【作者】 郜攀；

【导师】 黄岚；

【作者基本信息】 第三军医大学 ， 内科学， 2009， 博士

【摘要】 随着社会经济的发展和人口的老龄化,动脉粥样硬化(Atherosclerosis,AS)已经成为严重危害人类健康的一大类疾病,它是造成心脏、脑及肢体缺血的主要原因。动脉粥样硬化是通过脂质的累积、平滑肌细胞的增殖和钙化而引起的一种动脉损伤为特征的疾病。在动脉粥样硬化过程中血管壁增厚,形成动脉粥样硬化斑块。随着动脉粥样硬化病变和机理的研究更加透彻,现在有了更多的方法阻止动脉粥样硬化,并且已经有很多治疗动脉粥样硬化疾病的相关药物使用。内肽酶Meprin是哺乳动物细胞表面的一种含锌金属肽酶,在肾脏、肠刷状缘细胞、白细胞及一些肿瘤细胞中含量最丰富。meprin包含α、β两个亚单位,能够形成双桥结构的同二聚体和异二聚体,从而对不同的底物发挥作用。Meprin-α通常由多聚体组成,能够分泌到细胞外。meprin-β通常为四聚体结构,能够结合meprin低分子寡聚体到细胞膜。Meprin底物主要为生物活性肽和细胞外基质蛋白,既往的研究提示meprin可能参与癌症和消化道炎症过程。与中性内肽酶类似,Meprin具有水解内源性生长因子,血管活性肽、细胞因子和细胞外基质的功能,且在循环系统,如外周血细胞和血管壁中也发挥作用。内皮meprin能够降解血管活性肽类物质,使血管活性肽在血管壁局部浓度表达,从而稀释血管活性肽物质对血管壁局部作用。抑制Meprin作用则能加强内源性活性肽在局部血管壁的反应。利钠肽(natriuretic peptides, NPs)家族作为内分泌激素,能够通过调节心脏和肾脏的功能达到维持机体内稳态的作用。它们共有的生理作用是利尿、利钠、舒张血管、抗纤维化,抗内皮细胞过度增殖、抗炎和调节神经内分泌等,包括对抗肾素-血管紧张素-醛固酮系统(RAAS)、阻断交感神经、对抗内皮素。实验证明NPs,尤其是心房利钠肽(ANP)和脑钠肽(BNP)是内肽酶meprin在血管壁的重要作用底物。在既往的研究中发现,NPs能刺激内皮细胞释放血管舒张因子,如前列腺素,内皮衍生舒张因子和NO等。这些血管舒张因子具有共同的作用,即都能抑制血管平滑肌细胞(VSMC)的增殖和凋亡,调节细胞内活性氧簇(reactive oxygen species,ROS)生成。因而NP能够对某些重要的心血管疾病的病理生理过程发挥重要作用,如高血压、心肌肥大、血栓、再狭窄和动脉粥样硬化。NPs与动脉粥样硬化的关系有大量的实验证实:在AS发生发展过程中血管平滑肌细胞(VSMC)发挥了重要作用,VSMC功能改变是AS发病的重要环节之一。利钠肽家族成员中, ANP和BNP为心肌细胞源性,CNP为内皮源性。ANP和BNP结构和功能相似,都有抑制血管平滑肌增殖和迁移的作用,是体内调节血压和容量状态的重要激素。Casco等发现ANP、BNP和CNP在人冠状动脉粥样斑块中都有表达,尤其是BNP在进展期斑块中含量显著增高:这提示利钠肽家族成员可能影响动脉粥样斑块的形成以及血管壁重构。Meprin是体内调节NP家族(尤其是ANP、BNP)表达的一种重要内肽酶。Meprin可能通过调节NP家族成员在血管壁局部表达影响AS发生、发展过程。在AS病理过程中,VSMC增殖是一个重要的病理过程。既往研究已经证实既往研究已经证实NP家族成员均能显著抑制VSMC增殖,尤其是ANP抑制VSMC增殖可能与调节细胞内活性氧簇(Reactive oxygen species,ROS)生成有关。因而研究meprin在体内、体外对ROS生成的调控有重要的意义。本实验拟用Meprin特异性抑制剂Actinonin(AC)从体内、体外实验两方面进行干预,运用分子生物学和常规生物生化技术,观察1.建立ApoE-/-小鼠动脉粥样硬化模型,体内观察Meprin对动脉粥样硬化斑块形成的影响,并分析粥样斑块斑块组成成分;2.观察Meprin对NP在血管壁局部表达影响;3. Meprin对NP抑制ROS生成和NADPH氧化酶活性的影响。通过本研究可初步解释Meprin与AS发生、发展的关系,以及NP抑制ROS生成过程在Meprin与AS发病中的重要作用,并探讨NADPH氧化酶分子机制。有助于从一个新的角度揭示内肽酶家族促进动脉粥样硬化和血管损伤后不良修复等相关血管疾病的发病机制,为进一步研究延缓或逆转内肽酶促进动脉粥样硬化发生和血管损伤后不良修复的防治途径提供研究基础。方法:1、ApoE-/-雄性小鼠40只,10周龄大小,体重在18-22g左右,同时高脂喂食10周。与10周末,随机选取8只小鼠处死,设为对照组(n=8,control组,下同)。剩余的小鼠再分为两组:于第11周开始,一组给予安慰剂(n=16,placebo组,下同),另一组给予Meprin抑制剂AC(5 mg/kg体重/天;腹腔注射i.p;n=16,meprin-I组,下同)。再同时喂养6周,于第16周末处死小鼠。2、血管壁局部NP检测:采用NP RIA检测试剂盒(北京北方生物技术研究所提供)检测。操作步骤按照说明书进行。小鼠眼球摘除后取全血,全血于5500g 4℃离心10 min得到血浆,并储存与-80℃。血浆胆固醇和甘油三酯浓度在全自动生化分析仪上分别测定其含量。3、斑块大小和数量的检测:小鼠用过量的戊巴比妥钠处死,解剖,去除腹颈动脉周围的脂肪组织和结缔组织,取小鼠一侧颈动脉用油红O染色,大体水平上确定粥样斑块的数量和大小。分离颈动脉成4段(每段约为0.2 cm)。分离的腹颈动脉立即固定、包埋,切成5um厚的小片。组织制片后,常规采用苏木素-伊红(HE)染色,镜下观察粥样斑块形成。为了明确粥样斑块进展情况,定义粥样斑块易损指数。4、斑块内细胞成分确定及NADPH氧化酶活性的检测:采用免疫组化的方法。斑块内巨噬细胞鉴定用Mac-2抗体标记,平滑肌细胞鉴定用SMC-actin抗体标记,胶原成分鉴定采用Masson三色染色法显色。同时采用检测血管壁NADPH氧化酶活性。5、体外实验:与体外培养巨噬细胞和血管平滑肌细胞,用AC干预细胞后,DCF-DA荧光探针标记细胞内ROS生成,检测NADPH氧化酶蛋白和mRNA水平变化。凋亡检测采用TUNEL法,增殖的检测采用[3H]-Tdr掺入法。检测细胞增殖用血清刺激,并设为对照组;检测细胞凋亡和ROS生成,细胞均用LPS(内毒素)刺激,并设为对照组。结果:1、AC能够显著增加血管壁局部ANP和BNP表达:与placebo组比较,meprin-I组BNP含量显著增高,其中在血浆中增加2.5倍,血管壁中增加3.3倍(P<0.05);与placebo组比较,Meprin-I组ANP含量也显著增高,血浆中增高1.9倍,血管壁中增高1.8倍(P<0.05)。与control组比较,placebo组和meprin-I组血浆总胆固醇、甘油三酯水平,低密度脂蛋白(LDL)和高密度脂蛋白(HDL)、体重等基础指标显著增高(P<0.05);与placebo组比较,meprin-I组体重、血压、甘油三酯、胆固醇、HDL、LDL等指标均无显著改变(P>0.05)。2、与control组比较,HE染色结果placebo组颈动脉粥样斑块面积显著增加(P<0.05);油红O染色(Oil-Red-O染色,ORO染色)结果显示placebo颈动脉内膜表面脂质沉淀区域显著增加(P<0.05)。AC能够显著抑制ApoE-/-小鼠血管壁粥样病变的发生: HE染色结果显示,与placebo组比较,meprin-I组颈动脉粥样斑块面积显著降低(P<0.05)。