APOE e 2 allele is an independent risk factor for vulnerable carotid plaque in ischemic stroke patients Beata Blazejewska-Hyzorek1, Grazyna Gromadzka1,2, Marta Skowronska1, Anna Czlonkowska1 1
2nd Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland, 2Department of Experimental and Clinical Pharmacology, Warsaw Medical University, Warsaw, Poland
Background and Aims: The data about apolipoprotein E gene (APOE) genotype and the risk of stroke are inconsistent. The APOE genotype is expected to influence the development of carotid plaques. Our aim was to look for association between APOE genotype and carotid plaque morphology in ischemic stroke patients. Methods and Results: Data of ischemic stroke patients was collected prospectively for 2 years. The degree of stenosis and plaque echogenicity and surface were assessed with ultrasound. Subsequent APOE genotypes were compared: APOE e 3/e 3 (E3 – reference), APOE e 2/e 3 (E2 group) and APOE e 3/e 4, APOE e 4/e 4 (E4 group). We included 388 patients with acute ischemic stroke. Patients in E2 group had more often hypoechogenic, ulcerated plaques and severe stenosis comparing to E3 patients. On logistic regression analysis, e 2 genotype remained an independent risk factor for vulnerable carotid plaque (OR 5 2.3 for ,60% stenosis and OR 5 2.7 for $60% stenosis; 95% CI). Conclusions: This study suggests that e 2 allele is an independent risk factor for echolucent and ulcerated carotid plaque. Keywords: Apolipoproteins, Carotid plaque, Ischemic stroke
Introduction The apolipoprotein E gene (APOE) is widely studied in vascular diseases, including stroke.1–3 Its product, apolipoprotein E (apoE), is a glycoprotein, having three common isoforms, encoded by the alleles e 2, e 3, and e 4, which give rise to six genotypes. The most common is e 3/e 3, occurring in about one-half to twothirds in most populations. The apoE isoforms interact differently with lipoprotein receptors. They alter levels of cholesterol through different hepatic binding, uptake and catabolism of remnants of chylomicrons, and very-low-density lipoprotein (VLDL). The e 4 allele carriers appear to have higher plasma cholesterol levels, whereas the e 2 allele carriers lower, so APOE genotype is expected to influence the development of atherosclerosis. Not only the presence of carotid plaques and the degree of carotid stenosis, but also plaque properties are important for vascular disease. Dark and low-echogenicity (echolucent)
Correspondence to: A. Członkowska, Second Department of Neurology, Institute of Psychiatry and Neurology, 9 Sobieskiego Street, 02-957 Warsaw, Poland. Email: [email protected]
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ß W. S. Maney & Son Ltd 2014 DOI 10.1179/1743132814Y.0000000385
plaques on ultrasound are thought to be more ‘vulnerable’ causing vascular events.4 Few authors have tested the association between carotid plaques and APOE polymorphism, reporting inconsistent results.5–10 A recent autopsy study showed that the risk of developing and dying from cardiovascular disease, including coronary heart disease and cerebrovascular disease, is influenced by the apoE polymorphism, with more e 4-carriers and fewer e 2-carriers.11 We aimed to investigate the association of carotid plaques and the APOE polymorphism in ischemic stroke population.
Materials and Methods Study population All consecutive ischemic stroke patients, admitted within 7 days from stroke onset to our department, in 2 years period were considered for the study. All patients were Caucasians. Stroke was defined according to World Health Organization12 as a neurological deficit lasting over 24 hours. All patients had brain imaging (Computed
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Tomography – CT or Magnetic Resonance – MR) to exclude other, than ischemic, causes of symptoms. Patients who were unable to cooperate during the ultrasound examination (e.g. patients with very severe stroke, anxious during examination) were excluded from the study. Data about medical history, personal habits, and basic laboratory tests were prospectively collected. Smokers were defined as ever smoking. Hypertension was defined as either current blood pressurelowering therapy or a systolic blood pressure $ 140 mmHg and/or a diastolic blood pressure $ 90 mmHg on two separate measures done after acute phase of stroke. Diabetes mellitus was defined as a history of known diabetes or currently diagnosed (as a fasting plasma glucose level $100 mg/dl or as 2hour plasma glucose $200 mg/dl blood collected 1 week after stroke). Hypercholesterolemia was defined as fasting total cholesterol level .200 mg/dl, high-density lipoprotein (HDL) cholesterol ,45 mg/ dl, low-density lipoprotein (LDL) cholesterol .130 mg/dl, and triglyceride level .150 mg/dl.13 Data about vascular events defined as: history of stroke (or TIA), myocardial infarction, and angina pectoris (previously diagnosed or diagnosed during current hospitalization) were collected.
APOE e 2 allele
Genotyping Genomic DNA was extracted from the whole frozen EDTA-blood using the TRI Reagent (SIGMA, Poznan, Poland). The genotyping of APOE was performed by polymerase chain reaction-restriction fragments length polymorphism (PCR-RFLP), as previously described.14 In the first step, PCR was performed with primers that allowed for amplification of the polymorphic region. PCR product was digested by HhaI restriction endonuclease and then separated with electrophoresis.
