【作者】 白洁；

【导师】 李汇华；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2022， 博士

【摘要】 研究背景:心脏淋巴管生成和完整性在维持心肌组织液平衡中起着重要的作用。淋巴管生成障碍与心肌缺血性损伤及压力超负荷引起的心脏水肿和重构密切相关。然而,淋巴管生成和完整性在血管紧张素II（Ang Ⅱ）诱导的心脏重构中的作用及机制仍不清楚。研究目的:明确在Ang Ⅱ诱导的小鼠心脏重构过程中淋巴管生成和通透性的改变情况;阐明淋巴管生成和通透性改变调控心脏重构的分子机制,为临床上心脏重构的防治提供新药靶点和新措施。研究方法:1.实验动物模型的建立和处理本研究以野生型C57BL/6J（WT）小鼠、淋巴内皮特异性VEGFR-3基因敲除(VEGFR-3^f/-)小鼠及同窝对照(VEGFR-3^f/f)小鼠为研究对象,应用Ang Ⅱ（1000 ng/kg/min）灌注小鼠3、7和14天诱导小鼠心脏重构模型。同时,腹腔注射蛋白酶体抑制剂环氧霉素（Epoxomicin,0.58 mg/kg/day）并持续至14天,探讨蛋白酶体活性在Ang Ⅱ诱导的心脏重构中的作用。2.小鼠血压和心功能的监测采用尾套法（Tail-cuff）监测小鼠基础血压和Ang Ⅱ灌注不同时间点的血压;采用M-型超声心动图谱分析各组小鼠心脏收缩及舒张功能,检测参数包括:射血分数（EF%）、短轴缩短率（FS%）、左室前壁及后壁的厚度（LVAW,LVPW）,左室内径（LVID）等。3.小鼠心脏水含量的测定应用干湿重的方法检测各组小鼠心脏水含量,计算公式为:小鼠心脏水含量（%）=（心湿重-干重）/湿重x 100%。4.组织病理学观察心脏组织切片进行以下染色:苏木素-伊红（H&E）、马松三色（Masson）、免疫组织化学（IHC）、小麦胚芽凝集素（WGA）、二氢乙锭（DHE）及免疫荧光染色（IF）等。5.淋巴管通透性的测定动物:将20μl伊文斯兰注射于小鼠足垫内,16小时后取出小鼠腘窝淋巴结,研磨后,应用酶标仪在620 nm下,测定其吸光度（OD）值。体外淋巴管内皮细胞（LECs）:分别用伊文思蓝和FITC-葡聚糖检测LECs的通透性。6.蛋白酶体活性的测定提取小鼠心脏组织及淋巴内皮细胞蛋白,加入荧光底物,在酶标仪中分别测定半胱氨酸氧蛋白酶、胰蛋白酶样及糜蛋白酶样的活性。7.基因及蛋白表达的分析应用实时荧光定量PCR检测各基因m RNA的水平。应用免疫印迹方法测定相关蛋白的表达水平。8.统计学分析本研究中的所有数据以均数±标准差表示。使用Graph Pad Prism 9软件进行统计学分析。首先,对所检测数据进行正态分布分析。若实验数据符合正态分布,则使用独立的t检验来确定两组之间的统计学差异;若数据不符合正态分布,则使用Mann-Whitney检验进行分析。采用单因素方差分析和双因素方差分析分析多组之间的差异。以P<0.05表示具有统计学差异。研究结果:1.Ang Ⅱ灌注促进血压升高、心脏重构以及淋巴管生成增加Ang Ⅱ灌注野生小鼠3、7和14天后,收缩压、心肌肥厚、纤维化、炎症反应、氧自由基水平、LYVE1⁺/VEGFR3⁺淋巴管数量、LYVE-1⁺淋巴管数量与心肌细胞数量的比值以及VEGF-C和VEGFR3水平均呈时间依赖性增高。2.Ang Ⅱ灌注促进心脏淋巴管通透性增高和心脏水肿Ang Ⅱ灌注野生小鼠3天、7天和14天后,与盐水组相比,淋巴管通透性（用腘窝淋巴结OD值表示）及其相关蛋白信号蛋白p38 MAPK呈时间依赖性升高;而淋巴管通透性负性调节蛋白MKP5和VE-cadherin的表达水平呈时间依赖性降低。另外,心肌组织的含水量（%）也呈时间依赖性增高。3.VEGFR-3基因敲除抑制Ang Ⅱ诱导的心脏淋巴管生成,加重心脏水肿、重构和功能障碍Ang Ⅱ灌注后14天后,与VEGFR3^f/f对照小鼠比较,VEGFR-3敲除组小鼠的收缩血压明显升高,心脏淋巴管生成（用LYVE1⁺/VEGFR3⁺淋巴管数量、LYVE-1⁺淋巴管数量与心肌细胞数量的比值及VEGFR3/ERK1/2/AKT表达量表示）明显降低。同时,心脏重构程度（包括心肌肥厚、纤维化、氧自由基产生和CD68⁺巨噬细胞数量）及其相关信号通路和心功能能障碍在VEGFR-3敲低小鼠进一步加重。4.Ang Ⅱ促进蛋白酶体活性和淋巴管内皮细胞通透性增高与盐水组相比,Ang Ⅱ灌注14天后明显升高心脏组织的蛋白酶体的胰蛋白酶样和糜蛋白酶样的活性以及催化亚基（β2i、β5i）的表达水平。体外实验也发现,Ang Ⅱ处理后可显著升高淋巴管内皮细胞（LECs）中胰蛋白酶样和糜蛋白酶样的活性以及β2i和β5i的表达水平。同时,Ang Ⅱ激活LECs中p38 MAPK,而降低MKP5和VE-cadherin的蛋白水平。5.体外应用蛋白酶体抑制剂环氧霉素处理减轻淋巴管内皮细胞的通透性与对照处理组相比,Ang Ⅱ引起的LECs通透性（用伊文思蓝和FITC-葡聚糖检测）增高和VE-cadherin荧光强度的降低完全被氯沙坦（AT1R抑制剂）或环氧霉素逆转。同时,氯沙坦或环氧霉素也可显著逆转Ang Ⅱ引起的p38 MAPK激活和MKP5和VE-cadherin的降低。6.给予环氧霉素处理小鼠减轻心脏水肿,改善心脏重构和心功能障碍与对照盐水组比较,环氧霉素处理小鼠后可明显降低Ang Ⅱ引起的蛋白酶体活性和血压升高,减轻Ang Ⅱ引起的心脏水肿程度,改善Ang Ⅱ引起的淋巴管通透性增高（降低p38 MAPK活性,升高MKP5和VE-cadherin水平）和抑制心脏重构程度（包括心肌肥厚、纤维化、氧自由基产生和CD68⁺巨噬细胞数量）以及改善心功能障碍。研究结论:本研究发现心脏淋巴管生成和淋巴屏障通透性增高均参与了Ang Ⅱ诱导的心脏水肿和重构。Ang Ⅱ通过MKP5/p38 MAPK/VE-cadherin信号通路诱导LECs通透性增高,进而加重心脏水肿、重构和功能障碍。因此,选择性刺激淋巴管生成或抑制蛋白酶体活性可能是治疗高血压心脏重构的新策略。但仍需要进一步研究MKP5/p38 MAPK/VE-cadherin信号在高盐饮食或压力超负荷引起的心脏重构中的作用。更多还原

【Abstract】 Background:Cardiac lymphangiogenesis and integrity play an important role in the maintenance of myocardial tissue fluid balance.The disturbance of lymphangiogenesis is closely related to cardiac edema and remodeling induced by myocardial ischemia/reperfusion（I/R）injury and pressure overload.However,the role of lymphangiogenesis and integrity in the regulation of Ang Ⅱ-induced cardiac remodeling and the underlying mechanism remain unclear.Objective:To clarify the changes of lymphangiogenesis and permeability during Ang Ⅱ-induced cardiac remodeling in mice,and to elucidate the molecular mechanism for cardiac lymphangiogenesis and