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Anti-phospholipid antibody prevalence and association with subclinical atherosclerosis and atherothrombosis in the general population
Journal Pre-proof Anti-phospholipid antibody prevalence and association with subclinical atherosclerosis and atherothrombosis in the general population Carlo Selmi, Maria De Santis, Pier Maria Battezzati, Elena Generali, Simone Aldo Lari, Angela Ceribelli, Natasa Isailovic, Paola Zermiani, Sandra Neidhöfer, Torsten Matthias, Carlo A. Scirè, Damiano Baldassarre, Massimo Zuin PII:
S0167-5273(19)32822-0
DOI:
https://doi.org/10.1016/j.ijcard.2019.10.042
Reference:
IJCA 28094
To appear in:
International Journal of Cardiology
Received Date: 31 May 2019 Revised Date:
15 October 2019
Accepted Date: 24 October 2019
Please cite this article as: C. Selmi, M. De Santis, P.M. Battezzati, E. Generali, S.A. Lari, A. Ceribelli, N. Isailovic, P. Zermiani, S. Neidhöfer, T. Matthias, C.A. Scirè, D. Baldassarre, M. Zuin, Antiphospholipid antibody prevalence and association with subclinical atherosclerosis and atherothrombosis in the general population, International Journal of Cardiology (2019), doi: https://doi.org/10.1016/ j.ijcard.2019.10.042. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Anti-phospholipid antibody prevalence and association with subclinical atherosclerosis and atherothrombosis in the general population
Carlo Selmi MD PhD a,b*, Maria De Santis MD PhD a*, Pier Maria Battezzati MD c, Elena Generali MD a, Simone Aldo Lari MD a, Angela Ceribelli MD PhDa, Natasa Isailovic MSc a, Paola Zermiani BSc c, Sandra Neidhöfer BSc d, Torsten Matthias PhD d, Carlo A. Scirè MD PhD e, Damiano Baldassarre PhD b,f, and Massimo Zuin MD c
Affiliations: a Rheumatology and Clinical Immunology, Humanitas Clinical and Research CenterIRCCS, Rozzano (MI), Italy; b Department of Biomedical Science and Translational Medicine, University of Milan, Italy; cLiver and Gastroenterology Unit, San Paolo Hospital Department of Health Sciences, University of Milan, Italy; dAesku Diagnostics GmbH & Co. KG, Wendelsheim, Germany; e Epidemiology Unit, Italian Society of Rheumatology, Milan, Italy; f Centro Cardiologico Monzino IRCCS, Milan, Italy. * These authors contributed equally to the study
Corresponding author: Carlo Selmi MD PhD, Division of Rheumatology and Clinical Immunology, Humanitas Clinical and Research Center, via A. Manzoni 56, 20089 Rozzano, Milan, Italy; tel +39-02-8224-5129, fax +39-02-8224-2298, email [email protected]
Disclosures: Dr. Torsten Matthias is the head of the non-profitable Aesku.Kipp Institute and the owner of Aesku.Diagnostics GmbH& Co.KG. Sandra Neidhöfer is employed by Aesku.Kipp Institute. Funding: Lombardia region (DG Sanità 08/07/2008 n. 7364).
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Highlights •
Positive aPL are common in the population but their CV significance is unclear;
•
CV events are more frequent in subjects with aPL and an elevated CV risk;
•
aPLs are associated with ultrasound signs of atherosclerosis.
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ABSTRACT Background. There is no agreement on the prevalence of anti-phospholipid antibodies (aPLs) and the correlation with atherosclerosis and cardiovascular (CV) events in the general population. Methods. We performed a cross-sectional study on 1,712 randomly enrolled subjects from a Northern Italian city to investigate the presence of aPLs and the association with subclinical atherosclerosis (using the carotid artery intima media thickness measured as inter-adventitia common carotid artery diameters - ICCAD) and retrospectively collected CV factors and events (i.e. acute myocardial infarction, stroke, and peripheral obliterans arterial vasculopathy) using physician-assisted questionnaires. We tested serum IgG, IgM, and IgA anti-cardiolipin, antibeta2glycoprotein I (aGPI), and anti-phosphatidylserine-prothrombin antibodies. Results. Positive aPLs were found in 15.1% of the subjects, with no differences between sex but with higher rates in older subjects. Carotid subclinical atherosclerosis was more frequent in aPL positive subjects; more specifically, aGPI IgA were associated with higher ICCAD average (adjusted beta 0.51, 95% confidence interval (CI)0.17-0.84; p= 0.003). A positive history of CV events was also more frequent in aPL positive subjects (odds ratio (OR) 1.67, 95%CI 1.08-2.54; p=0.012), particularly peripheral obliterans arterial vasculopathy (OR 2.02; 95%CI 1.14-3.57; p=0.015). Among subjects with a Framingham risk score >20, and/or diabetes, and/or body mass index >35Kg/m2, aPL positivity was associated to the highest risk of CV events (OR 2.52, 95%CI 1.24-5.11; p=0.011). Conclusions. APL prevalence in the general population is higher than previously reported. CV events and subclinical atherosclerosis are more frequent in the presence of aPL, particularly when a high CV risk coexists.
Key words. Cardiovascular events; antiphospholipid antibodies; epidemiology; serum biomarkers
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1. Introduction. Anti-phospholipids (aPLs) are a heterogeneous group of autoantibodies which may define the homonymous syndrome [1] manifesting with arterial/venous thrombosis and/or recurrent miscarriages/placental insufficiency [2]. However, the primary aPL syndrome is found in 40-50 cases /100,000 adults [3] and is significantly outnumbered by the frequency of aPL positive subjects. In fact, aPLs can be positive also in other autoimmune and non-autoimmune diseases, such as cancer or infections, as well as in healthy individuals [1]: aPLs have been detected in ~45% of the patients with systemic lupus erythematosus [4], 7% of the women with recurrent miscarriages [5, 6], and 8% of blood donors [1, 7]. APLs included in classification criteria for primary aPL syndrome are lupus anticoagulant, IgM and IgG anti-cardiolipin (aCL), and IgM and IgG anti-β2 glycoprotein I (aGPI) antibodies [8]; however, a clear estimate of their prevalence or sex predominance in the general population lacks. Additional aPLs, defined as non-criteria aPLs, include anti-phosphatidylserine/prothrombin (aSP) [9], anti-phosphatidic acid, antivimentin/cardiolipin complex, anti-protein C/S, anti-factor XII, anti-factor X, anti-annexin A5/A2, and anti-D1 [10]. The major causes of mortality in the aPL syndrome are thrombotic events, but a number of nonthrombotic events are frequently reported, such as migraine, livedo, ulcers, and heart valve diseases. These manifestations are thought to be associated with the aPL-mediated endothelial activation [11]. To date, it has not been investigated whether such endothelial activation could be also implicated in the development of atherosclerosis or contributes to atherothrombosis in patients with aPLs, as suggested by small cohorts [10]. We took advantage of a unique cross-sectional study performed in a Northern Italian population to study aPLs and (i) the prevalence in the adult general population, (ii) the correlation with subclinical atherosclerosis, and (iii) the association with CV events.
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2. Materials and methods 2.1 Study design and population The cross-sectional CA.ME.LI.A (CArdiovascular risk, MEtabolic syndrome, LIver, and Autoimmunity) study included 2,555 subjects (age 18-75 years) that were randomly selected from the active voting lists of the Northern Italian city of Abbiategrasso (Milan area, population 32,000 inhabitants) in 2009. Subjects considered for randomization responded to the following criteria: residence in Abbiategrasso; medical assistance in Abbiategrasso; born between 1934-1980. A freely available computer-based software (www.randomizer.org) has been used for randomization and the randomized subjects have been contacted by phone call. The study has been based on the STROBE document for observational study (attached as supplementary material). All subjects underwent physical examination and blood tests including C-reactive protein (CRP), total cholesterol, triglycerides, homocysteine, glucose, and complete blood count to assess CV risk. The medical history was assessed with a physician-assisted questionnaire (Supplemental material) and the following information were registered: arterial hypertension, diabetes, smoking habit, height and weight, physical activity, history of myocardial infarction, stroke, and/or peripheral limb arteriopathy. Within this cohort, a subgroup of 1,712 randomly selected subjects that did not differ significantly in terms of demographic and clinical characteristics (data not shown) was used for the present study, as the sample size was estimated sufficient to identify an expected aPL prevalence of 7.5% [1, 5-7], using an alpha error of 0.05, and a power of 80%. The primary study endpoints were to establish the prevalence of aPLs, the correlation with ultrasonographic parameters of subclinical atherosclerosis, and the association with history of CV
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events. The secondary endpoint was to identify possible differences among the three main aPL specificity and isotypes. The local institutional review board approved the present study and all subjects signed an informed consent.
