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Anti-apolipoprotein A-1 autoantibodies as risk biomarker for cardiovascular diseases in type 2 diabetes mellitus
Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
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Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M
Anti-apolipoprotein A-1 autoantibodies as risk biomarker for cardiovascular diseases in type 2 diabetes mellitus Dalia El-Lebedy ⁎, Enas Rasheed, Mona Kafoury, Dalia Abd-El Haleem, Eman Awadallah, Ingy Ashmawy Department of Clinical and Chemical Pathology, Medical Research Division, National Research Centre, Cairo, Egypt
a r t i c l e
i n f o
Article history: Received 31 December 2015 Received in revised form 4 February 2016 Accepted 16 February 2016 Available online xxxx Keywords: Anti-apolipoprotein-A1 Type 2 diabetes Cardiovascular disease Biomarkers Autoantibodies
a b s t r a c t Objective: Anti-Apolipoprotein A-1 autoantibodies (anti-ApoA-1 IgG) represent an emerging prognostic cardiovascular marker in patients with myocardial infarction or autoimmune diseases associated with high thrombotic events. The aim of this work is to investigate the incidence of anti-apoA-1 autoantibodies in type 2 diabetes (T2DM) patients with and without CVD and to study potential association with disease risk and its effect on plasma lipid parameters. Methods: Qualitative determination of anti-apoA-1 IgG was assayed in sera from 302 subjects classified into T2DM patients (n = 102), T2DM + CVD (n = 112) and healthy controls (n = 88). Results: The incidence of anti-apoA-1 IgG was significantly higher among CVD patients (35.7%) than T2DM patients (8.8%) or control subjects (6.1%), p b 0.0001. A significant association with CVD was identified (p b 0.0001) and subjects who were positive for anti-apoA-1 IgG were at 8.5 times increased risk to develop CVD when compared to controls. Diabetic patients who were positive for the antibodies showed 5.7 times increased CVD risk. ROC analysis indicated anti-apoA-1 IgG as a risk biomarker for CVD in T2DM patients with an AUC value of 0.76, sensitivity of 35.7% and specificity of 91.2%. Studying the effect on lipid parameters, anti-apoA-1 IgG associated with significantly higher serum concentrations of TC and non-HDL-C in all groups and with higher concentrations of LDL-C in diabetic patients and higher TC/HDL-C ratio in CVD patients. Conclusion: Our results indicate that anti-apoA-1 IgG is a cardiovascular risk biomarker in T2DM patients. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Cardiovascular disease (CVD) is the most common cause of morbidity and mortality among patients with type 2 diabetes mellitus (T2DM) (Lorber, 2014). Plasma lipids and lipoproteins have been powerful risk factors for predicting CVD (Smith, Benjamin, Bonow, Braun, & Creager, 2011). High-density lipoprotein cholesterol (HDL-C) is an independent inverse risk factor for CVD (Di Angelantonio et al., 2009) and accumulating evidence indicates that HDL composition, rather than HDL-C circulating levels determines its functions (Vaisar, 2012). "Dysfunctional HDL" or "pro-inflammatory HDL" has been indicated as a proatherogenic factor (Sahin, 2012) and, recently, autoimmune-mediated HDL dysfunction was reported to contribute in atherogenesis (Srivastava, Yu, Parks, Black, & Kabarowski, 2011). Apolipoprotein A-1 (apoA-I) is the major structural and functional constituent of HDL representing about 75% of its protein content (Huang et al., 2014). ApoA-1 plays a major role in cholesterol homeostasis and exerts anti-inflammatory, antioxidant, and anti-atherogenic properties Conflict of interest: The authors declare that they have no conflict of interest. ⁎ Corresponding author at: Department of Clinical and Chemical Pathology, National Research Centre, Al-Bohouth Street, Cairo 12311, Egypt.
(Getz, Wool, & Reardon, 2010). ApoA-1 also stabilizes and maintains optimum activity of paraoxonase-1 (PON-1), the main antioxidant enzyme of HDL (Costa, Vitalone, Colea, & Furlong, 2005; Narshi, Giles, & Rahman, 2011; Précourt et al., 2011). Autoantibodies directed against apoA-1 may lead to apoA-1 dysfunction with secondary HDL dysfunction (Hahn, 2010). It was evidenced that anti-apoA-1 antibodies contribute to reducing PON1 activity by triggering the formation of HDL immune complexes (Delgado Alves et al., 2002; Batuca, Ames, Isenberg, & Alves, 2007; Fuhrman, 2012; Srivastava et al., 2011) which might give a possible explanation for the notion of serum PON1 inactivation under oxidative stress (Deakin, Moren, & James, 2007; Franco-Pons, Marsillach, Joven, Camps, & Closa, 2008; Fuhrman, 2012; Moren, Deakin, Liu, Taskinen, & James, 2008; Nguyen, Hung, Cheon-Ho, Ree, & Dai-Eun, 2009; Nguyen & Sok, 2003; Tavori, Aviram, & Khatib, 2011). Oxidative stress reported in T2DM might be the biological mechanism for increased autoantibody production by causing protein structural modification and appearance of neo-epitopes which enhances the humoral immune response with subsequent production of autoantibodies including antibodies to apoA-1 (Profumo, Buttari, & Riganò, 2011; Srivastava et al., 2011). Such a vicious circuit with its components maintains oxidative stress and subsequent diabetes-related proatherogenic state.
http://dx.doi.org/10.1016/j.jdiacomp.2016.02.014 1056-8727/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: El-Lebedy, D., et al., Anti-apolipoprotein A-1 autoantibodies as risk biomarker for cardiovascular diseases in type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.02.014
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D. El-Lebedy et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
Anti-apoA-1 auto antibodies have been reported in antiphospholipid syndrome (APS) and systemic lupus erythematosous (SLE), two systemic diseases with an increased risk of thrombotic events (Vuilleumier, Montecucco, & Hartley, 2014) and have been associated with CVD and atherosclerosis-related complications (Montecucco et al., 2011; Pagano et al., 2012; Podrez, 2010; Vuilleumier, Bas, et al., 2010; Vuilleumier, Rossier, et al., 2010). The aim of this study is to investigate the presence of anti-apoA-1 autoantibodies in T2DM patients with and without CVD and to study potential association with disease risk and its effect on plasma lipid parameters.
Very low density lipoprotein cholesterol (VLDL-C) was calculated according to the equation VLDL-C = (TC – LDL-C – HDL-C). Non-HDL-C level was calculated by subtracting HDL-C from TC, as a candidate biometrical equivalent to apoB-100 in diabetes (Hermans, Sacks, Ahn, & Rousseau, 2011). Glycated hemoglobin (HbA1c) was assayed by ion-exchange high-performance liquid chromatographic (HPLC) method using Agilent 1200 series HPLC system (Agilent Technologies, USA) equipped with UV/VIS-Detector 415 nm using the commercially available HbA1c test kit (RECIPE Chemicals and Instruments GmbH, Germany), this HPLC method was certified by National Glycohemoglobin Standardization Program (NGSP) (Little & Sacks, 2009).