permeability changes in regulating cardiac remodeling.This study will provide new therapeutic target and strategy for the prevention and treatment of cardiac remodeling.Methods:1.Animal model and treatmentWild-type（WT）C57BL/6J mice,VEGFR-3^f/-mice and VEGFR-3^f/fmice were used to establish cardiac remodeling with infusion of Ang Ⅱ（1000 ng/kg/min）for 3,7 and 14 days.Epoxomicin（0.58mg/kg/day）was injected intraperitoneally for 14 days to investigate the role of proteasome activity in Ang Ⅱ-induced cardiac remodeling.2.Measurement of blood pressure and cardiac functionThe blood pressure at basal condition and at different time points of Ang Ⅱ infusion were monitored by Tail-cuff method.Cardiac function was evaluated by M-type echocardiography,Cardiac parameters include ejection fraction（EF%）,shortening of short axis（FS%）,thickness of anterior and posterior wall of left ventricle（LVAW,LVPW）,and left ventricular internal diameter（LVID）.3.GravimetryThe water content in the hearts of mice was measured by the dry and wet weight of hearts.The formula was:the water content in the hearts of mice（%）=（wet weight-dry weight）/wet weight of mice heart x 100%4.Histological examinationsHeart tissue sections were stained with hematoxylin-eosin（H&E）,Masson trichrome（Masson）,immunohistochemistry（IHC）,wheat germ agglutinin（WGA）,dihydroethidium（DHE）and immunofluorescence（IF）,respectively.5.Lymphatic permeability assays in vivoAnimals:20μl Evans Blue was injected into the foot pad of mice.The popliteal lymph nodes were removed after 16 hours.After grinding,the absorbance（OD）of the lymph nodes was measured at620 nm using a microplate reader.Lymphatic endothelial cells（LECs）in vitro:The permeability of LECs was measured with Evans blue and FITC-dextran.6.Measurement of proteasome activityThe proteins were extracted from mouse heart or LECs,added with fluorescent substrate.The activities of caspase-like,trypsin-like and chymotrypsin-like were measured using a microplate reader.7.Analysis of gene and protein expressionThe m RNA level of each gene was detected by real-time quantitative PCR.The protein levels were measured using Western blot analysis.8.StatisticsAll data in this study were expressed as mean±standard deviation.Statistical analysis using Graph Pad Prism 9 software.Firstly,the normal distribution of the detected data is analyzed.Independent t-test was used to determine the statistical differences between the two groups if the experimental data were in normal distribution,and Mann-Whitney test was used to analyze the data if they were not in normal distribution.One-way ANOVA and two-way ANOVA were used to analyze the differences between the two groups.P<0.05 showed that there was a statistical difference.Results:1.Ang Ⅱ infusion induces increase of systolic pressure,cardiac remodeling,and lymphangiogenesis.After 3,7,14 days of Ang Ⅱ infusion in WT mice,systolic blood pressure,myocardial hypertrophy,fibrosis,inflammatory response,superoxide production,Lyve1⁺/VEGFR3⁺lymphatics,the ratio of Lyve-1⁺lymphatics to cardiomyocytes as well as levels of VEGF-C and VEGFR3 increased in a time-dependent manner.2.Ang Ⅱ infusion enhances lymphatic permeability and cardiac edema.Compared with saline group,the lymphatic permeability（expressed