血清淀粉样蛋白A1对动脉粥样硬化斑块稳定性的影响及其机制的实验研究
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摘要
研究背景：
动脉粥样硬化(atherosclerosis,AS)作为心脑血管疾病的病理基础,目前已经成为威胁人类尤其是中老年人健康的最重要的疾病,由此导致的心脑血管意外发生率和死亡率极高,即使是在幸免的存活者中也仍然存在极高的致残率。动脉粥样硬化所涉及的致病因素极多,且其病理过程复杂,尽管此前已有诸多学说试图解释其发病机理,但其具体的发病及进展机制至今仍未明确。
诸多研究已经证实,动脉粥样硬化所致的临床急性缺血事件更多与斑块的稳定性密切相关,而非动脉管腔的狭窄程度。不稳定斑块破裂出血,并继发导致血栓形成已经成为临床急性心血管事件的主要病理机制。由此引入了易损斑块(Vulnerable Plaque)的概念,其作为具有极强破裂倾向的斑块,特点包括纤维帽在各种因素下逐渐变薄、坏死脂质核心逐渐增大、以巨噬细胞浸润为特征的斑块内活性炎症状态、平滑肌细胞逐渐减少以及胶原含量逐渐减少。并以此为依据引入易损指数,易损指数可以定量地评估斑块易损性,其定义为：易损指数＝(脂质占斑块面积百分比+巨噬细胞占斑块面积百分比)／(胶原占斑块面积百分比+平滑肌细胞占斑块面积百分比)。因此,探寻影响AS斑块稳定性的各种危险因素,并寻求能有效地稳定易损斑块的干预靶点,积极避免斑块破裂及继发的心脑血管事件成为近年来的研究热点。
既往研究证实血清淀粉样蛋白A (serum amyloid A, SAA)家族由SAA1、 SAA2、SAA3及SAA4四个家族成员构成。作为家族最主要组成成分的SAA1是SAA家族中表达最广泛、活性最强、反应最敏感的亚型。虽然在常态下SAA1表达水平较低,但在急性期状态下其血清浓度在极短时间内升高1000倍。血清SAA1主要由肝细胞合成并分泌,而其他组织细胞同样具有局部分泌SAA1的功能。在AS斑块内,SAA1可以由巨噬细胞合成分泌。此外,SAA在细胞膜上可能存在的受体包括：B型清道夫受体1(Scavenger receptor class B member1, SR-B I)、甲酰肽样受体1(formyl peptide receptor like-1, FPRL1)、Toll样受体(toll-like receptor, TLR)等。在不同细胞上SAA根据结合受体的类型,可能发挥不同的生物学作用。
近年来,随着对SAA1的深入研究,人们发现SAA1与AS之间存在密切的联系。SAA1可在AS斑块进展的全程出现,并且血清SAA1的浓度与冠状动脉粥样硬化性心脏病的发生成显著正相关。最新相关临床研究则发现,血清SAA1的水平可以作为一个独立的预测因素评估心血管急性事件的发生。这提示我们,SAA1对AS病程所发挥的作用可能不仅仅局限于斑块的形成阶段,而是通过某种作用机制进一步影响AS斑块的稳定性,并最终影响心血管急性事件的发生。影响AS斑块稳定性的因素是多方面的,涉及到巨噬细胞浸润、脂质沉积、平滑肌增殖迁移,细胞外基质降解及内皮细胞功能障碍等诸多环节。此前的研究一方面显示,SAA1可增加脂质沉积,加速脂核形成,通过促进单核巨噬细胞的趋化和粘附,增加了AS斑块中的炎症细胞浸润,这表明SAA1具有减弱斑块稳定性的作用；而另一些研究则表明SAA能增加平滑肌细胞的迁移和增殖,同时能刺激细胞外基质的表达增加,这又从另外方面表明SAA1具有增加斑块稳定性的作用。由此可见,SAA对AS斑块稳定性的影响是多方面的,甚至可能是双向的,基于以往的研究无法推断SAA对AS斑块稳定性的影响,目前尚存在以下问题亟待探讨和解决：①SAA1对动脉粥样硬化斑块稳定性的具体影响；②SAA1对动脉粥样硬化斑块内成分的影响；③SAA1发挥作用的信号通路。
研究目的：
1.通过转染SAA1慢病毒,明确SAA1高表达对AS斑块稳定性的影响；
2.探讨SAA1慢病毒转染对斑块内脂质、巨噬细胞、平滑肌细胞及胶原的影响；
3.通过体内外实验明确SAA1对胶原表达的影响及其相关信号通路。
研究方法：
1.SAA1高表达慢病毒的构建
获取目的基因后,经RT-PCR扩增后将目的基因片段与慢病毒载体plenti6.3-MSC-IRES-EGFP连接,构建成plenti6.3-SAA1-IRES-EGFP,并经转化、包装后测定慢病毒的滴度,用于实验。
2.动物饲养及建模
将100只6-8周龄的雄性ApoE-/-小鼠适应性喂养1周,给予颈动脉套管。继续高脂喂养8周后,随机将剩余的95只ApoE-/-小鼠分为4组:Control组(n=23)、lenti-null组(n=25、low-lenti-SAA1组(n=23)及high-lenti-SAA1组(n=24)。Lenti-null组给予1x107TU空慢病毒载体,low-lenti-SAA1组给予1×107TUSAA1高表达慢病毒,high-lenti-SAA1组1×109TUSAA1高表达慢病毒,control组给予相同体积生理盐水。继续高脂喂养4周后,小鼠空腹12小时,称体重,以0.8%戊巴比妥麻醉,打开胸腔,心脏取血,置于-80度冰箱保存,以备后续检测。以生理盐水灌洗心脏和主动脉,冲洗出残余血液。部分动物继以4%多聚甲醛固定直至肝脏变硬,收集小鼠套管近端的颈动脉,以4%多聚甲醛浸泡24小时。其余部分动物的颈动脉标本置于-80度冰箱保存。
3.酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)
以ELISA法检测血清总胆固醇、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、甘油三脂及TNF-α、IL-6的浓度。
4.颈动脉油红O染色
对小鼠颈动脉进行5μm连续切片,每10张取1张用于油红O染色,病变的严重程度用斑块占颈动脉斑块面积的百分比来表示。
5.组织免疫组织化学染色
对冰冻切片行SAA1、α-SMC actin、MOMA-2、胶原I、胶原Ⅲ、TGF-β1、MMP1、MMP8免疫组化染色观察SAA高表达对斑块SAA、α-SMC actin、 MOMA-2、胶原I、胶原Ⅲ、TGF-β1、MMP1、MMP8的影响。
6. Western blot检测
定量称取冻存组织,或收集细胞,提取蛋白,采用western blot法,以GAPDH为内参检测SAA1、胶原I、胶原Ⅲ、TGF-β1、MMP1、MMP8、MMP2、MMP9的表达；以相应非磷酸化状态为内参检测p-p38、ERK1/2、JNK、Smad2、Smad3的表达。
7.实时定量PCR (real-time PCR)检测
定量称取冻存组织,Trizol法提取总RNA,经过逆转录后以18S为内参检测SAA1的表达,以确定SAA1高表达慢病毒的转染效果。
8.免疫荧光染色
重组SAA1蛋白刺激平滑肌细胞后,免疫荧光法检测胶原I、胶原III、MMP1和MMP8的表达。
9.统计学分析所有数值均以均数±标准差表示。对数据进行正态分布检验,正态分布的计数资料应用Student t检验,多组采用方差分析(ANOVA),非正态分布的资料行Mann-Whitney检验。设P<0.05有统计学差异。
研究结果：
1.各组小鼠的一般情况
Control组(n=23)、lenti-null组(n=25)、low-lenti-SAA1组(n=23)及high-lenti-SAA1组(n=24) ApoE-/-小鼠的体重及血清TC、TG、LDL-C和HDL-C的水平浓度无差异(P>0.05)。表明SAA1高表达慢病毒的局部转染,没有影响小鼠体内整体的脂质代谢。
2. plenti6.3-SAA1-IRES-EGFP慢病毒转染对血清SAA1及其他炎症因子表达的影响
Control组、lenti-null组、1ow-lenti-SAA1组的血清SAA1表达几乎没有变化,high-lenti-SAA1组的血清SAA1表达略高于前三组,但尚未达到统计学差异(P>0.05)；这四个组的炎症因子TNF-α、IL-6在血清中的分泌表达也未达到统计学差异(P>0.05)。表明慢病毒的局部转染,没有引起小鼠体内显著的炎症反应。
3. plenti6.3-SAA1-IRES-EGFP;慢病毒转染后SAA1在颈动脉斑块内表达的检测
SAA1的免疫组化结果显示, control组和lenti-null组的阳性染色面积无差异(P>0.05), low-lenti-SAA1组和high-lenti-SAA1组的SAA1阳性染色区域显著高于lenti-null组及control组(P<0.05)。Western blot结果显示,control组和lenti-null组的SAA1表达量无差异(P>0.05), low-lenti-SAA1组和high-lenti-SAA1组的SAA1表达量显著高于lenti-null组及control组(P<0.05)。Real-time PCR的检测结果与蛋白检测结果一致。这一结果表明SAA1高表达慢病毒成功达到了使SAA1在斑块内表达的目的。
4. SAA1高表达慢病毒转染对颈动脉斑块内脂质聚集的影响