2. Materials and Methods 2.1. Subjects Our study included 302 subjects recruited from the Outpatients' Clinic of the National Research Centre and the National Diabetes & Endocrinology Institute, Cairo, Egypt. Medical and family history, smoking status and physical activity were obtained by questionnaire. Physical activity was defined as exercise for 2–3 days/week for at least 30 minutes. Anthropometric measurements (weight and height) were collected and used for BMI calculation according to the standard formula BMI = weight (kg)/[height (m)] 2. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured and hypertension was defined as blood pressure above 140/90 mmHg or under antihypertensive drugs. Dyslipidemia was defined as level of total cholesterol (TC) N200 mg/dL, low density lipoprotein cholesterol (LDL-C) N 130 mg/dL, HDL-C b40 mg/dL, triglycerides (TG) N 150 mg/dL, TC/HDL-C ratio N 4.0 or under lipid lowering drugs (Jellinger et al., 2000). Diagnosis of diabetes was based on the criteria of American Diabetes Association (2014). Our subjects were classified into: Normal healthy controls (n = 88) with fasting plasma glucose (FPG) b 100 mg/dL. The exclusion criteria were hyperlipidemia, hypertension, family history of diabetes mellitus or any form of CVD, hepatic, renal, endocrine or autoimmune diseases and those under medication. T2DM patients without CVD (n = 102) fulfilled the diagnostic criteria of diabetes mellitus with FPG ≥ 126 mg/dL or under diabetes medication (oral and/or insulin) with no history or signs of any CVD. T2DM complicated with CVD (n = 112) diagnosed to have diabetes with FPG ≥ 126 mg/dL or under diabetes medication and complicated with any of vascular diseases e.g. ischemic heart disease (IHD), macroangiopathy and/or cerebrovascular disease. IHD included definite myocardial infarction (MI), ischemic electrocardiographic (ECG) changes and angina pectoris. Macroangiopathy included arteriosclerosis obliterans and cerebrovascular disease (history of transient ischemic attack, reversible ischemic neurological deficit or stroke caused by cerebral infarction). The exclusion criteria for diabetic patients were renal, hepatic, endocrine or autoimmune diseases. Informed consent was obtained from all subjects and the study protocol was approved by the Ethics Committee of the National Research Centre. 2.2. Methods 2.2.1. Assay of biochemical markers Venous blood samples were collected from all subjects after 12 hours of overnight fast, centrifuged within 2 hours and assayed for biochemical markers. For ELISA assay, aliquots were frozen at −80 °C till time of assay to avoid erroneous results from repeated freeze/thaw cycles. TC, TG, HDL-C, LDL-C and fasting plasma glucose (FPG) were assayed on c311 clinical chemistry autoanalyzer (Roche Diagnostics, Germany).
2.2.2. Assay of apoA-1 concentrations ApoA-1 was assayed in serum by endpoint nephlometry method on MininephPlus Analyser using MININEPH Apolipoprotein A1 kit ZK085.R (Binding Site Group Ltd, Birmingham, UK). 2.2.3. Assay for anti-apoA-1 autoantibody positivity Serum aliquots were thawed and centrifuged just prior to assay. Positivity for anti-apoA-1 IgG was assayed using Human anti-Apo-A1 antibody ELISA Kit MyBioSource, Inc. USA, #MBS703406 for qualitative determination of anti-apo-A1 IgG in serum. The intra- and inter-assay precision CV% was b15% with no cross-reactivity or interference with analogues. Positivity was considered when sample had an absorbance value ≥ 2.1 OD (optical density). 2.3. Statistical analysis Statistical analyses were performed using IBM SPSS version 20.0 software (Statistical Package for Social Science). Data were expressed as mean ± SD for continuous variables with a normal distribution and as frequency for categorical variables. Intergroup significance was assessed by Student's t-test (continuous variables) and chi-square test (χ 2) (categorical variables). Student's t-test was used to compare 2 Table 1 Demographic, clinical and biochemical data of the study population. Variable
Controls (n = 88)
T2DM (n = 102)
T2DM + CVD (n = 112)
Age (years) Sex (male/female) BMI (kg/m2) SBP (mmHg) DBP (mmHg) Hypertension (%) Smokers (%) Physical activity (%) Diabetes duration (years) Dyslipidemia (%) Glucose (mg/dL) HbA1c (%) Triglycerides (mg/dL) TC (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) VLDL-C (mg/dL) Non-HDL-C (mg/dL) TC/HDL-C apoA-1 (mg/dL) Anti-apoA-1 IgG positivity (%)
50.2 ± 5.1 48/40 24.3 ± 5. 2 117.3 ± 8.5 74.2 ± 6.2 – 13 65.5 –
50.9 ± 7.5 57/45 26.6 ± 4.9⁎ 130.2 ± 18.6⁎ 77.3 ± 15.5⁎ 32.5 8.6 52.8 7.9 ± 4.5
58.3 ± 6.2⁎⁎ 82/30⁎⁎† 27.8 ± 5.4⁎⁎
– 84 ± 7.5 5.4 ± 0.7 128.3 ± 41.1 172.7 ± 14 101.7 ± 5.5 51.9 ± 8.7 18.1 ± 24.2 123.14 ± 16.4 3.1 ± 0.5 135.4 ± 14.3 6.1
73 149.8 ± 54.5⁎ 6.4 ± 1.4⁎ 155.6 ± 82⁎ 190.1 ± 46.4⁎ 115.22 ± 37.9⁎ 49.7 ± 10.2⁎ 29.6 ± 14.4⁎ 148.3 ± 42.9⁎ 4.5 ± 1.1⁎ 130 ± 23.2 8.8
144.3 ± 20.4⁎⁎† 92.7 ± 9.9⁎⁎† 92.6† 14.2† 28.5⁎⁎† 14.2 ± 5.4† 92† 163 ± 63.7⁎⁎ 6.9 ± 1.2⁎⁎ 174.5 ± 76.2⁎⁎ 215.7 ± 45.9⁎⁎† 128 ± 42.1⁎⁎ 44.9 ± 10.8⁎⁎† 39.6 ± 21.9⁎⁎† 162.7 ± 45.5⁎† 4.8 ± 1.2⁎⁎ 122.3 ± 20.4⁎⁎† 35.7⁎⁎†
BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; HbA1c: hemoglobin A1C; TC: total cholesterol; LDL-C: low density lipoprotein cholesterol; HDL-C: high density lipoprotein cholesterol; VLDL-C: very low density lipoprotein cholesterol; apo-A1: apolipoprotein A1. Bonferroni multiple comparison test was applied. ⁎ Significant p in comparison between controls and T2DM. ⁎⁎ Significant p in comparison between controls and T2DM + CVD. † Significant p in comparison between T2DM and T2DM + CVD.
Please cite this article as: El-Lebedy, D., et al., Anti-apolipoprotein A-1 autoantibodies as risk biomarker for cardiovascular diseases in type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.02.014
D. El-Lebedy et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx Table 2 Association of anti-apoA-1 IgG positivity with T2DM and CVD risk compared to controls.
Controls (n = 98) T2DM (n = 102) T2DM + CVD (n = 112)
Anti-apo-A1 IgG positivity (%)
Unadjusted OR (95% CI)
p-value
6.1 8.8 35.7
1.483 (0.507–4.336) 8.518 (3.422–21.201)
0.4 b0.0001⁎
⁎ p remained significant after adjusting for other covariates.
Table 3 Association of anti-apoA-1 IgG positivity with CVD in T2DM patients.