2.2 APL analysis The sera of the 1,712 selected subjects were tested for aCL, aGPI, and aSP antibodies using commercially available ELISA tests (AESKU diagnostic, Wendelsheim, Germany) to identify IgG, IgM, and IgA isotypes for each autoantibody. Results were expressed as international units (IU) and cut-off values were established at 15 IU, as recommended by the manufacturer instructions, according to the Clinical and Laboratory Standards Institute guideline for reference intervals [12]. The range of results was 0-300 IU and values >40 IU were considered as high titers [13, 14]. Lupus anticoagulant could not be tested since fresh samples were not available.
2.3 Carotid ultra-sonographic analysis The quantitative evaluation of carotid intima-media thickness (IMT) was performed by trained sonographers unaware of clinical information using a 7.5 MHz probe [15] on 1:3 randomly selected subjects (n=563, no significant differences compared to the cohort tested for aPLs in terms of demographic and clinical characteristics; data not shown). Briefly, the far walls of the left and right common carotids, bifurcations, and internal carotids were visualized in anterior, lateral, and posterior angles and recorded. Carotid IMT measurements were performed in a centralized laboratory (Centro Cardiologico Monzino IRCCS, Milan, Italy) using a dedicated software (M’Ath, Metris SRL France). The ultra-sonographic variables used in the statistical analyses were the mean
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maximum intima-media thickness (IMTmean-max), the atherosclerotic plaques (defined as maximum IMT (IMTmax) >1.5 mm) [15], and the inter-adventitia common carotid artery diameters (ICCAD).
2.4 CV risk factors and events CV risk factors, such as arterial hypertension, age (≥ 50 years, considering CV risk based on age [16]; the mean age of menopause in Italy based on ICARUS study group [17] and the risk of CV events based on menopause onset [18]), smoking habit (>1 cigarette/day for at least one year), body mass index (BMI) ≥25 kg/m2, physical inactivity (no physical activity except work-related activity), type 1 and 2 diabetes, CRP ≥ 0.2 mg/dL [19], hypercholesterolemia (total cholesterol > 200 mg/dL), hypertriglyceridemia (>150 mg/dL ) [20], hyperhomocysteinemia (> 15umol/L) [21], and history of CV clinical events, such as acute myocardial infarction (ST elevation myocardial infarction-STEMI or non ST elevation myocardial infarction-NSTEMI), hemorrhagic or ischemic stroke, and peripheral limb arteriopathy (obliterans vasculopathy requiring surgery or prostanoid therapy), were recorded for each subject by the study physicians. The overall Framingham risk score was calculated using established formulas [22]. The Framingham risk score was chosen over the ESC score because the latter score is not applicable beyond age 65, while the former is intended for use in patients up to 79 years [23, 24].
2.5 Statistical analysis Data were analyzed using descriptive and inferential methods. We used both parametric (χ2) and non-parametric (Mann-Whitney) tests, when appropriate. To assess the association of aPLs with carotid ultra-sonographic measurements and CV events, univariate and multivariate analyses were performed, after adjusting for pre-specified confounders including CV risk factors. Results were expressed as odds ratio (OR) and 95% confidence intervals
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(95% CI) for binary, and as beta coefficient (95% CI) for continuous outcomes. All analyses were performed using STATA 13 for Macintosh (StataCorp, College Station, TX, USA), and p values<0.05 were considered statistically significant.
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3. Results 3.1 aPL prevalence The demographic and clinical characteristics of the study population are shown in Table 1. The overall prevalence of any aPL (defined as at least one positive aPL) was 15.1% (95% CI 13.416.8%) with the highest frequency observed in older subjects (18.1%; p<0.002) (Table 2). Hightiter aPL were found in 3.3% of subjects, multiple specificities in 2%. ACL were detected in 1.5% of the subjects (Table 2) with 1% at high titer. AGPI were positive in 4.3% of the subjects (Table 2) with 1.2% at high titer and the highest prevalence in older subjects (6.7%; p<0.001). AGPI IgA were more frequently observed in women (3.1%, p=0.040) and in older subjects, independently of sex (4%, p<0.001). ASP were the most frequently detected aPL, being found in 11.7% of the cases (Table 2) with 1.7% at high titer and in 9.8% of the cases as the only positive test (data not shown). ASP IgA were not detected.
3.2 CV risk factors The subjects with hypercholesterolemia had more frequently high-titer aPLs (39, 69.6% versus 762, 52.4% in aPL negative subjects, p=0.025 adjusted for sex and age), while subjects with high triglycerides had more frequently aGPI (18, 24.7% vs 220, 15.1% in aPL negative subjects, p=0.035 adjusted for sex and age; supplementary Table A) Interestingly, higher LDL levels were found in subjects with high-titer aPLs (median 142.5 (range 120-169) vs 133 (113-156), p= 0.021). Conversely, aPL prevalence did not differ according to physical inactivity, smoking, arterial hypertension, diabetes, triglycerides, and CRP (data not shown).
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We found a significant association between elevated homocysteine and all aPL specificities, including IgG aCL (10, 66.7%; p<0.001), aGPI (27, 37%; p=0.026), and IgG aSP (53, 33.8%; p=0.022; data not shown). The presence of more than one aPL was associated to elevated homocysteine in 60% of the cases (19 versus 368, 25.3% in aPL negative subjects, p<0.001 adjusted for sex and age; supplementary Table A). Positive aPL subjects had higher Framingham risk score at 10 years compared to aPL negative subjects; moreover, Framingham risk score was higher in case of more aPL specificities and high titer aPLs (Table 3). Of note, the highest Framingham risk score (>20%) was most frequently observed with high titer IgM aCL, aGPI, and aSP (40%, p<0.001; 33.3%, p=0.018; 25%, p=0.024; respectively; Table 3).
3.3 Carotid ultra-sonographic measurements APL positive subjects had more frequently atherosclerotic plaque and higher mean values of IMT Mean-Max and ICCAD (Table 4). Specifically, aCL IgM positive subjects had a significantly higher frequency of atherosclerotic plaques (OR 5.41, 95%CI 0.61-65.2, p=0.042), while aGPI IgA positive subjects had significantly increased IMTMean-Max (beta 0.04, 95%CI 0.004-0.072; p=0.027) and ICCAD (beta 0.02, 95%CI 0.009-0.041; p=0.002; Table 4). Multivariate analyses for carotid ultrasound measurements adjusted for pre-specified confounders are shown in supplementary Table B: only IgA aGPI were independently associated with increased ICCAD (beta 0.51, 95%CI 0.17-0.84; p =0.003). Considering sex and age, we observed that women younger than 50 years with aPLs had an increased ICCAD (6.74 IQR 6.2-7.0 vs 6.47 IQR 6.19-7.75 in women < 50 years without aPLs; at multivariate analysis beta 0.22, 95%CI 0.03-0.41; p=0.025), especially, in case of IgG aSP (beta 0.24, 95%CI 0.02-0.46; p=0.03, data not shown).
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3.4 CV events Overall CV events were more frequently reported in the aPL positive population (34, 13.2% versus 121, 8.3% in aPL negative subjects, p=0.012; OR 1.67 95%CI 1.12-2.50). Interestingly, while in the aPL negative subjects AMI and peripheral arteriopathy had a similar frequency (3.4% compared to 1.6% of stroke cases), in aPL positive subjects peripheral arteriopathy (6.6%) almost doubled the prevalence in a PL negative subjects and was significantly associated to anti-PLs independently of other CV risk factors (OR 2.02, 95%CI 1.07-3.64, p =0.013; Table 4). AMI and stroke were also more frequent in aPL positive subjects (3.9% and 2.7%, respectively; Table 4). The subjects with high level of homocysteine reported CV events more frequently and independently of aPL status (43, 11.7% versus 60, 5.5%; OR 2.3 95%CI 1.5-3.5, p<0.001), but those with high homocysteine and coexisting aPLs had a higher risk (OR 3.01 95%CI1.4-6.0). Finally, when analyzing a high-risk population defined by Framingham risk score >20, and/or diabetes, and/or BMI >35, aPL positivity was associated with a higher risk for any CV event (OR 2.52, 95%CI 1.24-5.11, p=0.011).