Ultrasound examination The B-mode system (Acuson 128XP/10C, Siemens, Berlin and Munich, Germany) with 7 MHz probe was used for plaque assessment. This high-resolution, non-invasive technique can be applied for plaque identification and for assessment of plaque echogenicity and cap irregularity.11 The examination protocol involved scanning of the common carotid arteries (CCA), the carotid bifurcations, and the origin (first 2 cm) of the internal carotid arteries (ICA) and external carotid arteries (ECA). Near and far walls of all arterial segments were scanned longitudinally and transversally for the presence of the plaques. Both ipsi- and contralateral carotid arteries were examined and plaques were
Table 1 Population characteristics n 5 388 Age, years Sex, male, n (%) Hypertension, n (%) AF, n (%) DM, n (%) Smoking, n (%) Previous stroke, n (%) Previous TIA, n (%) CHD, n (%) MI, n (%) Total cholesterol (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) TG (mg/dl) Total cholesterol . 200 mg/dl, n (%) HDL-C # 45 mg/dl, n (%) LDL-C . 130 mg/dl, n (%) TG . 150 mg/dl Carotid plaques, n (%) APOE polymorphism, n (%) e 2/e 2 e 2/e 3 (E2) e 3/e 3 (E3) e 2/e 4 e 3/e 4 (E4) e 4/e 4 (E4) TOAST classification Large artery atherosclerosis, n (%) Cardioembolism, n (%) Small-vessel occlusion, n (%) Stroke of other determined etiology Stroke of undetermined etiology (two or more causes) Stroke of undetermined etiology (negative/incomplete evaluation)
68 ¡ 11.1* 203 (52) 257 (66.8) 61 (15.8) 73 (19.0) 192 (49.5) 54 (14.0) 49 (12.7) 114 (29.5) 58 (15.0) 205.0 ¡ 56.5* 43.8 ¡ 16.6* 142.5 ¡ 47.0* 111.0 ¡ 65.4* 207 (55.5) 207 (55.5) 218 (58.8) 49 (13.1) 223 (57.4) 0 39 273 5 69 2
(10.1) (71.3) (1.3) – excluded (17.8) (0.5)
86 69 84 12 122 15
(22.2) (17.8) (21.6) (3.1) (31.4) (3.9)
*
Mean ¡ SD. AF: atrial fibrillation; DM: diabetes mellitus; CHD: coronary heart disease; MI: myocardial infarction; LDL-C: low-density lipoprotein; HDL-C: high-density lipoprotein; TG: triglycerides; TOAST: trial of org 10172 in acute stroke treatment.
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assessed on both sides (data is presented for both sides). The plaque was defined as localized echo structures encroaching into the vessel lumen for which the thickness was $1 mm.15 Hyperechogenic plaques were identified if .50% of plaque surface was hyperechogenic (echogenicity of the bones of the vertebral column) other plaques were described as hypoechogenic (echolucent). Plaque surface was assessed as ulcerated when the cap rupture was $ 2 mm.16 The degree of stenosis was assessed with the velocity criteria. The degree $60% was considered as severe stenosis. All examinations were performed no later than 7 days from admission, in majority of cases in the first 3 days after admission. The intraobserver coefficients of variability were 4.0% for performing sonographer (BB-H). Informed consent was obtained from all participants and the study was approved by the institutional ethics committee.
Statistics Data were analyzed with the Statistica PL 7.0 software package. Genotype APOE e 3/e 3 (E3) was considered as a reference. It was compared with APOE e 2/e 3 (E2) and APOE e 3/e 4 and APOE e 4/e 4 (E4). We excluded APOE e 2/e 4 genotype from the analysis, as those two alleles are considered to be opposite in lipid metabolism. The chi-square test was used to compare proportions and Student t or Mann–Whitney U test was used to compare continuous variables. Multivariate logistic regression analysis was used to assess the independent contribution of variables statistically significant on univariate analysis in the prediction of degree of atherosclerosis.
Demographic characteristics, vascular risk factors, APOE polymorphism, and stroke etiology are given in Table 1. There was no association between APOE polymorphism and the prevalence of vascular risk factors. The laboratory results are presented in Table 2. Erythrocyte sedimentation rate (ESR) was significantly higher in e 2 carriers than in reference group, but not lower in e 4. There was no interaction shown between total cholesterol levels and APOE polymorphism, but we found that e 2 carriers had LDLC . 130 mg/dl less often while e 4 more often than the reference E3 population (40.5, 66.2, and 59.8% respectively. Carotid plaques were found in 57% of patients. Over 60% carotid stenosis (both ipsi- and contralateral) stenosis was found more often in E2 than E4 group. There was no association between presence of plaques, number of carotid plaques (data about number not shown), and APOE polymorphism. We found that e 2/e 3 carriers had more often echolucent plaques with ulcerated fibrous cap comparing to e 3/e 3 homozygotes (Table 3). In multivariate logistic regression analysis adjusted for the vascular risk factors age, sex, hypertension, diabetes, smoking, cholesterol level, and history of vascular event, e 2/e 3 genotype was an independent risk factor for: echolucent or ulcerated plaque with ,60% stenosis, OR 5 2.27, CI 95% (1.09–4.71), P 5 0.03; echolucent and ulcerated plaque with .60% stenosis, OR 5 2.72, CI 95% (1.25–5.92), P 5 0.01; and for echolucent or ulcerated plaque
Table 3 The apolipoprotein E gene (APOE) genotypes and carotid plaques characteristic in 388 ischemic stroke patients
Results
Plaque characteristics
E3 n 5 273 E2 n 5 39 E4 n 5 71
During the study period, 497 ischemic stroke patients were admitted to our department. We have examined 388 patients (11 had severe stroke and did not cooperate during the ultrasound exam or died during first 48 hours, 8 withdrew consent, 90 patients were nor scanned with the examination protocol).
