摘要
背景
急性冠脉综合征(acute coronary syndrome, ACS)是指在冠状动脉粥样硬化斑块破裂或糜烂的基础上,伴随不同程度的重叠血栓的形成甚至栓塞,导致冠状动脉内血流显著减少或完全中断,从而引发的以心肌低灌注缺血为主要特点的一组临床综合征。急性冠脉综合征是临床上的常见急症,一经确诊需要采取紧急、恰当与有效的处理措施才能最大程度地减轻心肌缺血产生的不良影响,减少心肌梗死的面积、挽救患者的生命。急性冠脉综合征患者住院及随访过程中,发生复发性心绞痛、急性心肌梗死和心源性死亡的风险相对较高,如何提高对急性冠脉综合征患者未来再发心血管事件的预测水平始终是临床医生面临的巨大挑战。
目前国际上有许多针对急性冠脉综合征危险评分的系统,其中全球急性冠脉综合征注册(the Global Registry of Acute Coronary Events, GRACE)评分是较为广泛应用的评分系统。GRACE研究是对急性冠脉综合征患者进行的前瞻性的临床研究。该研究涉及14个国家的近100家医院,共纳入22645例患者,入选标准包含了急性冠脉综合征疾病谱的各种类别的患者。GRACE评分的内容包括年龄、心率、收缩压、基线肌酐水平、充血性心衰病史、院内PCI、心梗病史、入院心电图ST段改变及心肌酶或者其他标志物水平。GRACE评分的内容来源于大量真实的临床病例中统计得出的危险因素,而且较容易获得,因此其评分方法更加合理有效。
动脉粥样硬化是目前已知导致心血管疾病最常见的原因,它是一种发生在大、中动脉的富含脂质的多灶性、淤积性炎症性疾病。病理学研究发现,粥样斑块中含有大量炎症细胞的出现,影响了斑块形态以及性质的改变。然而,动脉粥样硬化的发生并不仅仅是一个局部的炎症反应,大量临床研究发现,一些分泌型的促炎反应生化标志物可能可以用来预测未来的心血管事件,提示患者的预后情况,如IL-6、MPO、MCP-1等,其中研究最广泛和最有说服力的指标就是C反应蛋白(CRP)。 CRP是一个强有力的临床标志物。最近的研究表明Wnt信号家族在炎症的调节中发挥着重要的作用,而该通路受到多个分泌型拮抗剂的作用,包括可溶性卷曲蛋白相关受体和dickkopf家族,而后者研究最多的是DKK-1。有研究发现动脉粥样硬化斑块中DKK-1的表达量是增加的,DKK-1在动脉粥样硬化斑块中能够通过影响内皮细胞与血小板之间的炎症反应,从而发挥促动脉粥样硬化作用。脑血管疾病患者和年轻的急性心肌梗死患者体内的DKK-1水平均高于正常对照人群。目前的研究发现DKK-1与动脉粥样硬化疾病人群密切相关,但是DKK-1在ACS患者体内表达及其与心血管疾病预后的研究尚未见报道。
目的
1.观察血浆DKK-1水平与ACS患者的关系；
2.判断DKK-1与CRP及GRACE评分分层的相关性；
3.探讨DKK-1对急性冠脉综合征患者再发心血管事件的预测价值,是否可以作为GRACE评分对ACS患者远期不良预后风险评估的一个补充指标。
方法
1.病例选择
入选急性冠脉综合征(ACS)患者,患者均行冠脉造影检查。入选标准为：选择性冠状动脉造影检查证实主要冠状动脉狭窄大于50%,并排除冠状动脉痉挛所致；符合STEMI和NSTEMI的诊断标准；UA的诊断标准为相应临床症状和(或)NSTEMI类似心电图表现,但是心肌标记物水平正常。满足上述标准的患者签署临床研究知情同意书后入选该研究。排除标准为：(1)合并心脏瓣膜疾病；(2)重度心律失常；(3)中、重度心功能不全；(4)重度或难治性高血压；(5)活动性肝脏功能障碍,或者查体发现ALT或AST>3倍正常上限；(6)恶性疾病：肿瘤；(7)贫血；(8)急、慢性炎症疾病：包括风湿性关节炎、类风湿关节炎等。
2.冠脉造影检查(CAG)与经皮冠脉介入治疗(PCI)
入选患者使用飞利浦Allura FD20心血管数字剪影仪进行造影检查,记录冠状动脉造影图像,判断靶血管,记录冠状动脉靶病变的类型、部位、形态、狭窄程度。
3.血液生化指标检查
使用日本HITACHI公司生产的7170型全自动生化分析仪检测每位患者的外周血总胆固醇、甘油三酯、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、肌酐、尿酸、CK、及空腹血糖。应用西门子BN2特种蛋白分析仪检测外周血高敏C反应蛋白浓度。
4.血浆中炎症因子DKK-1的检测
入选患者清晨抽取空腹外周血5ml,抗凝处理后分离血浆,采用ELISA试剂盒(R&D Systems)检测外周血中DKK-1浓度。
5. GRACE评分的计算
GRACE评分计算变量包括：年龄、心率、收缩压、基线肌酐水平、充血性心衰病史、院内PCI、心梗病史、入院心电图ST段压低及心肌酶或者标志物水平升高。GRACE评分的危险分级包括低危、中危和高危。
6.院内及出院用药情况
合理使用硝酸酯类药物、p受体抑制剂、血管紧张素酶抑制剂(ACEI)、血管紧张素Ⅱ受体阻断剂(ARB)钙通道阻滞剂等。出院时,详细告知患者和家属继续用药的方法和剂量。
7.患者随访与终点事件的判断
入选患者出院后进行随访,包括门诊随访和电话随访。门诊随访时检查患者的空腹血糖、血脂四项(包括TC、TG、LDL-C、HDL-C),记录静息心电图,判断终点事件的发生、时间及诱因。主要不良心血管事件(MACEs)包括心源性死亡、非致死性心肌梗死、再发不稳定心绞痛和冠状动脉血运重建。
8.统计学分析