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4. Discussion Our study represents the largest population-based analysis of aPL prevalence and the first attempt to determine the association between aPLs, subclinical atherosclerosis, and clinical CV events in the general population. From an epidemiological point of view, we report a high prevalence of aPLs without sex differences and with an increasing trend with age. However, the prevalence was similar to other reports [1, 5-7] when considering only aPLs included in classification criteria, such as aCL and aGPI in our cases, and only high titer (not having a 12-week confirmation test). ASP was found to be the most abundant aPL and in up to 10% of the cases was the only aPL detectable; moreover, ASP were significantly associated with increased ICCAD in childbearing-age women. Furthermore, one not routinely tested aPL, i.e. aGPI IgA, was found to be more frequent in women and the only aPL significantly and independently associated to increased ICCAD. CV events were overall more frequently found among aPL positive subjects, even if only peripheral arteriopathy was significantly associated to the aPL; moreover, in subjects with higher Framingham score, diabetes, higher BMI or hyper-homocysteinemia the risk of CV events was 2.5-3 folds higher in presence of aPLs. Overall our data suggest a possible independent and additive role of aPLs in the atherogenesis. Being CV events registered retrospectively, it can be suspected that aPLs could be an epiphenomenon, but the association with subclinical atherosclerosis seems to support an important role of aPLs. The association between hyper-homocysteinemia and aPL is of particular interest given the pro-thrombotic action of homocysteine via phosphatidylserine exposure on endothelial cells, myocardiocytes, and red blood cells [25, 26], thus suggesting that the development of aPLs could contribute to the thrombophilic status in hyper-homocysteinemia. Conflicting lines of clinical evidence suggest a predisposition to accelerated atherosclerosis mainly in primary aPL syndrome [27-32]. Basic science supports a causative atherogenic link as early
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atherosclerosis can be induced in low density lipoprotein (LDL)-receptor-deficient mice immunized with GPI [33], suggesting that antibodies targeting GPI could suppress the anti-atherogenic role of GPI. Similarly, aCL increases atherogenesis in the LDL-knockout mice [34]. A number of additional immune-mediated mechanisms have been also described, including the cross reactivity between aPLs and anti–oxidized LDL [35] and the increased uptake of oxidized LDL/GPI complex by macrophages exposed to aGPI [33]. A meta-analysis including 668 subjects with aPL syndrome demonstrated the relationship between aPLs and subclinical atherosclerosis [36], and, most recently, a cross-sectional study reported subclinical atherosclerosis in asymptomatic aPL positive subjects together with a correlation with title, triple or double positivity, and with aGPI presence over aCL [37]. The observation that aPL are more frequent in older subjects is in line with what is reported for antinuclear antibody (ANA) in the general population [38], while the similar rates in men and women may reflect a different sex epidemiology for aPLs respect to ANA [39]. While the present study represents the largest population-based analysis for aPLs, we are also aware of study limitations, particularly, the lack of a confirmation test after 12 weeks as recommended for positive sera in clinical practice and of lupus anticoagulant determination as this cannot be performed on frozen sera. Moreover, we did not observe an increasing risk of CV events in subjects with more than one positive autoantibody or high titers, maybe due to the low prevalence of this positivity or to the small number of CV events. Further, we used a questionnaire for the medical history that was custom created to include all the major objectives and clinical areas of the study which would have not been possible at once; we cannot exclude a role of inflammation because high sensitivity CRP was not available. In the end, a prospective follow up of subjects with aPLs regarding CV events should be assessed, because from a cross-sectional study we were not able to evaluate CV events occurring in the meantime.
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5. Conclusions The prevalence of aPLs in the general population is higher than previously reported and increases with age while being equally represented among men and women. APLs are associated with subclinical atherosclerosis and a higher prevalence of CV events, especially in subjects with a higher CV risk.
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Table 1. Characteristics of the study population arrayed based on sex and age. Total 1,712 47.2 (3761.3)
Women 852 (49.8%) 47.2 (36.861.9)
BMI >25 kg/m2
875 (51.1)
356 (41.8)
519 (60.3)
369 (38.6)
506 (60)
Smoking
438 (25.6)
185 (21.7)
253 (29.4)
303 (31.7)
135 (17.9)
Ex-smokers
401 (23.4)
138 (16.2)
263 (30.6)
168 (17.6)
233 (30.9)
754 (44)
396 (46.5)
358 (41.6)
416 (43.5)
338 (44.8)
378 (22.2)
189 (22.2)
189 (22)
65 (6.8)
313 (41.6)
99 (5.8) 622 (36.3)
33 (3.9) 341(54.8)
66 (7.7) 281 (45.2)
11 (1.2) 296 (47.6)
88 (11.7) 326 (52.4)
268 (15.6)
89 (10.4)
179 (20.8)
134 (14)
134 (17.8)
902 (52.7)
474 (55.6)
427 (49.7)
412 (43.1)
490 (64.8)
448 (26.2)
137 (16.1)
310 (36)
207 (21.7)
241 (31.9)
128 (22.7)
54 (42.2)
74 (57.8)
14 (10.9)
114 (89.1)
0.99 (0.861.22) 7.06 (6.677.57)
0.96 (0.851.16) 6.78 (6.407.22)
1.02 (0.881.27) 7.31 (6.937.88)
0.88 (0.820.98) 6.80 (6.447.19)
1.22 (1.031.54) 7.45 (7.028.04)
AMI
59 (3.4)
20 (33.9)
39 (66.1)
10 (17)
49 (83)
Stroke
30 (1.8)
14 (46.7)
16 (53.3)
3 (10)
27 (90)
PA
66 (3.9)
30 (45.5)
36 (54.5)
3 (4.5)
63 (95.5)
Age, m (IQR)
Physical inactivity Arterial hypertension Diabetes CRP ≥ 2 mg/L Triglycerides >150 mg/dL Cholesterol >200 mg/dL Homocysteine >15 umol/L
Men Age<50 Age≥50 860 (50.2%) 956 (55.8%) 756 (44.2%) 47.2 (37.260.6)
Outcomes IMT Max avg>1.5 n (%) IMT Mean Max m (IQR) ICCAD Avg m (IQR)
m: median; IQR: inter-quantile range; BMI: body mass index; CRP: C-reactive protei; IMT: intima-media thickness; avg: average; ICCAD: inter-adventitia Common Carotid Artery Diameter; AMI: acute myocardial infarction; PA: peripheral arteriopathy. Data are expressed as numbers(percentages) or median (IQR)
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Table 2. Prevalence of aPLs in the population according to age and sex. Antibody
Total
Women
Men
Age<50
Age≥50
1,712
860 (50.2%) 739 (85.9)
956 (55.8%) 835 (87.3)
756 (44.2%) 619 (81.9)
aPL negative
1,454 (84.9)
852 (49.8%) 715 (83.9)
aPL positive
258 (15.1)
137 (16.1)
121 (14.1)
121 (12.7)
137(18.1)*
aCL
26 (1.5)
10 (1.2)
16 (1.9)
10 (1)
16 (2.1)
aGPI
73 (4.3)
44 (5.2)
29 (3.4)
22 (2.3)
51 (6.7)*
aSP 201 (11.7) 103 (12.1) 98 (11.4) 104 (10.9) 97 (12.8) Data are presented as number (%). aPL: antiphospholipid antibodies; aCL: anti-cardiolipin antibodies; aGPI: anti-β2-glycoprotein antibodies I; aSP: anti-serin/prothrombin antibodies. * p<0.05 age<50 versus age ≥50.
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Table 3. Prevalence of Framingham risk classes according to the aPL status.
<5%
6-10%
11-15%
16-20%
>20%
aPL negative
952 (65.5)
186 (12.8)
82 (5.6)
124 (8.5)
110 (7.6)
aPL positive
156 (60.5)
40 (15.5)
16 (6.2)
21 (8.1)
25 (9.7)
>1 aPLs
15 (42.9)
8 (22.9)
5 (14.3)
3 (8.6)
4 (11.2)
aPL HT
31 (55.5)
9 (16.1)
1 (1.8)
8 (14.3)
7 (12.5)
aCL
10 (38.5)*
6 (23.1)
2 (7.7)
3 (11.5)
5 (19.2)*
aCL IgG
7 (46.7)
3 (20)
1 (6.7)
1 (6.7)
3 (20)
aCL IgM
4 (22.2)*
5 (27.8)
1 (5.6)
3 (16.7)
5 (27.8)*
aCL IgA
3 (75)
0
0
0
1 (25)
42 (57.5)
12 (16.4)
5 (6.9)
5 (6.9)
9 (12.3)
aGPI IgG
11 (55)
3 (15)
0
2 (10)
4 (20)*
aGPI IgM
17 (60.7)
4 (14.3)
2 (7.1)
3 (10.7)
2 (7.1)
aGPI IgM HT
2 (33.3)
0
0
2 (33.3)*
2 (33.3)*
aGPI IgA
18 (51.4)
7 (20)
4 (11.4)
1 (2.9)
5 (14.3)
aSP
121 (60.2)
32 (15.9)
14 (7)
17 (8.5)
17 (8.5)
aSP IgG
92 (58.6)
23 (14.7)
13 (8.3)
14 (8.9)
15 (9.6)
aSP IgM
40 (61.5)
12 (18.5)
1 (1.5)
5 (7.7)
7 (10.8)
aSP IgM HT
7 (58.3)
2 (16.7)
0
0
3 (25)*
aGPI
Data are presented as number (%). aPL: antiphospholipid antibodies; HT: high titer; aCL: anti-cardiolipin antibodies; aGPI: anti-β2-glycoprotein antibodies I; aSP: anti-serin/prothrombin antibodies. *= p<0.05 compared to aPL negative subjects.