ORO染色提示颈动脉内膜表面脂质沉淀区域显著降低(P<0.05)。3、病理形态学改变:肉眼可见placebo组小鼠颈动脉血管内膜粥样病变形成显著;为了判定AC对粥样病变发生的影响,本实验观察了粥样斑块内巨噬细胞、平滑肌细胞和胶原成分等指标。与placebo组比较,meprin-I组内膜下巨噬细胞及T淋巴细胞等炎症细胞显著减少(P<0.05);与placebo组比较,meprin-I组中层平滑肌增殖降低,但无统计学差别(P>0.05);胶原成分检测采用Masson三色染色法,Masson三色染色结果显示,与placebo组相比较,meprin-I胶原成分增高(P>0.05)。4、AC干预后能够显著降低血管壁基质金属蛋白酶(MMP-9)活性:与control组比较,placebo组MMP-9活性并无显著增加(P>0.05);相较于placebo组,meprin-I组MMP-9活性显著降低(P<0.05)。MMP-9是血管壁局部细胞外基质成分、胶原主要分解蛋白,AC抑制MMP-9活性与AC增高斑块和血管壁组织胶原成分的结果一致。AC干预后能够抑制血管壁原位ROS生成和凋亡:与control组比较,placebo组血管壁原位ROS生成和NADPH氧化酶活性显著增高(P<0.05);与control组比较,placebo组血管壁原位TUNEL阳性细胞显著增多(P<0.05)。与placebo组比较,meprin-I组血管壁局部ROS生成显著降低(P<0.05);NADPH氧化酶是调控ROS生成重要的酶,与placebo组比较,meprin-I组血管壁局部NADPH氧化酶活性显著降低(P<0.05)。TUNEL法检测细胞凋亡的结果显示:与placebo组比较,meprin-I组TUNEL阳性细胞显著降低(P<0.05)。5、细胞增殖、凋亡和ROS生成:与对照组比较,外源性ANP对THP-1单核细胞增殖无显著影响(P>0.05);BNP能够显著抑制THP-1细胞增殖(P<0.05)。与对照组比较ANP和BNP均能显著抑制血清诱导的VSMC增殖(P<0.05);中性内肽酶抑制(NEPI)能够增强ANP和BNP抑制VSMC作用(P<0.05),AC干预后能够显著增强BNP抑制VSMC增殖作用:与BNP组比较,meprin-I组VSMC增殖显著降低(P<0.05)。细胞凋亡和ROS生成的结果显示:与L对照组比较,ANP和BNP能够显著抑制LPS预处理诱导的细胞凋亡(P<0.05);NEPI能够增强ANP和BNP对细胞凋亡的抑制作用(P<0.05);AC干预显著后加强BNP对细胞凋亡的抑制作用(P<0.05)。NEPI和AC对细胞内ROS生成影响与细胞凋亡的影响一致。结论:1、体内实验中,meprin抑制剂AC能够显著抑制ApoE-/-小鼠血管壁粥样斑块病变的发生,对血管壁功能起到重要的保护作用;在细胞实验中meprin抑制剂AC能够增强NP抑制VSMC增殖、THP-1细胞凋亡和细胞ROS生成,而VSMC增殖、细胞凋亡和ROS生成是致AS发生重要因素,因而体内、体外两方面实验证明抑制meprin作用(AC)能够延缓AS发生。2、meprin-I能够抑制血管壁原位氧化应激反应发生,降低血管壁ROS生成和NADPH氧化酶活性;在体外培养的VSMC和THP-1细胞中同样能够抑制ROS生成,提示meprin-I抗粥样病变进展与降低血管壁氧化应激反应相关。3、VSMC、内皮细胞和巨噬细胞凋亡能够加速粥样斑块进展,诱导粥样斑块破裂。而血管壁细胞凋亡与ROS生成密切相关。本实验证实meprin-I能够抑制血管壁ROS生成,并进一步证实meprin-I能够抑制血管壁细胞凋亡,提示meprin-I抑制ROS诱导细胞凋亡是meprin-I抑制血管粥样病变发生、发展的重要机制。4、与安慰剂组比较,meprin-I能够显著抑制ApoE-/-小鼠粥样病变大小,粥样斑块内单核/巨噬细胞含量,增加胶原成分含量。结果提示AC能够阻止ApoE-/-小鼠血管壁局部粥样斑块发生、发展。更多还原

【Abstract】 During atherosclerosis, the arterial wall gradually thickens to form an atherosclerotic plaque, resulting in the narrowing of the lumen of the artery. Consequently, the amount of blood supplied to the organ is reduced, most commonly affecting the heart and the brain. Plaques can abruptly rupture, causing a blood clot and often myocardial infarction (heart attack) or stroke. Intensive study of the cellular and molecular mechanisms that underlie atherogenesis (that is, the formation of atherosclerotic plaques) and plaque rupture has led to a consensus view of these processes. Initiation and progression of the lesion are highly complex processes, and many aspects of atherogenesis remain incompletely understood. Furthermore, in most cases, mechanistic insights have yet to be translated into therapeutic approaches. The insights of mechanism of atherosclerosis may lead to the development of successful therapeutic interventions for atherosclerotic cardiovascular disease.Meprin is a member of the astacin family of zinc metalloendopeptidases. It is highly expressed in the kidney, intestinal brush border membranes, and leukocytes and in some cancer cells. It consists of two subunits,αandβ, which form disulfide-bridged homodimers and heterodimers that differ in oligomerization potentials and substrate specificity. Meprin-αforms heterogeneous multimers and is secreted. Meprin-βrestricts the oligomerization