T2DM (n = 102) T2DM + CVD (n = 112)
Anti-apo-A1 IgG positivity (%)
Unadjusted OR (95% CI)
p-value
8.8 35.7
5.740 (2.616–12.596)
b0.0001⁎
3
lower levels of physical activity (p = 0.004) and longer durations of diabetes (p = 0.001) compared to T2DM patients or controls. Serum apoA-1 concentration was significantly lower in CVD patients than in T2DM patients (p = 0.004) or in control (p = 0.003). Though the mean concentration in T2DM patients was lower than that in control (130 ± 23.2 vs. 135.4 ± 14.3, respectively), the difference was of no statistical significance (p = 0.2). The incidence of anti-apoA-1 IgG positivity was significantly higher in CVD patients (35.7%) than in T2DM patients (8.8%) or in control subjects (6.1%), (p b 0.0001), and has been associated with significantly lower serum apoA-1 concentrations (p = 0.001). AntiapoA-1 IgG positivity was over presented among male gender and older age due to the high prevalence of males among CVD patients and the higher chances to develop CVD with increasing age. The demographic, clinical and biochemical data of the studied subjects are shown in Table 1. 3.2. Association of anti-apoA-1 autoantibodies with disease risk
⁎ p remained significant after adjusting for other covariates.
groups and ANOVA test for more than 2 groups followed by posthoc Bonferroni multiple comparison test. Univariable logistic regression analysis was used to test the association between disease and anti-apoA1- IgG and presented as unadjusted odds ratio (OR) and corresponding 95% confidence intervals (95% CI). Multivariate logistic regression analysis was used to determine the independent risk factors for development of diabetes and CVD after adjusting for potential covariates: age, gender, BMI, hypertension, smoking status, lipid parameters and physical activity and presented as adjusted OR. Receiver operating curve (ROC) analysis was performed to assess anti-apo-A1 IgG as a biomarker for CVD. Differences were considered significant with p value b0.05.
Association studies of anti-apoA-1 autoantibodies showed no significant association with T2DM (OR = 1.483, 95% CI = 0.507– 4.336, p = 0.4). However, a significant association with CVD was observed and subjects who were positive for anti-apoA-1 IgG were 8.5 times at risk to develop CVD compared to controls (OR = 8.518, 95% CI = 3.422–21.201, p b 0.0001) (Table 2). In diabetic patients, anti-apoA-1 autoantibodies significantly associated with CVD, diabetic patients who were positive for those antibodies showed 5.7 times higher risk to develop CVD (OR = 5.7, 95% CI = 2.616–12.596, p b 0.0001) (Table 3). Data remained significant after adjustment for other covariates: age, gender, BMI, hypertension, disease duration, lipid parameters, smoking status and physical activity. 3.3. Relationship between anti-apoA-1 IgG and lipid profile parameters
3. Results 3.1. Characteristics of the study population The study included 302 subjects categorized into 3 groups: T2DM (n = 102), T2DM complicated with CVD (n = 112) and control subjects (n = 88). Their age ranged from 40 to 72 years. The frequencies of CVDs in our patients were: 68% ischemic heart disease (IHD), 15% cerebrovascular disease, 10% macroangiopathy, 4% combined IHD and cerebrovascular disease, and 3% combined macroangiopathy and cerebrovascular disease. BMI, SBP and DBP were significantly higher in patient groups than in controls. CVD patients had higher frequency of hypertension (p b 0.0001), dyslipidemia (p b 0.0001) and smoking (p = 0.01),
The presence of anti-apo A-1 IgG was associated with higher serum concentrations of TC and non-HDL-C ratio in all groups (p = 0.013 and 0.017, respectively). A significant association with higher concentrations of LDL-C was observed in diabetic patients (p = 0.022), and with higher TC/HDL-C ratio in CVD patients (p = 0.031) (Table 4). 3.4. ROC analysis ROC analysis indicated anti-apoA-1 IgG as risk biomarker for CVD in T2DM patients with an area under the curve (AUC) value of 0.76. The sensitivity was 35.7% (95% CI 26.88–45.32) and the specificity was 91.2% (95% CI 83.91–95.89). Negative predictive value was 56.4% (95% CI 48.44–64.06) and positive predictive value was 81.6% (95% CI
Table 4 Relation between anti-apoA-1 IgG and Lipid profile parameters. Control
T2DM
T2DM + CVD
Parameter (mg/dl)
Positive for anti-apoA-1 Negative for anti-apoA-1 IgG (n = 6) IgG (n = 92)
Positive for anti-apoA-1 Negative for anti-apoA-1 IgG (n = 9) IgG (n = 93)
Positive for anti-apoA-1 IgG (n = 40)
Negative for anti-apoA-1 IgG (n = 72)
TC TG HDL-C LDL-C VLDL-C Non-HDL-C TC/HDL-C
188 131.28 55. 5 97.57 17.76 133 3.54
224 149.64 45 151.7 31 169 4.7
201.4 153.1 49 136 30.7 151 4.5
± ± ± ± ± ± ±
20.11 16.12 12.75 10.37 3.88 16.19 0.87
170 117.1 55.8 99.44 15.42 114.33 3.10
± ± ± ± ± ± ±
18.02⁎ 32.56 9.11 12.87 12.06 15.66⁎ 0.47
201 141.78 42 121.8 29.8 152.5 4.2
± ± ± ± ± ± ±
23.2 64.56 11.21 34.22 15 34.36 0.9
181.44 144.43 43.35 118.3 29.3 143.3 4
± ± ± ± ± ± ±
54.1⁎ 55.05 10.9 15.25⁎ 10.5 13.57⁎ 1.1
± ± ± ± ± ± ±
43.23 56.42 11 42.4 11.2 42.3 0.98
± ± ± ± ± ± ±
68.8⁎ 75.3 14.5 59.4⁎ 12.3 60⁎ 0.8⁎
Data presented as mean ± SD. Bonferroni multiple comparison test was applied. ⁎ p value b0.05.
Please cite this article as: El-Lebedy, D., et al., Anti-apolipoprotein A-1 autoantibodies as risk biomarker for cardiovascular diseases in type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.02.014
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D. El-Lebedy et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
67.98–91.24). Positive likelihood ratio was 4.05 (95% CI 2.07–7.92) and the disease prevalence was 52.3% (95% CI 45.42–59.19).
