【作者】 郑浩；

【导师】 李强； 季颖群；

【作者基本信息】 同济大学 ， 内科学（专业学位）， 2023， 博士

【摘要】 研究背景与目的:肺动脉高压（pulmonary artery hypertension,PH）是一种表现为肺血管阻力和肺动脉压力升高,以进行性肺血管和右心室重构为特征的疾病。适应性免疫系统在PH发病机制和进展中具有重要作用,其中巨噬细胞功能异常在免疫系统紊乱及介导血管内皮功能失调和血管重构中发挥关键调节作用,促进PH疾病发生发展。近年的多项研究表明N6腺苷甲基（N6-methyladenosine,m6A）修饰参与调控PH的肺血管病理性重构,但m6A调控子在PH中的功能作用及其潜在调节机制尚未阐明。本课题旨在通过分析m6A调控子在PH肺组织单细胞水平中的表达及生物学功能,探讨m6A甲基化修饰在PH中的潜在作用机制,进一步探索甲基转移酶样蛋白3（METTL3）调控巨噬细胞功能在PH发生进展以及病理性血管和右心室重构中的作用和机制。材料和方法:第一部分;GEO数据库下载野百合碱（Monocrotaline,MCT）诱导的肺动脉高压（MCT-PAH）大鼠模型肺组织的全转录组测序（RNAseq）微阵列数据（GSE149713）,筛选差异表达基因（differentially expressed genes,DEGs）并分析m6A调控子的表达;下载MCT-PAH肺组织单细胞测序（sc RNA）数据,分析巨噬细胞与血管结构细胞之间的细胞交互作用以及其中差异表达的m6A调控子。使用基因本体论（GO）功能分析以及京都基因和基因组百科全书（KEGG）途径对m6A靶基因与巨噬细胞DEGs的交集基因进行功能注释,探讨m6A修饰在巨噬细胞功能调控中的作用。第二部分;首先,培养人髓系白血病单核细胞（human myeloid leukemia mononuclear cells,THP-1）诱导巨噬细胞和分离培养人外周血单个核细胞（Peripheral blood mononuclear cell,PBMC）诱导的巨噬细胞以及分离提取培养小鼠骨髓来源的巨噬细胞（Bone marrow-derived macrophages,BMDM）,在Hypo/LPS（Hypoxia,0.5%O₂;Lipopolysaccharide,LPS 100ng/ml）刺激下,检测METTL3的m RNA和蛋白表达以及炎症因子白细胞介素1β（Interleukin 1 Beta,IL-1β）、诱导型一氧化氮合酶（Inducible Nitric Oxide Synthase,i NOS）的m RNA表达水平;通过mito-sox荧光探针检测活性氧（ROS）水平;通过q RT-PCR检测糖酵解关键酶6磷酸果糖2激酶3（6-phosphofructo-2-kinase 3,PFKFB3）、乳酸脱氢酶（Lactate Dehydrogenase A,LDHa）的m RNA表达水平。进一步构建METTL3小干扰RNA（small interfering RNA,si RNA）,并通过q RT-PCR及WB检测METTL3的m RNA和蛋白水平的敲减效率;另外,运用METTL3抑制剂STM2457抑制METTL3的功能,并通过q RT-PCR检测炎症因子IL-1β、i NOS等的表达,ELISA检测上清液中IL-1β的释放,mito-sox荧光探针检测ROS水平变化,q RT-PCR检测PFKFB3、LDHa的m RNA表达以及细胞培养上清液中乳酸的释放。进一步通过Crispr/cas9技术构建髓系METTL3条件基因敲除小鼠(METTL3^fl/fl Lyz2^cre+),提取BMDM,在LPS诱导下检测i NOS m RNA和蛋白表达,以及刺激后2h,4h,12h的IL-1βm RNA表达;在IL-4刺激下检测精氨酸酶1（Arginase-1,Arg1）的m RNA和蛋白的表达水平。最后,收集不同组的巨噬细胞上清液与肺动脉平滑肌细胞（Pulmonary arterial smooth muscle cells,PASMC）、成纤维细胞进行共培养,通过WB检测TGFβ（ransforming growth factor beta）、SMAD3、MMP9（matrix metalloprotein 9,MMP9）、vimentin等的表达,以及成纤维肌化相关指标,α-SMA,Col1a1,Col1a3等;并通过Transwell检测重组蛋白IL-1β对PASMC迁移的作用。