| Research backgroundIntracranial atherosclerosis(ICAS) represents one of the main causes of ischemic stroke, and is associated with a high risk of recurrent stroke. So far, there has been no effective preventive measure to decrease the risk of recurrence of stroke in patients with symptomatic ICAS, especially after the advantage of intracranial intervention treatment was denied by the stenting and aggressive medical management for preventing recurrent stroke in intracranial stenosis(SAMMPRIS) trial. Therefore, in addition to aggressive drug treatment, strictly controlling risk factors also plays an important role for prevention of ICAS.ICAS was reported frequently in Asians, blacks, and Hispanics, while extracranial atherosclerosis(ECAS) was more prevalent among whites than else. The mechanism of different distribution between ICAS and ECAS was still unclear. Except genetic factor, the distribution differences of the atherosclerotic risk factors and different susceptibility to the risk factors may also be related to the distribution differences of ICAS and ECAS. Clinical data from Japanese population showed that the incidence of symptomatic carotid artery stenosis increased in recent 20 years, while the incidence of ICAS has no obvious change. Analysis result suggested this may be associated with increased incidence of diabetes in Japanese population. So, it is particularly important to detect the specific risk factors for ICAS to prevent the occurrence of ICAS and decrease the incidence of ischemic stroke related to ICAS.However, only a few clinical studies had compared difference in the risk factors for ICAS and ECAS. In white and black patients with ischemic stroke, only found that male as a risk factor of ICAS, but only the white was a risk factor for ECAS. Age, smoking and hypertension were closely related to the risk of ICAS, while women and hyperlipidemia were significantly correlated with the risk of ECAS in South Korea population. In the white population, age, male, hypertension, diabetes, smoking and heavy drinking were closely associated with the risk of ICAS, but only age and diabetes were closely related to the risk of ECAS. Clinical data from Japanese population did not found differences in the risk factor for ICAS and ECAS. The results of different studies were not consistent. At present, there has no clinical research to compared differences in risk factor of ICAS and ECAS in Chinese population.In recent years, a few clinical researches also discussed the correlation of metabolic syndrome(Mets), plasma adiponectin level, and single nucleotide polymorphisms(SNPs) of genes with the risk of ICAS. Mets represents a constellation of metabolic and vascular risk factors, including obesity, glucose intolerance or diabetes, elevated triglyceride, reduced high density lipoprotein cholesterol, and elevated blood pressure, and is closely related to the pathological process of vascular atherosclerosis and inflammation. In South Korea patients with ischemic stroke, Mets was closely related to the risk of ICAS, and no correlation with the risk of ECAS. However, in the stroke-free white population, clinical data suggested that Mets was both associated with risk of ICAS and ECAS.Adiponectin is a special adipokine which express in adipocytes and exert insulin-sensitizing, anti-inflammatory, and anti-atherosclerotic effects. Because closely correlated to inflammatory pathological process, disorders of lipid metabolism, and glucose metabolism, adiponectin was regarded as a potential link of Mets and cardiovascular disease. Clinical evidences showed that blood concentration of adiponectin reduced in patients with Mets compared to patients without Mets. In addition, hypoadiponectinemia was also associated with risk of coronary and carotid atherosclerosis and cardiovascular disease. In South Korea patients with ischemic stroke, it was found that the plasma adiponectin levels of ICAS patients were significantly lower than that of ECAS patients; and high adiponectin level may be the protection factor for ICAS rather than ECAS.Clinical data suggested that adiponectin levels were different among various ethnic populations. It was lower in the young American black and Indian-Asian populations as compared to the young non-Hispanic whites. Many clinical studies have demonstrated that adiponectin gene(ADIPOQ) SNPs were closely associated with the reduced adiponectin levels. Moreover, ADIPOQ SNPs were closely related to the increased carotid artery intima thickness, serious coronary artery stenosis, and increased risk of cardiovascular