所有试验数据采用SPSS16.0软件进行统计分析。数值变量的描述统计采用均数±标准误表示,分类变量的统计描述采用频率或者百分率表示。分类变量采用卡方(chi-square)检验或者Fisher's确切检验。数据间的相关分析采用双变量相关分析,多组数据间的比较采用单因素方差分析。采用二元logistic回归评估DKK-1水平与MACE的相关性。P<0.05即为存在统计学意义。结果
1.入选病例的基本情况
331例患者符合入选标准,获得其中322例患者的全部资料。所有患者的最长随访时间为36个月,最短随访时间为18个月,中位随访时间为24个月。至随访结束时共有31例患者失访。完成随访的291例患者包括46例STEMI患者和245例NSTE-ACS患者(63例NSTEMI)。所有患者均接受冠脉造影检查,198例(68%)患者符合PCI治疗者并置入相应支架,随访期间共44例MACEs发生。
2.急性冠脉综合征患者血浆中DKK-1的基线表达水平
DKK-1的中位表达水平是713pg/ml。在ACS患者中,DKK-1的水平与高敏C反应蛋白水平呈正相关(r=0.308,P<0.001),同时STEMI患者的DKK-1基线表达水平显著高于NSTE-ACS患者(P=0.006)。而在NSTEMI和UA患者中DKK-1的表达量无明显的差异,且PCI治疗不会影响患者体内DKK-1的表达量。
3.急性冠脉综合征患者GRACE评分情况
所有患者的中位GRACE评分为88分,其中STEMI患者GRACE得分为108(63-149)分,NSTE-ACS患者得分为84(38-148)分。两组之间GRACE得分的差异存在显著统计学意义(P<0.001)。
4.根据DKK-1血浆水平分组患者测量指标的组间比较
根据DKK-1的血浆水平由低到高将所有患者三等分,分为T1、T2和T3组,每组97例患者。统计分析发现,DKK-1水平高的患者年龄较大,血糖和hs-CRP的水平也明显增高(P<0.05)。至随访结束,T1组MACEs发生率为2.1%,T2组为13.4%,T3组为29.9%,三组事件发生率差别有统计学意义(P<0.01)。
5.事件组患者和无事件组患者测量指标的组间比较
事件组患者的总胆固醇、甘油三酯、LDL-C、血糖、hs-CRP. DKK-1表达水平明显高于无事件组患者(P<0.05), HDL-C水平明显低于无事件组患者(P<0.05)。事件组患者的高血压患病率和糖尿病患病率明显高于无事件组患者(P<0.05)。事件组与无事件组的GRACE评分差异无统计学意义(P=0.570)。
6.急性冠脉综合征患者血浆DKK-1水平与GRACE评分的关系
DKK-1的表达水平与GRACE评分呈正相关(r=0.259,P<0.05)。对于GRACE评分低危、中危、高危三组,DKK-1的中位浓度分别为642、718和959pg/ml。统计学分析发现GRACE评分高危组患者血浆DKK-1的浓度要高于中危或低危组患者(P=0.002和P<0.001),而中危组DKK-1浓度尽管高于低危组,但是差异无统计学意义(P=0.100)。
7.DKK-1是急性冠脉综合征患者不良预后的独立预测指标
在校正心血管危险因素(年龄、性别、BMI、吸烟、血糖、血脂)后,二元logistic回归分析发现DKK-1和hs-CRP水平与ACS患者不良心血管事件的发生有显著的相关性。
8.DKK-1对急性冠脉综合征患者不良预后的预测价值
采用ROC (receiver-operating characteristic curve)曲线分析后发现,GRACE评分并预测ACS患者不良预后的发生AUC较低为0.524(P=0.605)。 GRACE评分联合DKK-1指标后,AUC增加到0.775(P<0.001); GRACE评分联合hs-CRP后AUC增加到0.791(P<0.001)。而当GRACE评分同时联合上述两个指标时,AUC显著提高到0.847(P<0.001),此时该模型在不良事件预测方面的敏感性为81.8%,特异性为71.7%。
结论
1.冠心病的传统危险因素中,年龄、高血压、糖尿病对急性冠脉综合征患者再发不良心血管事件有预测价值。
2.在校正传统心血管病危险因素(性别、年龄、吸烟、高血压、糖尿病)的影响后,血浆外周血hs-CRP水平、外周血DKK-1水平对再发不良心血管事件具有独立的预测价值。
3.DKK-1联合hs-CRP与GRACE危险评分可以显著提高对急性冠脉综合征患者再发不良心血管事件的预测价值
背景
急性冠脉综合征(acute coronary syndrome, ACS)主要是由于突发的冠状动脉斑块破裂伴血栓形成引发的冠脉管腔完全或部分闭塞导致,动脉粥样硬化(atherosclerosis, AS)是其主要的病理基础。经典病理学研究发现动脉粥样硬化斑块中有大量炎性细胞和炎性因子的存在,其活跃程度与斑块的易损性密切联系。炎症理论的提出让人们对动脉粥样硬化有了更深层次的认识,然而,由于其发病机制的复杂性与参与因素的多样性,炎症在该过程中的作用机制至今也未完全阐明,针对各种炎症因子与血管壁细胞成分之间相互关系的研究仍不断地进行。