4. Discussion Recent studies indicated high incidence of anti-apo-A1 autoantibodies in many diseases associated with thrombotic events, such as SLE (32.5%), APS (22.9%) (Vuilleumier et al., 2014), acute coronary syndrome (ACS) (21%) (Vuilleumier et al., 2004, 2008), rheumatoid arthritis (RA) (17%) (Vuilleumier, Bas, et al., 2010), severe carotid stenosis (SCS) (20%) (Montecucco et al., 2011; Vuilleumier et al., 2013) and end stage renal disease (20%) (Pruijm et al., 2012). T2DM is a major independent risk factor for cardiovascular disease (CVD), the most common cause of morbidity and mortality among diabetic patients. The aim of this work was to investigate the presence of anti-apoA-1 autoantibodies in T2DM patients with and without CVD and to study potential association with CVD risk and the effect on plasma lipid profile parameters. To our knowledge, this is the first study investigating anti-apoA-1 autoantibodies in T2DM. Our results showed a significantly higher incidence of anti-apoA-1 IgG among CVD patients and subjects who were positive for those antibodies were at 8.5 times increased risk to develop CVD when compared to controls. In diabetic patients, anti-apoA-1 IgG showed significant association with CVD (p b 0.0001) and diabetic patients who were positive for the antibodies were at 5.7 times higher risk of CVD. ROC analysis indicated anti-apoA-1 IgG as risk biomarker for CVD in T2DM patients with an AUC of 0.76, sensitivity of 35.7% and specificity of 91.2%. Anti-apoA-1 IgG autoantibodies also associated with significantly higher serum concentrations of TC and non-HDL-C in all groups, and with higher concentrations of LDL-C in diabetic patients and higher TC/HDL-C ratio in CVD patients. Anti-apo-A1 autoantibodies may be present in general population but in low titers (up to 6.5%) (Vuilleumier et al., 2008; Vuilleumier, Bas, et al., 2010) and may be related to the vascular and immune ageing processes (Batuca et al., 2007). In our study, anti-apo-A1 IgG were detected in 6.1% of our control subjects. Our findings were consistent with results from previous studies, an association between anti-apoA-1 auto antibodies and cardiovascular risk was reported in autoimmune and non autoimmune diseases (Batuca et al., 2007; Montecucco et al., 2011; Pruijm et al., 2012; Radwan, El-Lebedy, Fouda, Elsorougy, & Fakhry, 2014; Vuilleumier et al., 2004, 2008; Vuilleumier, Bas, et al., 2010; Vuilleumier, Rossier, et al., 2010; Vuilleumier et al., 2013, 2014). In MI patients, anti-apoA-1 IgG was identified in 21% vs. 1% in healthy controls (p = 0.001) (Vuilleumier et al., 2004) and was associated with 4-fold increased risk of major adverse cardiac events (MACE) with an AUC value of 0.65 (p = 0.007) (Vuilleumier, Rossier, et al., 2010; Vuilleumier et al., 2011). Those autoantibodies also associated with atherosclerosis-related complications (Pagano et al., 2012) and increased atherosclerotic plaque vulnerability (Montecucco et al., 2011). While in SCS, anti-apoA-1 IgG was associated with 5-fold increase risk of MACE with an AUC of 0.74 (Vuilleumier et al., 2013). In RA patients, anti-apoA-1 IgG increased the cardiovascular risk 4-folds and was reported as the strongest predictor of major cardiovascular events with an AUC of 0.73, specificity of 50%, and a sensitivity of 90% (Vuilleumier, Bas, et al., 2010). Anti-apoA-1 IgG has been also associated with higher levels of matrix metalloproteinase (MMP-9), interleukin-8 (IL-8) and oxidized LDL; the major three markers and mediators of atherosclerotic plaque destabilization (Vuilleumier, Bas, et al., 2010; Vuilleumier, Bratt, et al., 2010). In a study on obese but otherwise healthy individuals, anti-apoA-1 IgG was reported as an independent predictor of coronary artery calcification (CAC) and coronary endothelial dysfunction with a negative predictive value of 94% and low IgG titer was indicated as a screening parameter to
identify those at low cardiovascular risk in whom coronary investigations could be overlooked (Quercioli et al., 2012). Auto antibodies directed against apoA-1 might affect the normal atheroprotective function of HDL (Hahn, 2010). The anti-atherogenic function of HDL could be partly explained by its main constituents, apoA-1 and PON1 (Guo, Li, Zhong, Tu, & Xie, 2012). Reduced PON1 concentration and/or activity have been reported as an independent risk factor for CVD (Dasgupta et al., 2011; El-Lebedy et al., 2014; Fuhrman, 2012; Gugliucci, Kotani, & Kimura, 2012; Gupta et al., 2012; Shenhar-Tsarfaty et al., 2013). ApoA-1 maintains optimum activity and stability of PON1 enzyme (Narshi et al., 2011) and enhances its binding to phospholipids (James & Deakin, 2004). Serum PON1 activity from apoA-1 knockout mice was found to be less stable than that of wild type siblings (Sorenson et al., 1999) and anti-apoA-1 antibodies were associated with reduced PON activity in plasma and in vitro inhibition assay confirmed a direct inhibition of the enzyme activity (Batuca et al., 2007). This subsequently reduced PON1 activity might play a role in the pathogenesis of T2DM itself. It was suggested that oxidative stress induced by reduced PON1 activity results in reducing glucose uptake from blood by muscle cells and develops into insulin resistance, results from previous studies support this hypothesis (Barbieri, Bonafe, & Marfella, 2002; Kordonouri, James, & Bennetts, 2001; Leviev, Kalix, Brulhart-Meynet, & James, 2001; Senti, Tomas, & Fito, 2003). Immune-mediated inflammation was recently indicated to play an important role in atherogenesis (Vuilleumier et al., 2014) and results from previous studies suggest that anti-apoA-1 IgG might be a mediator of inflammation and atherogenesis (Batuca et al., 2009; Vuilleumier, Bas, et al., 2010). Significant associations between high levels of anti-apoA-1 IgG and markers of oxidative stress, inflammation and endothelial dysfunction have been reported. In SLE patients, anti-apoA-1 IgG levels positively correlated with nitric oxide and inversely correlated with PON-1 activity suggesting that those autoantibodies might affect the antioxidant properties of HDL giving rise to a pro-oxidative microenvironment facilitating atherogenesis (Batuca et al., 2009; Vuilleumier, Bas, et al., 2010; Vuilleumier, Bratt, et al., 2010). In ACS, patients who were positive for anti-apoA-1 IgG had 5-fold higher levels of oxidized LDL (Vuilleumier et al., 2008). In a study involving MI patients, anti-apoA-1 IgG positively associated with serum levels of amyloid A protein which is involved in inflammation and cholesterol homeostasis (Vuilleumier et al., 2004). Also, in vitro studies demonstrated anti-apoA-1 IgG as an active mediator of atherosclerosis and atherosclerotic plaque vulnerability by promoting sterile inflammation through toll-like receptor-2 (TLR2)/CD14 complex (Pagano et al., 2012) and inducing specific neutrophil chemotaxis toward IL-8 and TNF-α (cytokines expressed within atherosclerotic plaques) (Montecucco et al., 2011). Anti-apoA-1 IgG may also act as pro-arythmogenic molecules through an aldosterone-dependent L-type calcium channels activation (Rossier et al., 2012). In mice model, passive immunization with anti-apoA-1 IgG increased atherogenesis and atherosclerotic plaque vulnerability (Montecucco et al., 2011) and decreased the antioxidant properties of HDL by inhibiting PON-1activity (Srivastava et al., 2011). In conclusion, our results indicate anti-apoA-1 IgG as a risk biomarker for CVD in type 2 diabetes patients. Considering the importance of identifying new and reversible CVD risk factors, these auto antibodies might represent a new possible therapeutic target and could allow the identification of a subset of reversible CVD patients who could benefit from specific immunomodulation in the future.
Financial disclosures None to declare.
Please cite this article as: El-Lebedy, D., et al., Anti-apolipoprotein A-1 autoantibodies as risk biomarker for cardiovascular diseases in type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.02.014
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Funding disclosures This work was funded by the National Research Centre (Project No. 10010309).