Plaque presence, n (%) 156 (57.1) Hypoechogenicity, n (%) 84 (30.8) Stenosis .60%, n (%) 75 (27.5) Cap ulceration, n (%) 104 (38.1)
28 19 15 22
(71.8) (48.7)* (38.5) (56.4)*
39 18 15 22
(54.9) (25.4) (21.1)* (31.0)
* P , 0.05 comparing to E3. E3 is a e 3/e 3 genotype (the reference), E2 is e 2/e 3 genotype, E4 is e 3/e 4 and e 4/e 4 genotype.
Table 2 Laboratory results
ESR (mm/hour) – median (IQR) WBC (K/ml) – median (IQR) PLT (K/ml) – median (IQR) TC . 200 mg/dl, n (%) LDL-C . 130 mg/dl, n (%) Hyper-TG, n (%) HDL-C # 45 mg/dl, n (%)
E3
E2
E4
16.0 (18.0) 7.9 (3.6) 219.0 (96.0) 149 (57.1) 156 (59.8) 68 (26.1) 130 (49.6)
24.5 (20.0)* 8.5 (3.2) 228.0 (107.5) 18 (47.4) 15 (40.5)* 9 (23.7) 16 (42.1)
16 (19.1) 7.8 (3.1) 227 (104.0) 39 (56.6) 45 (66.2)* 10 (14.5) 33 (48.5)
ESR: erythrocyte sedimentation rate; WBC: white blood count; PLT: platelets; TC: total cholesterol; LDL-C: low-density lipoprotein; HDL-C: high-density lipoprotein; TG: triglycerides; IQR: interquartile range. * P , 0.05 comparing to E3 E3 is a e 3/e 3 genotype (the reference), E2 is e 2/e 3 genotype, E4 is e 3/e 4 and e 4/e 4 genotype.
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with .60% stenosis, OR 5 2.32, CI 95% (1.11–4.85), P 5 0.024.
Discussion This is the first study, to our knowledge, showing the association of APOE polymorphism and carotid plaque morphology. In our study, we did not observe statistically significant association between APOE genotype and the presence or number of carotid plaques. There have been only few studies exploring the association of APOE polymorphism and presence of carotid plaques. One recent study showed that e 4 allele was significantly associated with extracranial carotid artery, but not intracranial stenosis.4 Two studies showed higher frequency of carotid plaques in e 4 and lower in e 2 carriers,5,9 one showed higher frequency of plaques in hypertensive e 4 carriers,7 one lower frequency of plaques in e 2 women carriers and higher frequency of plaques in men e 4 carriers.6 There are also studies showing no such association.8 Recently, more attention is paid not only to the presence or absence of plaques but also to the plaque morphology and the term ‘vulnerable plaque’ has been introduced for coronary plaques.17 This term was extended for carotid plaques. Those plaques are considered to be high-risk, prone to thrombotic complication, and causing ischemic events due to cerebral emboli.11 Echolucent plaques containing more soft tissue (lipid and/or hemorrhagic core) are thought to be an independent risk factor for ischemic stroke.18 Plaque ulceration is also a predictor of future ischemic events.19 Both plaque thrombus and ulceration are more prevalent in symptomatic patients comparing to asymptomatic.20 In our study, e 2 allele was an independent risk factor for having a vulnerable plaque, though e 2 carriers had hypercholesterolemia (LDL-C . 130 mg/dl) less often than e 3/e 3 homozygotes. Trying to explain that phenomenon we have to look more carefully at lipid metabolism, beyond cholesterol levels. In e 2 carriers, catabolism of triglyceride-rich lipoproteins, VLDL, and chylomicrons remnants are impaired. Those subjects have lower receptor binding affinity and subsequent upregulation of hepatic LDL receptors resulting in VLDL and chylomicrons accumulation in plasma and lower LDL cholesterol levels. Accumulation of VLDL and chylomicrons remnants is considered to be an atherogenic factor, like in type III hyperlipoproteinemia, disease associated with the presence of E2/E2 phenotype.21 To understand apoE effect on plaque ulceration, we must look at lipid effect of apoE. Plaque ulceration is a complex process that requires chronic increase in shear stress at sites of stenosis and sudden changes in intraarterial pressure. Intraplaque inflammation is an
APOE e 2 allele
initiator of ulceration. Apolipoprotein E is a mediator in inflammatory process – an enhanced inflammatory reaction is associated with the APOE4 allele after cardiopulmonary bypass.22 Apolipoprotein E can stimulate anti-inflammatory processes by decreasing VCAM-1 expression.23 Apolipoprotein E have also several other properties like modulation of platelet function, proliferation and migration of smooth muscle cells, and different oxidative effects.4,24,25 Apolipoprotein E-deficient mice suffer from spontaneous plaque rupture and thrombosis.26 Both inflammation and cell migration are triggers for plaque’s cap erosion.27 In our study, e 2 carriers had higher ESR rate on admission than other groups, and ESR rate is one of the inflammation markers. Non-lipid effects of apoE and the influence on plaque characteristics might be the key to explain different results regarding the role of APOE polymorphism in stroke. The main limitation of our study is the small sample size, but there was no selection bias as all consecutively admitted patients were included. To conclude, our study suggests that the e 2 allele is an independent risk factor for having echolucent and/ or ulcerated plaque, no matter the degree of stenosis.
