Large Artery Atherosclerotic Stroke Research Articles

Association study of CACNA1C polymorphisms with large artery atherosclerotic stroke in Chinese Han population

Aims Ischemic stroke (IS) is one of the most common diseases of neurology and the main cause of death and disability in Chinese population. CACNA1C was considered to be involved in the process of atherosclerosis, but there was little information about the association between genotypic polymorphisms of CACNA1C and ischemic stroke. Our study was designed to elucidate the relationship between four single-nucleotide polymorphisms (SNPs) variants in CACNA1C gene and the risk of large-artery atherosclerotic (LAA) stroke patients.Methods A total of 384 subjects were enrolled in this study, including 192 LAA stroke cases and 192 healthy controls. And four SNPs variants in CNCNA1C gene were genotyped using in-house developed multiplex tagged-amplicon deep sequencing (TAm-Seq). Statistical analysis were conducted using χ2 test and binary logistic regression analysis.Results We found one variant was significantly associated with LAA stroke in the allele models (rs10848683, p = 0.036, OR = 1.371, 95%CI: 1.021–1.841). And rs10848683 was also found to associate with LAA stroke under recessive model (p = 0.027, OR = 0.618, 95% CI: 0.403–0.947) after adjustment for gender and age. We also found that significant difference existed between haplotypes (rs229961-rs215976-rs216008-rs10848683) and LAA stroke (C-T-C-C, p = 0.017, OR = 2.265, 95%CI: 1.136–4.518; G-C-C-C, p = 0.046, OR = 1.891, 95% CI: 1.003–3.565; C-T-C-T, p = 0.001, OR = 0.256, 95%CI: 0.101–0.645).Conclusion The results suggested that there was a potential association between CNCNA1C gene and the risk factor of LAA stroke in Chinese Han population.

Research progress of susceptibility genes associated with large artery atherosclerotic ischemic stroke

Research progress of susceptibility genes associated with large artery atherosclerotic ischemic stroke

GWAS-Supported CRP Gene Polymorphisms and Functional Outcome of Large Artery Atherosclerotic Stroke in Han Chinese.

Elevated C-reactive protein (CRP) levels increase the risk of poor functional disability in patients with ischemic stroke (IS). This study aimed to investigate the association between CRP gene polymorphisms and 3-month functional disability of large artery atherosclerotic (LAA) stroke in Han Chinese. Patients with first-ever LAA IS were prospectively enrolled in Nanjing Stroke Registry Program between August 2013 and October 2015. Five single-nucleotide polymorphisms (SNPs) (rs876537, rs2794520, rs3093059, rs7553007 and rs11265260) in CRP gene related to CRP levels in Asian by genome-wide association study were genotyped. The functional outcome at 3months after the index stroke was assessed by the modified Rankin scale. Associations between genotypes and functional outcome of LAA IS were analyzed with logistic regression model. A total of 690 eligible patients (507 males) were evaluated. SNPs rs11265260 (multivariate-adjusted, p = 0.022), rs2794520 (multivariate-adjusted, p = 0.036) and rs3093059 (multivariate-adjusted, p = 0.027) were significantly associated with elevated CRP in acute IS. Two SNPs, rs3093059 (dominant model: adjusted OR 2.49; 95% CI 1.55-4.00; recessive model: adjusted OR 3.67; 95% CI 1.22-11.03) and rs11265260 (dominant model: adjusted OR 2.51; 95% CI 1.56-4.02; recessive model: adjusted OR 4.70; 95% CI 1.63-13.56) independently predicted 3-month poor outcome of first-ever LAA IS, after adjusting for covariates. In addition, haplotype analysis indicated that haplotype GCTGC (adjusted OR 1.76; 95% CI 1.05-2.95; p = 0.031) increased the poor outcome risk. SNPs rs3093059 and rs11265260 in CRP gene may influence the 3-month functional outcome of first-ever LAA IS in Han Chinese.

Challenges and opportunities in stroke genetics.