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Table 4. IMT measurements and clinical cardiovascular outcomes in the cohort studied according to aPL status.
aPL negative aPL positive >1 aPLs aPL HT
IMT max avg>1.5
IMT mean max
ICCAD avg
AMI
Stroke
PA
n (%)
m (IQR)
m (IQR)
n (%)
n (%)
n (%)
0.98 (0.86-1.21)
7.05 (6.66-7.57)
49 (3.4)
23 (1.6)
49 (3.4)
1.03 (0.88-1.30) 0.97 (0.88-1.04) 1.03 (0.98-1.04)
7.12 (6.77-7.66) 6.91 (6.8-7.24) 7 (6.9-7.22)
10 (3.9) 1 (1.8) 0
7 (2.7) 0 0
17 (6.6)* 0 0
106/488 (21.7) 22/75 (29.3) 2/16 (12.5) 1/5 (20)
aCL aCL IgG aCL IgM aCL IgA
3/8 (37.5) 1/6 (16.7) 3/5 (60)* 0
1.01 (0.89-1.21) 0.94 (0.88-1.04) 1.03 (0.98-1.39) 0.87 (0.77-0.98)
7.03 (6.86-7.30) 7.26 (6.96-7.38) 7.10 (6.87-7.22) 7.41 (7.22-7.60)
1 (3.9) 0 1 (5.6) 0
0 0 0 0
1 (3.9) 0 1 (5.6) 0
aGPI aGPI IgG aGPI IgM aGPI IgA
7/21 (33.3) 1/6 (16.7) 3/6 (50) 3/9 (33.3)
1.12 (0.96-1.5)* 1 (0.90-1.12) 1.12 (0.95-1.37) 1.22 (1.02-1.66)*
7.22 (6.84-7.92) 7.04 (6.67-7.92) 7.05 (6.84-7.41) 7.48 (6.92-8.58)*
1 (1.4) 0 1 (3.6) 1 (2.9)
2 (2.7) 0 1 (3.6) 1 (2.9)
5 (6.9) 1 (5) 2 (7.1) 3 (8.6)
aSP aSP IgG aSP IgM
17/59 (28.8) 14/47 (29.8) 5/15 (33.3)
1.02 (0.85-1.27) 0.98 (0.85-1.28) 1.03 (0.85-1.30)
7.09 (6.73-7.49) 7.18 (6.78-7.72) 6.9 (6.33-7.26)
9 (4.5) 6 (3.8) 3 (4.6)
5 (2.5) 4 (2.6) 1 (1.5)
12 (6) 10 (6.4) 3 (4.6)
Data are expressed as number (%) or median (interquartile range) m: median; aPL: antiphospholipid antibodies; HT: high titer; aCL: anti-cardiolipin antibodies; aGPI: anti-β2-glycoprotein antibodies I; aSP: anti-serin/prothrombin antibodies; IMT: intima-media thickness; avg: average; ICCAD: inter-adventitia Common Carotid Artery Diameter; AMI: acute myocardial infarction; PA: peripheral arteriopathy. *= p<0.05 compared to aPL negative subjects.
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7. References [1] Garcia D, Erkan D. Diagnosis and Management of the Antiphospholipid Syndrome. N Engl J Med. 2018;378:2010-21. [2] Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL, Cervera R, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost. 2006;4:295-306. [3] Gomez-Puerta JA, Cervera R. Diagnosis and classification of the antiphospholipid syndrome. J Autoimmun. 2014;48-49:20-5. [4] Love PE, Santoro SA. Antiphospholipid antibodies: anticardiolipin and the lupus anticoagulant in systemic lupus erythematosus (SLE) and in non-SLE disorders. Prevalence and clinical significance. Ann Intern Med. 1990;112:682-98. [5] Bowman ZS, Wunsche V, Porter TF, Silver RM, Branch DW. Prevalence of antiphospholipid antibodies and risk of subsequent adverse obstetric outcomes in women with prior pregnancy loss. J Reprod Immunol. 2015;107:59-63. [6] Vinatier D, Dufour P, Cosson M, Houpeau JL. Antiphospholipid syndrome and recurrent miscarriages. Eur J Obstet Gynecol Reprod Biol. 2001;96:37-50. [7] McIntyre JA, Wagenknech DR, Waxman DW. Frequency and specificities of antiphospholipid antibodies (aPL) in volunteer blood donors. Immunobiology. 2003;207:59-63. [8] Pengo V, Bison E, Zoppellaro G, Padayattil Jose S, Denas G, Hoxha A, et al. APS - Diagnostics and challenges for the future. Autoimmun Rev. 2016. [9] Fabris M, Giacomello R, Poz A, Pantarotto L, Tanzi N, Curcio F, et al. The introduction of antiphosphatidylserine/prothrombin autoantibodies in the laboratory diagnostic process of antiphospholipid antibody syndrome: 6 months of observation. Auto Immun Highlights. 2014;5:63-7. [10] Arachchillage DJ, Cohen H. Use of new oral anticoagulants in antiphospholipid syndrome. Curr Rheumatol Rep. 2013;15:331.
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[11] Meroni PL, Borghi MO, Grossi C, Chighizola CB, Durigutto P, Tedesco F. Obstetric and vascular antiphospholipid syndrome: same antibodies but different diseases? Nat Rev Rheumatol. 2018;14:433-40. [12] Bor MV, Jacobsen IS, Gram JB, Sidelmann JJ. Revisiting the Phadia/EliA cut-off values for anticardiolipin and anti-beta2-glycoprotein I antibodies: a systematic evaluation according to the guidelines. Lupus. 2018;27:1446-54. [13] Lakos G, Favaloro EJ, Harris EN, Meroni PL, Tincani A, Wong RC, et al. International consensus guidelines on anticardiolipin and anti-beta2-glycoprotein I testing: report from the 13th International Congress on Antiphospholipid Antibodies. Arthritis Rheum. 2012;64:1-10. [14] Bertolaccini ML, Amengual O, Andreoli L, Atsumi T, Chighizola CB, Forastiero R, et al. 14th International Congress on Antiphospholipid Antibodies Task Force. Report on antiphospholipid syndrome laboratory diagnostics and trends. Autoimmun Rev. 2014;13:917-30. [15] Baldassarre D, Hamsten A, Veglia F, de Faire U, Humphries SE, Smit AJ, et al. Measurements of carotid intima-media thickness and of interadventitia common carotid diameter improve prediction of cardiovascular events: results of the IMPROVE (Carotid Intima Media Thickness [IMT] and IMT-Progression as Predictors of Vascular Events in a High Risk European Population) study. J Am Coll Cardiol. 2012;60:1489-99. [16] Lloyd-Jones DM, Leip EP, Larson MG, D'Agostino RB, Beiser A, Wilson PW, et al. Prediction of lifetime risk for cardiovascular disease by risk factor burden at 50 years of age. Circulation. 2006;113:791-8. [17] Meschia M, Pansini F, Modena AB, de Aloysio D, Gambacciani M, Parazzini F, et al. Determinants of age at menopause in Italy: results from a large cross-sectional study. ICARUS Study Group. Italian Climacteric Research Group Study. Maturitas. 2000;34:119-25. [18] Muka T, Oliver-Williams C, Kunutsor S, Laven JS, Fauser BC, Chowdhury R, et al. Association of Age at Onset of Menopause and Time Since Onset of Menopause With