小鼠颈动脉斑块冰冻切片油红O染色显示,lenti-null组及low-lenti-SAA1组与control组相比,脂质含量未见显著改变；high-lenti-SAA1组与control组相比,脂质含量增加(P<0.05)；同时,high-lenti-SAA1组与low-lenti-SAA1组相比,脂质含量也显著增加(P<0.05)。这一结果表明,SAA1低浓度表达不能显著改变斑块内脂质沉积,而高浓度表达则显著促进脂质在斑块内的聚集。
5.SAA1高表达慢病毒转染对颈动脉斑块内巨噬细胞的调节作用
对小鼠颈动脉斑块冰冻切片进行巨噬细胞染色,可以发现与control组相比,lenti-null组巨噬细胞比例未见显著改变(P>0.05); low-lenti-SAA1组巨噬细胞的比例显著增加(P<0.05); high-lenti-SAA1组巨噬细胞的比例显著增加(P<0.01)；high-lenti-SAA1组与low-lenti-SAA1组相比,巨噬细胞占斑块面积的比例虽然增加,但未达到统计学差异(P>0.05)。这一结果表明,两个浓度梯度的SAA1高表达慢病毒转染均能显著增加颈动脉斑块内巨噬细胞的聚集。
6.SAA1高表达慢病毒转染对颈动脉斑块内平滑肌细胞的影响
对小鼠颈动脉斑块冰冻切片进行平滑肌细胞染色,四组之间的平滑肌细胞聚集未见显著改变,尽管两个浓度梯度的SAA1高表达慢病毒转染有轻微地增加颈动脉斑块内平滑肌细胞聚集的趋势,但未达到统计学差异。这一结果表明,SAA1高表达慢病毒转染不能改变斑块内平滑肌细胞的聚集。
7.SAA1高表达慢病毒转染对颈动脉斑块内胶原表达的影响
通过对小鼠颈动脉斑块冰冻切片进行masson染色,发现与control组相比,lenti-null组胶原未见显著改变(P>0.05); low-lenti-SAA1组胶原的比例显著增加(P<0.01); high-lenti-SAA1组胶原的比例未见显著增加(P>0.05); high-lenti-SAA1组与low-lenti-SAA1组相比,胶原的比例显著减少,达到统计学差异(P<0.05)。这一结果表明,低浓度的SAA1高表达慢病毒转染能显著增加颈动脉斑块内胶原的聚集,但增加SAA1高表达慢病毒的浓度则使这种促进胶原增加的作用消失。
8.SAA1高表达慢病毒转染对颈动脉斑块易损性的影响
结果显示,与control组相比,lenti-null组易损指数未见显著改变(P>0.05)；low-lenti-SAA1组易损指数显著减低(P<0.05); high-lenti-SAA1组易损指数显著增加(P<0.05); high-lenti-SAA1组与low-lenti-SAA1组相比,易损指数显著增加达到统计学差异(P>0.05)。这一结果表明,低浓度的SAA1高表达慢病毒转染能显著降低斑块的易损性,但高浓度SAA1高表达慢病毒转染则显著增加斑块的易损性。
9.SAA1诱导胶原Ⅰ和胶原Ⅲ表达
对小鼠颈动脉冰冻切片进行胶原Ⅰ的免疫组化染色发现,与control组相比,lenti-null组胶原Ⅰ的百分比未见显著改变(P>0.05); low-lenti-SAA1组胶原Ⅰ显著提高(P<0.05); high-lenti-SAA1组胶原Ⅰ无显著变化(P>0.05); high-lenti-SAA1组与low-lenti-SAA1组相比,胶原Ⅰ显著减少达到统计学差异(P<0.05)。胶原Ⅲ的免疫组化染色发现,与control组相比,lenti-null组胶原Ⅲ占斑块面积的百分比未见显著改变(P>0.05); low-lenti-SAA1组胶原Ⅲ显著提高(P<0.05)；high-lenti-SAA1组胶原Ⅲ显著减少(P<0.05); high-lenti-SAA1组与low-lenti-SAA1组相比,胶原Ⅲ显著减少(P<0.05)。Western blot结果与免疫组化结果一致。这一结果表明,低浓度的SAA1高表达慢病毒转染能显著增加颈动脉斑块内胶原Ⅰ和胶原Ⅲ的聚集,但高浓度SAA1高表达慢病毒转染则逆转这一作用。体外培养的小鼠和人平滑肌细胞显示,SAA1刺激后,胶原Ⅰ和胶原Ⅲ表达呈剂量和时间依赖性增加,0.1μg/ml的浓度即可显著促进胶原Ⅰ和胶原Ⅲ表达(P<0.05)。免疫荧光结果表明,经过SAA1刺激后,胶原Ⅰ和胶原Ⅲ表达显著增加。
10. MAPKs信号通路不介导SAA1诱导的胶原Ⅰ和胶原Ⅲ表达上调
经过不同时间的SAA1刺激后,p-JNK和p-p38MAPK的表达未发生显著改变(P>0.05); p-ERK1/2的表达均随时间的延长发生改变,刺激可使p-ERK1/2显著上调。然后分别应用JNK、ERK1/2以及p38MAPK的特异性抑制剂预处理平滑肌细胞,这三种抑制剂均未能显著抑制SAA1诱导的胶原Ⅰ和胶原Ⅲ表达上调。上述结果充分说明了JNK、p38MAPK两条信号通路在平滑肌细胞内几乎未被SAA1激活,因此不参与下游的胶原Ⅰ和胶原Ⅲ表达的调控；而ERK1/2信号通路虽然能被SAA1激活,但却没有参与SAA1对胶原Ⅰ和胶原Ⅲ表达的调控。
11. Smad2/3信号通路介导SAA1诱导的胶原Ⅰ和胶原Ⅲ表达上调
经过不同时间的SAA1刺激后,p-Smad2和p-Smad3的表达均发生显著改变(P<0.05)。然后分别应用Smad2/3的siRNA预处理平滑肌细胞,这两种siRNA均能显著抑制SAA1诱导的胶原Ⅰ和胶原Ⅲ表达上调。说明Smad2/3信号通路在平滑肌细胞内参与了下游的胶原Ⅰ和胶原Ⅲ表达的调控。
12.SAA1诱导胶原I和胶原IⅡ表达上调不依赖于TGF-β1
对小鼠颈动脉斑块冰冻切片进行TGF-β1的免疫组化染色,四组未见统计学差异,表明SAA1高表达慢病毒转染在小鼠颈动脉斑块内没有引起TGF-β1表达的改变。SAA1重组蛋白刺激平滑肌细胞不同时间,TGF-β1的表达未发生显著改变(P>0.05)；接着经过TGF-β1的siRNA及其特异性抑制剂SB431542干预后,SAA1诱导的胶原I和胶原III表达上调仍没有改变。这些结果充分证明SAA1诱导胶原I和胶原Ⅲ表达上调的过程不依赖于TGF-β1。
13.SAA1通过增加MMP-2、MMP-8及MMP-9表达促进胶原降解
对小鼠颈动脉斑块冰冻切片进行免疫组化染色,MMP1在四组未见统计学差异(P>0.05)。MMP8结果表明,lenti-null组、low-lenti-SAA1组与control组相比,MMP8占斑块面积的百分比未见显著改变(P>0.05); high-lenti-SAA1组MMP8占斑块面积的百分比显著升高(P<0.01)。体外培养的小鼠RAW264.7巨噬细胞中,1μg/ml和10μg/ml浓度的SAA1蛋白刺激巨噬细胞后,免疫荧光检测表明两个浓度均无法刺激MMP1表达显著升高(P>0.05);1μg/ml的浓度也无法显著增加MMP8的表达,但10μg/ml的浓度则显著增强MMP8的表达。Western blot结果显示,SAA1刺激后,MMP1的表达无显著改变(P>0.05)。而MMP8表达则呈剂量依赖性增加,当浓度达到5μg/ml以上时达到统计学差异(P<0.01)。此外1μg/ml的浓度的SAA1重组蛋白即可显著增加MMP2和MMP9的表达(P<0.05),随刺激浓度的升高,MMP2和MMP9的表达展现出剂量依赖性。以上结果表明,SAA1能够刺激MMP8、MMP2和MMP9的表达,并显示出剂量依赖性。
结论：
1.SAA1与实验性动脉粥样硬化斑块的稳定性密切相关；
2.低浓度SAA1慢病毒转染能显著增加AS斑块的稳定性,而高浓度SAA1慢病毒转染则显著减低AS斑块的稳定性；
3.低浓度SAA1即可增加胶原I、Ⅲ的表达,且0.1μg/ml的浓度即可达到刺激胶原I、III表达的最大效应；
4.SAA1通过Smad2/3通路增加胶原I、III的表达,而与MAPKs和TGF-β1无关；
5.浓度为5μg/ml以上的高浓度SAA1通过激活MMP-8促进胶原降解从而减低AS斑块的稳定性。
Background:
As the pathological basis of cardiovascular diseases, atherosclerosis (AS) has now become a threat to human health, especially in the elderly as the most important diseases, leading to heart cerebrovascular accident morbidity and high mortality rate, and also high morbidity in survivors. Risk factors of atherosclerosis involve in many areas, and the pathological process is complex. Although there are many theories attempting to explain its pathogenesis, the specific mechanism has not yet been defined.