Wnt信号通路是一条非常保守的信号转导途径,参与了胚胎形成的许多发育过程,并且在维持成体组织的动态平衡中起到了重要作用。近年来,Wnt通路在心血管系统中的作用也越来越受到人们的重视和重视,在脂肪生成、血管新生、血管钙化、心肌梗死后愈合和缺血预适应中也发挥着重要的作用。1998年研究人员发现DKK-1作为Wnt通路的一种分泌型抑制剂,通过与胞膜上的LRP5/6、 Kremen等受体结合而介导内吞,从而关闭经典Wnt通路。DKK-1可以参与胚胎发育中的心内膜细胞、心肌细胞和心脏瓣膜等多种心血管系统成分的生成。Ueland等人发现DKK-1参与血小板诱导的内皮细胞的活化,内皮和血小板来源的DKK-1都可以增加两者之间的炎症反应,表明DKK-1在动脉粥样硬化斑块中能够增加炎症反应,从而发挥促动脉粥样硬化的作用。在前期ACS患者的临床研究中,我们发现DKK-1在ACS患者体内的表达不仅与病情的严重程度有关,而且对患者的预后有一定的预测价值,提示其可以反映冠状动脉粥样硬化的严重程度及不稳定状态。
近年来有关DKK-1与动脉粥样硬化斑块易损性的机制研究尚未见到报道。2001年, von der Thiisen等通过在ApoE-/-小鼠的颈总动脉分叉前放置缩窄性硅胶套管,在经历了长约8周左右的高脂饮食后发现套管的近心端能成功地诱发出与人类病变相似的易损斑块。本研究通过采用该模型模拟动脉粥样硬化斑块的形成,并通过慢病毒载体介导的RNA干扰在体内沉默DKK-1基因的表达,使用组织病理学检查及免疫组化技术观察AS斑块的形成和稳定性,研究DKK-1对AS斑块发生和发展及易损性的影响机制。
目的
1.体内实验通过慢病毒载体介导DKK-1基因的沉默,观察DKK-1的干扰对ApoE-/-小鼠动脉粥样硬化斑块发生的影响；
2.探讨干扰DKK-1的表达对颈动脉斑块稳定性的作用及其机制；
3.体外实验观察ox-LDL刺激下人脐静脉内皮细胞内DKK-1的表达情况；
4.研究ox-LDL刺激下DKK-1对HUVECs eNOS磷酸化、粘附分子表达和凋亡功能的影响。
方法
1. Lenti-DKKli'慢病毒的构建
构建干扰小鼠DKK-1基因表达的RNAi'慢病毒载体(lenti-DKKli),以携带绿色荧光蛋白的慢病毒空载体(Lenti-GFP)为空载体对照组。2. ApoE-/-小鼠AS模型的构建与分组
购买的80只8周龄雄性ApoE-/-小鼠在2周的适应性喂养并过渡到高脂饮食后,进行右侧颈总动脉硅胶套管放置术。
小鼠行颈总动脉硅胶套管放置术后高脂喂养8周,分三组：(1)NS组(n=27)：尾静脉注射200μl生理盐水；(2)阴性对照组(n=27)：尾静脉注射2×107ifu的Lenti-GFP;(3)干预组(n=26)：尾静脉注射2xl07ifu的Lenti-DKKli。病毒转染后继续高脂喂养4周,并于2周时于NS组和阴性对照组随机各选取2只小鼠进行颈动脉切片观察慢病毒转染效率。
3.血液指标检测
各组小鼠分别留取外周血,离心后分离上清检测总胆固醇、甘油三酯、血糖、低密度脂蛋白和高密度脂蛋白等生化指标。
4.组织学斑块病理学检测
留取腹主动脉、主动脉根和颈动脉等组织浸泡4%甲醛固定,在OCT包埋组织前用流水充分冲洗以去除甲醛的影响。腹主动脉行大体油红O染色观察脂质沉积；分离颈动脉套管后OCT包埋,留取5μm厚的冰冻切片,行H&E染色观察斑块形态,油红O染色观察斑块内脂质沉积,天狼猩红染色观察斑块内胶原纤维含量,免疫组化染色分析巨噬细胞、平滑肌的含量,并计算易损指数。同时颈动脉冰冻切片行免疫组化染色,观察斑块内粘附分子(ICAM-1、VCAM-1)、炎性因子(TNF-α、MCP-1、IL-1β、IL-6)、基质金属蛋白酶(MMP-2、MMP-9)、胶原合成酶P4Hα1以及凋亡因子RIP3的表达情况。TUNEL法检测斑块内细胞凋亡。
5.组织分子生物学检测
留取颈动脉等组织-80℃保存,RIPA蛋白裂解液提取蛋白后,Western Blot检测各组DKK-1和caspase-3的表达。
6.人脐静脉内皮细胞(HUVECs)内研究
观察氧化低密度脂蛋白(ox-LDL)刺激HUVECs后DKK-1的表达情况。通过siRNA和重组DKK-1的干预处理,观察ox-LDL刺激下HUVECs内eNOS磷酸化、粘附分子(ICAM-1、VCAM-1)表达和凋亡(cleaved caspase-3)功能的改变。
7.统计学分析
数值变量资料以均数±标准误表示。两样本间的比较采用独立样本t检验；多样本比较采用单因素方差分析(ANOVA),组间差异两两比较当方差齐时采用LSD法,方差不齐时采用Dunnett T3法。P<0.05认为有统计学意义。
结果
1.慢病毒靶位的筛选结果
RAW264.7细胞内转染包含三种不同DKK-1干扰靶点的慢病毒载体。RT-PCR和Western Blot检测结果显示靶点A的干扰效果最好。靶点1、靶点2和靶点3的干扰效率分别为72.1%、68.1%和36.5%。我们选择靶点1作为有效干扰靶点进一步合成终滴度为5×108TU/ml的病毒载体悬液。
2.实验动物一般情况
三组小鼠实验结束时体重无显著差异(P>0.05)。三组小鼠血清TC、LDL-C、HDL-C、TG和BS水平无显著差异(P>0.05),表明DKK-1干扰对ApoE-/-小鼠体内血脂、血糖的表达水平无影响。
3.慢病毒转染及DKK-1干扰效果检测
病毒转染后两周,小鼠颈动脉斑块内有显著GFP表达。对小鼠颈动脉斑块组织蛋白进行Westeron Blot检测。结果显示：Lenti-DKKli组DKK-1蛋白水平明显下降(P<0.05)。4. DKK-1干扰对ApoE-/-小鼠斑块负荷的影响