events. However, whether ADIPOQ gene polymorphisms are related to ICAS has not been clinically studied and remains unknown.In summary, further research is needed to discuss the correlation of metabolic syndrome, adiponectin level, and ADIPOQ SNPs with the risk of ICAS. Therefore, in this case-control study, we discuss the correlation of metabolic syndrome, adiponectin level and ADIPOQ gene SNPs with the risk of ICAS. In addition, we compared the association between these factors with risk of ICAS and ECAS to investigate the risk factors specific to ICAS.Subjects and methods1. Study populationDuring 2012 June to 2013 January, consecutive patients who visited in our center were recruited into this study. The following patients were excluded:(1) patients were < 50 years old;(2) patients with intracranial or extracranial stenosis caused by dissection, moya moya disease, vasculitis, radiation-induced vasculopathy, fibromuscular dysplasia, tortuosity, or extravascular oppress;(3) patients’ clinical data were incomplete.2. Definition of intracranial and extracranial atherosclerosisAll subjects underwent a computed tomography angiography(CTA) examination of the cervical and intracranial vessels to determine the presence of atherosclerotic lesions. The ICAS was defined as stenosis of 50% or greater in the large intracranial artery(intracranial segments of the internal carotid artery [ICA], middle cerebral artery [MCA], anterior cerebral artery [ACA], posterior cerebral artery [PCA], intracranial segments of vertebral artery [VA], and basilar artery [BA]). Intracranial arteries stenos is was measured according to the warfarin-aspirin symptomatic intracranial disease(WASID) trial criteria. The ECAS was defined as stenosis of 50% or greater in the large cervical artery(carotid artery and VA). Cervical arteries stenosis was measured according to North American symptomatic carotid endarterectomy(NASCET) trial criteria.The subjects were classified into four groups according to the location of atherosclerosis lesions:(1) ICAS group: at least one stenosis of 50% or greater was present in the major intracranial arteries and without any stenosis of 50% or greater in the cervical arteries.(2) ECAS group: at least one stenosis of 50% or greater was present in the cervical arteries and without any stenosis of 50% or greater in the intracranial arteries.(3) Combined ICAS/ECAS group: stenoses of 50% or greater were present in both intracranial and cervical arteries simultaneously.(4) Non-atherosclerosis group: no stenosis of 50% or greater was present in intracranial and cervical arteries.3. Collection of clinical data related to the atherosclerotic risk factorsThe data of demographic data, medical history, and vascular risk factors were collected from the subjects’ self-report information, physical examination and the medical records. Fasting blood samples were drawn and sent to our central laboratory to measure the level of blood glucose, total cholesterol, triglyceride, high density lipoprotein cholesterol, and low density lipoprotein cholesterol.4. Definition of metabolic syndromeThe Mets was defined according to the updated United States National Cholesterol Education Program: Adult Treatment Panel III criteria, which required the presence of at least three of the following components:(1) abdominal obesity: waist circumference ≥ 90 cm in men or ≥ 80 cm in women.(2) elevated blood pressure: systolic blood pressure(SBP) ≥ 130 mm Hg, diastolic blood pressure(DBP) ≥ 85 mm Hg, or treated hypertension;(3) elevated fasting glucose(FSG): FSG ≥ 5.6mmol/L or previously diagnosed with type 2 diabetes;(4) elevated TG: TG level ≥ 1.70mmol/L; and(5) reduced HDL-C: HDL-C level < 1.03mmol/L in men or < 1.29mmol/L in women.5. Measurement of plasma adiponectin concentrationAll venous blood samples were drawn after an overnight fast. Then plasma were immediately separated by centrifugation at 3,000 rpm at 4℃ for 5 minutes and stored in refrigerator at – 80℃. Plasma was diluted 100-fold before assay. The concentration of plasma adiponectin was determined by commercially available enzyme linked immunosorbent assay(ELISA) kit(Boster, Wuhan).6. ADIPOQ Tag SNPs selection and genotypingGenotype data of ADIPOQ in the Chinese Han sample were downloaded from the Hap Map database(http://www.hapmap.org). The data was introduced into Haploview software(version 4.2), and screened the tag SNPs from the SNPs whose minimum allele frequency(MAF) > 0.05. With reference to the Adipo Q tag SNPs in Japanese(http://snp.ims.u-tokyo.ac.jp/) and the literatures about ADIPOQ SNPs, we selected