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Cardiovascular Outcomes, Intermediate Vascular Traits, and All-Cause Mortality: A Systematic Review and Meta-analysis. JAMA Cardiol. 2016;1:767-76. [19] Halcox JP, Roy C, Tubach F, Banegas JR, Dallongeville J, De Backer G, et al. C-reactive protein levels in patients at cardiovascular risk: EURIKA study. BMC Cardiovasc Disord. 2014;14:25. [20] Nelson RH. Hyperlipidemia as a risk factor for cardiovascular disease. Prim Care. 2013;40:195-211. [21] Kang SS, Rosenson RS. Analytic Approaches for the Treatment of Hyperhomocysteinemia and Its Impact on Vascular Disease. Cardiovasc Drugs Ther. 2018;32:233-40. [22] D'Agostino RB, Sr., Vasan RS, Pencina MJ, Wolf PA, Cobain M, Massaro JM, et al. General cardiovascular risk profile for use in primary care: the Framingham Heart Study. Circulation. 2008;117:743-53. [23] Mortensen MB, Nordestgaard BG, Afzal S, Falk E. ACC/AHA guidelines superior to ESC/EAS guidelines for primary prevention with statins in non-diabetic Europeans: the Copenhagen General Population Study. Eur Heart J. 2017;38:586-94. [24] Mortensen MB, Falk E. Limitations of the SCORE-guided European guidelines on cardiovascular disease prevention. Eur Heart J. 2017;38:2259-63. [25] Xie R, Jia D, Gao C, Zhou J, Sui H, Wei X, et al. Homocysteine induces procoagulant activity of red blood cells via phosphatidylserine exposure and microparticles generation. Amino Acids. 2014;46:1997-2004. [26] Kam A, Li KM, Razmovski-Naumovski V, Nammi S, Chan K, Li GQ. Combination of TNFalpha, homocysteine and adenosine exacerbated cytotoxicity in human cardiovascular and cerebrovascular endothelial cells. Cell Physiol Biochem. 2012;30:805-14. [27] Belizna CC, Richard V, Primard E, Kerleau JM, Cailleux N, Louvel JP, et al. Early atheroma in primary and secondary antiphospholipid syndrome: an intrinsic finding. Semin Arthritis Rheum. 2008;37:373-80. 21
[28] Medina G, Casaos D, Jara LJ, Vera-Lastra O, Fuentes M, Barile L, et al. Increased carotid artery intima-media thickness may be associated with stroke in primary antiphospholipid syndrome. Ann Rheum Dis. 2003;62:607-10. [29] Der H, Kerekes G, Veres K, Szodoray P, Toth J, Lakos G, et al. Impaired endothelial function and increased carotid intima-media thickness in association with elevated von Willebrand antigen level in primary antiphospholipid syndrome. Lupus. 2007;16:497-503. [30] Margarita A, Batuca J, Scenna G, Alves JD, Lopez L, Iannaccone L, et al. Subclinical atherosclerosis in primary antiphospholipid syndrome. Ann N Y Acad Sci. 2007;1108:475-80. [31] Jimenez S, Garcia-Criado MA, Tassies D, Reverter JC, Cervera R, Gilabert MR, et al. Preclinical vascular disease in systemic lupus erythematosus and primary antiphospholipid syndrome. Rheumatology (Oxford). 2005;44:756-61. [32] Bilora F, Boccioletti V, Girolami B, Zanon E, Armani M, Petrobelli F, et al. Are antiphospholipid antibodies an independent risk factor for atherosclerosis? Clin Appl Thromb Hemost. 2002;8:103-13. [33] Matsuura E, Kobayashi K, Tabuchi M, Lopez LR. Oxidative modification of low-density lipoprotein and immune regulation of atherosclerosis. Prog Lipid Res. 2006;45:466-86. [34] George J, Afek A, Gilburd B, Levy Y, Blank M, Kopolovic J, et al. Atherosclerosis in LDLreceptor knockout mice is accelerated by immunization with anticardiolipin antibodies. Lupus. 1997;6:723-9. [35] Wu R, Svenungsson E, Gunnarsson I, Haegerstrand-Gillis C, Andersson B, Lundberg I, et al. Antibodies to adult human endothelial cells cross-react with oxidized low-density lipoprotein and beta 2-glycoprotein I (beta 2-GPI) in systemic lupus erythematosus. Clin Exp Immunol. 1999;115:561-6. [36] Ambrosino P, Lupoli R, Di Minno A, Iervolino S, Peluso R, Di Minno MN. Markers of cardiovascular risk in patients with antiphospholipid syndrome: a meta-analysis of literature studies. Ann Med. 2014;46:693-702. 22
[37] Di Minno MND, Emmi G, Ambrosino P, Scalera A, Tufano A, Cafaro G, et al. Subclinical atherosclerosis in asymptomatic carriers of persistent antiphospholipid antibodies positivity: A cross-sectional study. Int J Cardiol. 2019;274:1-6. [38] Selmi C, Ceribelli A, Generali E, Scire CA, Alborghetti F, Colloredo G, et al. Serum antinuclear and extractable nuclear antigen antibody prevalence and associated morbidity and mortality in the general population over 15 years. Autoimmun Rev. 2016;15:162-6. [39] Selmi C, Gershwin ME. Sex and autoimmunity: proposed mechanisms of disease onset and severity. Expert Rev Clin Immunol. 2019;15:607-15.
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Highlights Positive aPL are frequent in the population but the CV significance is unclear; CV events are more frequent in subjects with aPL and a high-risk CV profile; aPLs are associated with ultrasound signs of subclinical atherosclerosis.
S0167-5273(19)32822-0
DOI:
https://doi.org/10.1016/j.ijcard.2019.10.042
Reference:
IJCA 28094
To appear in:
International Journal of Cardiology
Received Date: 31 May 2019 Revised Date:
15 October 2019
Accepted Date: 24 October 2019
Please cite this article as: C. Selmi, M. De Santis, P.M. Battezzati, E. Generali, S.A. Lari, A. Ceribelli, N. Isailovic, P. Zermiani, S. Neidhöfer, T. Matthias, C.A. Scirè, D. Baldassarre, M. Zuin, Antiphospholipid antibody prevalence and association with subclinical atherosclerosis and atherothrombosis in the general population, International Journal of Cardiology (2019), doi: https://doi.org/10.1016/ j.ijcard.2019.10.042. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Anti-phospholipid antibody prevalence and association with subclinical atherosclerosis and atherothrombosis in the general population
Carlo Selmi MD PhD a,b*, Maria De Santis MD PhD a*, Pier Maria Battezzati MD c, Elena Generali MD a, Simone Aldo Lari MD a, Angela Ceribelli MD PhDa, Natasa Isailovic MSc a, Paola Zermiani BSc c, Sandra Neidhöfer BSc d, Torsten Matthias PhD d, Carlo A. Scirè MD PhD e, Damiano Baldassarre PhD b,f, and Massimo Zuin MD c
Affiliations: a Rheumatology and Clinical Immunology, Humanitas Clinical and Research CenterIRCCS, Rozzano (MI), Italy; b Department of Biomedical Science and Translational Medicine, University of Milan, Italy; cLiver and Gastroenterology Unit, San Paolo Hospital Department of Health Sciences, University of Milan, Italy; dAesku Diagnostics GmbH & Co. KG, Wendelsheim, Germany; e Epidemiology Unit, Italian Society of Rheumatology, Milan, Italy; f Centro Cardiologico Monzino IRCCS, Milan, Italy. * These authors contributed equally to the study
Corresponding author: Carlo Selmi MD PhD, Division of Rheumatology and Clinical Immunology, Humanitas Clinical and Research Center, via A. Manzoni 56, 20089 Rozzano, Milan, Italy; tel +39-02-8224-5129, fax +39-02-8224-2298, email [email protected]
Disclosures: Dr. Torsten Matthias is the head of the non-profitable Aesku.Kipp Institute and the owner of Aesku.Diagnostics GmbH& Co.KG. Sandra Neidhöfer is employed by Aesku.Kipp Institute. Funding: Lombardia region (DG Sanità 08/07/2008 n. 7364).
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Highlights •
Positive aPL are common in the population but their CV significance is unclear;
•
CV events are more frequent in subjects with aPL and an elevated CV risk;
•
aPLs are associated with ultrasound signs of atherosclerosis.