Numerous studies have demonstrated that atherosclerosis-induced acute clinical ischemic events associated more with the stability of the plaque, rather than the arterial lumen stenosis. Unstable plaque rupture and thrombosis has become a major clinical and pathological mechanism of acute cardiovascular events. The vulnerable plaque has a strong tendency to rupture of the plaque characterized by a fibrous cap becoming thinner, necrotic lipid core increasing macrophage infiltration within the plaques, smooth muscle cells and collagen content gradually reducing. And on this basis, the vulnerability index is introduced to evaluate quantitatively plaque ulnerability, defined as:vulnerability index=(lipid plaque area+macrophages area)/(collagen area+smooth muscle cells area). Therefore, exploring the impact of various risk factors on AS plaque stability, seeking effective intervention targeting on vulnerable plaque stability, and actively avoiding plaque rupture and secondary cardiovascular events in recent years have become a research hotspot.
Serum amyloid A (serum amyloid A, SAA) family consists of SAA1, SAA2, SAA3and SAA4four family members. SAA1, as the main component, is the most widely expressed, the most active and the most sensitive response subtype in SAA family. Although under normal conditions, expression level of SAA1is low, but in the acute phase state its serum concentration increases1000times in a very short time. Serum SAA1is mainly synthesized and secreted by the liver cell, but also can be secreted by other cells. In AS plaque, SAA1can be synthesized and secreted by macrophages. In addition, SAA receptors on the cell membrane may include these types:type B Scavenger receptor1(the SR-B I), formyl peptide receptor1(FPRL1), toll-like receptors (TLR) and so on. According to the type of receptor binding, SAA1 may result in different biological effects on different cells.
In recent years, with the in-depth study of SAA1, people found that there is a close link between SAA1and AS. SAA1can appear in the whole process of development in the AS plaque, and the concentration of serum SAA1positively correlated with coronary atherosclerotic heart disease. New clinical studies found that levels of serum SAA1may serve as an independent predictor of acute cardiovascular events. This suggests that, SAA1may not only affect the formation of AS plaque, but also further affect AS plaque stability through some mechanism and ultimately affect the occurrence of acute cardiovascular events. There are many factors affecting the stability of the AS plaque, involves the macrophage infiltration, lipid and migration of smooth muscle proliferation and extracellular matrix degradation, and endothelial dysfunction. Previous researches displayed that on the one hand, SAA1increased lipid deposition and accelerated the formation of lipid core, and increased inflammatory cell infiltration through the promotion of monocyte-macrophage chemotaxis and adhesion, which suggested that SAA1might diminish the plaque stability; while on the other hand, other studies showed that SAA could increase smooth muscle cell migration and proliferation, and could stimulate increased expression of extracellular matrix, which further showed that SAA1might increase the plaque stability. Thus, the impact of SAA on AS plaque stability is multifaceted, and may even be bidirectional. Based on the previous researches we can not simply infer the specific impact of SAA1on AS plaque stability. Direct evidences of SAA1influences the stability of the plaques have not been reported so far, therefore it is of great importance to discuss and solve this problem. Currently there are the following problems needed to be discussed and solved:the effects of SAA1on the stability of atherosclerotic plaque; effect of SAA1on atherosclerotic plaque composition; the signaling pathway involved the reaction.
Objective:
1. To certify the effects of SAA1overexpression on atherosclerotic plaque stability, through transfecting lentivirus carrying SAA1gene to ApoE-/-mice;
2. To certify the effects of SAA1lentiviral transfection on lipids, macrophages, smooth muscle cells and collagen in the internal carotid artery plaque;
3. To investigate the specific effect of SAA1on the expression of collagen and its associated signaling pathways.
Methods:
1. SAA1lentivirus construction
After obtaining the target gene by RT-PCR, we amplified gene fragments and connect the fragments with lentiviral vector plenti6.3-MSC-IRES-EGFP to construct plenti6.3-SAA1-IRES-EGFP. After transformation, packaging and determination of lentivirus titer, the lentiviral was used in the experiments.
2. Animals breeding and Modeling
The100male ApoE-/-mice of6-8week-old fed for one week, and accepted the carotid artery cannula operation after that. Continued high fat diet for8weeks, the remaining95ApoE-/-mice were randomly divided into four groups:Control group (n=23), lenti-null group (n=25), low-lenti-SAA1group (n=23) and high-lenti-SAA1group (n=24). Lenti-null group were given1×107TU empty lentiviral vector, and low-lenti-SAA1group were given1×107TU SAAl lentivirus, and high-lenti-SAA1group were given1×109TU SAA1lentivirus and control group was given the same volume of saline. After4weeks high fat fed, mice were weighed and anesthetized with0.8%pentobarbital. The thorax was opened, and the heart bloods were collected, stored in-80℃freezer for subsequent detection. Saline was used to irrigate the hearts and aortas to flush out the residual blood. Following, some animals were irrigated with4%paraformaldehyde until the liver hard and then collect the mouse carotid arteries, with4%paraformaldehyde for another24hours. The remaining part of the animals'carotid arteries were directly collected and stored at-80℃refrigerator.
3. ELISA (enzyme-linked immunosorbent assay, ELISA)
Total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, and TNF-a, IL-6concentration in serum were detected by ELISA.
4. Carotid oil red O staining
5mm serial sections of mouse carotid artery, each of the10and1for oil red O staining, the severity of the lesions represented by the ratio of red stained lipid to carotid plaque area.
5. Immunohistochemical staining tissue
On frozen sections, expression of SAA1, a-SMC actin, MOMA-2, collagen I, collagenⅢ, TGF-β1, MMP1, MMP8were evaluated by immunohistochemical staining.
6. Western blot analysis
Frozen tissue was quantitatively weighed, and cells were collected and extracted proteins. Western blot method was used to detect the expression of SAA1, collagen I, collagen Ⅲ, TGF-β1, MMP1, MMP8, MMP2, MMP9, p-p38, ERK1/2, JNK, Smad2and Smad3.
7. Real-time quantitative PCR analysis
Frozen tissue was quantitatively weighed, and Trizol extraction of total RNA. After reverse transcription, express of SAA1was determined with real-time quantitative PCR analysis.
8. Immunofluorescence staining
After the SAA1protein stimulates smooth muscle cells and macrophages, the expression of collagen I, collagenⅢ, MMP1and MMP8were detected with immunofluorescence.
9. Statistical analysis
All values are expressed as mean±standard deviation. Test for normally distributed data, the normal distribution of count data Student t test, groups using analysis of variance (ANOVA), a non-normal distribution of data rows Mann-Whitney test. Statistical significance was confirmed as P<0.05.