Disclaimer Statements Contributors B. Blazejewska-Hyzorek – data collection and revising the manuscript; G. Gromadzka – data collection, statistical analysis, and revising the manuscript; M. Skowronska – manuscript writing and revising the manuscript; A. Czlonkowska – study design and revising the manuscript. Funding None. Conflicts of interest All authors have read the manuscript. The paper has not been previously published, and it is not under simultaneous consideration by another journal. All authors played a role in preparing the manuscript, there was no ghost writing. Authors report no financial or other conflicts of interest. Ethics approval Our study and publication has received ethical approval from Ethics Committee of Institute of Psychiatry and Neurology.
Acknowledgements None
References 1 Paternoster L, Gonzalez NAM, Lewis S, Sudlow C. Association between apolipoprotein E genotype and carotid intima-media thickness may suggest a specific effect on large artery atherothrombotic stroke. Stroke. 2008;39:48–54. 2 Sudlow C, Gonzalez NAM, Kim J, Clark C. Does Apolipoprotein E Genotype influence the risk of ischemic stroke, intracerebral hemorrhage, or subarachnoid hemorrhage? Systematic review and meta-analyses of 31 studies among 5961 cases and 17 965 controls. Stroke. 2006;37:364–70.
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3 Grane´r M, Kahri J, Varpula M, Salonen RM, Nyysso¨nen K, Jauhiainen M, et al. Apolipoprotein E polymorphism is associated with both carotid and coronary atherosclerosis in patients with coronary artery disease. Nutr Metab Cardiovasc Dis. 2008;18:271–7. 4 Kwee RM, van Oostenbrugge RJ, Hofstra L, Teule GJ, van Engelshoven JM, Mess WH, et al. Identifying vulnerable carotid plaques by noninvasive imaging. Neurology. 2008;70:2401–9. 5 Chutinet A, Suwanwela NC, Snabboon T, Chaisinanunkul N, Furie KL, Phanthumchinda K. Association between genetic polymorphisms and sites of cervicocerebral artery atherosclerosis. J Stroke Cerebrovasc Dis. 2012;21(5):379–85. 6 Slooter AJ, Bots ML, Havekes LM, del Sol AI, Cruts M, Grobbee DE, et al. Apolipoprotein E and carotid atherosclerosis: the Rotterdam study. Stroke. 2001;32:1947–52. 7 Beilby JP, Hunt CC, Palmer LJ, Chapman CM, Burley JP, McQuillan BM, et al. Carotid Ultrasound Disease Assessment Study. Apolipoprotein E gene polymorphisms are associated with carotid plaque formation but not with intima-media wall thickening: results from the Perth Carotid Ultrasound Disease Assessment Study (CUDAS). Stroke. 2003;34:869–74. 8 Karvonen J, Kauma H, Kervinen K, Ukkola O, Rantala M, Pa¨iva¨nsalo M, et al. Apolipoprotein E polymorphism affects carotid artery atherosclerosis in smoking hypertensive men. J Hypertens. 2002;20:2371–8. 9 Asakimori Y, Yorioka N, Tanaka J, Kohno N. Effect of polymorphism of the endothelial nitric oxide synthase and apolipoprotein E genes on carotid atherosclerosis in hemodialysis patients. Am J Kidney Dis. 2003;41:822–32. 10 Debette S, Lambert JC, Garie´py J, Fievet N, Tzourio C, Dartigues JF, et al. New insight into the association of apolipoprotein E genetic variants with carotid plaques and intima-media thickness. Stroke. 2006;37:2917–23. 11 Kumar NT, Liestøl K, Løberg EM, Reims HM, Brorson SH, Maehlen J. The apolipoprotein E polymorphism and cardiovascular diseases – an autopsy study. Cardiovasc Pathol. 2012;21(6):461–9. 12 World Health Organization (WHO). International classification of impairments disabilities, and handicaps. Geneva: World Health Organization; 1980. 13 Expert Panel on the Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on the Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA 2001, 285, 2486–2497. 14 Hixson JE, Vernier DT. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI. J Lipid Res. 1990;31:545–8.