Stroke, ischaemic stroke and subtypes of ischaemic stroke display substantial heritability. When compared with related vascular conditions, the number of established risk loci reaching genome-wide significance for association with stroke is still in the lower range, particularly for aetiological stroke subtypes such as large artery atherosclerotic stroke or small vessel stroke. Nevertheless, for individual loci substantial progress has been made in determining the specific mechanisms mediating stroke risk. In this review, we present a roadmap for functional follow-up of common risk variants associated with stroke. First, we discuss in silico strategies for characterizing signals in non-coding regions and highlight databases providing information on quantitative trait loci for mRNA and protein expression, as well as methylation, focussing on those with presumed relevance for stroke. Next, we discuss experimental strategies for following up on non-coding risk variants and regions such as massively parallel reporter assays, proteome-wide association studies, and chromatin conformation capture (3C) assays. These and other approaches are relevant for gaining insight into the specific variants and mechanisms mediating genetic stroke risk. Finally, we discuss how genetic findings could influence clinical practice by adding to diagnostic algorithms and eventually improve treatment options for stroke.

Abstract TP136: Dual versus Mono Antiplatelet Therapy in Acute Ischemic Stroke Patients Due to Large Artery Atherosclerosis

Background: Although dual antiplatelet treatment with aspirin and clopidogrel is widely used in stroke patients due to large artery atherosclerosis (LAA), there are limited data to justify this. Methods: We used a multicenter, prospective stroke registry database (CRCS-K) to analyze acute ischemic stroke patients due to LAA who were treated with aspirin alone or combination of aspirin and clopidogrel from May, 2008 to May 2015. Results were analyzed by intention-to-treat (ITT), as-treated (AT), and per-protocol (PP) method. Primary endpoint was the 1-year composite outcome of stroke recurrence, myocardial infarction (MI), and all-cause death. To balance the differences between groups, a frailty model with inverse probability of treatment weighting was used. Results: A total of 5,934 patients due to LAA were treated either aspirin alone (n=3,031, 51%) or combination of aspirin and clopidogrel (n=2,903, 49%). Primary outcome was 14% (n=410) in aspirin alone group and 12% (n=353) in aspirin plus clopidogrel group. In ITT analysis, compared to aspirin alone, aspirin plus clopidogrel did not decrease the hazards of primary outcome (adjusted hazards ratio, 0.86; 95% confidence intervals, 0.74-1.01; p=0.06), but decreased all-cause mortality significantly (0.80; 0.66-0.98; p=0.03). Stroke recurrence and a composite of stroke recurrence, MI, and vascular death were not reduced by aspirin plus clopidogrel. Results of AT and PP analyses are shown in the table below. Conclusion: Addition of clopidogrel to aspirin may decrease all-cause death and perhaps the composite of stroke recurrence, MI and all-cause death in patients with large artery atherosclerotic stroke.

Abstract TP133: A Network Approach to Identifying New Candidates for Ischemic Stroke

Introduction: Recent success identifying genetic variation in ischemic stroke (IS) has yet to translate into actionable targets or pathways. Weighted Gene Co-Expression Network Analysis (WGCNA) uses an unsupervised approach to cluster genes into modules based on co-expression profiles. Modules likely contain biologically related genes. The correlation between a gene’s expression and the module’s overall expression pattern (eigengene) defines connectivity. Genes with high connectivity may inform and supplement genome-wide association studies (GWAS). Methods: Gene Expression in Carotid Artery GSE43292 (GEO) resource contains publically available Affymetrix Human GeneChip Gene 1.0 ST data from 34 carotid atherosclerotic plaques and paired normal carotid tissue from the same individual. WGCNA resulted in 16 co-expression modules tested against the atherosclerosis phenotype. Significant modules had strong correlation between module eigengene and atherosclerosis. We took the top genes from significantly associated modules and looked them up in two existing IS genetic datasets: Stroke Genetic Network (SiGN) (17,000 IS cases & 32,000 controls) and METASTROKE (10,307 cases & 19,326 controls). We conducted a secondary GWAS informed by WGCNA for only known functional variants in METASTROKE with replication in SiGN. Results: Five of 16 modules showed evidence of association with two standouts (“Turquoise” and “Brown”) both enriched for genes associated with vascular disease related traits (carotid IMT, coronary artery disease, etc.) and contain genes associated with IS, especially large artery atherosclerotic stroke (including HDAC9). We took the top 20 genes from these modules, not currently known to associate with IS, and looking them up in SiGN and METASTROKE, we found association with variants in three genomic regions (SLC7A7, ATXN2/SH2B3, ALDH2). The informed GWAS approach identified ADAMTSL3 as a potential novel translational target. Conclusion: Leveraging expression data in a convergent genomics approach holds promise to supplement on-going GWAS and accelerate identification of potential translational targets. Gene ontology analyses of genes from the top modules are underway.

