【作者】 王蕾；

【导师】 季晓平； 昌慧霞；

【作者基本信息】 山东大学 ， 内科学（心血管病）（专业学位）， 2022， 博士

【摘要】 研究背景动脉粥样硬化(Atherosclerosis,AS)作为冠状动脉疾病(Coronary artery disease,CAD)最常见的潜在病因,是目前导致全球人口死亡的主要原因。AS斑块易损、破裂和血栓形成是导致急性心脑血管事件的主要病理机制。易损斑块的早期积极干预,对于预防和减低急性心脑血管事件的发生具有重要意义。研究发现,斑块内新生血管增多是斑块内炎症的先决或并发因素,斑块内巨噬细胞浸润、斑块内出血和薄帽纤维粥瘤的形成都与新生滋养血管的密度密切相关,这种现象存在于心血管病患者整个动脉床中。在AS发生发展过程中,由于内膜缺氧,起源于外膜的血管外膜滋养管(Adventitial vasa vasorum,VV)增生以增加血管壁供氧,进而造成斑块内出血。传统观点认为AS起源于内膜损伤,内膜炎症扩展到外膜及血管周围脂肪组织(Perivascularadiposetissue,PVAT),PVAT的炎症状态可作为AS的标志。然而越来越多的证据表明PVAT以一种“由外而内”的方式促进血管疾病,管周脂肪细胞来源的脂肪因子可影响并促进血管外膜及内膜的炎症状态和细胞增殖。血管外膜及内膜之间存在复杂的相互作用关系。有证据表明,VV增生早于内膜增厚甚至早于血管内皮功能障碍的发生,而PVAT功能障碍与VV增生相关,增生的VV作为炎性细胞和炎症因子在PVAT与血管内膜的运输管道,增加了两者之间的相互作用,影响AS的发生发展。作为最强的天然抗血管新生因子,色素上皮衍生因子(Pigment epithelium-derived factor,PEDF)是Tombran-Tink等1989年首先从胎儿视网膜色素上皮细胞的培养液中提取的,其抑制血管生成的作用是内皮抑制素的7倍,且PEDF的抗血管生成作用只针对新生的异常血管,对已经形成的正常血管无损伤作用。PEDF是一种多功能蛋白,除了具备很强的抑制血管新生作用之外,还具有抗炎、抗氧化、抑制血栓形成等作用,因此在稳定斑块的作用中,除了强效的抗血管新生作用之外的多效性,使PEDF具备了更多的优势。目前已知的PEDF特异性受体包括脂肪甘油三酯脂酶(Adipose triglyceride lipase,ATGL)/独立磷脂酶 A2(independentphospholipase A2,iPLA2)及 37/67-kDa层粘连蛋白受体(Laminin receptor,LR)。LR最初被鉴定为层黏连蛋白的67-kDa结合蛋白,在生命领域广泛存在,具有多种生理作用。越来越多的证据表明,LR在细胞信号转导中起到中介分子的作用。研究证实,PEDF及其衍生物与细胞膜的LR结构域结合,发挥其抗血管新生作用,抑制LR后可减弱PEDF的作用。但PEDF与LR结合后对血管内皮细胞信号通路的作用尚无深入研究。根据目前研究,我们认为上调PVAT来源的PEDF是抑制血管外膜VV新生和稳定斑块的重要手段,然而目前尚未发现PEDF的有效激动剂。多项研究结果表明,经典中药人参的主要活性物质人参皂苷Rb1可抑制AS的发生发展。课题组前期实验证实,Rb1 能够抑制人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)的增殖、迁移及小管形成,并证明Rbl通过调节PEDF表达发挥抑制血管新生作用。但Rb1的抗动脉粥样硬化作用是否与PEDF及抑制血管新生相关,目前尚无定论。虽然多项研究结果表明PEDF很可能是抑制VV新生、稳定斑块的有效因子,但是目前并没有直接证据。同时PEDF抑制血管新生的分子机制尚待进一步的研究。因此,本研究通过干预ApoE-/-小鼠体内PEDF的表达探究PEDF在动脉粥样硬化中的作用,探索其具体机制,为抗动脉粥样硬化治疗靶点提供新的方向和证据。论文Ⅰ 管周脂肪PEDF通过抑制血管外膜滋养管生成抗动脉粥样硬化的机制研究研究目的1.探讨心包脂肪PEDF在AS进展中的表达变化;2.探讨全身敲减PEDF对AS的影响;3.探讨干预管周脂肪PEDF对血管外膜滋养管的影响,分析PEDF影响AS的机制;4.探讨PEDF抑制血管新生的分子机制。研究方法1.冠心病患者体内PEDF含量检测Western blot及RT-qPCR检测冠心病及非冠心病患者中心包脂肪组织中PEDF的mRNA及蛋白表达水平;ELISA检测两组患者血清中PEDF的含量。本实验已获得山东大学齐鲁医院科研伦理委员会批准。2.构建PEDF敲减的AS斑块动物模型8周龄ApoE-/-雄鼠尾静脉注射PEDF干扰慢病毒(PEDF shRNA)或空载体病毒(CTR shRNA),并给予高脂高胆固醇饮食(40%脂肪,1.25%胆固醇)喂养12 w,构建PEDF敲减的斑块模型。ELISA检测小鼠血清中PEDF的含量,免疫组化检测斑块内PEDF表达水平,明确PEDF敲减成功。3.构建管周脂肪移植的斑块模型在PEDF敲减的ApoE-/-小鼠的基础上构建颈动脉管周脂肪移植模型。8周龄ApoE-/-雄鼠随机分为非手术组及手术组。非手术组小鼠分为空载体组(CTR shRNA组,n=15)和PEDF敲减组(PEDF shRNA组,n=15)。手术组小鼠分为提供内脏脂肪的供体组和接受脂肪移植的受体组。根据提供的内脏脂肪中PEDF是否敲减,供体组小鼠分为空载体组(Donor-CTR shRNA 组,D-CTR shRNA 组,n=15)和 PEDF 敲减组(Donor-PEDF shRNA组,D-PEDF shRNA组,n=15);为排除循环水平PEDF对移植脂肪的颈动脉AS的影响,受体组小鼠均为PEDF敲减的小鼠(PEDF shRNA)。移植供体小鼠的10 mg内脏脂肪(Visceral adipose tissue,VAT)至受体小鼠左侧颈动脉。为研究PVAT中PEDF对局部血管AS的影响,移植术后2 w,在超声引导下向移植的脂肪中注射PEDF过表达腺相关病毒(Adeno-associated virus-PEDF,AAV-PEDF)或腺相关病毒空载体(AAV-CTR)。根据移植脂肪组织PEDF是否敲减,以及移植后脂肪组织PEDF是否过表达,手术组小鼠分为 D-CTR shRNA+AAV-CTR 组、D-CTR shRNA+AAV-PEDF 组、D-PEDF shRNA+AAV-CTR组和D-PEDF shRNA+AAV-PEDF 组(n=15/组)。4.探讨PEDF敲减对炎症因子的影响ELISA检测CTR shRNA组及PEDF shRNA组小鼠血清炎症因子肿瘤坏死因子α(Tumor Necrosis Factor-α,TNFα)、白介素 6(Interleukin-6,IL-6)、白介素 1β(Interleukin-1β,IL-1β)。免疫组化验证斑块中炎症因子的含量。5.探讨PEDF敲减对血脂的影响生化检测仪检测CTR shRNA组及PEDF shRNA组小鼠血清中总胆固醇(Total cholesterol,TC)、甘油三酯(Triacylglycerol,TG)、低密度脂蛋白胆固醇(Low-density lipoprotein cholesterol,LDL-C)及高密度脂蛋白胆固醇(High-density lipoprotein cholesterol,HDL-C)含量。6.探讨PEDF敲减对斑块稳定性的影响(1)HE染色:HE染色观察主动脉根部及颈动脉移植脂肪部位斑块的形态、结构成分和面积;(2)油红O染色:观察斑块内脂质成分及比例;(3)天狼星红染色:观察斑块内胶原成分及比例;(4)免疫荧光:对冰冻切片进行免疫荧光染色,检测斑块内的巨噬细胞(MOMA2)、平滑肌细胞(αSMA);(5)免疫组化:检测斑块及移植脂肪中的PEDF、TNFα、IL-6、IL-1β的表达及VV(Endomucin)密度。7.在ApoE-/-/PEDF-/-小鼠体内验证PEDF对动脉粥样硬化的影响(1)AS模型构建:ApoE-/-/PEDF-/-双敲小鼠由中山大学蔡卫斌教授捐赠。8周龄的ApoE-/-/PEDF-/-雄鼠,高脂高胆固醇喂养12 w。Western blot及免疫组化验证VAT及斑块内PEDF表达水平,验证PEDF敲除成功。(2)PEDF对炎症的影响:Western blot及免疫组化检测各组小鼠VAT中的炎症因子含量。(3)PEDF对血脂的影响:生化法检测各组小鼠血清中TC、TG、LDL-C和HDL-C的含量。(4)PEDF对斑块稳定性的影响:1)HE染色:HE染色观察斑块的形态、结构成分和面积;2)油红O染色:观察斑块内脂质成分及比例;3)天狼星红染色:观察斑块内胶原成分及比例;4)免疫荧光:对冰冻切片进行免疫荧光染色,检测斑块内的巨噬细胞(MOMA2)、平滑肌细胞(αSMA)。8.明确PEDF对小鼠主动脉内皮细胞(Mouse aortic endothelial cells,MAECs)的最佳作用时间PEDF重组蛋白(100ng/ml)作用于MAECs,作用时间分别为0、4、8、12、16、20及24 h,确定最佳作用时间,进行后续实验。9.MAECs血管新生功能检测为研究PEDF对MAECs血管新生的影响,向MAECs加入PEDF(100 ng/ml)及等量PBS(Vehicle)。为研究LR在PEDF抗血管新生中的作用,向MAECs转染LR siRNA及对照siRNA(CTR siRNA)。为研究自噬在PEDF抗血管新生中的作用,向MAECs 中转染 Atg5 siRNA及 CTR siRNA。细胞分为以下 4 组:①CTR siRNA+Vehicle组;②CTR siRNA+PEDF 组;③LR siRNA+PEDF 组;④Atg5 siRNA+PEDF 组。Western blot检测各组MAECs中VEGFA及VEGFR2的表达。5-乙炔基-2’-脱氧尿嘧啶核苷(5-ethynyl-2’-deoxyuridine,EdU)法检测MAECs增殖水平;划痕实验及Transwell细胞迁移实验检测细胞迁移功能;Matrigel基质胶检测MAECs的成管功能。