Results:
1. The general conditions of groups of mice
The weight serum TC, TG, LDL-C and HDL-C concentration levels did not differ (P>0.05) in control group(n=23), lenti-null group(n=25), low-lenti-SAAl group(n=23) and high-lenti-SAA1group(n=24) ApoE7-/-mice. The results showed that high expression of SAA1fragmentary lentivirus transfection did not affect the overall lipid metabolism in mice.
2. Plenti6.3-SAA1-IRES-EGFP lentivirus transfection effect on serum SAA1and other inflammatory factor expression
Serum SAA1concentration has changed little in the control group, lenti-null, low-lenti-SAA1group. High-lenti-SAA1group is slightly higher than the previous three groups of serum SAA1expression but has yet to reach statistical difference (P>0.05). Serum TNF-a and IL-6concentrations of the four groups did not reach statistical difference (P>0.05), which indicateed that the local transfection of slow virus, did not cause significant inflammation in mice.
3. Plenti6.3-SAA1-IRES-EGFP lentivirus transfection effect on SAA1expression in carotid plaques
SAA1immunohistochemical results showed that there was no difference between the control group and lenti-null group(P>0.05). SAA1positive staining area of low-lenti-SAA1group and high-lenti-SAA1were both significantly higher than lenti-null group and control group (P<0.05). Western blot and real-time PCR detection showed a consistent result with immunohistochemical results. The results indicate that SAA1lentivirus successfully achieved the purpose of the SAA1high expression within plaques.
4. SAA1lentiviral transfection effect on the carotid artery plaque lipid accumulation
Oil red O staining of frozen sections of mouse carotid artery plaque showed that lenti-null group and the low-lenti-SAA1group changeed in lipid content compared with the control group; high-lenti-SAAl group increased significantly compared with the control group and low-lenti-SAA1group (P<0.05). This result suggested that, low concentration of SAA1did not significantly alter the plaque lipid deposition, while high concentration significantly promoted lipid accumulation in the plaque.
5. SAA1lentiviral transfection effect on the carotid artery plaque macrophages accumulation
Macrophages staining of frozen sections of mouse carotid artery plaque showed that compared with the control group, the proportion of macrophages in lenti-null group did not change significantly (P>0.05); low-lenti-SAA1group (P<0.05) and high-lenti-SAA1group (P<0.01) increased significantly; compared with low-lenti-SAA1group, the increase of macrophages in high-lenti-SAA1group did not reach statistical significance (P>0.05). This result suggests that both the concentration gradient SAA1overexpression lentivirus could significantly increase the accumulation of macrophages in the carotid artery plaque.
6. SAA1lentiviral transfection effect on the carotid artery plaque smooth muscle cells accumulation
Smooth muscle cells staining of frozen sections of mouse carotid artery plaque showed that accumulation had no significant change of smooth muscle cells among the four groups. This result suggests that, SAA1overexpression lentivirus can not change the aggregation of smooth muscle cells in the plaque.
7. SAA1lentiviral transfection effect on the carotid artery plaque collagen accumulation
Masson staining of frozen sections showed that lenti-null collagen group had no significant change compared with the control group (P>0.05); collagen in low-lenti-SAA1group increased significantly (P<0.01); no significant increase in high-lenti-SAAl group compared with the control group (P>0.05) and a significant reduction compared with low-lenti-SAA1group (P<0.05). The results showed that low concentration of SAA1overexpression lenti virus can significantly increase the accumulation of collagen, but high concentration of SAA1lenti virus make this effect disappeared.
8. SAA1lenti viral transfection effect on vulnerability index
The results showed that, compared with the control group, vulnerability index of lenti-null group had no significant change (P>0.05); low-lenti-SAA1group was significantly lower (P<0.05); high-lenti-SAA1group increased significantly (P<0.05) compared with control and low-lenti-SAA1groups reaching statistical significance (P>0.05). The results showed that low concentration of SAAl could significantly reduce plaque vulnerability, but high concentration of SAA1significantly increased plaque vulnerability.
9. SAA1induced expression of collagen I and collagen III
Collagen I immunohistochemical staining showed that, compared with the control group, the percentage of collagen I in lenti-null group had no significant change (P>0.05); low-lenti-SAA1group was significantly improved (P<0.05); high-lenti-SAA1group did not change significantly compared with the control group (P>0.05), but reduced significantly compared with low-lenti-SAA1group (P<0.05). CollagenⅢ immunohistochemical staining showed that, compared with the control group, lenti-null group had no significant change (P>0.05); low-lenti-SAA1group III collagen was significantly increased (P<0.05); high-lenti-SAA1group significantly decreased (P<0.05) compared with control and low-lenti-SAA1group (P<0.05). Western blot results were consistent with the results of immunohistochemistry. The results showed that low concentrations of SAA1overexpression lenti virus could significantly increase the accumulation of collagen I and collagenⅢ, but high concentration of SAA1reversed this effect. Cultured smooth muscle cells in mice and humans showed that after SAA1stimulation, collagen I and collagenⅢ expression increase in dose-and time-dependent manners. Immunofluorescence results also showed that SAA1stimulation significantly increased the expression of collagen I and collagenⅢ.
lO.MAPKs pathways didn't mediate SAA1-induced collagens production
After different time of SAA1stimulation, the expression of p-JNK and p-p38MAPK did not change significantly (P>0.05); the expression of p-ERK1/2increased along with the time change. Then, JNK, ERK1/2and p38MAPK specific inhibitors pretreated smooth muscle cell, and the three inhibitors were not significantly inhibited SAA1induced collagen I and collagen III expression. The results show that the JNK, p38MAPK and ERK1/2signal pathways did not participate in the regulation of SAA1on the expression of collagen I and collagen III.
11. Smad2/3pathway mediated SAA1-induced collagens production
After different time of SAA1stimulation, the expression of p-Smad2and p-Smad3changed significantly (P<0.05). Then Smad2/3siRNA pretreated smooth muscle cells, and the two siRNA can significantly inhibit SAA1induced collagen I and collagen III expression. The Smad2/3signaling pathways involved in the regulation of the collagen I and collagen III expression in smooth muscle eells.
12. SAA1-induced collagens production was independent of TGF-β
TGF-β1immunohistochemical staining showed that four groups have no statistical difference, indicating that SAA1lentivirus transfection caused no change in expression of TGF-β1in mouse carotid artery plaques. After recombinant SAA1stimulated smooth muscle cells for different times, expression of TGF-β1has not changed significantly (P>0.05); and TGF-β1siRNA and its specific inhibitor SB431542still has not changed SAA1induced collagenⅠ and collagenⅢ expression. These results demonstrate the process of collagenl and collagenⅢ expression induced by SAA1does not depend on the TGF-β1.
13. SAA1increased MMP-8, MMP-2and MMP-9expression, but not MMP-1
MMP1immunohistochemical staining showed that there was no statistical difference in the four groups (P>0.05). MMP8immunohistochemical staining showed that, compared with control group, lenti-null group and low-lenti-SAAl group had no significant change (P>0.05); high-lenti-SAA1group significantly increased (P<0.01). In vitro cultured mouse RAW264.7macrophages were stimulated with1μg/ml and10μg/ml concentrations of SAA1protein, and immunofluorescence detection showed that SAA1was unable to significantly increase MMP1expression (P>0.05);1μg/ml SAA1could not significantly increase the expression of MMP8, but10μg/ml SAA1 significantly enhanced MMP8expression. Western blot results showed that, after SAA1stimulation, no significant change was found in the expression of MMP1(P>0.05). The expression of MMP8was dose-dependently increased under the stimulation of SAA1. When the concentration reached more than5μg/ml, the change of MMP8expression reached statistical significance (P<0.01). In addition, concentration of1μg/ml SAA1recombinant protein could significantly increase the expression of MMP2and MMP9(P<0.05). With increasing concentration of SAAl, the expression of MMP2and MMP9showed a dose-dependent manner. These results indicated that SAA1could dose-dependently stimulate the expression of MMP8, MMP2, and MMP9.
Conclusion:
1. SAA1is closely related to the the stability of experimental atherosclerosis plaque;
2. Low concentration of SAA1lentivirus transfection could significantly increase the stability of AS plaque, while high concentration SAA1of lentivirus transfection could significantly reduced the stability of AS plaque;
3. Low SAA1concentration can increase the expression of collagen Ⅰ, Ⅲ, and0.1μ g/ml concentration of SAA1reach the maximum effect;
4. SAA1increased the expression of collagen I, III through Smad2/3pathway independent MAPKs and TGF-β1;
5. High concentrations of SAA1,5μg/ml and above, promotes collagen degradation and thus lower the stability of AS plaque through activation of MMP-8.