Abstract TP132: Vascular HDAC9 Modulates Atherogenic Processes and Enhances NF-kB Activation

Background: Genome-wide association studies (GWAS) identified the histone deacytelase 9 (HDAC9) gene region as the leading risk locus for large artery atherosclerotic stroke. Hdac9 deficiency was shown to attenuate atheroprogression in Apoe -/- and Ldlr -/- mice, however the mechanisms linking HDAC9 to atherosclerosis remain elusive. Objective: We aimed to determine HDAC9-related pro-inflammatory and pro-atherogenic responses in vitro and employed an early-stage atherosclerosis model to study these effects in vivo. Methods and Results: Human endothelial (ECs) and smooth muscle cells (SMCs) were cultured and transfected with HDAC9 siRNA or scrambled RNA (control) and stimulated with 20 ng/ml human TNF-α. HDAC9 depletion resulted in reduced adhesion molecule (VCAM-1, ICAM-1) and cytokine (CCL2, IL-8) expression as determined by RT-PCR, ELISA and Western Blot. Hdac9 +/+ Apoe -/- and Hdac9 -/- Apoe -/- mice were fed a western-type diet for 2 weeks. Hdac9 -deficient mice showed reduced circulating levels of Ccl2 and reduced levels of Vcam-1 and Icam-1 in whole aorta. Two-photon imaging of whole-mount carotid arteries further revealed a significant reduction of Vcam-1 expression. Motivated by the pattern of pro-inflammatory and pro-atherogenic responses, we investigated the effects of HDAC9 on NF-κB signaling. HDAC9 promoted NF-κB luciferase activity, facilitated p65 nuclear translocation, and enhanced p65 phosphorylation as determined by luciferase gene reporter assay, Western Blot and confocal microscopy. Conclusion: We demonstrate that HDAC9 promotes pro-inflammatory and pro-atherogenic responses in vascular cells and identify an activating effect of HDAC9 on NF-kB signalling. Our findings identify HDAC9 as a promising target for therapeutic interventions.

Abstract WMP39: Clopidogrel Resistance Predicts Early Neurological Worsening In Patients With Acute Large Artery Atherosclerotic Stroke

Introduction: Although platelet reactivity on clopidogrel is related to outcomes in patients with acute coronary syndromes, few studies have investigated its relation to clinical outcomes in acute ischemic stroke. Hypothesis: This study evaluated the association between clopidogrel resistance and early neurological outcomes in patients diagnosed with acute large artery atherosclerotic stroke. Methods: 1067 consecutive patients with onset within 7 days and stroke classification of large artery atherosclerosis were included. Platelet reactivity on clopidogrel was assessed using VeryfyNow P2Y12 assay at least 72 hours after the time of first clopidogrel dosage. Optimal cutoff value of clopidogrel resistance was obtained by using receiver-operating characteristic curve. Early neurological deterioration was defined as an increment of motor NIHSS score ≥1 before P2Y12 assay. We assessed any hemorrhagic transformation on 5-day MRI. Results: A total of 235 patients who met inclusion criteria were analyzed. The median initial dose of clopidogrel was 300 mg, and their median P2Y12 % inhibition was 26.3. P2Y12 % inhibition <29 was the significant discriminator of clopidogrel resistance with an AUC of 0.632 (95% CI 0.48-0.67; p=0.048). A multivariate analysis revealed that clopidogrel resistance is an independent risk factor even after adjustment for the variables that may affect the outcome (OR 2.75; 95% CI 1.11 to 6.83; p=0.029). Hemorrhagic transformation was not influenced by clopidogrel resistance (OR 0.60, 95% CI 0.14 to 2.540; P=0.484). Conclusion: In conclusion, high platelet reactivity with clopidogrel is an independent risk factor of early neurological worsening in acute large artery atherosclerotic stroke. Evaluation of ischemic stroke patients regarding antiplatelet resistance might be of clinical importance and alternative pharmacological strategies could be investigated in patients with high platelet reactivity.