tag SNPs which associated with adiponectin levels, atherosclerosis or cardiovascular events.Collection and preservation blood samples: venous blood samples were drawn and separated according to the former method, and blood cells were stored in refrigerator at – 80℃. Genomic DNA was extracted from peripheral blood leukocytes using a commercial blood DNA extract kit(Axygen Axy Prep ? Mag Blood Genomic DNA Kits, USA) and was stored at-20 °C. The genotyping of tag SNPs were performed using ligase detection reaction-polymerase chain reaction(LDR-PCR) by the Shanghai Bio Wing Applied Biotechnology Company(http://www.biowing.com.cn/).7. Statistical AnalysisThe continuous data are presented as the means ± standard deviation(SD), and the categorical data are presented as counts and percentages. The Student’s t-test was used to analyze continuous variable, and chi-square test was used to analyze categorical variable. The binary logistic regression analyses were performed to identify the independent risk factors for ICAS and ECAS. Two-tailed P-value < 0.05 were considered statistically significant. Statistical analyses were performed with the SPSS 18.0 statistical package.Result1. The baseline characteristics of study population975 subjects were recruited into this study, including 178(18.3%) ICAS patients, 42(4.3%) ECAS patients, 62(6.4%) combined ICAS/ECAS patients, and 693(71.1%) NCAS subjects.The results of binary logistic regression analyses suggested that age, hypertension, diabetes, and current smoking were closely related to the risk of ICAS, while age, male, hypertension, diabetes, current smoking, and history of CHD were signi ficantly associated with the risk of ECAS.2. The association between Mets and risk of ICASThe incidence of Mets in all 908 subjects was 39.0%, and it was higher in women compared to men(47.0% vs 30.3%, P < 0.001). In addition, the prevalence of Mets in ICAS group was higher compared to non-ICAS group both in men and women.2.1 The correlation of Mets with the risk of ICASAfter adjusting for age, sex, hypertension, diabetes, dyslipidemia, current smoking, and history of coronary heart disease(CHD),the results of binary logistic regression analyses suggested that Mets was closely related to the risk of ICAS in all 908 subjects. In Mets components, only elevated FSG was closely associated with the risk of ICAS. Further analysis suggested that no association was found between Mets and the risk of ECAS.2.2 The correlation of Mets with the risk of ICAS in ischemic stroke patientsAfter adjusting for conventional atherosclerotic risk factors, the results of binary logistic regression analyses suggested that no association was found between Mets with the risk of ICAS in patients with ischemic stroke. No association was found between Mets and the risk of ECAS.2.3 The correlation of Mets with the risk of ICAS in stroke-free patientsIn stroke-free patients, Mets was closely related to the risk of ICAS. Moreover, elevated BP and elevated FSG were closely associated with the risk of ICAS. But, no correlation was found between Mets and the risk of ECAS.2.4 The correlation of Mets with the risk of ICAS in menAfter adjusting for confounding factors, the results of binary logistic regression analyses suggested that Mets and two Mets components, including elevated BP and elevated FSG, were closely related to the risk of ICAS in men. No association was found between Mets and the risk of ECAS.2.5 The correlation of Mets with the risk of ICAS in womenIn women, no association was found between Mets with the risk of ICAS. Mets was not related to the risk of ECAS.3. The association between plasma adiponectin level and risk of ICAS3.1 The correlation of plasma adiponectin level with risk of ICAS405 subjects were recruited into this study, including 151 consecutive ICAS patients, 29 consecutive ECAS patients, 53 consecutive combined ICAS/ECAS patients, and 172 consecutive NCAS patients. The mean levels of adiponectin in ICAS group, ECAS group, combined ICAS/ECAS group, and NCAS group were 9.77 ± 4.83μg/ml, 3.72 ± 2.96 μg/ml, 6.39 ± 4.58μg/ml, and 12.05 ± 6.77μg/ml, respectively. There were significant differences in adiponectin levels between all the groups(P < 0.01).After adjusting for traditional atherosclerotic risk factors, the results of binary logistic regression analyses suggested that low adiponectin level was closely related to the increased risk of ICAS. Further analysis suggested that low adiponectin level was also closely related to the increased risk of ECAS.3.2 The correlation of plasma adiponectin level with risk