动脉粥样硬化(atherosclerosis,AS)作为心脑血管疾病的病理基础,目前已经成为威胁人类尤其是中老年人健康的最重要的疾病,由此导致的心脑血管意外发生率和死亡率极高,即使是在幸免的存活者中也仍然存在极高的致残率。动脉粥样硬化所涉及的致病因素极多,且其病理过程复杂,尽管此前已有诸多学说试图解释其发病机理,但其具体的发病及进展机制至今仍未明确。
诸多研究已经证实,动脉粥样硬化所致的临床急性缺血事件更多与斑块的稳定性密切相关,而非动脉管腔的狭窄程度。不稳定斑块破裂出血,并继发导致血栓形成已经成为临床急性心血管事件的主要病理机制。由此引入了易损斑块(Vulnerable Plaque)的概念,其作为具有极强破裂倾向的斑块,特点包括纤维帽在各种因素下逐渐变薄、坏死脂质核心逐渐增大、以巨噬细胞浸润为特征的斑块内活性炎症状态、平滑肌细胞逐渐减少以及胶原含量逐渐减少。并以此为依据引入易损指数,易损指数可以定量地评估斑块易损性,其定义为：易损指数＝(脂质占斑块面积百分比+巨噬细胞占斑块面积百分比)／(胶原占斑块面积百分比+平滑肌细胞占斑块面积百分比)。因此,探寻影响AS斑块稳定性的各种危险因素,并寻求能有效地稳定易损斑块的干预靶点,积极避免斑块破裂及继发的心脑血管事件成为近年来的研究热点。
既往研究证实血清淀粉样蛋白A (serum amyloid A, SAA)家族由SAA1、 SAA2、SAA3及SAA4四个家族成员构成。作为家族最主要组成成分的SAA1是SAA家族中表达最广泛、活性最强、反应最敏感的亚型。虽然在常态下SAA1表达水平较低,但在急性期状态下其血清浓度在极短时间内升高1000倍。血清SAA1主要由肝细胞合成并分泌,而其他组织细胞同样具有局部分泌SAA1的功能。在AS斑块内,SAA1可以由巨噬细胞合成分泌。此外,SAA在细胞膜上可能存在的受体包括：B型清道夫受体1(Scavenger receptor class B member1, SR-B I)、甲酰肽样受体1(formyl peptide receptor like-1, FPRL1)、Toll样受体(toll-like receptor, TLR)等。在不同细胞上SAA根据结合受体的类型,可能发挥不同的生物学作用。
近年来,随着对SAA1的深入研究,人们发现SAA1与AS之间存在密切的联系。SAA1可在AS斑块进展的全程出现,并且血清SAA1的浓度与冠状动脉粥样硬化性心脏病的发生成显著正相关。最新相关临床研究则发现,血清SAA1的水平可以作为一个独立的预测因素评估心血管急性事件的发生。这提示我们,SAA1对AS病程所发挥的作用可能不仅仅局限于斑块的形成阶段,而是通过某种作用机制进一步影响AS斑块的稳定性,并最终影响心血管急性事件的发生。影响AS斑块稳定性的因素是多方面的,涉及到巨噬细胞浸润、脂质沉积、平滑肌增殖迁移,细胞外基质降解及内皮细胞功能障碍等诸多环节。此前的研究一方面显示,SAA1可增加脂质沉积,加速脂核形成,通过促进单核巨噬细胞的趋化和粘附,增加了AS斑块中的炎症细胞浸润,这表明SAA1具有减弱斑块稳定性的作用；而另一些研究则表明SAA能增加平滑肌细胞的迁移和增殖,同时能刺激细胞外基质的表达增加,这又从另外方面表明SAA1具有增加斑块稳定性的作用。由此可见,SAA对AS斑块稳定性的影响是多方面的,甚至可能是双向的,基于以往的研究无法推断SAA对AS斑块稳定性的影响,目前尚存在以下问题亟待探讨和解决：①SAA1对动脉粥样硬化斑块稳定性的具体影响；②SAA1对动脉粥样硬化斑块内成分的影响；③SAA1发挥作用的信号通路。
研究目的：
1.通过转染SAA1慢病毒,明确SAA1高表达对AS斑块稳定性的影响；
2.探讨SAA1慢病毒转染对斑块内脂质、巨噬细胞、平滑肌细胞及胶原的影响；
3.通过体内外实验明确SAA1对胶原表达的影响及其相关信号通路。
研究方法：
1.SAA1高表达慢病毒的构建
获取目的基因后,经RT-PCR扩增后将目的基因片段与慢病毒载体plenti6.3-MSC-IRES-EGFP连接,构建成plenti6.3-SAA1-IRES-EGFP,并经转化、包装后测定慢病毒的滴度,用于实验。
2.动物饲养及建模
将100只6-8周龄的雄性ApoE-/-小鼠适应性喂养1周,给予颈动脉套管。继续高脂喂养8周后,随机将剩余的95只ApoE-/-小鼠分为4组:Control组(n=23)、lenti-null组(n=25、low-lenti-SAA1组(n=23)及high-lenti-SAA1组(n=24)。Lenti-null组给予1x107TU空慢病毒载体,low-lenti-SAA1组给予1×107TUSAA1高表达慢病毒,high-lenti-SAA1组1×109TUSAA1高表达慢病毒,control组给予相同体积生理盐水。继续高脂喂养4周后,小鼠空腹12小时,称体重,以0.8%戊巴比妥麻醉,打开胸腔,心脏取血,置于-80度冰箱保存,以备后续检测。以生理盐水灌洗心脏和主动脉,冲洗出残余血液。部分动物继以4%多聚甲醛固定直至肝脏变硬,收集小鼠套管近端的颈动脉,以4%多聚甲醛浸泡24小时。其余部分动物的颈动脉标本置于-80度冰箱保存。
3.酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)
以ELISA法检测血清总胆固醇、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、甘油三脂及TNF-α、IL-6的浓度。
4.颈动脉油红O染色
对小鼠颈动脉进行5μm连续切片,每10张取1张用于油红O染色,病变的严重程度用斑块占颈动脉斑块面积的百分比来表示。
5.组织免疫组织化学染色
对冰冻切片行SAA1、α-SMC actin、MOMA-2、胶原I、胶原Ⅲ、TGF-β1、MMP1、MMP8免疫组化染色观察SAA高表达对斑块SAA、α-SMC actin、 MOMA-2、胶原I、胶原Ⅲ、TGF-β1、MMP1、MMP8的影响。
6. Western blot检测
定量称取冻存组织,或收集细胞,提取蛋白,采用western blot法,以GAPDH为内参检测SAA1、胶原I、胶原Ⅲ、TGF-β1、MMP1、MMP8、MMP2、MMP9的表达；以相应非磷酸化状态为内参检测p-p38、ERK1/2、JNK、Smad2、Smad3的表达。
7.实时定量PCR (real-time PCR)检测
定量称取冻存组织,Trizol法提取总RNA,经过逆转录后以18S为内参检测SAA1的表达,以确定SAA1高表达慢病毒的转染效果。
8.免疫荧光染色
重组SAA1蛋白刺激平滑肌细胞后,免疫荧光法检测胶原I、胶原III、MMP1和MMP8的表达。
9.统计学分析所有数值均以均数±标准差表示。对数据进行正态分布检验,正态分布的计数资料应用Student t检验,多组采用方差分析(ANOVA),非正态分布的资料行Mann-Whitney检验。设P<0.05有统计学差异。
研究结果：
1.各组小鼠的一般情况
Control组(n=23)、lenti-null组(n=25)、low-lenti-SAA1组(n=23)及high-lenti-SAA1组(n=24) ApoE-/-小鼠的体重及血清TC、TG、LDL-C和HDL-C的水平浓度无差异(P>0.05)。表明SAA1高表达慢病毒的局部转染,没有影响小鼠体内整体的脂质代谢。
2. plenti6.3-SAA1-IRES-EGFP慢病毒转染对血清SAA1及其他炎症因子表达的影响
Control组、lenti-null组、1ow-lenti-SAA1组的血清SAA1表达几乎没有变化,high-lenti-SAA1组的血清SAA1表达略高于前三组,但尚未达到统计学差异(P>0.05)；这四个组的炎症因子TNF-α、IL-6在血清中的分泌表达也未达到统计学差异(P>0.05)。表明慢病毒的局部转染,没有引起小鼠体内显著的炎症反应。
3. plenti6.3-SAA1-IRES-EGFP;慢病毒转染后SAA1在颈动脉斑块内表达的检测
SAA1的免疫组化结果显示, control组和lenti-null组的阳性染色面积无差异(P>0.05), low-lenti-SAA1组和high-lenti-SAA1组的SAA1阳性染色区域显著高于lenti-null组及control组(P<0.05)。Western blot结果显示,control组和lenti-null组的SAA1表达量无差异(P>0.05), low-lenti-SAA1组和high-lenti-SAA1组的SAA1表达量显著高于lenti-null组及control组(P<0.05)。Real-time PCR的检测结果与蛋白检测结果一致。这一结果表明SAA1高表达慢病毒成功达到了使SAA1在斑块内表达的目的。
4. SAA1高表达慢病毒转染对颈动脉斑块内脂质聚集的影响