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15 Zureik M, Ducimetie`re P, Touboul PJ, Courbon D, BonithonKopp C, Berr C, et al. Common carotid intima-media thickness predicts occurrence of carotid atherosclerotic plaques: longitudinal results from the Aging Vascular Study (EVA) study. Arterioscler Thromb Vasc Biol. 2000;20:1622–9. 16 De Bray JM, Baud JM, Dauzat M. Consensus concerning the morphology and the risk of carotid plaques. Cerebrovasc Dis. 1997;8:289–96. 17 Casscells W, Naghavi M, Willerson JT. Vulnerable atherosclerotic plaque. A multifocal disease. Circulation. 2003; 107:2072–5. 18 Mathiesen EB, Bonaa KH, Joakimsen O. Echolucent plaques are associated with high risk of ischemic cerebrovascular events in carotid stenosis: the Tromso Study. Circulation. 2001; 103:2171–5. 19 Prabhakaran S, Rundek T, Ramas R, Elkind MS, Paik MC, Boden-Albala B, et al. Carotid plaque surface irregularity predicts ischemic stroke: the Northern Manhattan study. Stroke. 2006;37:2696–701. 20 Fisher M, Paganini-Hill A, Martin A, Cosgrove M, Toole JF, Barnett HJ, et al. Carotid plaque pathology. Thrombosis, ulceration, and stroke pathogenesis. Stroke. 2005;36:253–7. 21 Utermann G, Vogelberg KH, Steinmetz A, Schoenborn W, Pruin N, Jaeschke M, et al. Polymorphism of apolipoprotein E, II: genetics of hyperlipoproteinemia type III. Clin Genet. 1979;15:37–62. 22 Grocott HP, Newman MF, El-Moalem H, Bainbridge D, Butler A, Laskowitz DT. Apolipoprotein E genotype differentially influences the proinflammatory and anti-inflammatory response to cardiopulmonary bypass. J Thorac Cardiovasc Surg. 2001;122:622–3. 23 Stannard AK, Riddell DR, Sacre SM, Tagalakis AD, Langer C, von Eckardstein A, et al. Cell-derived apolipoprotein E (ApoE) particles inhibit vascular cell adhesion molecule-1 (VCAM-1) expression in human endothelial cells. J Biol Chem. 2001;276:46011–6. 24 Mazzone T. Apolipoprotein E secretion by macrophages: its potential physiological functions. Curr Opin Lipidol. 1996;7:303–7. 25 Davignon J. Apolipoprotein E and atherosclerosis: beyond lipid effect. Arterioscler Thromb Vasc Biol. 2005;25:267–9. 26 Calara F, Silvestre M, Casanada F, Yuan N, Napoli C, Palinski W. Spontaneous plaque rupture and secondary thrombosis in apolipoprotein E-deficient and LDL receptor-deficient mice. J Pathol. 2001;195:257–63. 27 Redgrave JN, Lovett JK, Gallagher PJ, Rothwell PM. Histological assessment of 526 symptomatic carotid plaques in relation to the nature and timing of ischemic symptoms: the Oxford Plaque Study. Circulation. 2006;113:2320–8.
2nd Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland, 2Department of Experimental and Clinical Pharmacology, Warsaw Medical University, Warsaw, Poland
Background and Aims: The data about apolipoprotein E gene (APOE) genotype and the risk of stroke are inconsistent. The APOE genotype is expected to influence the development of carotid plaques. Our aim was to look for association between APOE genotype and carotid plaque morphology in ischemic stroke patients. Methods and Results: Data of ischemic stroke patients was collected prospectively for 2 years. The degree of stenosis and plaque echogenicity and surface were assessed with ultrasound. Subsequent APOE genotypes were compared: APOE e 3/e 3 (E3 – reference), APOE e 2/e 3 (E2 group) and APOE e 3/e 4, APOE e 4/e 4 (E4 group). We included 388 patients with acute ischemic stroke. Patients in E2 group had more often hypoechogenic, ulcerated plaques and severe stenosis comparing to E3 patients. On logistic regression analysis, e 2 genotype remained an independent risk factor for vulnerable carotid plaque (OR 5 2.3 for ,60% stenosis and OR 5 2.7 for $60% stenosis; 95% CI). Conclusions: This study suggests that e 2 allele is an independent risk factor for echolucent and ulcerated carotid plaque. Keywords: Apolipoproteins, Carotid plaque, Ischemic stroke
Introduction The apolipoprotein E gene (APOE) is widely studied in vascular diseases, including stroke.1–3 Its product, apolipoprotein E (apoE), is a glycoprotein, having three common isoforms, encoded by the alleles e 2, e 3, and e 4, which give rise to six genotypes. The most common is e 3/e 3, occurring in about one-half to twothirds in most populations. The apoE isoforms interact differently with lipoprotein receptors. They alter levels of cholesterol through different hepatic binding, uptake and catabolism of remnants of chylomicrons, and very-low-density lipoprotein (VLDL). The e 4 allele carriers appear to have higher plasma cholesterol levels, whereas the e 2 allele carriers lower, so APOE genotype is expected to influence the development of atherosclerosis. Not only the presence of carotid plaques and the degree of carotid stenosis, but also plaque properties are important for vascular disease. Dark and low-echogenicity (echolucent)
Correspondence to: A. Członkowska, Second Department of Neurology, Institute of Psychiatry and Neurology, 9 Sobieskiego Street, 02-957 Warsaw, Poland. Email: [email protected]
950
ß W. S. Maney & Son Ltd 2014 DOI 10.1179/1743132814Y.0000000385
plaques on ultrasound are thought to be more ‘vulnerable’ causing vascular events.4 Few authors have tested the association between carotid plaques and APOE polymorphism, reporting inconsistent results.5–10 A recent autopsy study showed that the risk of developing and dying from cardiovascular disease, including coronary heart disease and cerebrovascular disease, is influenced by the apoE polymorphism, with more e 4-carriers and fewer e 2-carriers.11 We aimed to investigate the association of carotid plaques and the APOE polymorphism in ischemic stroke population.