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Please cite this article as: El-Lebedy, D., et al., Anti-apolipoprotein A-1 autoantibodies as risk biomarker for cardiovascular diseases in type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.02.014
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Please cite this article as: El-Lebedy, D., et al., Anti-apolipoprotein A-1 autoantibodies as risk biomarker for cardiovascular diseases in type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.02.014
Contents lists available at ScienceDirect
Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M
Anti-apolipoprotein A-1 autoantibodies as risk biomarker for cardiovascular diseases in type 2 diabetes mellitus Dalia El-Lebedy ⁎, Enas Rasheed, Mona Kafoury, Dalia Abd-El Haleem, Eman Awadallah, Ingy Ashmawy Department of Clinical and Chemical Pathology, Medical Research Division, National Research Centre, Cairo, Egypt
a r t i c l e
i n f o
Article history: Received 31 December 2015 Received in revised form 4 February 2016 Accepted 16 February 2016 Available online xxxx Keywords: Anti-apolipoprotein-A1 Type 2 diabetes Cardiovascular disease Biomarkers Autoantibodies
a b s t r a c t Objective: Anti-Apolipoprotein A-1 autoantibodies (anti-ApoA-1 IgG) represent an emerging prognostic cardiovascular marker in patients with myocardial infarction or autoimmune diseases associated with high thrombotic events. The aim of this work is to investigate the incidence of anti-apoA-1 autoantibodies in type 2 diabetes (T2DM) patients with and without CVD and to study potential association with disease risk and its effect on plasma lipid parameters. Methods: Qualitative determination of anti-apoA-1 IgG was assayed in sera from 302 subjects classified into T2DM patients (n = 102), T2DM + CVD (n = 112) and healthy controls (n = 88). Results: The incidence of anti-apoA-1 IgG was significantly higher among CVD patients (35.7%) than T2DM patients (8.8%) or control subjects (6.1%), p b 0.0001. A significant association with CVD was identified (p b 0.0001) and subjects who were positive for anti-apoA-1 IgG were at 8.5 times increased risk to develop CVD when compared to controls. Diabetic patients who were positive for the antibodies showed 5.7 times increased CVD risk. ROC analysis indicated anti-apoA-1 IgG as a risk biomarker for CVD in T2DM patients with an AUC value of 0.76, sensitivity of 35.7% and specificity of 91.2%. Studying the effect on lipid parameters, anti-apoA-1 IgG associated with significantly higher serum concentrations of TC and non-HDL-C in all groups and with higher concentrations of LDL-C in diabetic patients and higher TC/HDL-C ratio in CVD patients. Conclusion: Our results indicate that anti-apoA-1 IgG is a cardiovascular risk biomarker in T2DM patients. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Cardiovascular disease (CVD) is the most common cause of morbidity and mortality among patients with type 2 diabetes mellitus (T2DM) (Lorber, 2014). Plasma lipids and lipoproteins have been powerful risk factors for predicting CVD (Smith, Benjamin, Bonow, Braun, & Creager, 2011). High-density lipoprotein cholesterol (HDL-C) is an independent inverse risk factor for CVD (Di Angelantonio et al., 2009) and accumulating evidence indicates that HDL composition, rather than HDL-C circulating levels determines its functions (Vaisar, 2012). "Dysfunctional HDL" or "pro-inflammatory HDL" has been indicated as a proatherogenic factor (Sahin, 2012) and, recently, autoimmune-mediated HDL dysfunction was reported to contribute in atherogenesis (Srivastava, Yu, Parks, Black, & Kabarowski, 2011). Apolipoprotein A-1 (apoA-I) is the major structural and functional constituent of HDL representing about 75% of its protein content (Huang et al., 2014). ApoA-1 plays a major role in cholesterol homeostasis and exerts anti-inflammatory, antioxidant, and anti-atherogenic properties Conflict of interest: The authors declare that they have no conflict of interest. ⁎ Corresponding author at: Department of Clinical and Chemical Pathology, National Research Centre, Al-Bohouth Street, Cairo 12311, Egypt.
(Getz, Wool, & Reardon, 2010). ApoA-1 also stabilizes and maintains optimum activity of paraoxonase-1 (PON-1), the main antioxidant enzyme of HDL (Costa, Vitalone, Colea, & Furlong, 2005; Narshi, Giles, & Rahman, 2011; Précourt et al., 2011). Autoantibodies directed against apoA-1 may lead to apoA-1 dysfunction with secondary HDL dysfunction (Hahn, 2010). It was evidenced that anti-apoA-1 antibodies contribute to reducing PON1 activity by triggering the formation of HDL immune complexes (Delgado Alves et al., 2002; Batuca, Ames, Isenberg, & Alves, 2007; Fuhrman, 2012; Srivastava et al., 2011) which might give a possible explanation for the notion of serum PON1 inactivation under oxidative stress (Deakin, Moren, & James, 2007; Franco-Pons, Marsillach, Joven, Camps, & Closa, 2008; Fuhrman, 2012; Moren, Deakin, Liu, Taskinen, & James, 2008; Nguyen, Hung, Cheon-Ho, Ree, & Dai-Eun, 2009; Nguyen & Sok, 2003; Tavori, Aviram, & Khatib, 2011). Oxidative stress reported in T2DM might be the biological mechanism for increased autoantibody production by causing protein structural modification and appearance of neo-epitopes which enhances the humoral immune response with subsequent production of autoantibodies including antibodies to apoA-1 (Profumo, Buttari, & Riganò, 2011; Srivastava et al., 2011). Such a vicious circuit with its components maintains oxidative stress and subsequent diabetes-related proatherogenic state.
http://dx.doi.org/10.1016/j.jdiacomp.2016.02.014 1056-8727/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: El-Lebedy, D., et al., Anti-apolipoprotein A-1 autoantibodies as risk biomarker for cardiovascular diseases in type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.02.014
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D. El-Lebedy et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
Anti-apoA-1 auto antibodies have been reported in antiphospholipid syndrome (APS) and systemic lupus erythematosous (SLE), two systemic diseases with an increased risk of thrombotic events (Vuilleumier, Montecucco, & Hartley, 2014) and have been associated with CVD and atherosclerosis-related complications (Montecucco et al., 2011; Pagano et al., 2012; Podrez, 2010; Vuilleumier, Bas, et al., 2010; Vuilleumier, Rossier, et al., 2010). The aim of this study is to investigate the presence of anti-apoA-1 autoantibodies in T2DM patients with and without CVD and to study potential association with disease risk and its effect on plasma lipid parameters.
Very low density lipoprotein cholesterol (VLDL-C) was calculated according to the equation VLDL-C = (TC – LDL-C – HDL-C). Non-HDL-C level was calculated by subtracting HDL-C from TC, as a candidate biometrical equivalent to apoB-100 in diabetes (Hermans, Sacks, Ahn, & Rousseau, 2011). Glycated hemoglobin (HbA1c) was assayed by ion-exchange high-performance liquid chromatographic (HPLC) method using Agilent 1200 series HPLC system (Agilent Technologies, USA) equipped with UV/VIS-Detector 415 nm using the commercially available HbA1c test kit (RECIPE Chemicals and Instruments GmbH, Germany), this HPLC method was certified by National Glycohemoglobin Standardization Program (NGSP) (Little & Sacks, 2009).