by the OD value of popliteal lymph nodes）and its related mediator p38 MAPK activity were time-dependenly increased in mice.But the protein levels of MKP5 and VE-cadherin were decreased in a time-dependent manner.In addition,myocardial water content（%）was also time-dependently increased in mice.3.VEGFR-3 knockout reduces cardiac lymphangiogenesis,and aggravates Ang Ⅱ-induced increase of lymphatic permeability,cardiac edema,remodeling,and dysfunction.After 14 days of Ang Ⅱ infusion,compared with VEGFR3^f/fcontrol mice,the systolic blood pressure was significantly higher,but the number of lymphangiogenesis（expressed by the Lyve1⁺/VEGFR3⁺lymphatics,the ratio of Lyve-1⁺lymphangiogenesis to cardiomyocytes,and the expression of VEGFR3,p-ERK1/2 and p-AKT）was markedly lower in VEGFR-3 knockout mice.Accordingly,the degree of cardiac remodeling（including myocardial hypertrophy,fibrosis,superoxide production and the number of CD68⁺macrophages）and the related signal pathways and cardiac dysfunction were further aggravated in VEGFR-3 knockout mice.4.Ang Ⅱ upregulates proteasome activity and lymphatic endothelial cell permeability.Compared with saline group,Ang Ⅱ infusion for 14 days significantly increased both trypsin-like and chymotrypsin-like activities as well as the expression of catalytic subunits（β2i andβ5i）in cardiac tissues.Similarly,Ang Ⅱ treatment also had the same effect on proteasome activities and the expressions ofβ2i andβ5i in LECs.In addition,Ang Ⅱ activates p38 MAPK in LECs,and decreased MKP5 and VE-cadherin protein levels.5.Proteasome inhibitor epoxomicin reduced the permeability of lymphatic endothelial cells In vitro.Compared with saline control group,Ang Ⅱ treatment-induced hyperpermeability in LECs（detected by Evans blue and FITC-dextran）and downregulation of VE-cadherin fluorescence intensity were completely reversed by treatment of AT1R inhibitor or epoxomicin.Meanwhile,losartan or epoxomicin could reverse Ang Ⅱ-induced activation of p38 MAPK and decrease of MKP5 and VE-cadherin protein levels.6.Administration of epoxomicin reduces cardiac edema,remodeling,and dysfunction in mice.Compared with the control group,administration of epoxomicin in mice significantly reduced Ang Ⅱ-induced elevation of proteasome activity and blood pressure,attenuated the degree of cardiac edema,lymphatic permeability（as reflected by decrease of p-p38 MAPK,and increase of MKP5 and VE-cadherin protein levels）and cardiac remodeling（as indicated by hypertrophy,fibrosis,superoxide production and number of CD68⁺macrophages）,and improved cardiac dysfunction.Conclusions:Here,our results reveal that both cardiac lymphangiogenesis and lymphatic hyperpermeability are involved in Ang Ⅱ-induced adaptive hypertrophy.Ang Ⅱ induce LEC hyperpermeability leading to cardiac edema,hypertrophic remodeling,and dysfunction likely through MKP5/p38 MAPK/VE-cadherin signaling pathway.Thus,selective stimulation of lymphangiogenesis and/or inhibition of proteasome activity may be novel therapeutic strategies for treating hypertension-induced cardiac remodeling.Further researches are needed to assess the role of MKP5/p38 MAPK/VE-cadherin signaling in other types of hypertrophic remodeling caused by high-salt or pressure overload.更多还原