Materials and Methods Study population All consecutive ischemic stroke patients, admitted within 7 days from stroke onset to our department, in 2 years period were considered for the study. All patients were Caucasians. Stroke was defined according to World Health Organization12 as a neurological deficit lasting over 24 hours. All patients had brain imaging (Computed
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Tomography – CT or Magnetic Resonance – MR) to exclude other, than ischemic, causes of symptoms. Patients who were unable to cooperate during the ultrasound examination (e.g. patients with very severe stroke, anxious during examination) were excluded from the study. Data about medical history, personal habits, and basic laboratory tests were prospectively collected. Smokers were defined as ever smoking. Hypertension was defined as either current blood pressurelowering therapy or a systolic blood pressure $ 140 mmHg and/or a diastolic blood pressure $ 90 mmHg on two separate measures done after acute phase of stroke. Diabetes mellitus was defined as a history of known diabetes or currently diagnosed (as a fasting plasma glucose level $100 mg/dl or as 2hour plasma glucose $200 mg/dl blood collected 1 week after stroke). Hypercholesterolemia was defined as fasting total cholesterol level .200 mg/dl, high-density lipoprotein (HDL) cholesterol ,45 mg/ dl, low-density lipoprotein (LDL) cholesterol .130 mg/dl, and triglyceride level .150 mg/dl.13 Data about vascular events defined as: history of stroke (or TIA), myocardial infarction, and angina pectoris (previously diagnosed or diagnosed during current hospitalization) were collected.
APOE e 2 allele
Genotyping Genomic DNA was extracted from the whole frozen EDTA-blood using the TRI Reagent (SIGMA, Poznan, Poland). The genotyping of APOE was performed by polymerase chain reaction-restriction fragments length polymorphism (PCR-RFLP), as previously described.14 In the first step, PCR was performed with primers that allowed for amplification of the polymorphic region. PCR product was digested by HhaI restriction endonuclease and then separated with electrophoresis.
Ultrasound examination The B-mode system (Acuson 128XP/10C, Siemens, Berlin and Munich, Germany) with 7 MHz probe was used for plaque assessment. This high-resolution, non-invasive technique can be applied for plaque identification and for assessment of plaque echogenicity and cap irregularity.11 The examination protocol involved scanning of the common carotid arteries (CCA), the carotid bifurcations, and the origin (first 2 cm) of the internal carotid arteries (ICA) and external carotid arteries (ECA). Near and far walls of all arterial segments were scanned longitudinally and transversally for the presence of the plaques. Both ipsi- and contralateral carotid arteries were examined and plaques were
Table 1 Population characteristics n 5 388 Age, years Sex, male, n (%) Hypertension, n (%) AF, n (%) DM, n (%) Smoking, n (%) Previous stroke, n (%) Previous TIA, n (%) CHD, n (%) MI, n (%) Total cholesterol (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) TG (mg/dl) Total cholesterol . 200 mg/dl, n (%) HDL-C # 45 mg/dl, n (%) LDL-C . 130 mg/dl, n (%) TG . 150 mg/dl Carotid plaques, n (%) APOE polymorphism, n (%) e 2/e 2 e 2/e 3 (E2) e 3/e 3 (E3) e 2/e 4 e 3/e 4 (E4) e 4/e 4 (E4) TOAST classification Large artery atherosclerosis, n (%) Cardioembolism, n (%) Small-vessel occlusion, n (%) Stroke of other determined etiology Stroke of undetermined etiology (two or more causes) Stroke of undetermined etiology (negative/incomplete evaluation)
68 ¡ 11.1* 203 (52) 257 (66.8) 61 (15.8) 73 (19.0) 192 (49.5) 54 (14.0) 49 (12.7) 114 (29.5) 58 (15.0) 205.0 ¡ 56.5* 43.8 ¡ 16.6* 142.5 ¡ 47.0* 111.0 ¡ 65.4* 207 (55.5) 207 (55.5) 218 (58.8) 49 (13.1) 223 (57.4) 0 39 273 5 69 2
(10.1) (71.3) (1.3) – excluded (17.8) (0.5)
86 69 84 12 122 15
(22.2) (17.8) (21.6) (3.1) (31.4) (3.9)
*
Mean ¡ SD. AF: atrial fibrillation; DM: diabetes mellitus; CHD: coronary heart disease; MI: myocardial infarction; LDL-C: low-density lipoprotein; HDL-C: high-density lipoprotein; TG: triglycerides; TOAST: trial of org 10172 in acute stroke treatment.