potential of meprin to tetramers and attaches meprin oligomers to the plasma membrane. Its substrates include bioactive peptides and extracellular matrix proteins. Meprin proteins have been implicated in cancer and intestinal inflammation. Like neutral endopeptidase 24.11 (NEP), meprin could hydrolyze and inactive several endogenous growth factors, vasoactive peptides, cytokines, and extracellular matrix proteins circulating in peripheral blood or produced at vascular walls. Endothelial meprin could therefore metabolize vasoactive peptides and reduce their local concentrations at the arterial walls, resulting in attenuation of the effects of the peptides on vascular functions. Meprin inhibition may potentiate vascular responsiveness to these endogenous vasoactive peptides.The natriuretic peptide (NP) family has an important role in the regulation of blood pressure homeostasis and salt and water balance. Studies have demonstrated that NPs and other factors in the vascular wall are the substrates of meprin, particularly atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) (meprin has no effect on C-type natriuretic peptide (CNP)). NPs can also stimulate the endothelial release of vasodilators such as prostaglandins, endothelium-derived relaxing factor(s), and nitric oxide, which can inhibit the proliferation and apoptosis of vascular smooth muscle cells (VSMC), and modulation of the level of reactive oxygen species (ROS) in different cell types. These activities have been strongly associated with experimental hypertension, cardiac hypertrophy, thrombosis, restenosis, and atherosclerosis.Considerable evidence suggests that NP signaling may have a direct role in the development of atherosclerosis: most data support the anti-proliferative action of NP on smooth muscle cells (SMCs), suggesting an anti-atherogenic action of NP. Mice lacking the NPs receptor type-A (NPRA) and ApoE (Npr1–/– ApoE–/–) have increased atherosclerosis compared with ApoE-deficient mice that are of the wild-type for Npr1 (Npr1+/+ ApoE–/–). One could therefore expect that a meprin inhibitor could potentiate the vascular actions of NPs or other kinins by inhibition of their degradation at the vascular wall.This study initially aimed to assess the influence of chronic meprin inhibition by daily administration of the meprin inhibitor Actinonin(AC) on the development of atherosclerotic changes in ApoE–/–, and then aimed to clarify elevation of the level of NPs contributed to the anti-atherogenic effects of meprin inhibition.Methods:1. Male ApoE–/– mice (C57/Bl6 genetic background; purchased from Beijing University, Beijing, China) were used for this study. Twenty-eight ApoE–/– mice were maintained in a room set at 22°C with a 12-hour light/dark cycle. They received drinking water ad libitum. We assessed the influence of chronic meprin inhibition by daily administration of actinonin (5 mg/kg body weight per day; i.p.) on development of atherosclerotic changes in ApoE–/– mice. Mice were fed a high-fat (21% fat), cholesterol-rich (1% cholesterol) Western-type diet for 16 weeks starting at 10 weeks of age. At 20 weeks of age, randomly selected ApoE–/– mice were treated with Western-type-diet chow pellets supplemented with commercially available actinonin (meprin-I group) for 6 weeks; the diet of control ApoE–/– mice was supplemented with saline (placebo group). 