Short-term exposure to traffic-related air pollution and ischemic stroke onset in Barcelona, Spain

ObjectiveTo assess the relationship between short-term exposure to outdoor ambient air pollutants (fine particulate matter [PM2.5] and black carbon [BC]), ischemic stroke (IS) and its different subtypes, and the potential modifying effect of neighborhood greenspace and noise. MethodsThis time-stratified case-crossover study was based on IS and transient ischemic attacks (TIA) recorded in a hospital-based prospective stroke register (BASICMAR 2005–2014) in Barcelona (Catalonia, Spain). Daily and hourly pollutant concentrations and meteorological data were obtained from monitoring stations in the city. Time-lags (from previous 72h to acute stroke onset) were analyzed. Greenness and noise were determined from the Normalized Difference Vegetation Index (NDVI) and daily average noise level at the street nearest to residential address, respectively. ResultsThe 2742 cases with known onset date and time, living in the study area, were analyzed. After adjusting for temperature, no statistically significant association between pollutants exposure and overall stroke risk was found. In subtype analysis, an association was detected between BC exposure at 24–47h (odds ratio, 1.251; 95% confidence interval [CI], 1.001–1.552; P = 0.042) and 48–72h (1.211; 95% CI, 0.988–1.484; P = 0.065) time-lag prior to stroke onset and large-artery atherosclerosis subtype. No clear modifying effect of greenness or noise was observed. ConclusionsOverall, no association was found between PM2.5 and BC exposure and acute IS risk. By stroke subtype, large-artery atherosclerotic stroke could be triggered by daily increases in BC, a diesel fuel-related pollutant in the study area.

Rs4612666 Polymorphism of the NLRP3 Gene Is Associated with the Occurrence of Large Artery Atherosclerotic Ischemic Strokes and Microembolic Signals.

Purpose Large artery atherosclerosis (LAA) ischemic stroke (IS) is the most common IS subtype, and microemboli are clinically important for indicating an increased risk of IS. Nucleotide-binding domain-like receptor protein 3 (NLRP3) plays a crucial role in the pathogenesis of atherosclerosis. The aim of this study is to investigate the relationship between NLRP3 gene polymorphisms and susceptibility for LAA IS and microembolic signals (MES) in the Chinese Han population. Methods We studied 293 patients diagnosed with LAA IS and 265 controls. Transcranial Doppler (TCD) was used to monitor the MES in all of the patients. Depending on the presence or absence of MES, the patients were divided into MES-positive and MES-negative subgroups. PCR-RFLP or direct sequencing were used to analyze three NLRP3 gene polymorphisms. Results Seventy-six patients presented with MES and the MES-positive rate was 25.94%. Logistic regression analysis showed that the TT genotype frequency for the rs4612666 gene polymorphism was higher in study patients than in the controls (adjusted P = 0.001) and higher in MES-positive patients compared to MES-negative patients (adjusted P = 0.015). The T allele of rs4612666 was associated with an increased risk for developing LAA IS and MES (P = 0.001; P = 0.015, resp.). Prevalence of the CCC haplotype was higher in the controls than in the patients (P = 0.009) and prevalence of the TGT haplotype was lower in the controls than in the patients (P = 0.019). Conclusions The NLRP3 rs4612666 gene polymorphism may be related to the occurrence of LAA IS and MES, suggesting that the NLRP3 gene polymorphism increases the susceptibility of LAA IS by changing the plaque vulnerability.