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ABSTRACT Background. There is no agreement on the prevalence of anti-phospholipid antibodies (aPLs) and the correlation with atherosclerosis and cardiovascular (CV) events in the general population. Methods. We performed a cross-sectional study on 1,712 randomly enrolled subjects from a Northern Italian city to investigate the presence of aPLs and the association with subclinical atherosclerosis (using the carotid artery intima media thickness measured as inter-adventitia common carotid artery diameters - ICCAD) and retrospectively collected CV factors and events (i.e. acute myocardial infarction, stroke, and peripheral obliterans arterial vasculopathy) using physician-assisted questionnaires. We tested serum IgG, IgM, and IgA anti-cardiolipin, antibeta2glycoprotein I (aGPI), and anti-phosphatidylserine-prothrombin antibodies. Results. Positive aPLs were found in 15.1% of the subjects, with no differences between sex but with higher rates in older subjects. Carotid subclinical atherosclerosis was more frequent in aPL positive subjects; more specifically, aGPI IgA were associated with higher ICCAD average (adjusted beta 0.51, 95% confidence interval (CI)0.17-0.84; p= 0.003). A positive history of CV events was also more frequent in aPL positive subjects (odds ratio (OR) 1.67, 95%CI 1.08-2.54; p=0.012), particularly peripheral obliterans arterial vasculopathy (OR 2.02; 95%CI 1.14-3.57; p=0.015). Among subjects with a Framingham risk score >20, and/or diabetes, and/or body mass index >35Kg/m2, aPL positivity was associated to the highest risk of CV events (OR 2.52, 95%CI 1.24-5.11; p=0.011). Conclusions. APL prevalence in the general population is higher than previously reported. CV events and subclinical atherosclerosis are more frequent in the presence of aPL, particularly when a high CV risk coexists.
Key words. Cardiovascular events; antiphospholipid antibodies; epidemiology; serum biomarkers
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1. Introduction. Anti-phospholipids (aPLs) are a heterogeneous group of autoantibodies which may define the homonymous syndrome [1] manifesting with arterial/venous thrombosis and/or recurrent miscarriages/placental insufficiency [2]. However, the primary aPL syndrome is found in 40-50 cases /100,000 adults [3] and is significantly outnumbered by the frequency of aPL positive subjects. In fact, aPLs can be positive also in other autoimmune and non-autoimmune diseases, such as cancer or infections, as well as in healthy individuals [1]: aPLs have been detected in ~45% of the patients with systemic lupus erythematosus [4], 7% of the women with recurrent miscarriages [5, 6], and 8% of blood donors [1, 7]. APLs included in classification criteria for primary aPL syndrome are lupus anticoagulant, IgM and IgG anti-cardiolipin (aCL), and IgM and IgG anti-β2 glycoprotein I (aGPI) antibodies [8]; however, a clear estimate of their prevalence or sex predominance in the general population lacks. Additional aPLs, defined as non-criteria aPLs, include anti-phosphatidylserine/prothrombin (aSP) [9], anti-phosphatidic acid, antivimentin/cardiolipin complex, anti-protein C/S, anti-factor XII, anti-factor X, anti-annexin A5/A2, and anti-D1 [10]. The major causes of mortality in the aPL syndrome are thrombotic events, but a number of nonthrombotic events are frequently reported, such as migraine, livedo, ulcers, and heart valve diseases. These manifestations are thought to be associated with the aPL-mediated endothelial activation [11]. To date, it has not been investigated whether such endothelial activation could be also implicated in the development of atherosclerosis or contributes to atherothrombosis in patients with aPLs, as suggested by small cohorts [10]. We took advantage of a unique cross-sectional study performed in a Northern Italian population to study aPLs and (i) the prevalence in the adult general population, (ii) the correlation with subclinical atherosclerosis, and (iii) the association with CV events.
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2. Materials and methods 2.1 Study design and population The cross-sectional CA.ME.LI.A (CArdiovascular risk, MEtabolic syndrome, LIver, and Autoimmunity) study included 2,555 subjects (age 18-75 years) that were randomly selected from the active voting lists of the Northern Italian city of Abbiategrasso (Milan area, population 32,000 inhabitants) in 2009. Subjects considered for randomization responded to the following criteria: residence in Abbiategrasso; medical assistance in Abbiategrasso; born between 1934-1980. A freely available computer-based software (www.randomizer.org) has been used for randomization and the randomized subjects have been contacted by phone call. The study has been based on the STROBE document for observational study (attached as supplementary material). All subjects underwent physical examination and blood tests including C-reactive protein (CRP), total cholesterol, triglycerides, homocysteine, glucose, and complete blood count to assess CV risk. The medical history was assessed with a physician-assisted questionnaire (Supplemental material) and the following information were registered: arterial hypertension, diabetes, smoking habit, height and weight, physical activity, history of myocardial infarction, stroke, and/or peripheral limb arteriopathy. Within this cohort, a subgroup of 1,712 randomly selected subjects that did not differ significantly in terms of demographic and clinical characteristics (data not shown) was used for the present study, as the sample size was estimated sufficient to identify an expected aPL prevalence of 7.5% [1, 5-7], using an alpha error of 0.05, and a power of 80%. The primary study endpoints were to establish the prevalence of aPLs, the correlation with ultrasonographic parameters of subclinical atherosclerosis, and the association with history of CV
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events. The secondary endpoint was to identify possible differences among the three main aPL specificity and isotypes. The local institutional review board approved the present study and all subjects signed an informed consent.
2.2 APL analysis The sera of the 1,712 selected subjects were tested for aCL, aGPI, and aSP antibodies using commercially available ELISA tests (AESKU diagnostic, Wendelsheim, Germany) to identify IgG, IgM, and IgA isotypes for each autoantibody. Results were expressed as international units (IU) and cut-off values were established at 15 IU, as recommended by the manufacturer instructions, according to the Clinical and Laboratory Standards Institute guideline for reference intervals [12]. The range of results was 0-300 IU and values >40 IU were considered as high titers [13, 14]. Lupus anticoagulant could not be tested since fresh samples were not available.
2.3 Carotid ultra-sonographic analysis The quantitative evaluation of carotid intima-media thickness (IMT) was performed by trained sonographers unaware of clinical information using a 7.5 MHz probe [15] on 1:3 randomly selected subjects (n=563, no significant differences compared to the cohort tested for aPLs in terms of demographic and clinical characteristics; data not shown). Briefly, the far walls of the left and right common carotids, bifurcations, and internal carotids were visualized in anterior, lateral, and posterior angles and recorded. Carotid IMT measurements were performed in a centralized laboratory (Centro Cardiologico Monzino IRCCS, Milan, Italy) using a dedicated software (M’Ath, Metris SRL France). The ultra-sonographic variables used in the statistical analyses were the mean
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maximum intima-media thickness (IMTmean-max), the atherosclerotic plaques (defined as maximum IMT (IMTmax) >1.5 mm) [15], and the inter-adventitia common carotid artery diameters (ICCAD).
2.4 CV risk factors and events CV risk factors, such as arterial hypertension, age (≥ 50 years, considering CV risk based on age [16]; the mean age of menopause in Italy based on ICARUS study group [17] and the risk of CV events based on menopause onset [18]), smoking habit (>1 cigarette/day for at least one year), body mass index (BMI) ≥25 kg/m2, physical inactivity (no physical activity except work-related activity), type 1 and 2 diabetes, CRP ≥ 0.2 mg/dL [19], hypercholesterolemia (total cholesterol > 200 mg/dL), hypertriglyceridemia (>150 mg/dL ) [20], hyperhomocysteinemia (> 15umol/L) [21], and history of CV clinical events, such as acute myocardial infarction (ST elevation myocardial infarction-STEMI or non ST elevation myocardial infarction-NSTEMI), hemorrhagic or ischemic stroke, and peripheral limb arteriopathy (obliterans vasculopathy requiring surgery or prostanoid therapy), were recorded for each subject by the study physicians. The overall Framingham risk score was calculated using established formulas [22]. The Framingham risk score was chosen over the ESC score because the latter score is not applicable beyond age 65, while the former is intended for use in patients up to 79 years [23, 24].
2.5 Statistical analysis Data were analyzed using descriptive and inferential methods. We used both parametric (χ2) and non-parametric (Mann-Whitney) tests, when appropriate. To assess the association of aPLs with carotid ultra-sonographic measurements and CV events, univariate and multivariate analyses were performed, after adjusting for pre-specified confounders including CV risk factors. Results were expressed as odds ratio (OR) and 95% confidence intervals
7
(95% CI) for binary, and as beta coefficient (95% CI) for continuous outcomes. All analyses were performed using STATA 13 for Macintosh (StataCorp, College Station, TX, USA), and p values<0.05 were considered statistically significant.
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3. Results 3.1 aPL prevalence The demographic and clinical characteristics of the study population are shown in Table 1. The overall prevalence of any aPL (defined as at least one positive aPL) was 15.1% (95% CI 13.416.8%) with the highest frequency observed in older subjects (18.1%; p<0.002) (Table 2). Hightiter aPL were found in 3.3% of subjects, multiple specificities in 2%. ACL were detected in 1.5% of the subjects (Table 2) with 1% at high titer. AGPI were positive in 4.3% of the subjects (Table 2) with 1.2% at high titer and the highest prevalence in older subjects (6.7%; p<0.001). AGPI IgA were more frequently observed in women (3.1%, p=0.040) and in older subjects, independently of sex (4%, p<0.001). ASP were the most frequently detected aPL, being found in 11.7% of the cases (Table 2) with 1.7% at high titer and in 9.8% of the cases as the only positive test (data not shown). ASP IgA were not detected.
