| Background:Stroke is a complex disease caused by synergy between genetic and environmental factors. It is a major health burden in China. Mortality rate is high for stroke victims. The disability rate after stroke is up to70%in stroke survivors with very high recurrence rates (3and5-year recurrence rates were28%and51%, respectively). In China, there are about6-million stroke remnants and1.5-million of the new cases per year. This incidence gradually increases with younger age. Stroke is not one disease but rather a heterogeneous group of disorders, three subtypes of stroke were included:atherothrombotic, intracerebral hemorrhage and lacunar infarct. The mechanisms underlying stroke are multifactorial. One of the most important pathological basis of stroke is atherosclerosis. Currently, atherosclerosisis is a complex vascular disease with chronic inflammatory responses due to vascular injury. C-reactive protein (CRP), as one of systemic inflammation markers, plays a key role in the inflammatory responses. CRP levels in serum may reflect the potentially inflammatory activity in the body, and is closely related to stroke and other arteriosclerosis diseases, such as coronary heart disease. However, there are disputes concerning CRP in the pathogenesis of cerebrovascular diseases, from both the mechanism and epidemiological studies. Some believed that CRP directly mediates inflammatory responses and plays an important role in the occurrence of cardiovascular and cerebrovascular diseases. Others supported that no direct relationship was found between CRP levels and the occurrence of cardiovascular and cerebrovascular diseases. The increase of CRP levels just reflects the extent of non-specific inflammatory responses in atherosclerotic lesions. CRP is not directly involved in the development of atherosclerosis.In this study, the relationships between stroke susceptibility and stroke recurrence with serum CRP levels due to CRP genetic variations and haplotypes were investigated by multi-center case-control sdudy and prospective study, respectively.Objectives:This study intended to reveal whether CRP genetic variatnts and haplotypes are genetic risk factors for stroke susceptibility and recurrence, and whether CRP can be causal factor for stroke.Methods:A multi-center case-control study was used to investigate the association of serum levels of CRP (determined by using ELISA) and CRP variants with stroke susceptibility. A prospective study was used to explore the causal relation of CRP level and the CRP variants with stroke recurrence. Total1735patients with stroke (lacunar infarct (n=475), atherothrombotic (n=794) and intracerebral hemorrhage (n=466)) were followed-up for a period of4.5years (mean), the correlation was evaluated by using Kaplan-Meier analysis and the Cox regression models. The degree of linkage disequilibrium between genetic markers was calculated using Haploview software.Results:1. Serum CRP levels were significantly higher in patients and three stroke subtypes than in control. CRP levels were (median (range)):0.7100(9.96)mg/L for control group,1.0113(9.97)mg/L for overall stroke group,(for three subtypes:0.9784(9.85)mg/L for atherothrombotic stroke,1.0094(9.97)mg/L for lacunar infarct,1.0381(9.95)mg/L for intracerebral hemorrhage), P<0.01.2. After adjustment for traditional cardiovascular risk factors including age, sex, body mass index, systolic blood pressure, diastolic blood pressure, total cholesterol, HDL-C, diabetes and smoking, serum CRP levels in patients with rs1205A and rs2808630G alleles were significantly increased in both case and control group. CRP levels (median (range),mg/L) with rs1205allele were0.8106(7.76) for GG,1.0113(9.96) for GA,1.4400(9.84) for AA, respectively(P<0.001) in case and0.6294(9.95) for GG,0.7144(9.05) for GA,1.0100(9.28) for AA, respectively(P<0.001) in control. CRP levels (median (range),mg/L) with rs2808630allele were:0.9109(9.97)for AA,1.0769(9.71) for AG,1.8713(9.61) for GG in case group(P<0.001) and0.6875(9.96) for AA,0.7119(9.39) for AG,3.6891(9.06) for GG (P=0.004) in the control.3. After adjustment for traditional cardiovascular risk factors, whether it is in the dominant model, recessive model or additive model, there was no significant association of CRP polymorphisms rs1205G/A and rs2808630A/G with any one of the subtypes of stroke; the adjusted odds ratio of rs1205(GA+AA vs.GG)(95%confidence interval, CI) were:0.97(0.81-1.15) for overall stroke group,1.01(0.81-1.26) for atherothrombotic,1.00(0.78-1.27) for lacunar infarct,1.01(0.77-1.34) for intracerebral hemorrhage. The adjusted odds ratio of rs2808630(AG+GG vs. AA)(95%CI) were0.96(0.80-1.15) for stroke overall,0.85(0.67-1.07) for patients with atherothrombotic,1.14(0.89-1.47) for patients with lacunar infarct,0.94(0.71-1.26) for patients with intracerebral hemorrhage. There was no significant association between stroke and any subtypes of stroke with haplotype frequency in all three haplotypes with frequency>5%(rs1205/rs2808630:GA, AA and AG).4. After adjustment for traditional cardiovascular risk factors, including age, sex, body mass index, systolic blood pressure, diastolic blood pressure, total cholesterol, HDL-C, diabetes and smoking, there is still no significant correlations of stoke recurrence with rs1205G/A (relative risk (95%CI)1.039(0.816-1.324), P=0.755) and rs2808630A/G (relative risk(95%CI)1.094(0.853-1.403), P=0.479).Conclusions:Although these variants and corresponding hyplotypes in the CRP gene are associated with serum CRP concentrations, our study does not support that variants and corresponding hyplotypes studied here have a major influence on risk of stroke and stroke recurrence.Therefore, we speculate that CRP is not a causal