小鼠颈动脉斑块冰冻切片油红O染色显示,lenti-null组及low-lenti-SAA1组与control组相比,脂质含量未见显著改变；high-lenti-SAA1组与control组相比,脂质含量增加(P<0.05)；同时,high-lenti-SAA1组与low-lenti-SAA1组相比,脂质含量也显著增加(P<0.05)。这一结果表明,SAA1低浓度表达不能显著改变斑块内脂质沉积,而高浓度表达则显著促进脂质在斑块内的聚集。
5.SAA1高表达慢病毒转染对颈动脉斑块内巨噬细胞的调节作用
对小鼠颈动脉斑块冰冻切片进行巨噬细胞染色,可以发现与control组相比,lenti-null组巨噬细胞比例未见显著改变(P>0.05); low-lenti-SAA1组巨噬细胞的比例显著增加(P<0.05); high-lenti-SAA1组巨噬细胞的比例显著增加(P<0.01)；high-lenti-SAA1组与low-lenti-SAA1组相比,巨噬细胞占斑块面积的比例虽然增加,但未达到统计学差异(P>0.05)。这一结果表明,两个浓度梯度的SAA1高表达慢病毒转染均能显著增加颈动脉斑块内巨噬细胞的聚集。
6.SAA1高表达慢病毒转染对颈动脉斑块内平滑肌细胞的影响
对小鼠颈动脉斑块冰冻切片进行平滑肌细胞染色,四组之间的平滑肌细胞聚集未见显著改变,尽管两个浓度梯度的SAA1高表达慢病毒转染有轻微地增加颈动脉斑块内平滑肌细胞聚集的趋势,但未达到统计学差异。这一结果表明,SAA1高表达慢病毒转染不能改变斑块内平滑肌细胞的聚集。
7.SAA1高表达慢病毒转染对颈动脉斑块内胶原表达的影响
通过对小鼠颈动脉斑块冰冻切片进行masson染色,发现与control组相比,lenti-null组胶原未见显著改变(P>0.05); low-lenti-SAA1组胶原的比例显著增加(P<0.01); high-lenti-SAA1组胶原的比例未见显著增加(P>0.05); high-lenti-SAA1组与low-lenti-SAA1组相比,胶原的比例显著减少,达到统计学差异(P<0.05)。这一结果表明,低浓度的SAA1高表达慢病毒转染能显著增加颈动脉斑块内胶原的聚集,但增加SAA1高表达慢病毒的浓度则使这种促进胶原增加的作用消失。
8.SAA1高表达慢病毒转染对颈动脉斑块易损性的影响
结果显示,与control组相比,lenti-null组易损指数未见显著改变(P>0.05)；low-lenti-SAA1组易损指数显著减低(P<0.05); high-lenti-SAA1组易损指数显著增加(P<0.05); high-lenti-SAA1组与low-lenti-SAA1组相比,易损指数显著增加达到统计学差异(P>0.05)。这一结果表明,低浓度的SAA1高表达慢病毒转染能显著降低斑块的易损性,但高浓度SAA1高表达慢病毒转染则显著增加斑块的易损性。
9.SAA1诱导胶原Ⅰ和胶原Ⅲ表达
对小鼠颈动脉冰冻切片进行胶原Ⅰ的免疫组化染色发现,与control组相比,lenti-null组胶原Ⅰ的百分比未见显著改变(P>0.05); low-lenti-SAA1组胶原Ⅰ显著提高(P<0.05); high-lenti-SAA1组胶原Ⅰ无显著变化(P>0.05); high-lenti-SAA1组与low-lenti-SAA1组相比,胶原Ⅰ显著减少达到统计学差异(P<0.05)。胶原Ⅲ的免疫组化染色发现,与control组相比,lenti-null组胶原Ⅲ占斑块面积的百分比未见显著改变(P>0.05); low-lenti-SAA1组胶原Ⅲ显著提高(P<0.05)；high-lenti-SAA1组胶原Ⅲ显著减少(P<0.05); high-lenti-SAA1组与low-lenti-SAA1组相比,胶原Ⅲ显著减少(P<0.05)。Western blot结果与免疫组化结果一致。这一结果表明,低浓度的SAA1高表达慢病毒转染能显著增加颈动脉斑块内胶原Ⅰ和胶原Ⅲ的聚集,但高浓度SAA1高表达慢病毒转染则逆转这一作用。体外培养的小鼠和人平滑肌细胞显示,SAA1刺激后,胶原Ⅰ和胶原Ⅲ表达呈剂量和时间依赖性增加,0.1μg/ml的浓度即可显著促进胶原Ⅰ和胶原Ⅲ表达(P<0.05)。免疫荧光结果表明,经过SAA1刺激后,胶原Ⅰ和胶原Ⅲ表达显著增加。
10. MAPKs信号通路不介导SAA1诱导的胶原Ⅰ和胶原Ⅲ表达上调
经过不同时间的SAA1刺激后,p-JNK和p-p38MAPK的表达未发生显著改变(P>0.05); p-ERK1/2的表达均随时间的延长发生改变,刺激可使p-ERK1/2显著上调。然后分别应用JNK、ERK1/2以及p38MAPK的特异性抑制剂预处理平滑肌细胞,这三种抑制剂均未能显著抑制SAA1诱导的胶原Ⅰ和胶原Ⅲ表达上调。上述结果充分说明了JNK、p38MAPK两条信号通路在平滑肌细胞内几乎未被SAA1激活,因此不参与下游的胶原Ⅰ和胶原Ⅲ表达的调控；而ERK1/2信号通路虽然能被SAA1激活,但却没有参与SAA1对胶原Ⅰ和胶原Ⅲ表达的调控。
11. Smad2/3信号通路介导SAA1诱导的胶原Ⅰ和胶原Ⅲ表达上调
经过不同时间的SAA1刺激后,p-Smad2和p-Smad3的表达均发生显著改变(P<0.05)。然后分别应用Smad2/3的siRNA预处理平滑肌细胞,这两种siRNA均能显著抑制SAA1诱导的胶原Ⅰ和胶原Ⅲ表达上调。说明Smad2/3信号通路在平滑肌细胞内参与了下游的胶原Ⅰ和胶原Ⅲ表达的调控。
12.SAA1诱导胶原I和胶原IⅡ表达上调不依赖于TGF-β1
对小鼠颈动脉斑块冰冻切片进行TGF-β1的免疫组化染色,四组未见统计学差异,表明SAA1高表达慢病毒转染在小鼠颈动脉斑块内没有引起TGF-β1表达的改变。SAA1重组蛋白刺激平滑肌细胞不同时间,TGF-β1的表达未发生显著改变(P>0.05)；接着经过TGF-β1的siRNA及其特异性抑制剂SB431542干预后,SAA1诱导的胶原I和胶原III表达上调仍没有改变。这些结果充分证明SAA1诱导胶原I和胶原Ⅲ表达上调的过程不依赖于TGF-β1。
13.SAA1通过增加MMP-2、MMP-8及MMP-9表达促进胶原降解
对小鼠颈动脉斑块冰冻切片进行免疫组化染色,MMP1在四组未见统计学差异(P>0.05)。MMP8结果表明,lenti-null组、low-lenti-SAA1组与control组相比,MMP8占斑块面积的百分比未见显著改变(P>0.05); high-lenti-SAA1组MMP8占斑块面积的百分比显著升高(P<0.01)。体外培养的小鼠RAW264.7巨噬细胞中,1μg/ml和10μg/ml浓度的SAA1蛋白刺激巨噬细胞后,免疫荧光检测表明两个浓度均无法刺激MMP1表达显著升高(P>0.05);1μg/ml的浓度也无法显著增加MMP8的表达,但10μg/ml的浓度则显著增强MMP8的表达。Western blot结果显示,SAA1刺激后,MMP1的表达无显著改变(P>0.05)。而MMP8表达则呈剂量依赖性增加,当浓度达到5μg/ml以上时达到统计学差异(P<0.01)。此外1μg/ml的浓度的SAA1重组蛋白即可显著增加MMP2和MMP9的表达(P<0.05),随刺激浓度的升高,MMP2和MMP9的表达展现出剂量依赖性。以上结果表明,SAA1能够刺激MMP8、MMP2和MMP9的表达,并显示出剂量依赖性。
结论：
1.SAA1与实验性动脉粥样硬化斑块的稳定性密切相关；
2.低浓度SAA1慢病毒转染能显著增加AS斑块的稳定性,而高浓度SAA1慢病毒转染则显著减低AS斑块的稳定性；
3.低浓度SAA1即可增加胶原I、Ⅲ的表达,且0.1μg/ml的浓度即可达到刺激胶原I、III表达的最大效应；
4.SAA1通过Smad2/3通路增加胶原I、III的表达,而与MAPKs和TGF-β1无关；
5.浓度为5μg/ml以上的高浓度SAA1通过激活MMP-8促进胶原降解从而减低AS斑块的稳定性。
Background:
As the pathological basis of cardiovascular diseases, atherosclerosis (AS) has now become a threat to human health, especially in the elderly as the most important diseases, leading to heart cerebrovascular accident morbidity and high mortality rate, and also high morbidity in survivors. Risk factors of atherosclerosis involve in many areas, and the pathological process is complex. Although there are many theories attempting to explain its pathogenesis, the specific mechanism has not yet been defined.