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assessed on both sides (data is presented for both sides). The plaque was defined as localized echo structures encroaching into the vessel lumen for which the thickness was $1 mm.15 Hyperechogenic plaques were identified if .50% of plaque surface was hyperechogenic (echogenicity of the bones of the vertebral column) other plaques were described as hypoechogenic (echolucent). Plaque surface was assessed as ulcerated when the cap rupture was $ 2 mm.16 The degree of stenosis was assessed with the velocity criteria. The degree $60% was considered as severe stenosis. All examinations were performed no later than 7 days from admission, in majority of cases in the first 3 days after admission. The intraobserver coefficients of variability were 4.0% for performing sonographer (BB-H). Informed consent was obtained from all participants and the study was approved by the institutional ethics committee.
Statistics Data were analyzed with the Statistica PL 7.0 software package. Genotype APOE e 3/e 3 (E3) was considered as a reference. It was compared with APOE e 2/e 3 (E2) and APOE e 3/e 4 and APOE e 4/e 4 (E4). We excluded APOE e 2/e 4 genotype from the analysis, as those two alleles are considered to be opposite in lipid metabolism. The chi-square test was used to compare proportions and Student t or Mann–Whitney U test was used to compare continuous variables. Multivariate logistic regression analysis was used to assess the independent contribution of variables statistically significant on univariate analysis in the prediction of degree of atherosclerosis.
Demographic characteristics, vascular risk factors, APOE polymorphism, and stroke etiology are given in Table 1. There was no association between APOE polymorphism and the prevalence of vascular risk factors. The laboratory results are presented in Table 2. Erythrocyte sedimentation rate (ESR) was significantly higher in e 2 carriers than in reference group, but not lower in e 4. There was no interaction shown between total cholesterol levels and APOE polymorphism, but we found that e 2 carriers had LDLC . 130 mg/dl less often while e 4 more often than the reference E3 population (40.5, 66.2, and 59.8% respectively. Carotid plaques were found in 57% of patients. Over 60% carotid stenosis (both ipsi- and contralateral) stenosis was found more often in E2 than E4 group. There was no association between presence of plaques, number of carotid plaques (data about number not shown), and APOE polymorphism. We found that e 2/e 3 carriers had more often echolucent plaques with ulcerated fibrous cap comparing to e 3/e 3 homozygotes (Table 3). In multivariate logistic regression analysis adjusted for the vascular risk factors age, sex, hypertension, diabetes, smoking, cholesterol level, and history of vascular event, e 2/e 3 genotype was an independent risk factor for: echolucent or ulcerated plaque with ,60% stenosis, OR 5 2.27, CI 95% (1.09–4.71), P 5 0.03; echolucent and ulcerated plaque with .60% stenosis, OR 5 2.72, CI 95% (1.25–5.92), P 5 0.01; and for echolucent or ulcerated plaque
Table 3 The apolipoprotein E gene (APOE) genotypes and carotid plaques characteristic in 388 ischemic stroke patients
Results
Plaque characteristics
E3 n 5 273 E2 n 5 39 E4 n 5 71
During the study period, 497 ischemic stroke patients were admitted to our department. We have examined 388 patients (11 had severe stroke and did not cooperate during the ultrasound exam or died during first 48 hours, 8 withdrew consent, 90 patients were nor scanned with the examination protocol).
Plaque presence, n (%) 156 (57.1) Hypoechogenicity, n (%) 84 (30.8) Stenosis .60%, n (%) 75 (27.5) Cap ulceration, n (%) 104 (38.1)
28 19 15 22
(71.8) (48.7)* (38.5) (56.4)*
39 18 15 22
(54.9) (25.4) (21.1)* (31.0)
* P , 0.05 comparing to E3. E3 is a e 3/e 3 genotype (the reference), E2 is e 2/e 3 genotype, E4 is e 3/e 4 and e 4/e 4 genotype.
