【作者】 李敏；

【导师】 刘乃丰；

【作者基本信息】 东南大学 ， 临床医学（内科学）（专业学位）， 2023， 博士

【摘要】 背景:冠脉迂曲表现为心外膜血管呈连续样或盘旋样弯曲,是血管造影时常见但病因及临床意义尚不明确的血管异常。从血流力学角度来看,迂曲血管可导致局部血流紊乱,形成以血流分离、再循环和低振荡剪应力为特征的微环境,已被证明与动脉粥样硬化发生发展密切相关,然而其对血管钙化(矿物基质在血管壁的异位沉积过程)的影响还不甚明确。为了进一步了解冠脉迂曲的影响因素及对血管钙化的潜在影响机制,本文分别从临床研究和基础实验两个层面进行探讨,(1)冠脉迂曲临床危险因素分析;(2)剪应力对血管内膜钙化的影响,以加深对该血管异常的判断,探明其隐含的病理生理意义。具体研究如下:第一部分冠脉迂曲的危险因素分析方法:回顾性收集2020年1月1日至2022年6月9日就诊东南大学附属中大医院行冠脉CTA检查患者的病例资料,包括一般资料、冠脉CTA检查结果、心脏彩超结果、生化指标。采用专用软件计算钙化积分并量化为Agatston评分。经纳入排除标准筛选后本研究共纳入1533例患者。冠脉迂曲定义为:在心脏收缩和舒张期,沿主干走形的三支血管中至少有一支血管存在≥3个的连续弯曲,弯曲角度≥45°。计算冠脉CTA检查患者中冠脉迂曲的患病率。分析并比较不同性别、不同年龄段患者冠脉迂曲患病率的高低。采用logistic回归分析探讨冠脉分支迂曲患病的危险因素。结果:本研究中冠脉迂曲的患病率为38.6%,其中,单支迂曲患病率为22.5%,合并两支迂曲患病率为12.6%;三支均迂曲患病率为3.6%。单支及多支病变中,左前降支(left anterior descending coronary artery,LAD)迂曲发生比例最高,为13.4%,左回旋支(left circumflex coronary artery,LCX)&右冠状动脉(right coronary artery,RCA)迂曲患病率最低,为0.7%。男性冠脉迂曲患病率低于女性为30.9%vs.46.5%,其中,LAD是最常受累动脉,患病率男女分别为11.3%、15.6%,LCX&RCA患病率最低,分别为0.5%、0.8%。两性在冠脉各分支中,迂曲患病率的高低依次为(男性vs.女性)LAD 23.8%vs.34.0%,LCX 13.1%vs.25.7%,RCA 10.4%vs.10.1%。18-29岁年龄段无冠脉迂曲患者,单支及多支冠脉迂曲多集中于70-80岁人群。不同年龄段冠脉三支中LAD受累居多,其次是LCX,RCA最少受累。男性在60-69岁年龄段单支及多支冠脉迂曲患病率最高,为11.1%,而女性在70-80岁迂曲患病率最高,为46.5%。单因素logistic回归分析发现,年龄、BMI、高血压、糖尿病、Agatston score是不同冠脉分支迂曲的共同影响因素,但校正混杂因素后发现,仅年龄、BMI、高血压、Agatston score是不同冠脉分支血管迂曲的共同危险因素,即高龄(LAD:OR 1.04,95%CI 1.03-1.05,P<0.001;LCX:OR 1.03,95%CI 1.02-1.04,P<0.001;RCA:OR 1.03,95%CI 1.01-1.04,P<0.001)、高血压(LAD:OR 1.61,95%CI1.23-2.09,P<0.001;LCX:OR 1.46,95%CI 1.08-1.97,P=0.014;RCA:OR 2.47,95%CI 1.62-3.75,P<0.001)、中度的Agatston score患者(LAD:OR 1.49,95%CI1.01-2.18,P=0.043;LCX:OR 2.32,95%CI 1.07-5.09,P=0.034;RCA:OR 0.91,95%CI 0.81-1.01,P=0.041)冠脉血管迂曲发生风险更高,肥胖与体重正常患者相比,冠脉迂曲发生风险较低(LAD:OR 0.55,95%CI 0.37-0.82,P=0.003;LCX:OR 0.51,95%CI 0.30-0.85,P=0.010;RCA:OR 0.46,95%CI 0.23-0.85,P=0.021)。而女性(OR 1.36,95%CI 1.06-1.74,P=0.014)、EF值(OR 1.01,95%CI 1.00-1.03,P=0.038)与LAD迂曲发生风险独立相关;女性(OR 2.09,95%CI 1.58-2.78,P<0.001)、糖尿病(OR 1.59,95%CI 1.12-2.19,P=0.004)是LCX迂曲的危险因素;吸烟(OR 1.79,95%CI 1.09-2.95,P=0.025)则是RCA迂曲的独立危险因素。结论:冠脉迂曲有较高的患病率,女性多于男性,且随着年龄的增加呈现上升趋势。年龄、BMI、高血压、Agatston score是不同冠脉分支迂曲的共同影响因素,而性别、吸烟、EF值、糖尿病是不同冠脉分支迂曲的独立危险因素。第二部分剪应力对血管内膜钙化的影响方法:本研究使用德国Ibidi流动泵系统对HUVEC细胞施加不同模式的流体力学,即层流剪应力(laminar shear stress,LSS)和振荡剪应力(oscillatory shear stress,OSS)强度分别为12 dyn/cm~2和0.5±6 dyn/cm~2,干预24 h后,通过RNA-seq检测两组差异基因的表达,并进行GO富集分析筛选靶基因。沉默和过表达该基因,通过茜素红、Western blot、RT-q PCR、免疫荧光、免疫共沉淀等证实该基因对HUVEC钙化的影响。结果:对HUVEC加载剪应力处理后,通过RNA-seq检测LSS组和OSS组差异表达基因3583个,其中,上调基因1956个,下调基因1627个。GO分析显示差异表达基因富集了与Cellular response to fluid shear stress和Positive regulation of bone mineralization等相关通路。进一步筛选出Mst1作为靶基因。LSS干预条件下,随着时间延长,Mst1的m RNA表达水平升高;相反,随着OSS干预时间的延长,Mst1的m RNA表达水平显著下降。与RT-q PCR结果相一致,OSS干预下随时间延长Mst1的蛋白表达水平也呈现下降。此外,与LSS组相比,OSS组Mst1的免疫荧光强度降低。接下来,构建慢病毒表达载体介导sh RNA沉默Mst1并转染HUVEC,与NC-sh RNA组相比,Mst1-sh RNA组中的成骨分化转录因子RUNX2、BMP2、Msx2的蛋白表达水平升高;而过表达Mst1基因,与NC-OE+β-GP组相比,Mst1-OE+β-GP组RUNX2、BMP2、Msx2蛋白水平下降。一致的是,β-GP诱导下,Mst1-OE组橘红色颗粒状钙化结节明显减少,HUVEC钙含量降低。此外,免疫荧光染色观察到Mst1与RUNX2存在共定位。使用CHX和MG132分别去抑制RUNX2的合成和降解进一步证实,过表达Mst1能够降低RUNX2的蛋白稳定性和增强RUNX2的泛素化降解。使用慢病毒表达载体介导sh RNA敲低WWP2,过表达Mst1下RUNX2的蛋白表达水平变化不明显。CO-IP结果显示,过表达Mst1能够促进WWP2与RUNX2的结合,而敲低Mst1抑制了WWP2与RUNX2的结合,从而影响VC的发生。结论:OSS诱导下Mst1的降低与VC的发生密切相关。过表达Mst1能够通过WWP2促进RUNX2的泛素化降解,进而减少EC钙盐沉积。更多还原