Numerous studies have demonstrated that atherosclerosis-induced acute clinical ischemic events associated more with the stability of the plaque, rather than the arterial lumen stenosis. Unstable plaque rupture and thrombosis has become a major clinical and pathological mechanism of acute cardiovascular events. The vulnerable plaque has a strong tendency to rupture of the plaque characterized by a fibrous cap becoming thinner, necrotic lipid core increasing macrophage infiltration within the plaques, smooth muscle cells and collagen content gradually reducing. And on this basis, the vulnerability index is introduced to evaluate quantitatively plaque ulnerability, defined as:vulnerability index=(lipid plaque area+macrophages area)/(collagen area+smooth muscle cells area). Therefore, exploring the impact of various risk factors on AS plaque stability, seeking effective intervention targeting on vulnerable plaque stability, and actively avoiding plaque rupture and secondary cardiovascular events in recent years have become a research hotspot.
Serum amyloid A (serum amyloid A, SAA) family consists of SAA1, SAA2, SAA3and SAA4four family members. SAA1, as the main component, is the most widely expressed, the most active and the most sensitive response subtype in SAA family. Although under normal conditions, expression level of SAA1is low, but in the acute phase state its serum concentration increases1000times in a very short time. Serum SAA1is mainly synthesized and secreted by the liver cell, but also can be secreted by other cells. In AS plaque, SAA1can be synthesized and secreted by macrophages. In addition, SAA receptors on the cell membrane may include these types:type B Scavenger receptor1(the SR-B I), formyl peptide receptor1(FPRL1), toll-like receptors (TLR) and so on. According to the type of receptor binding, SAA1 may result in different biological effects on different cells.
In recent years, with the in-depth study of SAA1, people found that there is a close link between SAA1and AS. SAA1can appear in the whole process of development in the AS plaque, and the concentration of serum SAA1positively correlated with coronary atherosclerotic heart disease. New clinical studies found that levels of serum SAA1may serve as an independent predictor of acute cardiovascular events. This suggests that, SAA1may not only affect the formation of AS plaque, but also further affect AS plaque stability through some mechanism and ultimately affect the occurrence of acute cardiovascular events. There are many factors affecting the stability of the AS plaque, involves the macrophage infiltration, lipid and migration of smooth muscle proliferation and extracellular matrix degradation, and endothelial dysfunction. Previous researches displayed that on the one hand, SAA1increased lipid deposition and accelerated the formation of lipid core, and increased inflammatory cell infiltration through the promotion of monocyte-macrophage chemotaxis and adhesion, which suggested that SAA1might diminish the plaque stability; while on the other hand, other studies showed that SAA could increase smooth muscle cell migration and proliferation, and could stimulate increased expression of extracellular matrix, which further showed that SAA1might increase the plaque stability. Thus, the impact of SAA on AS plaque stability is multifaceted, and may even be bidirectional. Based on the previous researches we can not simply infer the specific impact of SAA1on AS plaque stability. Direct evidences of SAA1influences the stability of the plaques have not been reported so far, therefore it is of great importance to discuss and solve this problem. Currently there are the following problems needed to be discussed and solved:the effects of SAA1on the stability of atherosclerotic plaque; effect of SAA1on atherosclerotic plaque composition; the signaling pathway involved the reaction.
Objective:
1. To certify the effects of SAA1overexpression on atherosclerotic plaque stability, through transfecting lentivirus carrying SAA1gene to ApoE-/-mice;
2. To certify the effects of SAA1lentiviral transfection on lipids, macrophages, smooth muscle cells and collagen in the internal carotid artery plaque;
3. To investigate the specific effect of SAA1on the expression of collagen and its associated signaling pathways.
Methods:
1. SAA1lentivirus construction
After obtaining the target gene by RT-PCR, we amplified gene fragments and connect the fragments with lentiviral vector plenti6.3-MSC-IRES-EGFP to construct plenti6.3-SAA1-IRES-EGFP. After transformation, packaging and determination of lentivirus titer, the lentiviral was used in the experiments.
2. Animals breeding and Modeling
The100male ApoE-/-mice of6-8week-old fed for one week, and accepted the carotid artery cannula operation after that. Continued high fat diet for8weeks, the remaining95ApoE-/-mice were randomly divided into four groups:Control group (n=23), lenti-null group (n=25), low-lenti-SAA1group (n=23) and high-lenti-SAA1group (n=24). Lenti-null group were given1×107TU empty lentiviral vector, and low-lenti-SAA1group were given1×107TU SAAl lentivirus, and high-lenti-SAA1group were given1×109TU SAA1lentivirus and control group was given the same volume of saline. After4weeks high fat fed, mice were weighed and anesthetized with0.8%pentobarbital. The thorax was opened, and the heart bloods were collected, stored in-80℃freezer for subsequent detection. Saline was used to irrigate the hearts and aortas to flush out the residual blood. Following, some animals were irrigated with4%paraformaldehyde until the liver hard and then collect the mouse carotid arteries, with4%paraformaldehyde for another24hours. The remaining part of the animals'carotid arteries were directly collected and stored at-80℃refrigerator.
3. ELISA (enzyme-linked immunosorbent assay, ELISA)
Total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, and TNF-a, IL-6concentration in serum were detected by ELISA.
4. Carotid oil red O staining
5mm serial sections of mouse carotid artery, each of the10and1for oil red O staining, the severity of the lesions represented by the ratio of red stained lipid to carotid plaque area.
5. Immunohistochemical staining tissue
On frozen sections, expression of SAA1, a-SMC actin, MOMA-2, collagen I, collagenⅢ, TGF-β1, MMP1, MMP8were evaluated by immunohistochemical staining.
6. Western blot analysis
Frozen tissue was quantitatively weighed, and cells were collected and extracted proteins. Western blot method was used to detect the expression of SAA1, collagen I, collagen Ⅲ, TGF-β1, MMP1, MMP8, MMP2, MMP9, p-p38, ERK1/2, JNK, Smad2and Smad3.
7. Real-time quantitative PCR analysis
Frozen tissue was quantitatively weighed, and Trizol extraction of total RNA. After reverse transcription, express of SAA1was determined with real-time quantitative PCR analysis.
8. Immunofluorescence staining
After the SAA1protein stimulates smooth muscle cells and macrophages, the expression of collagen I, collagenⅢ, MMP1and MMP8were detected with immunofluorescence.
9. Statistical analysis
All values are expressed as mean±standard deviation. Test for normally distributed data, the normal distribution of count data Student t test, groups using analysis of variance (ANOVA), a non-normal distribution of data rows Mann-Whitney test. Statistical significance was confirmed as P<0.05.
Results:
1. The general conditions of groups of mice
The weight serum TC, TG, LDL-C and HDL-C concentration levels did not differ (P>0.05) in control group(n=23), lenti-null group(n=25), low-lenti-SAAl group(n=23) and high-lenti-SAA1group(n=24) ApoE7-/-mice. The results showed that high expression of SAA1fragmentary lentivirus transfection did not affect the overall lipid metabolism in mice.