factor for stroke. Backgroud Stroke is a complex disease caused by synergy between environmental and genetic factors. It is a major health burden in China. Although the pathological processes of stroke included atherosclerosis, vascular inflammation, vascular permeability changes, its underlying pathogenesis is not clear.Previous reports indicated that angiogenic factors and its receptor involve in a number of closely related pathological process of stroke. This signal system regulates not only angiogenesis, but also endothelial cell inflammatory responses. Both angiopoietin1(Angl) and angiopoietin2(Ang2) are natural ligands of tyrosine kinase receptor Tie2(tyrosine kinase with immunoglobulin and EGFR growth factor homology domain2). They can interact with Tie2. Ang2and Angl is natural antagonist each other. Ang2can bind to the vascular endothelial Tie2receptor, prevent the binding of Ang1to Tie2and inhibit phosphorylation of Tie2. It can destabilize the blood vessels, loose the vascular structure, and release the inhibition of the perivascular cells and endothelial extracellular matrix to endothelial cells. Therefore, Ang2is the dynamic regulater of Ang-Tie2system and maintain the dynamic equilibrium of blood vessel growth and degradation.Ang2also involved in the regulation of inflammation responses. It, with the synergized with TNFα during vascular rapid response, can promote TNFa-induced expression of inflammation-related factors, such as ICAM1and VCAM1. Clinical studies have shown that Ang2is upregulated in patients with acute coronary syndrome and hypertension. Both acute coronary syndrome and hypertension are risk factors for stroke. Althought it is not clear that increased serum levels of Ang2are the cause or result of the acute coronary syndrome and hypertension, Ang2has been considered as the risk biomarker for high blood pressure in patients with cardiovascular disease (myocardial infarction, stroke and death).In summary, we proposed a hypothesis: there is association of susceptibility and recurrence of stroke with the plasma Ang2levels and/or genetic variants of ANGPT2(gene coding Ang2).Objectives:The purpose of this study was to test the hypothesis that plasma levels of Ang2and variants of ANGPT2will confer susceptibility to stroke and stroke recurrence. Methods:Amulti-center case-control study was used to investigate the association of ANGPT2variants with stroke susceptibility. A prospective study was used to explore the the association of plasma Ang2and the variants in ANGPT2promoter with stroke recurrence.Total1513patients with stroke of three subtypes(lacunar infarct (n=419), atherothrombotic(n=674) and intracerebral hemorrhage (n=420))and1485control in case-control study were genotyped by restriction fragment length polymorphism(RFLP)); in the prospective study, the association of plasma Ang2(determined by using ELISA) and the variants in ANGPT2promoter with stroke recurrence was tested in1735patients with stroke (lacunar infarct (n=475), atherothrombotic (n=794) and hemorrhage (n=466)). The patients were followed-up for a period of4.5years (mean); the association was evaluated by using Kaplan-Meier analysis and the Cox regression models. The degree of linkage disequilibrium between genetic markers was calculated using Haploview software. To further probe the potential mechanism, a luciferase reporter system was used to evaluation the effect of the variation in the promoter region of ANGPT2gene on its transcriptional activity.Results:1. Allele C of rs3739390conferred a1.42-fold risk for lacunar infarction{adjusted odds ratio [OR],1.42(95%CI,1.08-1.87); P=0.012)and a2.10-fold higher transcriptional activity as compared with the corresponding G allele (P=0.014)2. Elevated Ang2levels were strongly associated with risk of stroke recurrence only in the patients with lacunar infarct and the association remained after adjustment for age, sex, hypertension, or diabetes, BMI, cigarette smoking, alcohol consuming, glucose, TG and TC. As compared with the lowest quartile (the first), the hazard ratio (HR)(95%CI) for recurrence of stroke was1.48(0.74-2.95) for the second quartile (middle level),2.56(1.35-4.86) for the third quartile,2.15(1.11-4.17) for the fourth quartile (the highest level). Additional adjustment for variants in the promoter of ANGPT2did not substantially change the results.3. Allele T of rs3739391was associated with elevated Ang2levels. After adjustment for traditional risk factors, plasma Ang2level in the+398T allele carriers (n=126, the average concentration was4.26ng/ml, range:0.13-59.44ng/ml) is higher than the+398C allele carriers(n=234, the average concentration of3.95ng/ml, range:0.12-49.41ng/ml,P=0.033).4. Allele T of rs3739391was associated with risk of stroke recurrence in the patients with lacunar infarct. Compared with+398C,+398T allele led a risk ratio of1.67(95%CI) (1.06-2.63) for lacunar infarction recurrence; After further adjustment for plasma Ang2levels, the result remains similar.5. The haplotype G-G-T conferred a1.54-fold risk for atherothrombotic stroke and a1.64-fold risk for hemorrhagic stroke, while the haplotype G-C-C conferred a1.84-fold risk for atherothrombotic stroke,2.10fold risk for hemorrhagic stroke and2.16fold risk for lacunar infarction. Our results indicate that haplotypes in the promoter of ANGPT2gene conferred high risk of stroke in a Chinese population.Conclusions:In this study, we found that haplotypes in the promoter of the ANGPT2conferred high risk of stroke in a Chinese population. The plasma levels of Ang2and genetic polymorphism in ANGPT2promoter region may be used as a new predicting maker for recurrence of stroke (especially lacunar stroke).
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