【Abstract】 Objective:Coronary tortuosity is defined as continuous or spiral curvature of epicardial vessels,which is a common vascular abnormality in angiography,but its etiology and clinical significance are not yet clear.From the biomechanical point of view,vascular tortuosity can lead to the disturbance of local hemodynamic environment characterized by flow separation or recirculation and low oscillatory wall shear stress that have been extensively demonstrated to associate with the initiation and progression of atherosclerosis.However,its effects on vascular calcification,the process of ectopic deposition of mineral matrix in the vascular wall,are still unclear.In order to further understand the influencing factors of coronary tortuosity and its impact on vascular calcificationis defined as abnormal deposits of mineral matrix in the vessel wall,we explored them from two levels of clinical and basic experiments,so as to improve our understanding of the disease,and learn the deep meaning behind its existence.The specific research contents are as follows:(1)The clinical risk factors affecting coronary tortuosity;(2)The effects of shear stress on intimal calcification.Specific researches are as follows:PartⅠ:The risk factors affecting coronary tortuosityMethods:The case data of patients who underwent coronary CTA examination at Zhongda Hospital Affiliated to Southeast University from January 1,2020 to June 9,2022 were retrospectively collected,including general information,coronary CTA examination results,cardiac color Doppler ultrasound results,and biochemical indicators.The CAC score was calculated by special software and quantified as Agatston score.After screening by inclusion and exclusion criteria,1533 patients were included in the study.Coronary tortuosity is defined as the presence of at least three consecutive curvatures of more than 45°measured during systole or diastole of a major epicardial coronary artery.The population prevalence of coronary tortuosity in patients with coronary CTA was calculated.Tortuous prevalence of coronary branches and their concomitant branches in patients of different genders and ages was analyzed and compared.Logistic regression analysis was used to explore the risk factors of coronary branch tortuosity.Results:In this study,the prevalence of coronary artery tortuosity was 38.6%,among which,the prevalence rate of single-vessel tortuosity was 