2. Materials and Methods 2.1. Subjects Our study included 302 subjects recruited from the Outpatients' Clinic of the National Research Centre and the National Diabetes & Endocrinology Institute, Cairo, Egypt. Medical and family history, smoking status and physical activity were obtained by questionnaire. Physical activity was defined as exercise for 2–3 days/week for at least 30 minutes. Anthropometric measurements (weight and height) were collected and used for BMI calculation according to the standard formula BMI = weight (kg)/[height (m)] 2. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured and hypertension was defined as blood pressure above 140/90 mmHg or under antihypertensive drugs. Dyslipidemia was defined as level of total cholesterol (TC) N200 mg/dL, low density lipoprotein cholesterol (LDL-C) N 130 mg/dL, HDL-C b40 mg/dL, triglycerides (TG) N 150 mg/dL, TC/HDL-C ratio N 4.0 or under lipid lowering drugs (Jellinger et al., 2000). Diagnosis of diabetes was based on the criteria of American Diabetes Association (2014). Our subjects were classified into: Normal healthy controls (n = 88) with fasting plasma glucose (FPG) b 100 mg/dL. The exclusion criteria were hyperlipidemia, hypertension, family history of diabetes mellitus or any form of CVD, hepatic, renal, endocrine or autoimmune diseases and those under medication. T2DM patients without CVD (n = 102) fulfilled the diagnostic criteria of diabetes mellitus with FPG ≥ 126 mg/dL or under diabetes medication (oral and/or insulin) with no history or signs of any CVD. T2DM complicated with CVD (n = 112) diagnosed to have diabetes with FPG ≥ 126 mg/dL or under diabetes medication and complicated with any of vascular diseases e.g. ischemic heart disease (IHD), macroangiopathy and/or cerebrovascular disease. IHD included definite myocardial infarction (MI), ischemic electrocardiographic (ECG) changes and angina pectoris. Macroangiopathy included arteriosclerosis obliterans and cerebrovascular disease (history of transient ischemic attack, reversible ischemic neurological deficit or stroke caused by cerebral infarction). The exclusion criteria for diabetic patients were renal, hepatic, endocrine or autoimmune diseases. Informed consent was obtained from all subjects and the study protocol was approved by the Ethics Committee of the National Research Centre. 2.2. Methods 2.2.1. Assay of biochemical markers Venous blood samples were collected from all subjects after 12 hours of overnight fast, centrifuged within 2 hours and assayed for biochemical markers. For ELISA assay, aliquots were frozen at −80 °C till time of assay to avoid erroneous results from repeated freeze/thaw cycles. TC, TG, HDL-C, LDL-C and fasting plasma glucose (FPG) were assayed on c311 clinical chemistry autoanalyzer (Roche Diagnostics, Germany).
2.2.2. Assay of apoA-1 concentrations ApoA-1 was assayed in serum by endpoint nephlometry method on MininephPlus Analyser using MININEPH Apolipoprotein A1 kit ZK085.R (Binding Site Group Ltd, Birmingham, UK). 2.2.3. Assay for anti-apoA-1 autoantibody positivity Serum aliquots were thawed and centrifuged just prior to assay. Positivity for anti-apoA-1 IgG was assayed using Human anti-Apo-A1 antibody ELISA Kit MyBioSource, Inc. USA, #MBS703406 for qualitative determination of anti-apo-A1 IgG in serum. The intra- and inter-assay precision CV% was b15% with no cross-reactivity or interference with analogues. Positivity was considered when sample had an absorbance value ≥ 2.1 OD (optical density). 2.3. Statistical analysis Statistical analyses were performed using IBM SPSS version 20.0 software (Statistical Package for Social Science). Data were expressed as mean ± SD for continuous variables with a normal distribution and as frequency for categorical variables. Intergroup significance was assessed by Student's t-test (continuous variables) and chi-square test (χ 2) (categorical variables). Student's t-test was used to compare 2 Table 1 Demographic, clinical and biochemical data of the study population. Variable
Controls (n = 88)
T2DM (n = 102)
T2DM + CVD (n = 112)
Age (years) Sex (male/female) BMI (kg/m2) SBP (mmHg) DBP (mmHg) Hypertension (%) Smokers (%) Physical activity (%) Diabetes duration (years) Dyslipidemia (%) Glucose (mg/dL) HbA1c (%) Triglycerides (mg/dL) TC (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) VLDL-C (mg/dL) Non-HDL-C (mg/dL) TC/HDL-C apoA-1 (mg/dL) Anti-apoA-1 IgG positivity (%)
50.2 ± 5.1 48/40 24.3 ± 5. 2 117.3 ± 8.5 74.2 ± 6.2 – 13 65.5 –
50.9 ± 7.5 57/45 26.6 ± 4.9⁎ 130.2 ± 18.6⁎ 77.3 ± 15.5⁎ 32.5 8.6 52.8 7.9 ± 4.5
58.3 ± 6.2⁎⁎ 82/30⁎⁎† 27.8 ± 5.4⁎⁎
– 84 ± 7.5 5.4 ± 0.7 128.3 ± 41.1 172.7 ± 14 101.7 ± 5.5 51.9 ± 8.7 18.1 ± 24.2 123.14 ± 16.4 3.1 ± 0.5 135.4 ± 14.3 6.1
73 149.8 ± 54.5⁎ 6.4 ± 1.4⁎ 155.6 ± 82⁎ 190.1 ± 46.4⁎ 115.22 ± 37.9⁎ 49.7 ± 10.2⁎ 29.6 ± 14.4⁎ 148.3 ± 42.9⁎ 4.5 ± 1.1⁎ 130 ± 23.2 8.8
144.3 ± 20.4⁎⁎† 92.7 ± 9.9⁎⁎† 92.6† 14.2† 28.5⁎⁎† 14.2 ± 5.4† 92† 163 ± 63.7⁎⁎ 6.9 ± 1.2⁎⁎ 174.5 ± 76.2⁎⁎ 215.7 ± 45.9⁎⁎† 128 ± 42.1⁎⁎ 44.9 ± 10.8⁎⁎† 39.6 ± 21.9⁎⁎† 162.7 ± 45.5⁎† 4.8 ± 1.2⁎⁎ 122.3 ± 20.4⁎⁎† 35.7⁎⁎†
BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; HbA1c: hemoglobin A1C; TC: total cholesterol; LDL-C: low density lipoprotein cholesterol; HDL-C: high density lipoprotein cholesterol; VLDL-C: very low density lipoprotein cholesterol; apo-A1: apolipoprotein A1. Bonferroni multiple comparison test was applied. ⁎ Significant p in comparison between controls and T2DM. ⁎⁎ Significant p in comparison between controls and T2DM + CVD. † Significant p in comparison between T2DM and T2DM + CVD.
Please cite this article as: El-Lebedy, D., et al., Anti-apolipoprotein A-1 autoantibodies as risk biomarker for cardiovascular diseases in type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.02.014
D. El-Lebedy et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx Table 2 Association of anti-apoA-1 IgG positivity with T2DM and CVD risk compared to controls.
Controls (n = 98) T2DM (n = 102) T2DM + CVD (n = 112)
Anti-apo-A1 IgG positivity (%)
Unadjusted OR (95% CI)
p-value
6.1 8.8 35.7
1.483 (0.507–4.336) 8.518 (3.422–21.201)
0.4 b0.0001⁎
⁎ p remained significant after adjusting for other covariates.
Table 3 Association of anti-apoA-1 IgG positivity with CVD in T2DM patients.