Serum levels of endocan and endoglin are associated with large-artery atherosclerotic stroke

BackgroundAccumulating evidence has suggested that endocan and endoglin may play important roles in cardiovascular disease. However, no previous study has focused on these circulating levels in patients with large-artery atherosclerotic (LAA) stroke. MethodsSerum levels of endocan and endoglin in 114 patients with LAA stroke and 114 age- and sex-matched controls were measured by ELISA. Serum samples from patients were available on day 1, day 6 and in the 4th week after ischaemic stroke(IS). Stroke severity was determined based on the NIHSS score and the stroke volume. An unfavourable outcome was defined as a mRS score>2 on day 90 after IS. ResultsThe endocan levels were significantly higher in patients with LAA stroke compared with the controls (p=0.001), and after adjustment for other factors (p=0.001). In addition, higher endocan levels were independently associated with unfavourable outcomes on both day 1 and day 6 after IS (p=0.018 and p=0.011). Endoglin levels were decreased on day 6 (p=0.002) and then recovered in the 4th week after IS. No correlation was found between endocan or endoglin and stroke severity. ConclusionsEndocan levels are higher in patients with LAA stroke and can help in predicting the short-term unfavourable outcome. Endoglin levels are changed after stroke.

Association of SNPs in the TIMP-2 gene and large artery atherosclerotic stroke in southern Chinese Han population.

Tissue inhibitor of matrix metalloproteinase 2 (TIMP-2) regulates the extracellular matrix degradation, which involved in vascular remodeling and dysfunction, destabilization of atherosclerotic plaque and many other pathological processes. The rupture of atherosclerotic plaque is the trigger of Large artery atherosclerotic (LAA) stroke. We speculate that the Single nucleotide polymorphisms (SNPs) in TIMP-2 may have an association with LAA stroke. To prove this hypothesis, we conducted this case-control study. 250 LAA stroke patients and 250 healthy controls were collected for the analysis of TIMP-2 polymorphisms. Among six SNPs, we detected no deviation from Hardy-Weinberg equilibrium in control group. There was a significant difference in rs4789936 T allele frequency between patient and control groups (OR = 0.68, 95% CI = 0.51–0.91, P = 0.009), which means lower risk of LAA stroke. We observed the rs4789936 had a decreased risk of LAA stroke according to the codominant (OR = 0.64, 95% CI = 0.44–0.92, P = 0.026), dominant (OR = 0.62, 95% CI = 0.43-0.88, P = 0.008), overdominant (OR = 0.68, 95% CI = 0.48–0.98, P = 0.039), log-additive (OR = 0.68, 95% CI = 0.51–0.91, P = 0.009) models analyses. However, these findings could only validate under dominant model (OR = 0.65, 95% CI = 0.42–1.00, P = 0.049) after adjustment of gender and age. The results indicate a potential association between TIMP-2 variants and LAA stroke risk in southern Chinese Han population.

Distinct predictive role of collateral status on clinical outcome in variant stroke subtypes of acute large arterial occlusion.