Lenti-DKKli组主动脉表面AS病变范围显著下降(P<0.05), Lenti-DKKli组的小鼠主动脉根部AS斑块的病变范围显著下降(P<0.05)。
5.不同组别斑块易损性的情况
Lenti-DKKli组斑块内巨噬细胞含量明显低于NS组和Lenti-GFP组(P<0.05),而平滑肌细胞和胶原含量高于NS组和Lenti-GFP组(P<0.05). Lenti-DKKli组斑块内脂质的含量虽然有下降趋势,但在三组之间无显著差异(P>0.05)。Lenti-DKKli组的斑块易损指数明显低于NS组并Lenti-GFP组(P<0.05)。斑块纤维帽的厚度在Lenti-DKKli组也有明显增加(P<0.05)。
6.DKK-1干扰对颈动脉斑块内炎症因子和基质金属蛋白酶的影响
Lenti-DKKli组斑块内的炎症因子(MCP-1、IL-6、IL-lβ、ICAM-1、VCAM-1)含量明显低于NS组和Lenti-GFP组(P<0.05)。Lenti-DKKli组斑块内MMP-2和MMP-9的含量显著减少,而P4Hal含量高于NS组和Lenti-GFP组(P<0.05)。
7.DKK-1干扰对颈动脉斑块内细胞凋亡的影响
Lenti-DKKli组斑块内TUNEL阳性细胞比例明显低于NS组和Lenti-GFP组(P<0.05)。Lenti-DKKli组斑块内凋亡因子RIP3含量明显低于NS组和Lenti-GFP组(P<0.05)。 Western Blot结果显示Lenti-DKKli组斑块组织cleaved caspase-3的表达量下降,cleaved caspase-3与caspase-3比值下降(P<0.05)。
8.DKK-1对ox-LDL刺激下HUVECs细胞功能的影响
Ox-LDL刺激HUVECs后DKK-1的表达上调,HUVECs、DKK-1的表达对ox-LDL具有浓度和时间依赖性。与ox-LDL+NC组比较,ox-LDL+siRNA组内DKK-1和粘附分子(ICAM-1、VCAM-1)的表达水平下降(P<0.05),而rDKK-1组粘附分子(ICAM-1、VCAM-1)无明显变化(P>0.05)。与空白对照组比较,ox-LDL+siRNA组内粘附分子(ICAM-1、VCAM-1)的表达水平升高(P<0.05)。表明DKK-1参与ox-LDL刺激HUVECs粘附分子产生的过程。
与ox-LDL+NC组比较,ox-LDL+siRNA组内磷酸化eNOS的表达水平上升(P<0.05),而rDKK-1组磷酸化eNOS无明显变化(P>0.05)。与空白对照组比较,ox-LDL+siRNA组内磷酸化eNOS的表达水平下降(P<0.05)。表明DKK-1参与ox-LDL刺激HUVECs内eNOS活性下降的过程。
与ox-LDL+NC组比较,ox-LDL+siRNA组内cleaved caspase-3的表达水平下降(P<0.05),而rDKK-1组cleaved caspase-3的表达水平无明显变化(P>0.05)。与空白对照组比较,ox-LDL+siRNA组内cleaved caspase-3的表达水平升高(P<0.05)。表明DKK-1参与ox-LDL刺激HUVECs的凋亡过程。
结论
1.DKK-1干扰能够减少ApoE-/-小鼠AS斑块的发生；
2.DKK-1干扰能够减少ApoE-/-小鼠AS斑块中巨噬细胞含量,增加平滑肌细胞和胶原成分,增加斑块的稳定性；
3.DKK-1干扰能够减少ApoE-/-小鼠AS斑块内炎症和凋亡的发生；
4.DKK-1参与ox-LDL刺激下HUVECs eNOS磷酸化、粘附分子表达和凋亡功能的改变。
Background
Acute coronary syndrome (ACS) is a group of myocardium ischemia charactized syndrome caused by atherosclerotic coronary artery plaque rupture, hemorrhage and local thrombosis, or vasospasm, with subsequent total or subtotal occlusion of coronary arteries. ACS is a common emergency and warrants urgent, proper and effective treatment once diagnosed to minimize the harmful effects of myocardial ischemia and areas of myocardial infarction and save patients' lives. The risks of recurrent angina, acute cardiac infarction and cardiac death during hospitalization and follow-up are relatively high, and how to improve the prognosis of recurrent cardiac events remains as a great challenge for the clinical physicians.