3.2 CV risk factors The subjects with hypercholesterolemia had more frequently high-titer aPLs (39, 69.6% versus 762, 52.4% in aPL negative subjects, p=0.025 adjusted for sex and age), while subjects with high triglycerides had more frequently aGPI (18, 24.7% vs 220, 15.1% in aPL negative subjects, p=0.035 adjusted for sex and age; supplementary Table A) Interestingly, higher LDL levels were found in subjects with high-titer aPLs (median 142.5 (range 120-169) vs 133 (113-156), p= 0.021). Conversely, aPL prevalence did not differ according to physical inactivity, smoking, arterial hypertension, diabetes, triglycerides, and CRP (data not shown).
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We found a significant association between elevated homocysteine and all aPL specificities, including IgG aCL (10, 66.7%; p<0.001), aGPI (27, 37%; p=0.026), and IgG aSP (53, 33.8%; p=0.022; data not shown). The presence of more than one aPL was associated to elevated homocysteine in 60% of the cases (19 versus 368, 25.3% in aPL negative subjects, p<0.001 adjusted for sex and age; supplementary Table A). Positive aPL subjects had higher Framingham risk score at 10 years compared to aPL negative subjects; moreover, Framingham risk score was higher in case of more aPL specificities and high titer aPLs (Table 3). Of note, the highest Framingham risk score (>20%) was most frequently observed with high titer IgM aCL, aGPI, and aSP (40%, p<0.001; 33.3%, p=0.018; 25%, p=0.024; respectively; Table 3).
3.3 Carotid ultra-sonographic measurements APL positive subjects had more frequently atherosclerotic plaque and higher mean values of IMT Mean-Max and ICCAD (Table 4). Specifically, aCL IgM positive subjects had a significantly higher frequency of atherosclerotic plaques (OR 5.41, 95%CI 0.61-65.2, p=0.042), while aGPI IgA positive subjects had significantly increased IMTMean-Max (beta 0.04, 95%CI 0.004-0.072; p=0.027) and ICCAD (beta 0.02, 95%CI 0.009-0.041; p=0.002; Table 4). Multivariate analyses for carotid ultrasound measurements adjusted for pre-specified confounders are shown in supplementary Table B: only IgA aGPI were independently associated with increased ICCAD (beta 0.51, 95%CI 0.17-0.84; p =0.003). Considering sex and age, we observed that women younger than 50 years with aPLs had an increased ICCAD (6.74 IQR 6.2-7.0 vs 6.47 IQR 6.19-7.75 in women < 50 years without aPLs; at multivariate analysis beta 0.22, 95%CI 0.03-0.41; p=0.025), especially, in case of IgG aSP (beta 0.24, 95%CI 0.02-0.46; p=0.03, data not shown).
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3.4 CV events Overall CV events were more frequently reported in the aPL positive population (34, 13.2% versus 121, 8.3% in aPL negative subjects, p=0.012; OR 1.67 95%CI 1.12-2.50). Interestingly, while in the aPL negative subjects AMI and peripheral arteriopathy had a similar frequency (3.4% compared to 1.6% of stroke cases), in aPL positive subjects peripheral arteriopathy (6.6%) almost doubled the prevalence in a PL negative subjects and was significantly associated to anti-PLs independently of other CV risk factors (OR 2.02, 95%CI 1.07-3.64, p =0.013; Table 4). AMI and stroke were also more frequent in aPL positive subjects (3.9% and 2.7%, respectively; Table 4). The subjects with high level of homocysteine reported CV events more frequently and independently of aPL status (43, 11.7% versus 60, 5.5%; OR 2.3 95%CI 1.5-3.5, p<0.001), but those with high homocysteine and coexisting aPLs had a higher risk (OR 3.01 95%CI1.4-6.0). Finally, when analyzing a high-risk population defined by Framingham risk score >20, and/or diabetes, and/or BMI >35, aPL positivity was associated with a higher risk for any CV event (OR 2.52, 95%CI 1.24-5.11, p=0.011).
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4. Discussion Our study represents the largest population-based analysis of aPL prevalence and the first attempt to determine the association between aPLs, subclinical atherosclerosis, and clinical CV events in the general population. From an epidemiological point of view, we report a high prevalence of aPLs without sex differences and with an increasing trend with age. However, the prevalence was similar to other reports [1, 5-7] when considering only aPLs included in classification criteria, such as aCL and aGPI in our cases, and only high titer (not having a 12-week confirmation test). ASP was found to be the most abundant aPL and in up to 10% of the cases was the only aPL detectable; moreover, ASP were significantly associated with increased ICCAD in childbearing-age women. Furthermore, one not routinely tested aPL, i.e. aGPI IgA, was found to be more frequent in women and the only aPL significantly and independently associated to increased ICCAD. CV events were overall more frequently found among aPL positive subjects, even if only peripheral arteriopathy was significantly associated to the aPL; moreover, in subjects with higher Framingham score, diabetes, higher BMI or hyper-homocysteinemia the risk of CV events was 2.5-3 folds higher in presence of aPLs. Overall our data suggest a possible independent and additive role of aPLs in the atherogenesis. Being CV events registered retrospectively, it can be suspected that aPLs could be an epiphenomenon, but the association with subclinical atherosclerosis seems to support an important role of aPLs. The association between hyper-homocysteinemia and aPL is of particular interest given the pro-thrombotic action of homocysteine via phosphatidylserine exposure on endothelial cells, myocardiocytes, and red blood cells [25, 26], thus suggesting that the development of aPLs could contribute to the thrombophilic status in hyper-homocysteinemia. Conflicting lines of clinical evidence suggest a predisposition to accelerated atherosclerosis mainly in primary aPL syndrome [27-32]. Basic science supports a causative atherogenic link as early
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atherosclerosis can be induced in low density lipoprotein (LDL)-receptor-deficient mice immunized with GPI [33], suggesting that antibodies targeting GPI could suppress the anti-atherogenic role of GPI. Similarly, aCL increases atherogenesis in the LDL-knockout mice [34]. A number of additional immune-mediated mechanisms have been also described, including the cross reactivity between aPLs and anti–oxidized LDL [35] and the increased uptake of oxidized LDL/GPI complex by macrophages exposed to aGPI [33]. A meta-analysis including 668 subjects with aPL syndrome demonstrated the relationship between aPLs and subclinical atherosclerosis [36], and, most recently, a cross-sectional study reported subclinical atherosclerosis in asymptomatic aPL positive subjects together with a correlation with title, triple or double positivity, and with aGPI presence over aCL [37]. The observation that aPL are more frequent in older subjects is in line with what is reported for antinuclear antibody (ANA) in the general population [38], while the similar rates in men and women may reflect a different sex epidemiology for aPLs respect to ANA [39]. While the present study represents the largest population-based analysis for aPLs, we are also aware of study limitations, particularly, the lack of a confirmation test after 12 weeks as recommended for positive sera in clinical practice and of lupus anticoagulant determination as this cannot be performed on frozen sera. Moreover, we did not observe an increasing risk of CV events in subjects with more than one positive autoantibody or high titers, maybe due to the low prevalence of this positivity or to the small number of CV events. Further, we used a questionnaire for the medical history that was custom created to include all the major objectives and clinical areas of the study which would have not been possible at once; we cannot exclude a role of inflammation because high sensitivity CRP was not available. In the end, a prospective follow up of subjects with aPLs regarding CV events should be assessed, because from a cross-sectional study we were not able to evaluate CV events occurring in the meantime.
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5. Conclusions The prevalence of aPLs in the general population is higher than previously reported and increases with age while being equally represented among men and women. APLs are associated with subclinical atherosclerosis and a higher prevalence of CV events, especially in subjects with a higher CV risk.