of ICAS in ischemic stroke patientsThe results of binary logistic regression analyses suggested that adiponectin level was negatively related to the risk of ICAS in patients with ischemic stroke. In addition, adiponectin level was also negatively related to the risk of ECAS.3.3 The correlation of plasma adiponectin level with risk of ICAS in stroke-free patientsIn stroke-free patients, no association was found between adiponectin level and the risk of ICAS. But, negative correlation was found between adiponectin level and risk of ECAS.3.4 The correlation of plasma adiponectin level with risk of ICAS in menAfter adjusting for confounding factors, the results of binary logistic regression analyses suggested that adiponectin level was negatively related to the risk of ICAS in men. Moreover, the similar association was found between adiponectin level and risk of ECAS.3.5 The correlation of plasma adiponectin level with risk of ICAS in womenAfter adjusting for confounding factors, the results of binary logistic regression analyses suggested that adiponectin level was negatively related to the risk of ICAS in women. Further analysis suggested that adiponectin level was also negatively related to the risk of ECAS.3.6 The association between plasma adiponectin level, Mets and risk of ICASThe adiponectin level in Mets subjects was lower compared to non-Mets subjects. The similar result was found in men(7.09 ± 4.24μg/ml vs 10.69 ± 6.78μg/ml, P < 0.001), whereas no significant difference in adiponectin level was found between Mets patients and non-Mets subjects in women(10.06 ± 5.85μg/ml vs 11.54 ± 6.21μg/ml, P = 0.121). In addition, the adiponectin level in Mets subjects was lower than that of non-Mets subjects in NCAS group(P = 0.019), while no difference in adiponectin levels were found between Mets and non-Mets subjects in ICAS and ECAS groups.After adjusting for Mets, the results of binary logistic regression analyses suggested that both adiponectin level and Mets were closely related to the risk of ICAS in all 405 subjects. But, the correlation of adiponectin level with the risk of ICAS vanished after adjusting for Mets, while the association between Mets and risk of ICAS still remained in men. However, the reverse result was found in female.4. The association between ADIPOQ SNPs and risk of ICAS4.1 ADIPOQ Tag SNPs selection and genotypingAccording to the former method, 10 tag SNPs of ADIPOQ were selected in this study: rs822396 A > G, rs7649121 A > T, rs3774261 A > G, rs1063539 G > C, rs2241767 A > G, rs1501299 C > A, rs16861194 A > G, rs12495941 G > T, rs182052 G > A, rs266729 C > G. 602 subjects were successfully detected the 10 tag SNPs, including 147 consecutive ICAS patients, 31 consecutive ECAS patients, 52 consecutive combined ICAS/ECAS patients, and 372 consecutive NCAS patients.4.2 The correlation of ADIPOQ SNPs with risk of ICASAfter adjusting for conventional atherosclerotic risk factors, the results of binary logistic regression analyses suggested that the AG/AA genotype of rs2241767 and AG/GG genotype of rs182052 were closely correlated to the increased risk of ICAS. Further haplotype analysis of rs2241767 and rs182052 suggested that the frequency of haplotype A-G in ICAS group was significantly higher compared to control group. No association was found between ADIPOQ SNPs and risk of ECAS.ConclusionFirst, the results of this study suggested that metabolic syndrome, lower plasma adiponectin level, and ADIPOQ gene SNPs at rs2241767 and rs182052 were closely related to the increased risk of ICAS.Second, the associations between these factors and risk of ICAS were not the same in different population. Mets was significantly associated with risk of ICAS in stroke-free patients, but no association between Mets and risk of ICAS was found in ischemic stroke patients. Mets was closely related to risk of ICAS in men, while no association between Mets and risk of ICAS was found in women. The plasma adiponectin level was closely associated with risk of ICAS in ischemic stroke patients, whereas no association between adiponectin level and risk of ICAS was found in stroke-free patients.Third, no associations were found between metabolic syndrome and ADIPOQ gene SNPs with the risk of ECAS. Metabolic syndrome and ADIPOQ gene SNPs may be associated with ICAS distribution differences.These maybe not only help to provide a scientific basis for development of ICAS prevention strategies, but also to provide clues for the studies of the mechanisms in distribution difference of ICAS. |
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