The Global Registry of Acute Coronary Events (GRACE) is a widely used scoring system evaluating risks of ACS. The GRACE research is the first prospective study of ACS patients worldwide, with22645subjects from about100hospitals of14countries enrolled. The inclusion criteria were all types of ACS. The variables required for calculation of GRACE score include age, heart rate, systolic blood pressure, baseline creatinine level, history of congestive heart failure, in-hospital percutaneous coronary intervention, history of MI, ST-segment depression on admission electrocardiography (ECG) and elevated cardiac enzyme or marker levels. The variables for GRACE score calculation were based on risk factors from large scale objective clinical trials and data of the variables are easy to obtain, which make GRACE score rational and effective.
Atherosclerosis, a multifocal lipidstatic disease characterized by lipid accumulation and inflammation in aortas and median arteries, is the most common known cause of cardiovascular diseases. Pathological studies showed that atherosclerotic plaques are filled with inflammatory cells, which alter morphology and characters of the plaques. However, atherosclerosis is more than a localized inflammatory disease. Various clinical trials claimed that secretary proinflammatory biochemical markers, such as IL-6, MPO, MCP-1and so on, are effective for cardiovascular events prediction and prognosis evaluation. C Reactive Protein (CRP) is one of the most widely studied and convincible clinical biochemical markers. Resent researches clarified that the Wnt signaling family plays an important role in inflammation modulation. Several secretory antagonists were confirmed to suppress the Wnt pathway, including FIZZLE and dickkopf family, and the mostly studied of the latter is DKK-1. Serous DKK-1level was showed effective to predict prognosis of osteoarthritis and some malignant cancers such as multiple myeloma. DKK-1was found upregulated in atherosclerotic plaques and playing a proinflammatory role to promote atherosclerosis and plaque vulnerability.
Objectives
1. To investigate the expression of DKK-1in ACS patients;
2. To investigate the relevance between DKK-1and CRP or GRACE score;
3. To investigate the role of peripheral concentration of DKK-1in predicting recurrent cardiovascular events of ACS patients and the feasibility of taking DKK-1as one supplementary variable for GRACE score predicting long-term adverse cardiac events.
Methods
1. Study population
We included consecutive patients hospitalized in the Department of Cardiology of Qilu Hospital, Shandong University, from March2008to January2010. Inclusion criteria were diagnosis of ACS, including ST-segment elevation myocardial infarction (STEMI) and non-ST elevated ACS (NSTE-ACS); all patients underwent coronary angiography. The diagnosis of STEMI was typical chest pain with serum cardiac enzyme levels twice that of the upper level of normal or cardiac troponin I (cTnl) level≥0.1ng/ml, both with persistent electrocardiographic ST segment elevation>1mm in2or more contiguous leads or newly occurred left bundle branch block. NSTE-ACS included non-STEMI (NSTEMI) and unstable angina (UA). The diagnosis of NSTEMI was angina or discomfort at rest with ST segment depression or transient elevation and/or prominent T-wave inversion, with cardiac enzyme levels twice that of the upper level of normal or cTnI≥0.1ng/ml. Patients with clinical features and/or electrocardiographic expression of NSTEMI but normal cardiac biomarker levels were diagnosed as having UA. Exclusion criteria were valvular heart disease, severe arrhythmias, severe heart failure, severe or refractory hypertension, active hepatosis, malignant diseases, anemia and acute or chronic inflammatory diseases.
2. Coronary angiogram (CAG) and percutaneous conronary intervention (PCI)
The patients enrolled in the study all received coronary angiography (CAG) examination. Imaging of coronary arteries was taken and target arteries were diagnosed. Type, location, morphology and degree of stenosis of the target coronary artery lesions were recorded.
3. Laboratory analysis
All laboratory data, including total cholesterol (TC), triglycerides (TG), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), blood glucose, uric acid level, creatinine level, creatinine kinase activity, and cTnl and hs-CRP levels were measured in the biochemical department of Qilu Hospital.