22.5%,the prevalence of combined two-vessel tortuosity was 12.6%,and the prevalence of three-vessel tortuosity was 3.6%.Among single-vessel and multi-vessel lesions,the highest prevalence of LAD tortuosity was 13.4%,and the lowest prevalence of LCX&RCA tortuosity was 0.7%.The prevalence of coronary tortuosity in males was lower than in females(30.9%and 46.5%,respectively).Among them,LAD was the most commonly involved artery,with a prevalence of 11.3%in males and 15.6%in females.The prevalence of LCX&RCA was the lowest,0.5%and 0.8%,respectively.The prevalence of tortuosity in each branch of the coronary arteries in both genders(male vs.female)was LAD 23.8%vs.34.0%,LCX 13.1%vs.25.7%,RCA 10.4%vs.10.1%.Patients without coronary tortuosity were in the 18-29 age group,while patients with single-vessel and multi-vessel lesions were mostly in those aged 70-80 years.In different age groups,the LAD was most involved,followed by the LCX,and the RCA was the least affected.For men aged 60-69,the prevalence of coronary tortuosity is the highest(11.1%)among single-vessel and multi-vessel lesions,while for women aged70-80,that prevalence is the highest(46.5%).Univariate logistic regression analysis found that age,BMI,hypertension,diabetes and Agatston score were the main factors affecting coronary tortuosity,but after adjusting confounding factors,it was found that the age,BMI,hypertension and Agatston score were the independent risk factors influencing coronary tortuosity.Elderly patients(LAD:OR 1.04,95%CI 1.03-1.05,P<0.001;LCX:OR 1.03,95%CI 1.02-1.04,P<0.001;RCA:OR 1.03,95%CI1.01-1.04,P<0.001)and patients with hypertensive(LAD:OR 1.61,95%CI 1.23-2.09,P<0.001;LCX:OR 1.46,95%CI 1.08-1.97,P=0.014;RCA:OR 2.47,95%CI 1.62-3.75,P<0.001)and moderate Agatston score(LAD:OR 1.49,95%CI1.01-2.18,P=0.043;LCX:OR 2.32,95%CI 1.07-5.09,P=0.034;RCA:OR 0.91,95%CI 0.81-1.01,P=0.041)were particularly prone to coronary tortuosity.Obese patients(LAD:OR 0.55,95%CI 0.37-0.82,P=0.003;LCX:OR 0.51,95%CI 0.30-0.85,P=0.010;RCA:OR 0.46,95%CI 0.23-0.85,P=0.021)were less likely to have coronary tortuosity than those with normal weight.Female(OR 1.36,95%CI 1.06-1.74,P=0.014)and EF value(OR 1.01,95%CI 1.00-1.03,P=0.038)were risk factors for LAD tortuosity;Female(OR 2.09,95%CI 1.58-2.78,P<0.001)and diabetes(OR1.59,95%CI 1.12-2.19,P=0.004)were risk factors for LCX tortuosity;Smoking(OR1.79,95%CI 1.09-2.95,P=0.025)was significantly correlated with RCA tortuosity.Conclusion:The prevalence of coronary tortuosity is higher,and more prevalent in males than females.Coronary tortuosity tends to increase with age.Age,BMI,hypertension and Agatston score were the common influencing factors affecting coronary tortuosity,while gender,smoking,EF value and diabetes were the independent risk factors in different coronary branch