10.MAECs自噬流检测Western blot 检测各组 MAECs 中 LC3、Atg5、SQSTM1/p62、Beclin1 等自噬相关蛋白的表达水平。向各组MAECs转染mRFP-GFP-LC3双荧光标记腺病毒。正置荧光显微镜下观察自噬体及自噬溶酶体形成情况。11.探究脂肪细胞PEDF对MAECs细胞迁移的影响为模拟体内PVAT与血管内皮的解剖学位置关系,体外共培养成熟脂肪细胞与MAECs,下室为成熟脂肪细胞,上室为MAECs。为研究脂肪细胞PEDF对MAECs的作用,向成熟的脂肪细胞中转染PEDF siRNA及CTR siRNA。ELISA检测各组细胞上清中PEDF含量;Transwell检测MAECs的细胞迁移。12.统计学分析定量数据表示为均数±标准误(Mean±SEM)。对所有数据进行正态分布性检验。两组数据间的比较应用独立样本t检验,三组及以上的数据比较应用单因素方差分析。P<0.05被认为有统计学差异。研究结果1.冠心病患者血清及心包脂肪中PEDF含量降低冠心病患者心包脂肪组织及血清中PEDF含量较非冠心病患者降低。2.PEDF敲减的ApoE-/-小鼠模型构建成功PEDF shRNA组小鼠斑块及VAT中PEDF表达显著降低。ELISA结果证实PEDF敲减小鼠血清PEDF含量较对照组明显减少。3.PEDF敲减增加炎症水平ELISA结果显示,PEDF敲减的小鼠血清中IL-6及IL-1β含量增加,TNFα含量无明显差异。4.PEDF敲减对血脂无显著影响生化检测结果显示,CTR shRNA组及PEDF shRNA组小鼠血清TC、TG、LDL-C及HDL-C含量无显著差异。5.PEDF敲减加速AS进展主动脉大体油红O染色结果显示,PEDF敲减小鼠主动脉斑块面积增大。主动脉根部冰冻切片染色结果显示PEDF敲减小鼠斑块面积增大,且斑块内脂质沉积增加、巨噬细胞比例明显增加、平滑肌细胞减少、胶原成分减少,即PEDF敲减增加斑块易损性。同时PEDF敲减小鼠主动脉根部斑块内TNFα、IL-6、IL-1β等炎症因子表达较对照组增加。6.管周脂肪移植加速颈动脉AS进展,管周脂肪组织来源的PEDF改善颈动脉局部斑块负荷大体油红O染色结果显示,与非手术组小鼠相比,手术组小鼠左侧颈总动脉斑块面积显著增加。连续冰冻切片染色结果显示,管周脂肪过表达PEDF组小鼠颈动脉斑块面积较非过表达PEDF组小鼠降低,同时斑块内巨噬细胞及脂质减少、平滑肌细胞及胶原纤维成分增加,斑块稳定性增加。术后4 w颈动脉超声结果显示,D-CTR shRNA+AAV-PEDF组小鼠颈动脉脉搏波传导速度(Pulse wave velocity,PWV)较D-CTR shRNA+AAV-CTR 组降低。7.管周脂肪组织来源的PEDF改善颈动脉局部斑块炎症水平颈动脉及移植PVAT的冰冻切片免疫组化染色结果显示,PVAT过表达PEDF组小鼠颈动脉斑块TNFα和IL-6表达水平较非过表达PEDF组降低。8.管周脂肪来源的PEDF抑制VV新生颈动脉及移植PVAT的冰冻切片免疫组化染色结果显示,PVAT过表达PEDF组小鼠颈动脉斑块处血管外膜VV密度较非过表达PEDF组减少。9.PEDF敲除加速AS进展主动脉大体油红O染色结果显示,PEDF敲除的ApoE-/-小鼠主动脉斑块面积增加。与ApoE-/-小鼠相比,PEDF敲除的小鼠斑块内巨噬细胞比例明显增加、平滑肌细胞减少、胶原纤维含量减少,即PEDF敲除增加斑块易损性。10.PEDF敲除增加炎症水平PEDF敲除小鼠VAT中IL-6及TNFα表达显著增加,IL-1β蛋白表达有增加趋势。11.PEDF敲除增加血脂水平PEDF敲除小鼠血清TC、TG及LDL-C水平显著增加,HDL-C水平有降低趋势。12.PEDF抑制MAECs的血管新生能力PEDF抑制MAECs中VEGFA蛋白表达,同时降低VEGFA/VEGFR2比例,该作用具有时间依赖性。PEDF可显著抑制MAECs的增殖、迁移及小管形成。13.PEDF通过LR抑制MAECs的血管新生与 CTR siRNA+PEDF 组相比,LR siRNA+PEDF 组 MAECs 中 VEGFA 蛋白含量明显增加、VEGFR2蛋白表达明显减少,VEGFA/VEGFR2比例有增加的趋势;同时抑制LR表达可减轻PEDF对MAECs增殖、迁移和小管形成的抑制作用。免疫共沉淀证实,LR可与RACK1结合。干预MAECs中的RACK1可抑制PEDF的抗血管新生作用。14.PEDF通过作用于LR促进自噬进而抑制MAECs的血管新生能力PEDF增加MAECs中的LCⅡ蛋白表达、降低SQSTM1/p62蛋白表达,且该作用具有时间依赖性。PEDF增加MAECs自噬溶酶体的形成。干扰Atg5的表达,抑制PEDF对MAECs自噬的促进作用,MAECs中LC3 Ⅱ及Beclin1蛋白表达降低,SQSTM1/p62蛋白表达增加,自噬溶酶体数量降低。与CTR siRNA+PEDF组相比,Atg5 siRNA+PEDF组MAECs中VEGFA蛋白含量明显增加、EdU阳性细胞明显增加、迁移细胞数明显增加、划痕愈合面积增加、小管形成数目增加。干预LR后,PEDF对MAECs自噬的促进作用被抑制。15.脂肪细胞PEDF可抑制MAECs的迁移脂肪细胞与MAECs共培养体系中,PEDF siRNA组上清PEDF含量较CTR siRNA组显著降低;Transwell迁移实验结果证实,干扰脂肪细胞中PEDF的分泌可促进MAECs的迁移。研究结论1.冠心病患者心包脂肪及血清中PEDF含量较非冠心病患者降低;2.PEDF敲减增加斑块内炎症水平,降低斑块稳定性,加速动脉动脉粥样硬化的发展;3.管周脂肪组织来源的PEDF,抑制血管外膜滋养管新生,降低局部血管的斑块内炎症水平,增加斑块稳定性,抑制动脉粥样硬化的发展;4.PEDF通过作用于LR和RACK1,促进内皮细胞自噬,进而抑制内皮细胞的血管新生。论文Ⅱ 人参皂苷Rb1通过调节miR-33与PEDF抑制外膜滋养管新生并稳定斑块研究目的1.验证PEDF是mi-R33的靶基因;2.验证Rbl对动脉粥样硬化发生发展的作用;3.探讨Rb1对血管外膜滋养管新生的作用;4.验证Rb1调控血管外膜滋养管的分子机制。研究方法1.验证PEDF是miR-33的靶基因灵长类动物表达两种类型的miR-33,即miR-33a和miR-33b;而啮齿类动物仅表达一种类型的miR-33,即miR-33a。为验证PEDF是miR-33的靶基因,将PEDF的3’-UTR构建入带有荧光素酶的表达载体(pLightSwitch-3’-UTR vector),构建荧光素酶质粒(WT);同时对预测的结合位点进行突变,构建突变型质粒(MUT)。将构建的野生型和突变型质粒分别与miR-33a的类似物(mimic)及类似物的对照物(CTR)转染至HUVECs中,同时将带有海肾荧光素酶基因的质粒(phRL-TK)作为对照质粒与报告基因质粒共同转染至细胞。细胞分为4组:①WT+CTR;②WT+mimic;③MUT+CTR;④MUT+mimic。裂解细胞,并加入底物荧光素,检测不同组别细胞的荧光素酶活性。2.小鼠动脉粥样硬化斑块模型构建45只8周龄的ApoE-/-小鼠高脂喂养(0.25%胆固醇、15%可可脂)20 w。3.探讨Rb1对PEDF表达及动脉粥样硬化斑块稳定性的影响(1)HE染色:HE染色观察主动脉根部斑块的形态、结构成分和面积。(2)油红O染色:观察斑块内脂质成分及比例。(3)天狼星红染色:观察斑块内胶原成分及比例。(4)免疫组化:检测斑块中的PEDF、TNFα、IL-6、IL-1β、MOMA2及αSMA的表达。4.验证Rb1通过调节miR-33增加PEDF表达并抑制血管外膜滋养管新生将 ApoE-/-小鼠随机分为 3 组(n=15 只/组),即 Vehicle+LV-vector 组,Rb1+LV-vector组,Rb1+LV-miR-33组。Rb1+LV-miR-33组小鼠尾静脉注射过表达miR-33的慢病毒,同时连续腹腔注射Rb1(50mg/kg/d)4 w;Rb1+LV-vector组小鼠尾静脉注射等量空载体慢病毒并连续腹腔注射Rb1;Vehicle+LV-vector组小鼠给予等量的生理盐水及空载体慢病毒。RT-qPCR验证各组斑块处miR-33表达水平。小鼠取材前通过体内灌注生物素标记的番茄凝集素标记血管外膜滋养管,评估各组小鼠VV密度。免疫组化染色检测各组小鼠斑块中PEDF的表达水平。5.探讨Rb1对血脂的影响生化检测仪检测各组小鼠血清TC、TG、LDL-C及HDL-C含量。6.统计学分析定量数据表示为均数±标准误(Mean±SEM)。对所有数据进行正态分布性检验。两组数据间的比较应用独立样本t检验,三组及以上的数据比较应用单因素方差分析。P<0.05被认为有统计学差异。研究结果1.PEDF是miR-33的靶基因转染野生型质粒的细胞中,过表达miR-33a可显著抑制荧光素酶的活性;将PEDF 3’-UTR的结合位点突变后,过表达miR-33a对细胞中的荧光素酶活性无影响。证实,PEDF是miR-33的靶基因。2.Rb1抑制ApoE-/-小鼠动脉粥样硬化的发展、增加斑块稳定性Rbl+LV-vector组小鼠斑块负荷较Vehicle+LV-vector组明显减小,同时斑块内平滑肌细胞增加、胶原纤维成分增加、巨噬细胞减少、脂质沉积减少,即Rbl增加斑块稳定性。同时,Rbl+LV-vector组小鼠斑块内炎症因子IL-1β、IL-6及TNFα等的含量较Vehicle+LV-vector组显著降低。3.Rb1抑制ApoE-/-小鼠斑块处血管外膜滋养管增生番茄凝集素染色结果显示,Rb1+LV-vector组小鼠主动脉根部动脉粥样硬化斑块周围血管外膜滋养管密度较Vehicle+LV-vector组减少,提示Rbl可抑制斑块处血管外膜滋养管增生。4.miR-33过表达抑制Rb1对斑块的改善作用与Rbl+LV-vector组小鼠相比,Rb1+LV-miR-33组小鼠斑块内巨噬细胞及脂质含量增加、平滑肌细胞及胶原纤维含量减少,斑块易损性增加。Rbl+LV-miR-33组小鼠斑块内炎症因子含量较Rbl+LV-vector组显著增加。同时,过表达miR-33可显著减弱Rb1对血管外膜滋养管的抑制作用。5.miR-33参与Rb1对PEDF的促进作用与Vehicle+ LV-vector组相比,Rbl+LV-vector组小鼠斑块内PEDF含量显著增加。同时,过表达miR-33可显著抑制Rb1对PEDF表达的促进作用。6.Rb1对血脂无影响Vehicle+LV-vector 组、Rb1+LV-vector 组和 Rb1+LV-miR-33 组小鼠血清中 TC、TG、LDL-C及HDL-C含量无统计学差异,Rb1对血脂无显著影响。研究结论1.PEDF是miR-33的靶基因;2.Rbl通过抑制血管外膜滋养管新生及斑块内炎症,增加斑块稳定性;3.Rbl通过调控miR-33及PEDF表达,抑制血管外膜滋养管新生及斑块内炎症反应。更多还原