4. Measurement of DKK-1in serum
Blood samples of5ml each were collected in EDTA-containing tubes and then centrifuged at4℃.The collected plasma was stored in aliquots at-80℃. DKK-1concentration was measured by use of an ELISA kit (R&D Systems, Minneapolis, USA).
5. Calculation of GRACE risk scores
The variables required for calculation of the score include age, heart rate, systolic blood pressure, baseline creatinine level, history of congestive heart failure, in-hospital percutaneous coronary intervention, history of MI, ST-segment depression on admission electrocardiography (ECG) and elevated cardiac enzyme or marker levels. The risk categories of GRACE score were divided into low, medium and high.
6. Hospital and post-discharge medications
Nitrates, beta receptor inhibitors, angiotensin enzyme inhibitors (ACEI), angiotensin II receptor blockers (ARB), calcium channel blockers and vasodilators were applied according to the pharmacopeia.
7. Follow-up
Endpoints after discharge were MACEs, including sudden cardiac death, MI, percutaneous coronary intervention, coronary artery bypass grafting and recurrent unstable angina pectoris. Patients were followed up by researchers from Qilu Hospital, Shandong University after discharge. Follow-up examinations included fasting blood glucose, TC, TG, LDL-C, HDL-C and resting electrocardiogram.
8. Statistical analysis
All data were analyzed by use of SPSS v16.0(SPSS Inc., Chicago, IL, USA). Numeric variables are expressed as mean±SD. Categorical variables are expressed as frequencies and percentages. Kolmogorov-Smirnov test was used to assess normal distribution of quantitative variables, with log transformation for non-normal distribution. Categorical data were compared by chi-square test or Fisher's exact test as appropriate. Bivariate correlation was used for correlation analysis. One-way ANOVA was used for comparison of multiple groups. Binary logistic regression was used to assess the independent association of DKK-1level with MACE. Differences in the predictive values were estimated by comparing the area under the receiver-operating characteristic curve (ROC). The level of statistical significance was set at P<0.05.
Results
1. Baseline characteristics of study subjects
A total of331patients with ACS met the inclusion criteria, and we had complete data for322. At the end of the study, data for291patients (193males,66.3%) with complete follow-up data were analyzed, including46with STEMI and245with NSTE-ACS (63with NSTEMI,182with UA), and68%of our patients underwent percutaneous coronary intervention. CAG was performed for all the subjects and among which198patients (68%) were qualified and treated with PCI. During the follow-up,44MACEs took place.
2. Plasma levels of DKK-1in patients with ACS
Median plasma DKK-1level was713pg/ml (range129-2139pg/ml). DKK-1levels were correlated with hs-CRP level(r=0.308, P<0.001). DKK-1was significantly higher in patients with STEMI than those with NSTE-ACS at baseline. DKK-1level did not differ between patients with NSTEMI and those with UA. Treatment with PCI did not alter the DKK-1level in patients.
3. Risk stratification by GRACE score
The median GRACE risk score was88(range38-149) for the whole population, 108(63-149) for patients with STEMI,84(38-148) for patients with NSTE-ACS. The GRACE scores were significantly higher for patients with STEMI than NSTE-ACS at baseline.
4. Clinical index among groups divided by concentrations of DKK-1in plasma
The patients were divided into three groups (T1, T2and T3) according to the concentrations of DKK-1, with97patients in each group. The patients in T3were much older and the levels of blood sugar and hs-CRP were higher than the other two groups (P<0.05). Till the end of follow-up, the rate of MACEs was2.1%in T1,13.4%in T2and29.9%in T3. There was significant difference in the rate of MACEs among the three groups (P<0.05).
5. Clinical index between event and non-event groups
The levels of TC, TG, LDL-C, BS, hs-CRP and DKK-1are significantly higer in event group than in non-event group (P<0.05); the level of HDL-C decreased in the event group (P<0.05). The rates of hypertension and diabetes were higher in the event group than in non-event group (P<0.05). There was no difference in the GRACE score between the two groups (P>0.05).
6. The relationship of plasma levels of DKK-1and GRACE scores
DKK-1levels were correlated with GRACE scores (r=0.259, P<0.05). The medium concentrations of DKK-1were642,718and959pg/ml for low, intermediate and high GRACE category, respectively. The concentrations of DKK-1were elevated with high-risk than intermediate-or low-risk GRACE score (P=0.002and P<0.001). DKK-1levels were higher but not significantly with intermediate than low risk (P=0.100).
7. DKK-1is an independent predictor of long-term MACE for patients with ACS
After adjustment for cardiovascular risk factors (age, sex, BMI, smoking, blood glucose and blood lipids), binary logistic regression revealed a significant association of DKK-1and hs-CRP levels and MACE for ACS patients.
8. DKK-1has Better prognostic value for patients with ACS
Receiver-operating characteristic curve (ROC) analysis showed that the GRACE score alone was a poor predictor of MACE, the area under the ROC (AUC) was0.524(P=0.605). With the addition of DKK-1level, the AUC was increased to0.775(P<0.001) and to0.791with hs-CRP level (P<0.001). With both biomarkers added, the AUC was significantly increased to0.847(P<0.001), and the sensitivity of this model in evaluating prognosis was81.8%, with specificity71.7%.