2. Plenti6.3-SAA1-IRES-EGFP lentivirus transfection effect on serum SAA1and other inflammatory factor expression
Serum SAA1concentration has changed little in the control group, lenti-null, low-lenti-SAA1group. High-lenti-SAA1group is slightly higher than the previous three groups of serum SAA1expression but has yet to reach statistical difference (P>0.05). Serum TNF-a and IL-6concentrations of the four groups did not reach statistical difference (P>0.05), which indicateed that the local transfection of slow virus, did not cause significant inflammation in mice.
3. Plenti6.3-SAA1-IRES-EGFP lentivirus transfection effect on SAA1expression in carotid plaques
SAA1immunohistochemical results showed that there was no difference between the control group and lenti-null group(P>0.05). SAA1positive staining area of low-lenti-SAA1group and high-lenti-SAA1were both significantly higher than lenti-null group and control group (P<0.05). Western blot and real-time PCR detection showed a consistent result with immunohistochemical results. The results indicate that SAA1lentivirus successfully achieved the purpose of the SAA1high expression within plaques.
4. SAA1lentiviral transfection effect on the carotid artery plaque lipid accumulation
Oil red O staining of frozen sections of mouse carotid artery plaque showed that lenti-null group and the low-lenti-SAA1group changeed in lipid content compared with the control group; high-lenti-SAAl group increased significantly compared with the control group and low-lenti-SAA1group (P<0.05). This result suggested that, low concentration of SAA1did not significantly alter the plaque lipid deposition, while high concentration significantly promoted lipid accumulation in the plaque.
5. SAA1lentiviral transfection effect on the carotid artery plaque macrophages accumulation
Macrophages staining of frozen sections of mouse carotid artery plaque showed that compared with the control group, the proportion of macrophages in lenti-null group did not change significantly (P>0.05); low-lenti-SAA1group (P<0.05) and high-lenti-SAA1group (P<0.01) increased significantly; compared with low-lenti-SAA1group, the increase of macrophages in high-lenti-SAA1group did not reach statistical significance (P>0.05). This result suggests that both the concentration gradient SAA1overexpression lentivirus could significantly increase the accumulation of macrophages in the carotid artery plaque.
6. SAA1lentiviral transfection effect on the carotid artery plaque smooth muscle cells accumulation
Smooth muscle cells staining of frozen sections of mouse carotid artery plaque showed that accumulation had no significant change of smooth muscle cells among the four groups. This result suggests that, SAA1overexpression lentivirus can not change the aggregation of smooth muscle cells in the plaque.
7. SAA1lentiviral transfection effect on the carotid artery plaque collagen accumulation
Masson staining of frozen sections showed that lenti-null collagen group had no significant change compared with the control group (P>0.05); collagen in low-lenti-SAA1group increased significantly (P<0.01); no significant increase in high-lenti-SAAl group compared with the control group (P>0.05) and a significant reduction compared with low-lenti-SAA1group (P<0.05). The results showed that low concentration of SAA1overexpression lenti virus can significantly increase the accumulation of collagen, but high concentration of SAA1lenti virus make this effect disappeared.
8. SAA1lenti viral transfection effect on vulnerability index
The results showed that, compared with the control group, vulnerability index of lenti-null group had no significant change (P>0.05); low-lenti-SAA1group was significantly lower (P<0.05); high-lenti-SAA1group increased significantly (P<0.05) compared with control and low-lenti-SAA1groups reaching statistical significance (P>0.05). The results showed that low concentration of SAAl could significantly reduce plaque vulnerability, but high concentration of SAA1significantly increased plaque vulnerability.
9. SAA1induced expression of collagen I and collagen III
Collagen I immunohistochemical staining showed that, compared with the control group, the percentage of collagen I in lenti-null group had no significant change (P>0.05); low-lenti-SAA1group was significantly improved (P<0.05); high-lenti-SAA1group did not change significantly compared with the control group (P>0.05), but reduced significantly compared with low-lenti-SAA1group (P<0.05). CollagenⅢ immunohistochemical staining showed that, compared with the control group, lenti-null group had no significant change (P>0.05); low-lenti-SAA1group III collagen was significantly increased (P<0.05); high-lenti-SAA1group significantly decreased (P<0.05) compared with control and low-lenti-SAA1group (P<0.05). Western blot results were consistent with the results of immunohistochemistry. The results showed that low concentrations of SAA1overexpression lenti virus could significantly increase the accumulation of collagen I and collagenⅢ, but high concentration of SAA1reversed this effect. Cultured smooth muscle cells in mice and humans showed that after SAA1stimulation, collagen I and collagenⅢ expression increase in dose-and time-dependent manners. Immunofluorescence results also showed that SAA1stimulation significantly increased the expression of collagen I and collagenⅢ.
lO.MAPKs pathways didn't mediate SAA1-induced collagens production
After different time of SAA1stimulation, the expression of p-JNK and p-p38MAPK did not change significantly (P>0.05); the expression of p-ERK1/2increased along with the time change. Then, JNK, ERK1/2and p38MAPK specific inhibitors pretreated smooth muscle cell, and the three inhibitors were not significantly inhibited SAA1induced collagen I and collagen III expression. The results show that the JNK, p38MAPK and ERK1/2signal pathways did not participate in the regulation of SAA1on the expression of collagen I and collagen III.
11. Smad2/3pathway mediated SAA1-induced collagens production
After different time of SAA1stimulation, the expression of p-Smad2and p-Smad3changed significantly (P<0.05). Then Smad2/3siRNA pretreated smooth muscle cells, and the two siRNA can significantly inhibit SAA1induced collagen I and collagen III expression. The Smad2/3signaling pathways involved in the regulation of the collagen I and collagen III expression in smooth muscle eells.
12. SAA1-induced collagens production was independent of TGF-β
TGF-β1immunohistochemical staining showed that four groups have no statistical difference, indicating that SAA1lentivirus transfection caused no change in expression of TGF-β1in mouse carotid artery plaques. After recombinant SAA1stimulated smooth muscle cells for different times, expression of TGF-β1has not changed significantly (P>0.05); and TGF-β1siRNA and its specific inhibitor SB431542still has not changed SAA1induced collagenⅠ and collagenⅢ expression. These results demonstrate the process of collagenl and collagenⅢ expression induced by SAA1does not depend on the TGF-β1.
13. SAA1increased MMP-8, MMP-2and MMP-9expression, but not MMP-1
MMP1immunohistochemical staining showed that there was no statistical difference in the four groups (P>0.05). MMP8immunohistochemical staining showed that, compared with control group, lenti-null group and low-lenti-SAAl group had no significant change (P>0.05); high-lenti-SAA1group significantly increased (P<0.01). In vitro cultured mouse RAW264.7macrophages were stimulated with1μg/ml and10μg/ml concentrations of SAA1protein, and immunofluorescence detection showed that SAA1was unable to significantly increase MMP1expression (P>0.05);1μg/ml SAA1could not significantly increase the expression of MMP8, but10μg/ml SAA1 significantly enhanced MMP8expression. Western blot results showed that, after SAA1stimulation, no significant change was found in the expression of MMP1(P>0.05). The expression of MMP8was dose-dependently increased under the stimulation of SAA1. When the concentration reached more than5μg/ml, the change of MMP8expression reached statistical significance (P<0.01). In addition, concentration of1μg/ml SAA1recombinant protein could significantly increase the expression of MMP2and MMP9(P<0.05). With increasing concentration of SAAl, the expression of MMP2and MMP9showed a dose-dependent manner. These results indicated that SAA1could dose-dependently stimulate the expression of MMP8, MMP2, and MMP9.
Conclusion:
1. SAA1is closely related to the the stability of experimental atherosclerosis plaque;
2. Low concentration of SAA1lentivirus transfection could significantly increase the stability of AS plaque, while high concentration SAA1of lentivirus transfection could significantly reduced the stability of AS plaque;
3. Low SAA1concentration can increase the expression of collagen Ⅰ, Ⅲ, and0.1μ g/ml concentration of SAA1reach the maximum effect;
4. SAA1increased the expression of collagen I, III through Smad2/3pathway independent MAPKs and TGF-β1;
5. High concentrations of SAA1,5μg/ml and above, promotes collagen degradation and thus lower the stability of AS plaque through activation of MMP-8.
引文
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