【Abstract】 BackgroundAs the most common underlying cause of coronary artery disease(CAD),atherosclerosis(AS)is the leading cause of death worldwide.AS is a complex pathological process which leads to artery stenosis and results in cerebrovascular disease and CAD.The rupture of vulnerable AS plaque and thrombogenesis are the main pathological mechanisms of acute vascular event.Early intervention on vulnerable plaque is necessary for reducing the incidence of acute cardiovascular events.The classical studies show that the AS starts from the intimal injury,and the intimal inflammation expands to the adventitia and perivascular adipose tissue(PVAT).The inside-out signaling paradigm has been widely accepted.However,more and more studies reveal that PVAT contributes to the pathogenesis of AS through the outside-in model.The adipokines derived from PVAT can act in the adventitia and intima.There are complex interactions between PVAT and intima.Studies illustrate that increase of angiogenesis is the prerequisite of the vulnerable plaque.The invasion of macrophages,intraplaque hemorrhage,the thin fibrous cap and inflammatory infiltration are closely related to the neovascularization of the adventitial vasa vasorum(VV).During the development of AS,the VV which arises from the vascular adventitia proliferates to adapt to the vascular hypoxia caused by intimal thickening and results in intraplaque hemorrhage.However,the recent evidence shows that the proliferation of VV is earlier than the intima thickening,and the dysfunction PVAT is responsible for the proliferative VV.The VV acts as the pipage for the interaction between PVAT and intima,and transports inflammatory factors and cells to the intima during AS process.PVAT,the special adipose tissue,is also an endocrine organ which secrets amounts of proand anti-inflammatory factors and influences the development of AS.The dysfunction of PVAT caused by obesity can aggravate adventitial inflammatory infiltration and angiogenesis,and accelerate intimal hyperplasia and VV proliferation.Based on these studies,inhibition of adventitia vasa vasorum angiogenesis to weaken the effects of inflammatory cytokines from PVAT on vessels can be the new therapeutic target against atherosclerosis.Our previous research demonstrated that pigment epithelium derived factor(PEDF),a potent endogenous anti-angiogenic factor,could protect against atherosclerosis.Compared with exogenetic angiogenesis inhibitors,PEDF possesses high security which only effects on the abnormal neovascularization without injuring the normal vessels.As a multifunctional factor,PEDF can also inhibit inflammation,resist oxidation and inhibit thrombosis besides the antiangiogenesis effect.The presently known receptors of PEDF include adipose triglyceride lipase/independent phospholipase A2(iPLA2)and 37/67-kDa laminin receptor(LR).Studies shows that LR acts as a mediator of cellular signal transduction,and reacts with many signal pathways.LR is firstly recognized as the 67-kDa binding protein of laminin and distributes in many domains of life.LR is the plasma membrane protein,and participates in many biology processes including cell proliferation,migration and protein synthesis.LR is suggested the real receptor mediated the anti-angiogenetic function.However,the molecular mechanisms of PEDF and LR in anti-antiangiogenesis are still unclear.On the basis of available data,upregulation of PEDF might be an important manner to intervene VV proliferation and stabilize atherosclerosis plaque.The effective agonist of PEDF will be a new therapy for AS.As the representative component of ginseng,Ginsenoside Rb1 exhibits many protective properties,including anti-obesity,anti-oxidative stress,antiangiogenesis and anti-fatigue.Our group proved that