Conclusions
1. Traditional CHD risk factors age, hypertension and diabetes are predictive for long-term MACE for patients with ACS.
2. After adjustment for cardiovascular risk factors (age, sex, BMI, smoking, blood glucose and blood lipids), peripheral plasma DKK-1and hs-CRP levels are independent predictors of long-term MACE for patients with ACS.
3. The long-term predictive ability of post-discharge GRACE score combined with hs-CRP may be enhanced by adding DKK-1level.
Background
The complete or part occlusion of coronary artery, generated by sudden rupture of atherosclerotic plaque and the following thrombosis, is the main cause of acute coronary syndrome (ACS). Atherosclerosis is the dominant pathological etiology of ACS. Canonical pathological research revealed that amount of inflammatory cells and cytokines existed in the atherosclerotic plaque, the activities of these factors correlated closely with stability of the plaque. The theory of inflammation provided us with a deeper recognition of atherogenesis. However, the complexity of pathogenesis and diversity of factors involved in atherosclerosis signify the difficulty in totally elucidating the role of inflammation in atherogenesis. The researches of relationship between inflammatory factors and vascular cells are still carried on all over the world.
Wnt pathway is a conserved signaing transduction pathway. It participates in many aspects during embryonic development and plays a great role in maintaining the dynamic balance in adult tissue. The role of wnt pathway in cardiovascular system has become more and more important during recent years, it takes part in adipogenesis, angiogenesis, vascular calcification, myocardial infarction and ischemic adaption. In1998, researchers found that a secretory glycoprotein, DKK-1, can block wnt pathway by competitively bind to the receptors (LRP5/6, Kremen) on the cellular member. DKK-1takes part in the formation of cardiovascular system including endocardium, myocardial cells and cardiac valve, etc. Ueland et al. found that DKK-1participated in the activation of endothelial cells deduced by platelet. DKK-1derived from endothelial cells and platelet can enhance the inflammation between them, indicating that DKK-1promotes the inflammatory reaction in atherosclerotic plaque and DKK-1is an atherogenic factor. In the clinical study of ACS patients, we found that the expression of DKK-1in plasma was not only correlated with the severity of disease, but also predicted the prognosis of disease. The expression of DKK-1implies its ability in reflecting the severity and stability of coronary atherosclerosis.
There is no report about the role and mechanism of DKK-1in atherogenesis. According to the results discovered in the clinical research, we tried to study the role of DKK-1in atherosclerotic animal model. The pathological changes of atherosclerosis in mice carotid are similar to those changes in human, including stenos is, formation of fibrous cap and plaque rupture, etc. By placement of a constrictive perivascular collar around the common carotid artery in ApoE-/-mice and high fat diet for about8weeks, von der Thusen et al. successfully induced vulnerable plaque similar to human lesion in the proximal part of the collar. In the animal study, we used this model to implement the formation of atherosclerotic plaque, and interfered the expression of DKK-1by RNAi mediated with lentiviral vector. Histopathological and immunohistochemical measurement were used to observe the formation and stability of atherosclerotic plaque. The research about mechanism of DKK-1in the study of atherosclerosis might provide a new target for the prevention and treatment of atherosclerosis.
Objectives
1. To study the effects of DKK-1interference on the development and stability of atherosclerotic plaque in ApoE-/-mice;
2. To study the mechanisms of DKK-1interference on stability of atherosclerotic plaque;
3. To observe the expression of DKK-1in HUVECs under stimulation of ox-LDL in vitro;
4. To study the influence and mechanism about DKK-1on functions of HUVECs under stimulation of ox-LDL.
Methods
1. Construction of lentiviral vector
The DKKli lentiviruses (KD sites:1,2,3) were transfected into RAW264.7cells. Three days after the transfection, cells were collected for real-time PCR analysis to select the most effective targeting site for the DKK-1gene to amplify. The siRNA NS vector containing lentivirus only was used as a control. Finally, two lentiviral vectors were produced:Lenti-DKKli and Lenti-GFP.
2. Atherosclerosis animal experimental protocol
80ApoE-/-mice (male,8-week old) were fed on an atherogenic chow after two weeks'adaptive feed. Atherosclerotic plaques were induced by placement of a perivascular collar around the right common carotid artery. All mice were randomly divided into3groups8weeks after the placement of collar:NS group (n=27) injected with normal saline; Lenti-GFP group (n=27) transfected with Lenti-GFP; Lenti-DKKli group (n=26) transfected with Lenti-DKKli. A total suspension of200ul was injected into the mice through tail vein. Four mice from NS and Lenti-GFP group were selected at week12for detection of lentiviral transfection efficiency. All mice were euthanized at the end of week14.
3. Measurement of serum lipid and blood sugar
Blood samples were collected and centrifuged. The serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and blood sugar were measured.