Clinical trials have shown that robust collateral flow has a relationship with good clinical outcome; however, different stroke subtypes were lumped together. This study explored the relationship between baseline collaterals and the onset-to-imaging time (OIT) and the correlation between pre-treatment collateral status and clinical outcome amongst different subtypes. Prospectively collected data from consecutive acute ischaemic stroke patients with acute middle cerebral artery occlusion who received reperfusion therapy were reviewed. The regional leptomeningeal score (20 points) was based on the scoring extent of contrast opacification in the six Alberta Stroke Program Early CT Score (ASPECTS) cortical regions (M1-6), parasagittal anterior cerebral artery territory and the basal ganglia by perfusion-derived dynamic four-dimensional computed tomography angiography (4D CTA). Stroke subtype was determined by the TOAST classification criteria. A 3-months modified Rankin Scale score of 0-2 was defined as a good outcome. The analysis included 158 patients: 30 (19.0%) patients had large artery atherosclerotic stroke (LAA), 87 (55.1%) cardioembolic stroke (CE) and 41 (25.9%) stroke of undetermined etiology. Baseline collateral was negatively correlated with OIT (P = 0.0205) in the CE group after adjusting for female sex, smoking, hyperlipidemia, baseline National Institutes of Health Stroke Scale (NIHSS) and baseline mismatch ratio, but not in the LAA group. Baseline collateral showed a strong relationship with good clinical outcome after adjusting for recanalization, baseline NIHSS, age and female sex (odds ratio 1.120, confidence interval 1.013-1.238, P = 0.027) in all patients and in the CE group (odds ratio 3.223, confidence interval 1.212-8.570, P = 0.019), but not in the LAA patients. Based on 4D CTA, sustained good leptomeningeal collaterals may predict good outcome in CE but not in LAA patients. Moreover, the extent of collaterals was associated with OIT in the CE patients, which indicates prompt reperfusion therapy in this group of patients.

Association of FOXF2 gene polymorphisms with ischemic stroke in Chinese Han population.

Recently, a novel locus at chromosome 6p25 (rs12204590, near FOXF2) associated with an increased risk of stroke in European populations was identified. However, whether polymorphisms in FOXF2 are also associated with the incidence of ischemic stroke in other populations remains unknown. In this case-control study, 803 Chinese Han patients with ischemic stroke and 803 matched control individuals were enrolled. Four tag SNPs and rs12204590 located in or near FOXF2 were selected, and the associations between genotypes/alleles and ischemic stroke were analyzed. In our study, we did not detect an association between the previously reported locus rs12204590 and ischemic stroke. By the genotype analysis, a novel SNP rs1711972, near FOXF2, was observed to be associated with an increased risk of ischemic stroke(CA genotype, adjusted OR = 1.35; 95% CI, 1.07 to 1.70), but not significantly after Bonferroni corrections for multiple tests. However, in the subgroup analysis, we discovered that rs1711972 was associated with an increased risk of large-artery atherosclerotic stroke in the additive model (P = 0.020; CA genotype, adjusted OR = 1.50; 95%CI, 1.09 to 2.07) and dominant model (P = 0.010; OR = 1.47; 95%CI, 1.09 to 1.99). Collectively, these results indicate that a novel SNP near FOXF2 may influence the risk of large-artery atherosclerotic stroke in Chinese Han population.

Impact of Tag Single Nucleotide Polymorphisms (SNPs) in CCL11 Gene on Risk of Subtypes of Ischemic Stroke in Xinjiang Han Populations.

BackgroundCCL11 is an important inflammatory cytokine associated with inflammation-related diseases such as atherosclerosis and stroke. The aim of this study was to investigate the relationship between CCL11 gene polymorphism with subtypes of ischemic stroke in Xinjiang Han populations.Material/MethodsThe improved multiple ligase detection reaction (iMLDR) method was used to analyze the genotypes of 6 tag SNPs in the CCL11 gene (rs1129844, rs17809012, rs1860183, rs1860184, rs4795898, and rs4795895) in a case-control study of 406 lacunar stroke patients, 214 large-artery atherosclerotic (LAA) stroke patients, and 425 controls.ResultsWe found the GG genotype of rs4795895 was significantly associated with increased risk of lacunar stroke (adjusted OR=1.676, 95%CI=1.117–2.515), and the GA genotype of rs17809012 was associated with a significant increase in risk of LAA stroke (adjusted OR=1.337, 95%CI=1.127–1.585). Hypertension stratification analyses showed that the GA genotype of rs17809012 was significantly associated with LAA stroke in the hypertensive group (adjusted OR=1.274, 95%CI=1.015–1.601). In the non-hypertensive group, the GA genotype of rs17809012 was significantly associated with LAA stroke (adjusted OR=1.361, 95%CI=1.041–1.780). The GG genotype of rs4795895 (adjusted OR=1.147, 95%CI=1.115–4.134) and the TT genotype of rs1860184 were significantly associated with lacunar stroke (adjusted OR=2.440, 95%CI=1.550–3.840).ConclusionsThis study demonstrates that the CCL11 gene could play an important role in the pathogenesis of lacunar stroke and LAA stroke in the Han population of China.