Rb1 inhibited HUVECs angiogenesis in vitro through modulating miR-33a and PEDF.However,it remains unclear whether Rbl could inhibit VV proliferation in AS.Although a number of studies have shown that PEDF may be an effective factor in antiangiogenesis of VV and stabilization of plaque,there is no direct evidence at present.Meanwhile,the mechanism of the PEDF anti-angiogenesis effect remains unclear.We will investigate the role of PEDF in PVAT during the development of AS and whether PEDF could inhibit the angiogenesis of VV by modulating PEDF expression in ApoE-/-mice.Dissertation Ⅰ Anti-atherosclerotic Effect of PEDF Secreted by Perivascular Adipose Tissue is Exerted via Inhibiting Adventitia Vasa Vasorum AngiogenesisObjectives1.To investigate the alterations of PEDF expression of pericardial adipose tissue in AS procession.2.To investigate whether PEDF affects the development of AS.3.To investigate whether PEDF in PVAT affects the development of AS through VV.4.To investigate the molecular mechanism of PEDF.Methods1.Explore the change of PEDF expression in CAD patientsA total of 6 patients undergoing thoracotomy(3 CAD patients and 3 without CAD patients)were evaluated.The pericardial adipose tissues were obtained at the beginning of extracorporeal circulation.The serum was collected after preoperative examination for the research.The experiment obtained approval from the ethics board.2.Establish the mouse model of PEDF-knockdown atherosclerosisMale ApoE-/-mice(8 weeks old)were treated with lentivirus PEDF shRNA or lentivirus vector(CTR shRNA).The mice were fed with high-fat diet(HFD,40%fat and 1.25%cholesterol)for 12 weeks.3.Establish the mouse model of transplanted perivascular adipose tissueThe models of transplanted perivascular adipose tissue were build based on the PEDFknockdown mice.The ApoE-/-mice(8 weeks old)were randomly divided into 2 groups:the non-operation group and the operation group.The non-operation group mice were randomly divided into CTR shRNA group(n=15)and PEDF shRNA group(n=15).The operation group mice were randomly divided into the donator group and the receptor group.The donator group mice were randomly divided into D-CTR shRNA and D-PEDF shRNA.The receptor group mice were all treated with PEDF shRNA.Mice in donator groups were anesthetized and separated 10mg VAT.The separated adipose tissues were transplanted to encircle the left carotid artery of the mice in the receptor group.Mice in the receptor group were locally injected with adeno-associated virus(AAV)to over express PEDF(AAV-PEDF)or the AAV vector(AAV-CTR)in the transplanted adipose tissue under ultrasound guidance 2 weeks after the transplant.According to the PEDF expression levels of transplanted adipose tissues,mice in receptor groups were divided into four groups:D-CTR shRNA+AAV-CTR group,D-CTR shRNA+AAV-PEDF group,D-PEDF shRNA+AAV-CTR group,and D-PEDF shRNA+AAV-PEDF group(n=15 per group).All mice were fed with HFD for total 12 weeks.4.Explore the effect of reduced PEDF on inflammationSerum levels of PEDF,tumor necrosis factor-α(TNFα),interleukin-6(IL-6)and interleukin-1β(IL-1β)in CTR shRNA and PEDF shRNA groups were measured by enzyme linked immunosorbent assay(ELISA).The content of inflammatory factors in plaque were detected by IHC.5.Explore the effect of PEDF on lipid levelsThe serum levels of total cholesterol(TC),triacylglycerol(TG),low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C)were measured by biochemical detection.6.Explore the effect of PEDF on atherosclerotic plaque stability(1)HE stainingTissue sections were stained with hematoxylin and eosin to show the morphology of the plaques.(2)Oil O stainingTo evaluate the plaque burden of the aortas and the content of lipid in plaque,the aortas,carotid artery,and aortic root sections were stained with oil-red O solution.(3)Sirius red stainingThe sections of carotid artery and aorta were stained by Sirius red to quantified the collagen content in plaque.(4)Immunofluorescent stainingImmunofluorescent staining was used to analyze macrophages(MOMA2)and smooth muscle cells(αSMA)in plaque.