4. Histopathological and immunohistochemical measurement
The mice were perfused through the left ventricle with saline, followed by4%formaldehyde. The whole aorta, aortic root and right common carotid artery were excised and immersed in4%formaldehyde overnight at4°C. The vessel tissue was rinsed before embedded in OCT, and stored at-40℃until use. OCT-embedded aortic root and carotid artery were cross-sectioned into slices about5μm thick. The whole aorta was stained with oil red O. Sections were stained with hematoxylin and eosin for plaque morphology, and also stained with oil red O for lipids and picrosirius red for collagen. Smooth muscle cells, macrophages, inflammatory cytokines (MCP-1, IL-6, IL-1β, TNF-a), adhesive factors (ICAM-1, VCAM-1), MMP-2, MMP-9, P4Hal and RIP3were immuno-stained with corresponding antibodies. TUNEL test was used to detect cellular apoptosis in the plaque. Vulnerability index was calculated with the following formula:(macrophage staining%+lipid staining%)/(SMC staining%+collagen staining%). The content of each index in the plaque was quantified with the Image-Pro Plus6.0software.
5. Molecular biological analysis
The expressions of DKK-1in each group were detected by Western Blot with specific antibodies.
6. The role of DKK-1in HUVECs
HUVECs were stimulated by ox-LDL. In order to elucidate the role of DKK-1in regulating oxidative stress, secretion of adhesive factors and apoptosis of HUVECs under stimulation of ox-LDL, siRNA for DKK-1and recombinant DKK-1were used according to the procedure.
7. Statistical analysis
Quantitative values are expressed as mean±SEM. And the data were analyzed by independent sample t-test or one-way ANOVA as appropriate. The level of P<0.05was considered significant.
Results
1. Screening the most effective targeting sites for DKK-1genes silencing by real-time PCR
RAW264.7cell line was transfected with lentivirus-based vectors expressing three different DKK-1siRNAs and gene silencing analysis showed that the DKKli-KD1lentivirus was the most effective vector in blocking DKK-1expression. The DKK1-RNAi KD1, KD2, and KD3exhibited72.1%,68.1and36.5%reduction, respectively. The terminal virus titer was5x108IFU/mL.
2. General condition of the mice
There were no significant differences in body weight, levels of blood sugar and serum lipids among the three groups at the end of experiment.
3. The efficiency of lentivirus transfection
Significant amount of GFP was expressed in carotid plaques2weeks after the injection of lentivirus. Western blot analysis of DKK-1expression in tissue protein from carotid artery exhibited that level of DKK-1in Lenti-DKKli group was significantly down-regulated.
4. The influence of DKK-1interference on plaque burden of ApoE-/-mice
The gross oil red O staining revealed that atherosclerotic area of the whole aorta in Lenti-DKKli group significantly decreased (P<0.05). H&E staining revealed that cross-section of the aortic root was also reduced in the Lenti-DKKli group (P<0.05).
5. Vulnerability index in different groups
Specific staining revealed that the content of macrophage in carotid plaque in the Lenti-DKKli group significantly decreased compared with NS and Lenti-GFP groups (P<0.05), yet the contents of smooth muscle cells and collagen fibers increased (P<0.05). Though the content of lipids in the interference group slightly reduced, no significant differences existed among the three groups (P>0.05). Vulnerability index in the Lenti-DKKli group significantly diminished compared with the other two groups (P<0.05). The thickness of fibrous cap increased in the interference group (P<0.05).
6. The effect of DKK-1interference on expression of inflammatory factors and matrix metalloproteinase in carotid plaques
The content of inflammatory factors (MCP-1, IL-6, IL-1β, ICAM-1and VCAM-1) in Lenti-DKKli group decreased compared with NS and Lenti-GFP groups (P<0.05). The content of MMP-2and MMP-9decreased in the Lenti-DKKi group, yet the content of P4Hα1increased compared with NS and Lenti-GFP groups (P<0.05).
7. The effect of DKK-1interference on cellular apoptosis in carotid plaques
The content of apoptotic factor RIP3decreased in Lenti-DKKli group compared with NS and Lenti-GFP groups (P<0.05). The percentage of TUNEL positive cells in Lenti-DKKli group also decreased compared with NS and Lenti-GFP groups (P<0.05). Western Blot results showed the Lenti-DKKli group had less cleaved caspase-3expression in the carotid plaque tissue (P<0.05).
8. The roles of DKK-1in HUVECs stimulated by ox-LDL
The expression of DKK-1in HUVECs was elevated after stimulation of ox-LDL. In the ox-LDL+siRNA group, the expression of adhesive factors (ICAM-1, VCAM-1) and apoptotic factor cleave caspase-3declined, yet the level of p-eNOS increased compared with the ox-LDL group and ox-LDL+NC group(P<0.05). The changes of the above factors weren't different between recombinate DKK-1group and ox-LDL group. Though the levels of adhesive and apoptotic facters declined and p-eNOS increased in the ox-LDL+siRNA group, they were still higher or lower than those in the empty group (P<0.05). Gain and loss of function tests showed DKK-1participated in the stimulation of HUVECs by ox-LDL.
Conclusions
1. DKK-1interference can effectively decrease the formation of atherosclerotic plaque in ApoE-/-mice;
2. By decreasing the content of macrophage and increasing the content of smooth muscle cells and collagen, DKK-1interference can effectively improve the stability of atherosclerotic plaque;
3. DKK-1interference can decrease the extent of inflammation and apoptosis in the atherosclerotic plaque in ApoE-/-mice;
4. DKK-1participates in the influences of expression of adhesive factors, eNOS and apoptosis of HUVECs under stimulation of ox-LDL.
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