Integrative studies implicate matrix metalloproteinase-12 as a culprit gene for large-artery atherosclerotic stroke.

Ischaemic stroke and coronary heart disease are important contributors to the global disease burden and share atherosclerosis as the main underlying cause. Recent evidence from a genome-wide association study (GWAS) suggested that single nucleotide polymorphisms (SNP) near the MMP12 gene at chromosome 11q22.3 were associated with large-vessel ischaemic stroke. Here, we evaluated and extended these results by examining the relationship between MMP12 and atherosclerosis in clinical and experimental studies. Plasma concentrations of MMP12 were measured at baseline in 3394 subjects with high-risk for cardiovascular disease (CVD) using the Olink ProSeek CVD I array. The plasma MMP12 concentration showed association with incident cardiovascular and cerebrovascular events (130 and 67 events, respectively, over 36 months) and carotid intima-media thickness progression (P = 3.6 × 10-5 ). A GWAS of plasma MMP12 concentrations revealed that SNPs rs499459, rs613084 and rs1892971 at chr11q22.3 were independently associated with plasma MMP12 (P < 5 × 10-8 ). The lead SNPs showed associations with mRNA levels of MMP12 and adjacent MMPs in atherosclerotic plaques. MMP12 transcriptomic and proteomic levels were strongly significantly increased in carotid plaques compared with control arterial tissue and in plaques from symptomatic versus asymptomatic patients. By combining immunohistochemistry and proximity ligation assay, we demonstrated that MMP12 localizes to CD68 + macrophages and interacts with elastin in plaques. MMP12 silencing in human THP-1-derived macrophages resulted in reduced macrophage migration. Our study supports the notion that MMP12 is implicated in large-artery atherosclerotic stroke, functionally by enhancing elastin degradation and macrophage invasion in plaques.

Association of GWAS-Reported Variant rs11196288 near HABP2 with Ischemic Stroke in Chinese Han Population.

A recent genome-wide association analysis identified a novel single nucleotide polymorphism locus on chromosome 10q25.3 (rs11196288, near HABP2) associated with the risk of early-onset ischemic stroke (IS) in European population, but not with late-onset IS. However, the role of this genome-wide association study (GWAS)-reported variant in ischemic stroke in Chinese Han population remained unknown. In our study, 389 adult ischemic stroke patients with an age of onset <60years and 389 matched healthy controls were enrolled to investigate association of rs11196288 genotypes with early-onset ischemic stroke and its subtypes; the association was further examined in another independent population consisting of 349 ischemic stroke patients with an age of onset ≧60years and 349 matched healthy individuals. Logistic regression analysis showed no significant association between rs11196288 and early-onset ischemic stroke (IS), large artery atherosclerotic (LAA) stroke, or small vessel disease (SVD) stroke (all P>0.050). Nevertheless, in subgroup analysis of the older population, rs11196288 presented significant effect on late-onset SVD stroke susceptibility in the dominant model (GG/GA vs AA, OR 1.70; 95%CI 1.02 to 2.85; P=0.042). The results indicated that the role of rs11196288 polymorphism in ischemic stroke susceptibility in Chinese Han population may be different from that in European. Larger studies with diverse populations are warranted to confirm and extend our findings.

NINJ2– A novel regulator of endothelial inflammation and activation

Previous genetic studies suggested that variants in NINJ2 (encode ninjurin2) confer risk to ischemic stroke or large artery atherosclerotic stroke. However, the underlying mechanisms of NINJ2 in ischemic stroke or atherosclerosis are still unknown. In this study, we hypothesized that NINJ2 may play a role in endothelial inflammation and activation, and regulate the process of atherosclerosis. Here, we demonstrated that NINJ2 can regulate the expression of a panel of genes that are associated with inflammation and atherosclerosis (e.g. IL-1β, TNF-α, IL-8, IL-6, ICAM-1 and E-selectin) in human vascular endothelial cells (HUVECs). Moreover, we found the expression of ninjurin2 is upregulated in LPS stimulated HUVECs and mouse aorta, and it can regulate LPS-induced endothelial activation and the adhesion of monocytes to endothelial cells. We also found that NINJ2 can regulate NF-κB and c-jun through interacting with TLR4. In conclusion, our study suggests that ninjurin2 is a novel regulator of endothelia inflammation and activation through TLR4 signaling pathways, and these data provided new insights into the mechanisms between NINJ2 and atherosclerosis.