Table 2 Laboratory results
ESR (mm/hour) – median (IQR) WBC (K/ml) – median (IQR) PLT (K/ml) – median (IQR) TC . 200 mg/dl, n (%) LDL-C . 130 mg/dl, n (%) Hyper-TG, n (%) HDL-C # 45 mg/dl, n (%)
E3
E2
E4
16.0 (18.0) 7.9 (3.6) 219.0 (96.0) 149 (57.1) 156 (59.8) 68 (26.1) 130 (49.6)
24.5 (20.0)* 8.5 (3.2) 228.0 (107.5) 18 (47.4) 15 (40.5)* 9 (23.7) 16 (42.1)
16 (19.1) 7.8 (3.1) 227 (104.0) 39 (56.6) 45 (66.2)* 10 (14.5) 33 (48.5)
ESR: erythrocyte sedimentation rate; WBC: white blood count; PLT: platelets; TC: total cholesterol; LDL-C: low-density lipoprotein; HDL-C: high-density lipoprotein; TG: triglycerides; IQR: interquartile range. * P , 0.05 comparing to E3 E3 is a e 3/e 3 genotype (the reference), E2 is e 2/e 3 genotype, E4 is e 3/e 4 and e 4/e 4 genotype.
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with .60% stenosis, OR 5 2.32, CI 95% (1.11–4.85), P 5 0.024.
Discussion This is the first study, to our knowledge, showing the association of APOE polymorphism and carotid plaque morphology. In our study, we did not observe statistically significant association between APOE genotype and the presence or number of carotid plaques. There have been only few studies exploring the association of APOE polymorphism and presence of carotid plaques. One recent study showed that e 4 allele was significantly associated with extracranial carotid artery, but not intracranial stenosis.4 Two studies showed higher frequency of carotid plaques in e 4 and lower in e 2 carriers,5,9 one showed higher frequency of plaques in hypertensive e 4 carriers,7 one lower frequency of plaques in e 2 women carriers and higher frequency of plaques in men e 4 carriers.6 There are also studies showing no such association.8 Recently, more attention is paid not only to the presence or absence of plaques but also to the plaque morphology and the term ‘vulnerable plaque’ has been introduced for coronary plaques.17 This term was extended for carotid plaques. Those plaques are considered to be high-risk, prone to thrombotic complication, and causing ischemic events due to cerebral emboli.11 Echolucent plaques containing more soft tissue (lipid and/or hemorrhagic core) are thought to be an independent risk factor for ischemic stroke.18 Plaque ulceration is also a predictor of future ischemic events.19 Both plaque thrombus and ulceration are more prevalent in symptomatic patients comparing to asymptomatic.20 In our study, e 2 allele was an independent risk factor for having a vulnerable plaque, though e 2 carriers had hypercholesterolemia (LDL-C . 130 mg/dl) less often than e 3/e 3 homozygotes. Trying to explain that phenomenon we have to look more carefully at lipid metabolism, beyond cholesterol levels. In e 2 carriers, catabolism of triglyceride-rich lipoproteins, VLDL, and chylomicrons remnants are impaired. Those subjects have lower receptor binding affinity and subsequent upregulation of hepatic LDL receptors resulting in VLDL and chylomicrons accumulation in plasma and lower LDL cholesterol levels. Accumulation of VLDL and chylomicrons remnants is considered to be an atherogenic factor, like in type III hyperlipoproteinemia, disease associated with the presence of E2/E2 phenotype.21 To understand apoE effect on plaque ulceration, we must look at lipid effect of apoE. Plaque ulceration is a complex process that requires chronic increase in shear stress at sites of stenosis and sudden changes in intraarterial pressure. Intraplaque inflammation is an
APOE e 2 allele
initiator of ulceration. Apolipoprotein E is a mediator in inflammatory process – an enhanced inflammatory reaction is associated with the APOE4 allele after cardiopulmonary bypass.22 Apolipoprotein E can stimulate anti-inflammatory processes by decreasing VCAM-1 expression.23 Apolipoprotein E have also several other properties like modulation of platelet function, proliferation and migration of smooth muscle cells, and different oxidative effects.4,24,25 Apolipoprotein E-deficient mice suffer from spontaneous plaque rupture and thrombosis.26 Both inflammation and cell migration are triggers for plaque’s cap erosion.27 In our study, e 2 carriers had higher ESR rate on admission than other groups, and ESR rate is one of the inflammation markers. Non-lipid effects of apoE and the influence on plaque characteristics might be the key to explain different results regarding the role of APOE polymorphism in stroke. The main limitation of our study is the small sample size, but there was no selection bias as all consecutively admitted patients were included. To conclude, our study suggests that the e 2 allele is an independent risk factor for having echolucent and/ or ulcerated plaque, no matter the degree of stenosis.
Disclaimer Statements Contributors B. Blazejewska-Hyzorek – data collection and revising the manuscript; G. Gromadzka – data collection, statistical analysis, and revising the manuscript; M. Skowronska – manuscript writing and revising the manuscript; A. Czlonkowska – study design and revising the manuscript. Funding None. Conflicts of interest All authors have read the manuscript. The paper has not been previously published, and it is not under simultaneous consideration by another journal. All authors played a role in preparing the manuscript, there was no ghost writing. Authors report no financial or other conflicts of interest. Ethics approval Our study and publication has received ethical approval from Ethics Committee of Institute of Psychiatry and Neurology.
Acknowledgements None
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