血浆细胞外囊泡表面高迁移率族蛋白B1与颈动脉斑块易损性的相关性研究OA

Objective To investigate the association between high mobility group box 1 protein on the surface of extracellular vesicles(EV-HMGB1)and plaque vulnerability in patients with carotid atherosclerotic stenosis(CAS)undergoing carotid artery stenting.Methods Consecutive patients with CAS who underwent carotid artery stenting in the Department of Neurosurgery,Xuanwu Hospital,Capital Medical University from February 2023 to October 2024 were prospectively enrolled.Clinical data,including age,sex,body mass index,medical history(hypertension,diabetes,hyperlipidemia,coronary heart disease,atrial fibrillation,peripheral vascular disease),smoking history,drinking history,symptomatic status(symptomatic was defined as ipsilateral ocular or cerebral ischemic symptoms occurring within the past 6 months prior to hospitalization,with or without anterior circulation infarction;asymptomatic was defined as no ipsilateral ocular or cerebral ischemic symptoms related to the carotid artery territory within the past 6 months prior to hospitalization),medication use upon admission(antiplatelet drugs,statins,and antihypertensive drugs),and laboratory indicators upon admission(total cholesterol,high-density lipoprotein cholesterol,low-density lipoprotein cholesterol,triglycerides,and fasting blood glucose),were collected from the patients.All patients underwent preoperative carotid CT angiography(CTA)and three-dimensional time-of-flight MR angiography(3D TOF-MRA)to assess the side of the affected CAS,the stenosis rate of the affected CAS,contralateral carotid artery stenosis rate(＞50％,≤50％),and whether the affected carotid artery plaque was a vulnerable plaque.Plaque vulnerability was classified according to the plaque-reporting and data system(plaque-RADS),with grades ranging from 1 to 4.In this study,patients with plaque-RADS grades 3a-3c were defined as the stable plaque group,and those with plaque-RADS grades 4a-4c were defined as the vulnerable plaque group.The vulnerable plaque group was further divided into 4a,4b,and 4c subgroups.EV morphology was observed using transmission electron microscopy.Nanoparticle tracking analysis was used to determine the particle size distribution and concentration of EV.Western blotting was used to detect the expression of typical positive EV markers(tumor susceptibility gene 101[TSG101],cluster of differentiation 9[CD9],CD81)and the negative marker calreticulin.Human umbilical vein endothelial cell(HUVEC)lysate was used as a positive control to characterize the isolated EV.Plasma EV-HMGB1 levels were measured by enzyme-linked immunosorbent assay.Western blot analysis was performed on EV samples and HUVEC.Factors with statistically significant differences in univariate analysis were included in multivariate logistic regression analysis to explore whether EV-HMGB1 was an influencing factor for vulnerable plaques in CAS patients undergoing carotid artery stenting.Receiver operating characteristic(ROC)curve analysis was used to evaluate the predictive efficacy of EV-HMGB1 for vulnerable plaques in these patients.Results A total of 311 CAS patients who underwent carotid artery stenting were enrolled,including 258 males and 53 females,with a mean age of(67±7)years.There were 201 patients in the stable plaque group and 110 in the vulnerable plaque group.Among the vulnerable plaque group,there were 57 patients in 4a,47 in 4b,and 6 in 4c.(1)Compared to the stable plaque group,the vulnerable plaque group had a higher proportion of male patients(92.7％[102/110]vs.77.6％[156/201],P=0.001).No statistically significant differences were observed in other clinical and imaging data between the groups(all P＞0.05).(2)Differences in the proportion of male patients(77.6％[156/201],98.2％[56/57],89.4％[42/47],4/6,P＜0.01)and peripheral vascular disease(1.5％[3/201],8.8％[5/57],2.1％[1/47],0/6,P=0.047)were statistically significant among the stable plaque group,4a,4b,and 4c subgroups.Differences in other clinical and imaging data among the four groups were not statistically significant(all P＞0.05).Post-hoc pairwise comparisons showed that the proportion of males in the 4a subgroup was significantly higher than that in the stable plaque group(98.2％[56/57]vs.77.6％[156/201],adjusted P=0.000 4),while differences between other pairs were not statistically significant(all adjusted P＞0.05).The prevalence of peripheral vascular disease showed no statistically significant differences in any pairwise comparisons(adjusted P＞0.05).(3)Transmission electron microscopy revealed typical cup-shaped vesicular structures with intact or partially enclosed dense material in both stable and vulnerable plaque groups,with no obvious morphological differences observed between the two groups.Nanoparticle tracking analysis showed particle sizes of(139.1±60.6)nm in the vulnerable plaque group and(131.4±50.4)nm in the stable plaque group,with no statistically significant difference(P=0.23).The concentration was(3.01±1.24)× 1 09 particles/ml in the vulnerable plaque group and(2.87±1.09)× 109 particles/ml in the stable plaque group,with no statistically significant difference(P=0.31).Western blotting showed positive expression of EV markers TSG101,CD9,and CD81 in both stable and vulnerable plaque groups from CAS patients compared to the HUVEC cell lysate positive control,while the EV negative marker calreticulin was not detected in either group.(4)Plasma EV-HMGB1 concentration was significantly higher in the vulnerable plaque group compared to the stable plaque group(6.11[4.66,7.33]μg/L vs.4.06[3.49,4.78]μg/L,P＜0.01).EV-HMGB1 levels differed significantly among the four groups(H=96.34,P＜0.01).Post-hoc pairwise comparisons revealed that EV-HMGB1 concentration in the stable plaque group(4.06[3.49,4.78]μg/L)was significantly lower than that in 4a(5.68[4.25,6.66]μg/L,adjusted P＜0.01),4 b(6.37[5.19,7.62]μg/L,adjusted P＜0.01),and 4c(5.87[5.38,7.19]μg/L,adjusted P=0.002 9)subgroups.However,pairwise comparisons among the 4a,4b,and 4c subgroups showed no statistically significant differences in EV-HMGB1 concentration(all adjusted P＞0.05).(5)Multivariate Logistic regression analysis identified male sex(OR,3.39,95％CI 1.36-9.74,P=0.014)and higher EV-HMGB1 level(OR,3.68,95％CI 2.76-5.12,P＜0.01)as independent risk factors for vulnerable plaques in CAS patients.(6)ROC curve analysis showed that plasma EV-HMGB1 identified vulnerable plaques in CAS patients with an area under the curve of 0.831(95％CI 0.779-0.883,P＜0.01).The optimal cut-off value was 5.344 μg/L,yielding a sensitivity of 65.5％and specificity of 95.5％.Conclusion Plasma EV-HMGB1 level is associated with plaque vulnerability in CAS patients who underwent carotid artery stenting and may serve as a potential biomarker for identifying vulnerable carotid plaques.