2. Natriuretic peptides were measured in snap-frozen carotid arteries and plasma from experimental animals. Levels of natriuretic peptides were measured by RIA commercial kits specific for ANP and mouse BNP (Phoenix Biotech, Beijing, China) according to the manufacturer’s instructions. Plasma was obtained through centrifugation of the blood for 10 minutes at 5500g at 4°C and stored at -80°C. Total plasma cholesterol and triglyceride concentrations were measured using enzymatic assayon a Cobas Mira Plus automated analyzer.3. For morphometric studies, at 26 weeks of age, rats were anesthetized and killed. Left carotid arteries were serially sectioned (5-μm) and, beginning from a random start site within the first 75μm, a section was stained every 75μm with hematoxylin and eosin (H&E). Images were captured with a Leica microscope and lesion area quantified using an image analysis system.4. Carotid artery superoxide levels were measured with Dihydroethidium (DHE) on serial frozen sections (10μm). NADPH oxidase activity in vascular was assessed by lucigenin-enhanced chemiluminescence as described previously with some modifications. An in-situ cell death detection POD kit was used with slight modification. After development using diaminobenzidine, sections were counterstained with methyl green. Four serial sections from each mouse were stained.5. Cells were cultured in 25-mm plastic tissue culture flasks and grown in DMEM supplemented with 10% fetal calf serum (FCS), 100μg/ml streptomycin and 100 U/ml penicillin in a humidified atmosphere of 5% CO2 and 95% air at 37 ?C. Cells, harvested once a week with 0.25% trypsin-0.02% EDTA and refed every other day, were used as confluent monolayers after 6–8 days at passage level 4–9 and cell number was plated at a density of 5×105 cells/cm2. Intracellular ROS were detected by flow cytometry using 2’,7’-dichlorodihydrofluorescein diacetate (H2-DCF-DA). Apoptosis was measured by TUNEL assay according to the manufacturer’s instructions. Proliferation was measured by [3H]-thymidine incorporation assay as previously described.Results:1. We first aimed to confirm that ApoE–/– administrated with actinonin resulted in increased levels of NPs in plasma and vascular tissues. BNP levels of meprin inhibition (meprin-I) in ApoE–/– mice were significantly increased by 2.5-fold in plasma and 3.3-fold in the vascular wall compared with those observed in ApoE–/– control mice. ANP levels were also significantly increased by 1.9-fold in plasma and 1.8-fold in the vascular wall. Total cholesterol, triglyceride levels, body weights and blood pressure were similar in meprin-I ApoE–/– mice and ApoE–/– mice.2. Treatment with actinonin changed the size of atherosclerotic lesions in carotid arteries compared with placebo group (meprin-I group), which suggested actinonin exerted apparent anti-atherogenic actions. These inhibitory effects of actinonin