T2DM (n = 102) T2DM + CVD (n = 112)
Anti-apo-A1 IgG positivity (%)
Unadjusted OR (95% CI)
p-value
8.8 35.7
5.740 (2.616–12.596)
b0.0001⁎
3
lower levels of physical activity (p = 0.004) and longer durations of diabetes (p = 0.001) compared to T2DM patients or controls. Serum apoA-1 concentration was significantly lower in CVD patients than in T2DM patients (p = 0.004) or in control (p = 0.003). Though the mean concentration in T2DM patients was lower than that in control (130 ± 23.2 vs. 135.4 ± 14.3, respectively), the difference was of no statistical significance (p = 0.2). The incidence of anti-apoA-1 IgG positivity was significantly higher in CVD patients (35.7%) than in T2DM patients (8.8%) or in control subjects (6.1%), (p b 0.0001), and has been associated with significantly lower serum apoA-1 concentrations (p = 0.001). AntiapoA-1 IgG positivity was over presented among male gender and older age due to the high prevalence of males among CVD patients and the higher chances to develop CVD with increasing age. The demographic, clinical and biochemical data of the studied subjects are shown in Table 1. 3.2. Association of anti-apoA-1 autoantibodies with disease risk
⁎ p remained significant after adjusting for other covariates.
groups and ANOVA test for more than 2 groups followed by posthoc Bonferroni multiple comparison test. Univariable logistic regression analysis was used to test the association between disease and anti-apoA1- IgG and presented as unadjusted odds ratio (OR) and corresponding 95% confidence intervals (95% CI). Multivariate logistic regression analysis was used to determine the independent risk factors for development of diabetes and CVD after adjusting for potential covariates: age, gender, BMI, hypertension, smoking status, lipid parameters and physical activity and presented as adjusted OR. Receiver operating curve (ROC) analysis was performed to assess anti-apo-A1 IgG as a biomarker for CVD. Differences were considered significant with p value b0.05.
Association studies of anti-apoA-1 autoantibodies showed no significant association with T2DM (OR = 1.483, 95% CI = 0.507– 4.336, p = 0.4). However, a significant association with CVD was observed and subjects who were positive for anti-apoA-1 IgG were 8.5 times at risk to develop CVD compared to controls (OR = 8.518, 95% CI = 3.422–21.201, p b 0.0001) (Table 2). In diabetic patients, anti-apoA-1 autoantibodies significantly associated with CVD, diabetic patients who were positive for those antibodies showed 5.7 times higher risk to develop CVD (OR = 5.7, 95% CI = 2.616–12.596, p b 0.0001) (Table 3). Data remained significant after adjustment for other covariates: age, gender, BMI, hypertension, disease duration, lipid parameters, smoking status and physical activity. 3.3. Relationship between anti-apoA-1 IgG and lipid profile parameters
3. Results 3.1. Characteristics of the study population The study included 302 subjects categorized into 3 groups: T2DM (n = 102), T2DM complicated with CVD (n = 112) and control subjects (n = 88). Their age ranged from 40 to 72 years. The frequencies of CVDs in our patients were: 68% ischemic heart disease (IHD), 15% cerebrovascular disease, 10% macroangiopathy, 4% combined IHD and cerebrovascular disease, and 3% combined macroangiopathy and cerebrovascular disease. BMI, SBP and DBP were significantly higher in patient groups than in controls. CVD patients had higher frequency of hypertension (p b 0.0001), dyslipidemia (p b 0.0001) and smoking (p = 0.01),
The presence of anti-apo A-1 IgG was associated with higher serum concentrations of TC and non-HDL-C ratio in all groups (p = 0.013 and 0.017, respectively). A significant association with higher concentrations of LDL-C was observed in diabetic patients (p = 0.022), and with higher TC/HDL-C ratio in CVD patients (p = 0.031) (Table 4). 3.4. ROC analysis ROC analysis indicated anti-apoA-1 IgG as risk biomarker for CVD in T2DM patients with an area under the curve (AUC) value of 0.76. The sensitivity was 35.7% (95% CI 26.88–45.32) and the specificity was 91.2% (95% CI 83.91–95.89). Negative predictive value was 56.4% (95% CI 48.44–64.06) and positive predictive value was 81.6% (95% CI
Table 4 Relation between anti-apoA-1 IgG and Lipid profile parameters. Control
T2DM
T2DM + CVD
Parameter (mg/dl)
Positive for anti-apoA-1 Negative for anti-apoA-1 IgG (n = 6) IgG (n = 92)
Positive for anti-apoA-1 Negative for anti-apoA-1 IgG (n = 9) IgG (n = 93)
Positive for anti-apoA-1 IgG (n = 40)
Negative for anti-apoA-1 IgG (n = 72)
TC TG HDL-C LDL-C VLDL-C Non-HDL-C TC/HDL-C
188 131.28 55. 5 97.57 17.76 133 3.54
224 149.64 45 151.7 31 169 4.7
201.4 153.1 49 136 30.7 151 4.5
± ± ± ± ± ± ±
20.11 16.12 12.75 10.37 3.88 16.19 0.87
170 117.1 55.8 99.44 15.42 114.33 3.10
± ± ± ± ± ± ±
18.02⁎ 32.56 9.11 12.87 12.06 15.66⁎ 0.47
201 141.78 42 121.8 29.8 152.5 4.2
± ± ± ± ± ± ±
23.2 64.56 11.21 34.22 15 34.36 0.9
181.44 144.43 43.35 118.3 29.3 143.3 4
± ± ± ± ± ± ±
54.1⁎ 55.05 10.9 15.25⁎ 10.5 13.57⁎ 1.1
± ± ± ± ± ± ±
43.23 56.42 11 42.4 11.2 42.3 0.98
± ± ± ± ± ± ±
68.8⁎ 75.3 14.5 59.4⁎ 12.3 60⁎ 0.8⁎
Data presented as mean ± SD. Bonferroni multiple comparison test was applied. ⁎ p value b0.05.
Please cite this article as: El-Lebedy, D., et al., Anti-apolipoprotein A-1 autoantibodies as risk biomarker for cardiovascular diseases in type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.02.014
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D. El-Lebedy et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
67.98–91.24). Positive likelihood ratio was 4.05 (95% CI 2.07–7.92) and the disease prevalence was 52.3% (95% CI 45.42–59.19).