(5)Immunohistochemical(IHC)stainingIHC staining was used to detect the expression levels of PEDF,TNFα,IL-6 and IL-1β in plaque and transplanted PVAT.The density of VV was analyzed by IHC staining with endomucin primary antibody.The levels of PEDF,TNFα,IL-6,IL-1β and CD31 in VAT were analyzed by IHC staining.7.Verify the PEDF impact on AS progress in ApoE-/-/PEDF-/-mice(1)Establish the PEDF-knockout mouse modelMale ApoE-/-/PEDF-/-mice(8 weeks old)were all fed with high-fat diet(HFD)for 12 weeks.Measure the PEDF expression in plaque and VAT.(2)Explore the effect of PEDF on inflammation:Western blot and Immunohistochemistry staining were used to detect the content of inflammatory factors in VAT of each group.(3)Explore the effect of PEDF on serum lipids:TC,TG,LDL-C and HDL-C levels in serum were detected.(4)Explore the effect of PEDF on AS plaque stability1)HE stainingTissue sections were stained with hematoxylin and eosin to show the morphology of the plaques.2)Oil O stainingTo evaluate the plaque burden of the aortas and the content of lipid in plaque,the aortas and aortic root sections were stained with oil-red O solution.3)Sirius red stainingThe sections of aorta were stained by Sirius red to quantified the collagen content in plaque.4)Immunofluorescent stainingImmunofluorescent staining was used to analyze macrophages(MOMA2)and smooth muscle cells(αSMA)in plaque.8.Explore the incubation time of PEDF on Mouse aortic endothelial cells(MAECs)The primary MAECs was purchased from Procell company and cultured with endothelial cell medium(ECM).The MAECs were treated with PEDF recombinant protein for 0,4,8,12,16,20 and 24 h to observe the optimal action time.9.Explore the effect of PEDF on neovascularization of MAECsThe MACEs were treated with PEDF(100 ng/ml)or equal volume PBS(Vehicle)to explore the effect of PEDF on angiogenesis.The MAECs were transfected with LR siRNA or CTR siRNA to study the effect of LR.The MAECs were transfected with Atg5 siRNA or CTR siRNA to study whether autophagy was involved in the anti-angiogenesis of PEDF.The MAECs were divided into four groups:①CTR siRNA + Vehicle group;②CTR siRNA +PEDF group;③LR siRNA+PEDF group;④Atg5 siRNA+PEDF group。Western blot was used to detect the protein levels of VEGFA and VEGFR2 in MAECs.The proliferation of MAECs was measured by 5-ethynyl-2’-deoxyuridine(EdU)method.24-well inserts with 8-μm pores were used for Transwell assay.Cells migration were evaluated by scratch wound healing assay.MAECs were seeded in the 24-well plate coated with Matrigel for analyzing the ability of tube formation.10.Explore the effect of PEDF on autophagy flux of MAECsWestern blot was used to detect the expression of autophagy proteins,including LC3、Atg5、SQSTM1/p62 and Beclin1.The MAECs in four groups were transfected with mRFPGFP-LC3 adenovirus.The number of autophagosomes and autolysosomes were measured under fluorescence microscope.11.Explore the effect of PEDF in adipocytes on MAECsThe mature adipocytes differentiated from 3T3-L1 cells and MAECs were co-cultured to simulate the anatomical location of PVAT and intima.The adipocytes were transfected with PEDF siRNA or CTR siRNA,and co-cultured with MAECs.The PEDF levels in supernatant were detected by ELISA.The MAECs migration were measured by Tranwell assay.12.Statistical analysesAll data were presented as the Mean ± SEM of three independent experiments at least.Statistical analysis was carried out using one-way analysis-of-variance(one-way ANOVA)followed by Turkey’s post hoc test(GraphPad,CA USA).The student’s t-test was used for statistical comparisons between two groups.P<0.05 was considered significant.Results1.The PEDF level was down-regulated in pericardial adipose tissue and serum of CAD patientsThe results showed that protein level of PEDF was significantly decreased in the PC AT of CAD patients.Although there was no statistical difference,mRNA expression of PEDF was also tended to be less in PCAT with CAD.The PEDF levels were reduced in patients with CAD.2.The PEDF level was decreased in PEDF shRNA micePEDF shRNA was injected via tail veins to knock down the PEDF in the whole body.The knock-down efficiency was verified through detect PEDF levels in serum and AS plaque after mice sacrificed.It was illustrated that the PEDF expression was significantly reduced in AS plaque and VAT with PEDF shRNA