test. The normality distribution was assessed using the Kolmogorov-Smirnov test. Survival curves were performed using the Kaplan-Meier method.
Given the fact that anti-ApoB-D autoantibodies (Abs) seropositivity cut-offs or correlate of the protection were not defined nor validated, continuous Abs levels and reactivity index (RI) were categorized according to percentile limit to evaluate the association with clinical outcomes. For this study we used percentile 75 (p75) of RI considered more rigorous analysis of associations among Abs and outcomes.
Univariate analysis was performed to analyze the individual factors associated with primary outcome through Cox regression and was expressed with hazard ratios (HR) and 95% confidence interval (CI).
Multivariate analyses were performed only with variables that presented a p-value <0.05 in the univariate analysis. Statistical analyzes were performed using SPSS 24.0 software (SPSS® Inc., Chicago, IL, USA), with a significance level below 5%.
Between March 2001 and December 2011, 294 patients were recruited, with a median follow-up of 8.2 years. The study subjects’ baseline characteristics and data are summarized in Supplementary Table 1S.
Immunoglobulin M (IgM) and G (IgG) anti-ApoB-D Abs titers did not differ between men and women at baseline (p = 0.12 and p = 0.74, respectively). IgM anti-oxLDL Abs titers did not differ between men and women at baseline (p = 0.75), but baseline anti-oxLDL IgG Abs titers were higher in men than women (p = 0.005). Total IgG and IgM antibodies did not differ between men and women at baseline (p = 0.55 and p = 0.45, respectively). Eighty-seven primary outcome events occurred during follow-up, 30 in women and 57 in men.
IgM and IgG anti-ApoB-D and anti-oxLDL titers were not associated with MACE occurrence in the cohort general subjects (Fig. 1S).
We found different anti-ApoB-D Abs responses between sexes. IgM anti-ApoB-D Abs titers were associated with MACE only in men. In men, a higher risk of cardiovascular events was associated with lower IgM Abs titers at baseline (hazard ratio [HR] = 2.86, 95% confidence interval [CI]: 1.21–6.78; p = 0.017; Fig. 1C) but not with IgG anti-ApoB-D Abs titers (HR = 1.03, 95% CI: 0.55–1.95; p = 0.92; Fig. 1D). In women, higher MACE risk was associated with higher IgG anti-ApoB-D Abs titers at baseline (HR = 2.41, 95% CI: 1.13–5.14; p = 0.023; Fig. 1B) but not with IgM anti-ApoB-D Abs titers (HR = 0.78, 95% CI: 0.34–1.77; p = 0.54; Fig. 1A).
Fig. 1. Kaplan-Meier event curves using Cox regression for primary outcome as time-to-first event analysis for MACE, according to 75 percentile (p75) of reactivity index (RI) of autoantibodies related to sexes. (A) IgM anti-ApoB-D in women group; (B) IgG anti-ApoB-D in women; (C) IgM anti-ApoB-D in male group; (D) IgG anti-ApoB-D autoantibodies in male group. HR, hazard ratio; MACE; outcome composite of cardiovascular death, myocardial infarction or stroke.
The IgG and IgM anti-oxLDL Abs and the total IgG and IgM antibodies were not associated with MACE risk related to sexes during follow-up (Fig. 2S and 3S). Univariable analysis of Abs titers associated with MACE among sexes and in the total population are shown in Table 2S. Other clinical, laboratory, and medication uses are described in Supplementary Table 3S and Table 4S shows the correlations between measured IgM and IgG ApoB-D and oxLDL reactive Abs titers.
In women, MACE risk remained associated with previous MI (adjusted HR = 10.95, 95% CI: 4.15–28.91; p < 0.001) and IgG anti-ApoB-D Abs titers (adjusted HR = 2.20, 95% CI: 1.01–4.80; p = 0.048). In men, after Cox regression, MACE risk remained associated with previous MI (adjusted HR = 6.40, 95% CI: 2.62–15.63; p < 0.001) and smoking (adjusted HR = 3.22, 95% CI: 1.55–6.69; p = 0.002). IgM anti-ApoB-D Abs titer was not significant in the regression model for men.
Our findings corroborate and complement the initial observation of Bjorkbacka et al. [7], who used a different set of peptides with or without MDA modification to investigate associations between anti-apoB Abs and CVD risk in a large cohort aged 46–68 years with a follow-up >15 years.
Previous studies have shown a relationship between high IgG anti-oxLDL and ApoB-D titers with atherosclerosis progression in animal models [8], consistent with our clinical findings. The exact mechanisms by which natural autoantibodies play a protective role against cardiovascular outcomes remain unknown. However, this ability is believed to be due to these autoantibodies facilitating uptake and removing apoptotic cells and oxidized LDL particles, decreasing the inflammatory process involved in atherosclerosis through natural immune responses by IgM Abs [9].
The ApoB peptides are more stable and independent of the in vitro oxidation of LDL. In addition, these Abs could be biomarkers for predicting MACE risk in patients with T2DM and may be significant in identifying individuals needing immune-modulatory therapy. Therefore, further studies are needed to identify the mechanisms involved in this process and whether some therapeutic interventions may modulate these Abs to reduce CVD incidence.
Our study had some limitations. First, our baseline data showed differences in LDL-cholesterol concentrations between sexes that might have affected our anti-oxLDL Abs results. Second, our sample size was small for the final humoral response against apoB immunodominant regions final conclusions about DM patients. However, the data are consistent with another Swedish study on a cohort with similar outcomes and follow-up in a high-risk population [10]. Finally, larger sample sizes are needed to clarify the dynamic changes of antibodies against other apoB regions related to LDL particle oxidation and degradation. However, we have examined a stable apoB peptide fragment that is associated with LDL particle changes related to the atherosclerosis process and MACE in high-risk subjects.
Financial support
VARS received fellowship grant by Coordination for the Improvement of Higher Education Personnel (CAPES). MCOI received research grants of National Council for Scientific and Technological Development (CNPq).
Credit authorship contribution statement
Henrique Andrade R. Fonseca and Viviane Aparecida R. Sant'Anna: Project administration, Formal analysis, Writing – Original draft and Final manuscript, Funding acquisition. Viviane Aparecida R. Sant'Anna and Esteferson Fernandes Rodrigues: Laboratory analyses. Henrique T. Bianco, Francisco Antonio Fonseca and Maria Cristina O. Izar: Outcome adjudication, data curation, and manuscript final revision. Henrique Andrade R. Fonseca and Magnus Gidlund: Conceptualization, Laboratory methodology conceptualization, writing and review final manuscript.
Declaration of the Competing Interest
HARF received research grants of Ministry of Health of Brazil, AstraZeneca, Aché, and Pfizer; The authors declare no conflict of interest.
Acknowledgments
National Institute of Science and Technology Complex Fluids (INCT-FCx).
Vaccine Heart Group: Henrique Tria Bianco; Esteferson Fernandes Rodrigues; Francisco Antônio H. Fonseca.