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Table 1. Characteristics of the study population arrayed based on sex and age. Total 1,712 47.2 (3761.3)
Women 852 (49.8%) 47.2 (36.861.9)
BMI >25 kg/m2
875 (51.1)
356 (41.8)
519 (60.3)
369 (38.6)
506 (60)
Smoking
438 (25.6)
185 (21.7)
253 (29.4)
303 (31.7)
135 (17.9)
Ex-smokers
401 (23.4)
138 (16.2)
263 (30.6)
168 (17.6)
233 (30.9)
754 (44)
396 (46.5)
358 (41.6)
416 (43.5)
338 (44.8)
378 (22.2)
189 (22.2)
189 (22)
65 (6.8)
313 (41.6)
99 (5.8) 622 (36.3)
33 (3.9) 341(54.8)
66 (7.7) 281 (45.2)
11 (1.2) 296 (47.6)
88 (11.7) 326 (52.4)
268 (15.6)
89 (10.4)
179 (20.8)
134 (14)
134 (17.8)
902 (52.7)
474 (55.6)
427 (49.7)
412 (43.1)
490 (64.8)
448 (26.2)
137 (16.1)
310 (36)
207 (21.7)
241 (31.9)
128 (22.7)
54 (42.2)
74 (57.8)
14 (10.9)
114 (89.1)
0.99 (0.861.22) 7.06 (6.677.57)
0.96 (0.851.16) 6.78 (6.407.22)
1.02 (0.881.27) 7.31 (6.937.88)
0.88 (0.820.98) 6.80 (6.447.19)
1.22 (1.031.54) 7.45 (7.028.04)
AMI
59 (3.4)
20 (33.9)
39 (66.1)
10 (17)
49 (83)
Stroke
30 (1.8)
14 (46.7)
16 (53.3)
3 (10)
27 (90)
PA
66 (3.9)
30 (45.5)
36 (54.5)
3 (4.5)
63 (95.5)
Age, m (IQR)
Physical inactivity Arterial hypertension Diabetes CRP ≥ 2 mg/L Triglycerides >150 mg/dL Cholesterol >200 mg/dL Homocysteine >15 umol/L
Men Age<50 Age≥50 860 (50.2%) 956 (55.8%) 756 (44.2%) 47.2 (37.260.6)
Outcomes IMT Max avg>1.5 n (%) IMT Mean Max m (IQR) ICCAD Avg m (IQR)
m: median; IQR: inter-quantile range; BMI: body mass index; CRP: C-reactive protei; IMT: intima-media thickness; avg: average; ICCAD: inter-adventitia Common Carotid Artery Diameter; AMI: acute myocardial infarction; PA: peripheral arteriopathy. Data are expressed as numbers(percentages) or median (IQR)
15
Table 2. Prevalence of aPLs in the population according to age and sex. Antibody
Total
Women
Men
Age<50
Age≥50
1,712
860 (50.2%) 739 (85.9)
956 (55.8%) 835 (87.3)
756 (44.2%) 619 (81.9)
aPL negative
1,454 (84.9)
852 (49.8%) 715 (83.9)
aPL positive
258 (15.1)
137 (16.1)
121 (14.1)
121 (12.7)
137(18.1)*
aCL
26 (1.5)
10 (1.2)
16 (1.9)
10 (1)
16 (2.1)
aGPI
73 (4.3)
44 (5.2)
29 (3.4)
22 (2.3)
51 (6.7)*
aSP 201 (11.7) 103 (12.1) 98 (11.4) 104 (10.9) 97 (12.8) Data are presented as number (%). aPL: antiphospholipid antibodies; aCL: anti-cardiolipin antibodies; aGPI: anti-β2-glycoprotein antibodies I; aSP: anti-serin/prothrombin antibodies. * p<0.05 age<50 versus age ≥50.
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Table 3. Prevalence of Framingham risk classes according to the aPL status.
<5%
6-10%
11-15%
16-20%
>20%
aPL negative
952 (65.5)
186 (12.8)
82 (5.6)
124 (8.5)
110 (7.6)
aPL positive
156 (60.5)
40 (15.5)
16 (6.2)
21 (8.1)
25 (9.7)
>1 aPLs
15 (42.9)
8 (22.9)
5 (14.3)
3 (8.6)
4 (11.2)
aPL HT
31 (55.5)
9 (16.1)
1 (1.8)
8 (14.3)
7 (12.5)
aCL
10 (38.5)*
6 (23.1)
2 (7.7)
3 (11.5)
5 (19.2)*
aCL IgG
7 (46.7)
3 (20)
1 (6.7)
1 (6.7)
3 (20)
aCL IgM
4 (22.2)*
5 (27.8)
1 (5.6)
3 (16.7)
5 (27.8)*
aCL IgA
3 (75)
0
0
0
1 (25)
42 (57.5)
12 (16.4)
5 (6.9)
5 (6.9)
9 (12.3)
aGPI IgG
11 (55)
3 (15)
0
2 (10)
4 (20)*
aGPI IgM
17 (60.7)
4 (14.3)
2 (7.1)
3 (10.7)
2 (7.1)
aGPI IgM HT
2 (33.3)
0
0
2 (33.3)*
2 (33.3)*
aGPI IgA
18 (51.4)
7 (20)
4 (11.4)
1 (2.9)
5 (14.3)
aSP
121 (60.2)
32 (15.9)
14 (7)
17 (8.5)
17 (8.5)
aSP IgG
92 (58.6)
23 (14.7)
13 (8.3)
14 (8.9)
15 (9.6)
aSP IgM
40 (61.5)
12 (18.5)
1 (1.5)
5 (7.7)
7 (10.8)
aSP IgM HT
7 (58.3)
2 (16.7)
0
0
3 (25)*
aGPI
Data are presented as number (%). aPL: antiphospholipid antibodies; HT: high titer; aCL: anti-cardiolipin antibodies; aGPI: anti-β2-glycoprotein antibodies I; aSP: anti-serin/prothrombin antibodies. *= p<0.05 compared to aPL negative subjects.
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Table 4. IMT measurements and clinical cardiovascular outcomes in the cohort studied according to aPL status.
aPL negative aPL positive >1 aPLs aPL HT
IMT max avg>1.5
IMT mean max
ICCAD avg
AMI
Stroke
PA
n (%)
m (IQR)
m (IQR)
n (%)
n (%)
n (%)
0.98 (0.86-1.21)
7.05 (6.66-7.57)
49 (3.4)
23 (1.6)
49 (3.4)
1.03 (0.88-1.30) 0.97 (0.88-1.04) 1.03 (0.98-1.04)
7.12 (6.77-7.66) 6.91 (6.8-7.24) 7 (6.9-7.22)
10 (3.9) 1 (1.8) 0
7 (2.7) 0 0
17 (6.6)* 0 0
106/488 (21.7) 22/75 (29.3) 2/16 (12.5) 1/5 (20)
aCL aCL IgG aCL IgM aCL IgA
3/8 (37.5) 1/6 (16.7) 3/5 (60)* 0
1.01 (0.89-1.21) 0.94 (0.88-1.04) 1.03 (0.98-1.39) 0.87 (0.77-0.98)
7.03 (6.86-7.30) 7.26 (6.96-7.38) 7.10 (6.87-7.22) 7.41 (7.22-7.60)
1 (3.9) 0 1 (5.6) 0
0 0 0 0
1 (3.9) 0 1 (5.6) 0
aGPI aGPI IgG aGPI IgM aGPI IgA
7/21 (33.3) 1/6 (16.7) 3/6 (50) 3/9 (33.3)
1.12 (0.96-1.5)* 1 (0.90-1.12) 1.12 (0.95-1.37) 1.22 (1.02-1.66)*
7.22 (6.84-7.92) 7.04 (6.67-7.92) 7.05 (6.84-7.41) 7.48 (6.92-8.58)*
1 (1.4) 0 1 (3.6) 1 (2.9)
2 (2.7) 0 1 (3.6) 1 (2.9)
5 (6.9) 1 (5) 2 (7.1) 3 (8.6)
aSP aSP IgG aSP IgM
17/59 (28.8) 14/47 (29.8) 5/15 (33.3)
1.02 (0.85-1.27) 0.98 (0.85-1.28) 1.03 (0.85-1.30)
7.09 (6.73-7.49) 7.18 (6.78-7.72) 6.9 (6.33-7.26)
9 (4.5) 6 (3.8) 3 (4.6)
5 (2.5) 4 (2.6) 1 (1.5)
12 (6) 10 (6.4) 3 (4.6)
Data are expressed as number (%) or median (interquartile range) m: median; aPL: antiphospholipid antibodies; HT: high titer; aCL: anti-cardiolipin antibodies; aGPI: anti-β2-glycoprotein antibodies I; aSP: anti-serin/prothrombin antibodies; IMT: intima-media thickness; avg: average; ICCAD: inter-adventitia Common Carotid Artery Diameter; AMI: acute myocardial infarction; PA: peripheral arteriopathy. *= p<0.05 compared to aPL negative subjects.
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Highlights Positive aPL are frequent in the population but the CV significance is unclear; CV events are more frequent in subjects with aPL and a high-risk CV profile; aPLs are associated with ultrasound signs of subclinical atherosclerosis.