injection.The PEDF level in serum was also decreased in the PEDF shRNA mice.3.The decrease of PEDF increased the inflammatory factorsThe ELISA analyses illustrated that IL-6 and IL-1β levels in serum were increased in PEDF knock-down mice.4.The decrease of PEDF did not influence the lipid levelsThere were no significant differences in the serum levels of TC,TG,LDL-C and HDL-C between CTR shRNA and PEDF shRNA.5.The decrease of PEDF accelerated AS processionAfter HFD for 12 weeks,the relative en face atherosclerotic area was significant enlarged in PEDF knock-down mice.The sectional lesion size at aortic root was also augmented.It demonstrated the decrease of PEDF accelerated AS procession.The accumulation of macrophages and lipids were increased while the content of SMCs and collagen were decreased in the plaque.These data demonstrated that the plaque vulnerability index was dramatically increased in PEDF knock-down mice.Meanwhile,the results indicated that the expressions of TNFα,IL-6 and IL-1β were significantly increased in the plaque of PEDF knock-down mice.6.The PEDF in PVAT ameliorated the AS plaqueOil O red staining showed that the en face atherosclerosis area of left carotid artery surrounded by PVAT in the operation group was significantly augment compared with the nonoperation group.Oil O red staining showed that the sectional lesion size of carotid artery in operation mice with AAV-PEDF injection were decreased compared with the operation mice with AAV-CTR injection.The accumulation of macrophages and lipid in plaque were decreased and the content of smooth muscle cells and collagen were increased in the operation mice with PEDF overexpression in transplanted PVAT compared with the mice without PEDF overexpression in transplanted PVAT.7.PEDF in PVAT ameliorated the inflammatory state in carotid plaqueTo investigate the mechanisms of PEDF anti-atherosclerosis action,we detected the inflammatory levels in carotid artery plaque.The results of IHC staining showed that the expression of TNFa and IL-6 in plaque were reduced with the overexpression of PEDF in PVAT.8.PEDF in PVAT inhibited the proliferation of VVWe stained the frozen sections of carotid arteries and the surrounded PVAT with endomucin primary antibody to evaluate the angiogenesis of VV.The positive areas of endomucin were smaller in the operation mice with PEDF overexpression in transplanted PVAT compared with the mice without PEDF overexpression in transplanted PVAT.9.The knockout of PEDF accelerated AS processionThe area of plaque of aorta in ApoE-/-/PEDF-/-mice was decreased compared with the ApoE-/-mice.The accumulation of macrophages and lipids were increased while the content of SMCs and collagen were decreased in the plaque of ApoE-/-/PEDF-/-mice.These data demonstrated that the plaque vulnerability index was dramatically increased in ApoE-/-/PEDF/-mice.10.The knockout of PEDF increased the inflammatory factors in VATThe results showed that the expressions of IL-6 and TNFa were significantly increased in VAT of ApoE-/-/PEDF-/-mice compared with ApoE-/-mice.The IL-1β protein level trended to be increased in VAT of ApoE-/-/PEDF-/-mice.11.The knockout of PEDF increased lipid levels in serumThe serum levels of TC,TG and LDL-C were significantly increased in ApoE-/-/PEDF-/mice compared with ApoE-/-mice.The HDL-C levels trended to be decreased in ApoE-/-/PEDF/-mice.12.PEDF inhibited the neovascularization of MAECsVEGFA expression was markedly reduced in MAECs with PEDF treatment.The decrease was depended on incubation time.The ratio of VEGFA to VEGFR2 of MAECs was significantly diminished at 8,12,16,20,24 h of PEDF treatment.To investigate the influence of PEDF on MAECs,the capacity for proliferation,migration and tube formation were evaluated in vitro.The EdU positive ratio was reduced by PEDF treatment.PEDF also inhibited cell migration.Compared with the control group,tubular morphogenesis was also inhibited by PEDF treatment.13.PEDF inhibited the angiogenesis of MAECs through LRThe down-regulation of VEGFA by PEDF was inhibited by LR siRNA transfection.LR also repressed the effect of PEDF on VEGFR2.PEDF reduced the ratio of VEGFA/VEGFR2 of MAECs,and interference of the LR expression could