4. Discussion Recent studies indicated high incidence of anti-apo-A1 autoantibodies in many diseases associated with thrombotic events, such as SLE (32.5%), APS (22.9%) (Vuilleumier et al., 2014), acute coronary syndrome (ACS) (21%) (Vuilleumier et al., 2004, 2008), rheumatoid arthritis (RA) (17%) (Vuilleumier, Bas, et al., 2010), severe carotid stenosis (SCS) (20%) (Montecucco et al., 2011; Vuilleumier et al., 2013) and end stage renal disease (20%) (Pruijm et al., 2012). T2DM is a major independent risk factor for cardiovascular disease (CVD), the most common cause of morbidity and mortality among diabetic patients. The aim of this work was to investigate the presence of anti-apoA-1 autoantibodies in T2DM patients with and without CVD and to study potential association with CVD risk and the effect on plasma lipid profile parameters. To our knowledge, this is the first study investigating anti-apoA-1 autoantibodies in T2DM. Our results showed a significantly higher incidence of anti-apoA-1 IgG among CVD patients and subjects who were positive for those antibodies were at 8.5 times increased risk to develop CVD when compared to controls. In diabetic patients, anti-apoA-1 IgG showed significant association with CVD (p b 0.0001) and diabetic patients who were positive for the antibodies were at 5.7 times higher risk of CVD. ROC analysis indicated anti-apoA-1 IgG as risk biomarker for CVD in T2DM patients with an AUC of 0.76, sensitivity of 35.7% and specificity of 91.2%. Anti-apoA-1 IgG autoantibodies also associated with significantly higher serum concentrations of TC and non-HDL-C in all groups, and with higher concentrations of LDL-C in diabetic patients and higher TC/HDL-C ratio in CVD patients. Anti-apo-A1 autoantibodies may be present in general population but in low titers (up to 6.5%) (Vuilleumier et al., 2008; Vuilleumier, Bas, et al., 2010) and may be related to the vascular and immune ageing processes (Batuca et al., 2007). In our study, anti-apo-A1 IgG were detected in 6.1% of our control subjects. Our findings were consistent with results from previous studies, an association between anti-apoA-1 auto antibodies and cardiovascular risk was reported in autoimmune and non autoimmune diseases (Batuca et al., 2007; Montecucco et al., 2011; Pruijm et al., 2012; Radwan, El-Lebedy, Fouda, Elsorougy, & Fakhry, 2014; Vuilleumier et al., 2004, 2008; Vuilleumier, Bas, et al., 2010; Vuilleumier, Rossier, et al., 2010; Vuilleumier et al., 2013, 2014). In MI patients, anti-apoA-1 IgG was identified in 21% vs. 1% in healthy controls (p = 0.001) (Vuilleumier et al., 2004) and was associated with 4-fold increased risk of major adverse cardiac events (MACE) with an AUC value of 0.65 (p = 0.007) (Vuilleumier, Rossier, et al., 2010; Vuilleumier et al., 2011). Those autoantibodies also associated with atherosclerosis-related complications (Pagano et al., 2012) and increased atherosclerotic plaque vulnerability (Montecucco et al., 2011). While in SCS, anti-apoA-1 IgG was associated with 5-fold increase risk of MACE with an AUC of 0.74 (Vuilleumier et al., 2013). In RA patients, anti-apoA-1 IgG increased the cardiovascular risk 4-folds and was reported as the strongest predictor of major cardiovascular events with an AUC of 0.73, specificity of 50%, and a sensitivity of 90% (Vuilleumier, Bas, et al., 2010). Anti-apoA-1 IgG has been also associated with higher levels of matrix metalloproteinase (MMP-9), interleukin-8 (IL-8) and oxidized LDL; the major three markers and mediators of atherosclerotic plaque destabilization (Vuilleumier, Bas, et al., 2010; Vuilleumier, Bratt, et al., 2010). In a study on obese but otherwise healthy individuals, anti-apoA-1 IgG was reported as an independent predictor of coronary artery calcification (CAC) and coronary endothelial dysfunction with a negative predictive value of 94% and low IgG titer was indicated as a screening parameter to
identify those at low cardiovascular risk in whom coronary investigations could be overlooked (Quercioli et al., 2012). Auto antibodies directed against apoA-1 might affect the normal atheroprotective function of HDL (Hahn, 2010). The anti-atherogenic function of HDL could be partly explained by its main constituents, apoA-1 and PON1 (Guo, Li, Zhong, Tu, & Xie, 2012). Reduced PON1 concentration and/or activity have been reported as an independent risk factor for CVD (Dasgupta et al., 2011; El-Lebedy et al., 2014; Fuhrman, 2012; Gugliucci, Kotani, & Kimura, 2012; Gupta et al., 2012; Shenhar-Tsarfaty et al., 2013). ApoA-1 maintains optimum activity and stability of PON1 enzyme (Narshi et al., 2011) and enhances its binding to phospholipids (James & Deakin, 2004). Serum PON1 activity from apoA-1 knockout mice was found to be less stable than that of wild type siblings (Sorenson et al., 1999) and anti-apoA-1 antibodies were associated with reduced PON activity in plasma and in vitro inhibition assay confirmed a direct inhibition of the enzyme activity (Batuca et al., 2007). This subsequently reduced PON1 activity might play a role in the pathogenesis of T2DM itself. It was suggested that oxidative stress induced by reduced PON1 activity results in reducing glucose uptake from blood by muscle cells and develops into insulin resistance, results from previous studies support this hypothesis (Barbieri, Bonafe, & Marfella, 2002; Kordonouri, James, & Bennetts, 2001; Leviev, Kalix, Brulhart-Meynet, & James, 2001; Senti, Tomas, & Fito, 2003). Immune-mediated inflammation was recently indicated to play an important role in atherogenesis (Vuilleumier et al., 2014) and results from previous studies suggest that anti-apoA-1 IgG might be a mediator of inflammation and atherogenesis (Batuca et al., 2009; Vuilleumier, Bas, et al., 2010). Significant associations between high levels of anti-apoA-1 IgG and markers of oxidative stress, inflammation and endothelial dysfunction have been reported. In SLE patients, anti-apoA-1 IgG levels positively correlated with nitric oxide and inversely correlated with PON-1 activity suggesting that those autoantibodies might affect the antioxidant properties of HDL giving rise to a pro-oxidative microenvironment facilitating atherogenesis (Batuca et al., 2009; Vuilleumier, Bas, et al., 2010; Vuilleumier, Bratt, et al., 2010). In ACS, patients who were positive for anti-apoA-1 IgG had 5-fold higher levels of oxidized LDL (Vuilleumier et al., 2008). In a study involving MI patients, anti-apoA-1 IgG positively associated with serum levels of amyloid A protein which is involved in inflammation and cholesterol homeostasis (Vuilleumier et al., 2004). Also, in vitro studies demonstrated anti-apoA-1 IgG as an active mediator of atherosclerosis and atherosclerotic plaque vulnerability by promoting sterile inflammation through toll-like receptor-2 (TLR2)/CD14 complex (Pagano et al., 2012) and inducing specific neutrophil chemotaxis toward IL-8 and TNF-α (cytokines expressed within atherosclerotic plaques) (Montecucco et al., 2011). Anti-apoA-1 IgG may also act as pro-arythmogenic molecules through an aldosterone-dependent L-type calcium channels activation (Rossier et al., 2012). In mice model, passive immunization with anti-apoA-1 IgG increased atherogenesis and atherosclerotic plaque vulnerability (Montecucco et al., 2011) and decreased the antioxidant properties of HDL by inhibiting PON-1activity (Srivastava et al., 2011). In conclusion, our results indicate anti-apoA-1 IgG as a risk biomarker for CVD in type 2 diabetes patients. Considering the importance of identifying new and reversible CVD risk factors, these auto antibodies might represent a new possible therapeutic target and could allow the identification of a subset of reversible CVD patients who could benefit from specific immunomodulation in the future.
Financial disclosures None to declare.
Please cite this article as: El-Lebedy, D., et al., Anti-apolipoprotein A-1 autoantibodies as risk biomarker for cardiovascular diseases in type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.02.014
D. El-Lebedy et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
Funding disclosures This work was funded by the National Research Centre (Project No. 10010309).
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Please cite this article as: El-Lebedy, D., et al., Anti-apolipoprotein A-1 autoantibodies as risk biomarker for cardiovascular diseases in type 2 diabetes mellitus, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.02.014