Association of cytochrome P4502E1 and NAD(P)H:quinone oxidoreductase 1 genetic polymorphisms with susceptibility to large artery atherosclerotic ischemic stroke: a case-control study in the Turkish population.

Stroke, a major cause of death and disability, is described as interruption or severe reduction of blood flow in cerebral arteries. Oxidative stress plays an important role in the pathogenesis of atherosclerosis and carotid atherosclerosis is a risk factor for stroke. Combination of multiple environmental and genetic risk factors is thought to increase stroke. Therefore, investigation of the polymorphisms of enzymes is of crucial importance to determine the molecular etiology of the disease. To test this hypothesis, we performed a case-control study in which we compared the distribution of CYP2E1 and NQO1 genotypes between 245 large artery atherosclerotic ischemic stroke patients and 145 controls, using PCR-RFLP. A significant difference was observed between stroke patients and controls with respect to the CYP2E1*5B genotype (odds ratio; OR 8.069, P=0.011) and allele (OR 7.876, P=0.011) distribution. However, this polymorphism was not a significant predictor of disease status in logistic regression analysis. NQO1*2 polymorphism genotype distribution was significantly different between patients and controls (P=0.027) and heterozygote *1*2 genotype was found to be a protective factor against large artery atherosclerotic ischemic stroke in logistic regression analysis (OR 0.562, P=0.018). This is the first study conducted regarding the association of CYP2E1 and NQO1 genetic polymorphisms and large artery atherosclerotic ischemic stroke risk in Turkish population.

Association of eNOS and Cav-1 gene polymorphisms with susceptibility risk of large artery atherosclerotic stroke.

Endothelial nitric oxide synthase (eNOS) is localized in caveole and has important effects on caveolar coordination through its interaction with caveolin-1 (Cav-1), which supports normal functioning of vascular endothelial cells. However, the relationship between genotypic polymorphisms of e-NOS and Cav-1 genes and ischemic stroke (IS) remains lesser reported. This hospital-based case-control study aimed to determine the genetic polymorphisms of the eNOS (Glu298Asp) and Cav-1 (G14713A and T29107A) genes in association with susceptibility risk in patients who had suffered from a large artery atherosclerotic (LAA) stroke. Genotyping determination for these variant alleles was performed using the TaqMan assay. The distributions of observed allelic and genotypic frequencies for the polymorphisms were in Hardy-Weinberg equilibrium in healthy controls. The risk for an LAA stroke in the Asp298 variant was 1.72 (95% CI = 1.09–2.75) versus Glu298 of the eNOS. In the GA/AA (rs3807987) variant, it was 1.79 (95% CI = 1.16–2.74) versus GG and in TA/AA (rs7804372) was 1.61 (95% CI = 1.06–2.43) versus TT of the Cav-1, respectively. A tendency toward an increased LAA stroke risk was significant in carriers with the eNOS Glu298Asp variant in conjunction with the G14713 A and T29107A polymorphisms of the Cav-1 (aOR = 2.03, P-trend = 0.002). A synergistic effect between eNOS and Cav-1 polymorphisms on IS risk elevation was significantly influenced by alcohol drinking, heavy cigarette smoking (P-trend<0.01), and hypercholesterolemia (P-trend < 0.001). In conclusion, genotypic polymorphisms of the eNOS Glu298Asp and Cav-1 14713A/29107A polymorphisms are associated with the elevated risk of LAA stroke among Han Chinese in Taiwan.