debilitated this regulation.The inhibition effects of PEDF on the proliferation,migration and tube formation were alleviated by LR siRNA transfection.Co-IP results showed that LR combined with RACK1,and inhibition of RACK1 could also reverse the anti-angiogenesis effect of PEDF on MAECs.14.PEDF inhibited the angiogenesis of MAECs by changing the autophagy level through LR pathwayTo investigate the possible molecular mechanism,the Atg5 siRNA and CTR siRNA were transfected into MAECs.We detected the autophagy levels of MAECs with PEDF treatment.The results implicated that PEDF increased the expression of LC Ⅱ and decreased the expression of SQSTM1/p62.This stimulative action was in a time dependent manner.The number of autophagosomes and autolysosomes was increased with PEDF treatment.VEGFA expression was increased in Atg5 siRNA+PEDF group compared with the CTR siRNA+PEDF group.The inhibition effects of PEDF on the proliferation,migration and tube formation were alleviated by Atg5 siRNA transfection,which demonstrated that PEDF inhibited the neovascularization of MAECs through regulating autophagy.The number of autolysosomes was reduced in Atg5 siRNA+PEDF group compared with the CTR siRNA+PEDF group.The accelerative effect of PEDF on autophagy flux was evidently inhibited with LR knockdown.15.PEDF in adipocytes inhibited the migration of MAECsThe PEDF level in the supernatant of PEDF siRNA adipocytes was decreased compared with CTR siRNA adipocytes.The decreased PEDF of adipocytes increased the number of migrated MAECs in the co-culture system.Conclusion1.The PEDF levels in PVAT were reduced in CAD patients.2.PEDF knock-down accelerated AS procession.3.Transplanted PVAT promoted the development of AS.4.PEDF in PVAT inhibited VV proliferation and ameliorated AS procession.5.PEDF inhibited the angiogenesis of MAECs by regulating the autophagy level through LR pathway.Dissertation Ⅱ Ginsenoside Rbl Enhances Plaque Stability and Inhibits Adventitial Vasa Vasorum via the Modulation of miR-33 and PEDFObjectives1.To investigate PEDF as a target gene of mi-33;2.To investigate the effect of Rbl on atherosclerosis.3.To investigate the effect of Rb1 on adventitia vasa vasorum angiogenesis.4.To verify the mechanism of Rbl regulating adventitia vasa vasorum angiogenesis.Methods1.Identification of PEDF as a target gene of miR-33 in HUVECsThe 3’-UTR of SERPINF1 was cloned into the pLightSwitch-3’-UTR vector to construct the wild type plasmid(WT).To confirm the predicted binding site,a construct with mutated complementary to seed region of miR-33a(MUT)were generated.Cells seeded were cotransfected with WT or MUT plasmid,phRL-TK,and miR-33a mimic or control mimic.After 24 h transfection,cells were lysed by lysis buffer and luciferase activity was measured.2.Establish the mouse model of atherosclerosis45 female ApoE-/-mice at 8 week-age with a C57BL/6 background were fed with a highfat diet for 20 weeks.3.Explore the effect of Rbl on PEDF expression and atherosclerotic plaque stability(1)HE stainingTissue sections were stained with hematoxylin and eosin to show the morphology of the plaques.(2)Oil O stainingTo evaluate the plaque burden of the aortas and the content of lipid in plaque,the aortas and aortic root sections were stained with oil-red O solution.(3)Sirius red stainingThe sections of carotid artery and aorta were stained by Sirius red to quantified the collagen content in plaque.(4)Immunohistochemical(IHC)stainingIHC staining was used to detect the expression levels of PEDF,TNFα,IL-6 and IL-lβ in plaque.The positive areas of macrophages(MOMA2)and smooth muscle cells(αSMA)in plaque were also analyzed by IHC staining.4.Verify the specific mechanism of Rbl anti-angiogenesis actionAll ApoE-/-mice were randomly divided into 3 groups(n=15 per group),including Vehicle+LV-vector group,Rb1+LV-vector group,and Rb1+LV-miR-33 group.Mice in Rbl+LV-vector group were treated with lentivirus to overexpress miR-33 and intraperitoneal injection of Rb1(50mg/kg/d for 4 weeks).The mice in Rbl+LV-vector group received empty lentivirus by tail vein injection and Rbl treatment by intraperitoneal injection.The mice in Vehicle+LV-vector group were treated with equal amount of saline and empty