tortuosity.PartⅡ:Effect of shear stress on intimal calcificationMethods:Laminar flow and oscillatory shear flowgenerated by Ibidi flow system(Ibidi,Germany)were applied to HUVECs with shear stress of 12 dyn/cm~2(LSS),0.5±6 dyn/cm~2(OSS),respectively.After 24 h,RNA-seq was used to detect genes with differential expression,and GO enrichment analysis was performed to screen target gene.Subsequently,the effect of this gene on HUVEC calcification after being silenced or overexpressed was confirmed by Alizarin red staining,Western blotting,RT-q PCR,immunofluorescence,and co-immunoprecipitation.Results:Under shear loading,RNA‐seq analysis identified 3583 differentially expressed genes,among which 1956 genes were up‐regulated and 1627 genes were down‐regulated in the OSS group compared to the LSS group.GO enrichment analysis showed that the differentially expressed genes were mainly enriched in shear stress and ossification related pathways.Additionally,Mst1 was screened as the target gene.Under the condition of LSS intervention,the m RNA expression level of Mst1 increased with the extension of time.In contrast,the m RNA expression level of Mst1 was significantly decreased with the extension of OSS intervention time.Consistent with the RT-q PCR results,the protein expression level of Mst1 also decreased over time after OSS intervention.In addition,compared with the LSS group,the immunofluorescence intensity of Mst1 was decreased in the OSS group.Next,HUVECs were transfected with lentivirus-mediated short hairpin RNA(sh RNA)targeting Mst1.Compared with NC-sh RNA group,the protein expression levels of RUNX2,BMP2 and Msx2 in the Mst1-sh RNA group were increased.When MST1was overexpressed,compared with the NC-OE+β-GP group,the protein levels of RUNX2,BMP2,and Msx2 were decreased.Consistent with this,the number of calcified nodules,and the calcium content of HUVEC in the Mst1-OE+β-GP group were significantly decreased.In addition,co-localization of Mst1 and RUNX2 was observed by immunofluorescence staining.Inhibition of RUNX2 synthesis and degradation by CHX and MG132,respectively,further confirmed that overexpression of Mst1 reduced the protein stability and enhanced the ubiquitination and degradation of RUNX2.Knockdown of WWP2 was achieved by lentiviral vector-mediated sh RNA expression,the protein expression level of RUNX2 was not significantly changed under Mst1 overexpression.CO-IP results showed that overexpression of Mst1promoted the binding of WWP2 to RUNX2,while knockdown of Mst1 inhibited the binding of WWP2 to RUNX2,thereby affecting the occurrence of VC.Conclusion:The decrease of Mst1 induced by OSS is closely related to the occurrence of VC.Overexpression of Mst1 can promote the ubiquitination and degradation of RUNX2 through WWP2,thereby reducing calcium deposition in EC.更多还原