on atherosclerosis were significantly displayed among the three groups when entire right carotid arteries were stained by Oil-red-O.3. To evaluate lesion composition in the three groups, we immunostained lesions for macrophages, SMCs, as well as collagen and calculated the percentage of lesional area occupied. Treatment of ApoE–/– mice with actinonin resulted in dramatic reduction in lesion size, monocyte/macrophage content, and augmentation of collagen deposition compared with placebo group. These results suggest that treatment with actinonin halted genesis/progression of plaques, and altered the phenotype of lesions in ApoE–/– mice.4. In mice treated with actinonin, MMP-9 activity was significantly reduced compared with placebo. This result was consistent with actinonin-induced augmentation of collagen deposition. Actinonin markedly suppressed superoxide levels in the carotid arteries of ApoE-deficient mice. Consistent with decreased in-situ production of ROS, NADPH oxidase activity was also significantly reduced in tissues from actinonin-treated ApoE–/– mice. Actinonin treatment significantly reduced the number of TUNEL-positive cells in the carotid arteries of ApoE–/– mice fed a high-fat diet.5. Exogenous ANP failed to affect proliferation rates of THP-1 cells. BNP significantly inhibited proliferation of THP-1 cells. Exogenous ANP and BNP significantly affected serum-induced VSMC proliferation compared with untreated cells. Neutral endopeptidase inhibition (NEP-I) increased ANP- and BNP-inhibited VSMC proliferation rate, but actinonin increased only BNP-inhibited VSMC proliferation rate. The rate of lipopolysaccharide-induced apoptosis, as detected by TUNEL assay in THP-1 cells and VSMC, was reduced by treatment with ANP and BNP compared with untreated cells. NEP-I increased ANP- and BNP-inhibited cell apoptosis, and actinonin increased the effects of BNP on cell apoptosis only. These effects of NEP-I and actinonin work in parallel with ROS production in VSMC and THP-1 cells. Conclusions:1. The present study demonstrated that meprin inhibition by actinonin suppresses formation of atherosclerotic plaques, and preserves vascular wall function in ApoE–/– mice fed a high-cholesterol diet.2. In the current study, meprin inhibition for 6 weeks reduced ROS production and NAD(P)H oxidase activity in the vascular wall by 25%. Reduced oxidant stress may contribute to the anti-atherogenic effects of meprin inhibition.3. Apoptosis of VSMCs, endothelial cells and macrophages may promote plaque growth, pro-coagulation and may induce rupture, the major consequence of atherosclerosis in humans. The fact that increased levels of ROS induce cell apoptosis has been demonstrated in these cells. In the present study, meprin inhibition also reduced apoptosis measured by an in-situ cell death assay.4. Treatment of ApoE–/– mice with actinonin resulted in dramatic reduction in lesion size, monocyte/macrophage content, and augmentation of collagen deposition compared with placebo group. Treatment with actinonin may halt genesis/progression of plaques, and altered the phenotype of lesions in ApoE–/– mice.更多还原