lentivirus.RTqPCR was used to detect the miR-33 expression levels in the three groups.VV in adventitia were identified through the perfusion with biotinylated Lycopersicon Esculentum(Tomato)lectin.5.Explore the effect of Rb1 on lipidThe serum levels of TC,TG,LDL-C and HDL-C were measured by biochemical detection.6.Statistical analysesAll data were presented as the Mean ± SEM of three independent experiments at least.Statistical analysis was carried out using one-way analysis-of-variance(one-way ANOVA)followed by Turkey’s post hoc test(GraphPad,CA USA).The student’s t-test was used for statistical comparisons between two groups.P<0.05 was considered significant.Results1.PEDF is a target gene of miR-33Overexpression of miR-33a but not the control mimics substantially repressed the luciferase activity with WT plasmid transfection.In contrast,no effect was found when miR33a-binding sites in the 3’-UTR of PEDF were mutated,which suggested that miR-33a could directly bind to the 3’-UTR of PEDF and regulate its expression.2.Rbl inhibited atherosclerotic plaque growth and enhanced plaque stability in ApoE-/miceThere is a significant reduction of the relative en face atherosclerotic lesion area as well as the cross-sectional lesion size at the aortic root,which demonstrated that the extent of atherosclerosis was significantly decreased in Rbl+LV-vector mice.Furthermore,immunohistochemistry analysis also revealed that Rbl treatment exerted notable effects on upregulating the contents of SMCs and collagen.Conversely,Rbl treatment could also reduce the accumulation of macrophages and lipid.As a consequence,the plaque vulnerability index was dramatically decreased in Rbl treated mice.Meanwhile,the immunohistochemical studies of inflammatory cytokines showed that Rbl treatment significantly reduced the expression levels of IL-1β,IL-6 and TNFa in plaque.All the above results indicated that Rbl treatment could enhance AS plaque stability.3.Rbl inhibited VV proliferation in atherosclerotic plaque of ApoE-/-miceConsidering the anti-angiogenic role of Rbl,average number of vasa vasorum in adventitia surrounding the atherosclerosis plaques in aortic roots were counted to uncover whether Rb1 could inhibit neovascularization in ApoE-/-mice.The results showed that Rbl+LV-vector group had less vasa vasorum,indicating that Rbl could inhibit proliferation of VV in plaques of ApoE-/-mice.4.The beneficial effects of Rb1 on atherosclerosis were attenuated by miR-33 overexpressionIn this study,ApoE-/-mice injected with miR-33 lentivirus were applied to study that miR33 overexpression attenuated the beneficial effects of Rbl on atherosclerosis.Compared with Rbl+LV-vector group,miR-33 overexpression significantly increased the accumulation of macrophages and lipid,decreased the contents of SMCs and collagen.Therefore,the plaque vulnerability index was dramatically increased in miR-33 overexpression mice.Moreover,upregulation of miR-33 could significantly attenuate the anti-inflammation effects of Rbl by decreasing the expression levels of IL-1β,IL-6,and TNFa within AS plaques in ApoE-/-mice.Rbl-mediated VV inhibition was abrogated by overexpression of miR-33.Together,these results demonstrated that miR-33 has profound negative impact on the Rbl-mediated enhancement of plaque stability,reduction of plaque burden,anti-inflammation and inhibition of VV in ApoE-/-mice.5.miR-33 was involved in Rbl-induced PEDF expression in atherosclerotic plaque in ApoE-/-miceRbl treatment led to the induction of PEDF expression in AS plaque.Interestingly,miR33 expression was also significantly downregulated in Rbl treated ApoE-/-mice.Overexpression of miR-33 results in a significant regression of Rbl-mediated PEDF elevation in AS plaque in ApoE-/-mice.6.Rbl had no effect on lipidThere were no significant differences in serum levels of TC,TG,LDL-C and HDL-C among the three groups.Conclusion1.PEDF is a target gene of miR-33.2.Ginsenoside Rbl attenuates plaque growth and enhances plaque stability partially through inhibiting adventitial vasa vasorum proliferation and inflammation in ApoE-/-mice.3.The anti-angiogenic and anti-inflammation effects of Rbl are exerted via the modulation of miR-33 and its target gene PEDF.更多还原