第三部分;分析PH患者肺组织病理标本METTL3表达;建立小鼠Sugen/Hypoxia（Su Hx）PH动物模型,分为腹腔注射METTL3抑制剂STM2457组以及对照溶剂组。建立METTL3^fl/fl和METTL3^fl/fl Lyz2^cre+Su Hx PH模型。完善心脏超声和血流动力学检测;评估PASMC增殖以及血管重塑;检测肺组织IL-1β、i NOS、Arg1、IL-10等相关基因的表达;通过肺组织CD86、CD206免疫荧光染色检测巨噬细胞分化状态,免疫荧光染色检测肺组织i NOS的表达和细胞核转录因子P65（Nuclear factor NF-kappa-B p65,P65-NFκB）通路变化。结果:第一部分:一、m6A甲基化调控子在MCT-PAH大鼠模型肺组织中表达具有差异性。高表达的DEGs显著富集在细胞因子-受体相互作用、ECM（extracellular matrix）-受体相互关系、粘附、p53信号通路和磷脂酰肌醇3-激酶和AKT/蛋白激酶b（PI3K-Akt）等信号通路。二、MCT-PAH肺组织sc RNA数据鉴定存在PAEC、PASMC、成纤维细胞、巨噬细胞、T细胞、NK细胞、中性粒细胞、树突状细胞（dendritic cell,DC）等亚群。三、METTL3在巨噬细胞中呈现差异性表达,m6A甲基化阅读蛋白HNRNPC在巨噬细胞中表达上调,YTHDC1,ZC3H13,YTHDF2在巨噬细胞中表达下调。四、巨噬细胞与PASMC、PAEC、成纤维细胞和T细胞通过受体配体对发生交互作用。与对照组相比,MCT-PAH中巨噬细胞和PASMC之间的相互作用相关的COL1A1/CD44、COL4A1/CD44,FN1/CD44和LAMC1/CD44以及COL6A2/CD44信号通路增加。五、m6A甲基化可参与巨噬细胞的重要生物学功能,包括氧化应激、肿瘤坏死因子（tumor necrosis factor,TNF）信号通路,凋亡,氧化磷酸化,IL-17信号通路,丝裂原活化蛋白激酶（MAPK）信号通路等。第二部分:一、Hypo/LPS诱导BMDM促炎因子IL-1β、iNOS显著升高（p<0.01）。低氧刺激THP-1巨噬细胞和BMDM的METTL3 m RNA以及蛋白的表达水平较对照组显著升高（p<0.01）,同时总RNA的m6A甲基化水平上调（p<0.01）。二、PBMC巨噬细胞在低氧刺激下IL-1β表达升高（p<0.01）,并且STM2457组显著抑制IL-1β的表达（p<0.01）;另外,在THP-1巨噬细胞敲减METTL3或STM2457抑制METTL3（p<0.01）,均抑制炎症因子IL-1β的表达（p<0.01）。三、在LPS刺激下Mettl3^fl/flLyz2^cre+组BMDM IL-1β的表达较Mettl3^fl/flLyz2^cre-组显著下降（p<0.01）。STM2457可抑制Hypo/LPS诱导的BMDM炎症因子IL-1β和i NOS m RNA和蛋白水平（p<0.01）。LPS刺激Mettl3^fl/fl Lyz2^Cre的BMDM i NOS的m RNA水平在2h,4h较对照组显著下降（p<0.05）,同时显著抑制i NOS蛋白表达。Mettl3^fl/flLyz2^Cre+BMDM在IL-4诱导下Arg1的m RNA和蛋白表达显著升高（p<0.01）。四、低氧诱导巨噬细胞线粒体ROS水平升高,且STM2457抑制低氧诱导的巨噬细胞线粒体ROS水平的升高。五、PH肺组织巨噬细胞中多种糖酵解关键酶表达失调,其中己糖激酶2（hexokinase 2,HK2）、PFKFB3、LDHa在巨噬细胞中表达上调（p<0.05）;低氧刺激BMDM糖酵解关键酶HK2、PFKFB3、LDHa高表达（p<0.05）;另外,较常氧组,低氧促进BMDM的乳酸水平产生和释放（p<0.01）,且STM2457抑制低氧诱导BMDM乳酸的释放（p<0.05）。五、Hypo/LPS巨噬细胞上清液促进PASMC的TGFβ/SMAD3的表达,STM2457组上清液抑制PASMC的TGFβ/SMAD3的表达。重组细胞生长因子IL-1β促进PASMC的迁移。STM2457组的巨噬细胞上清液可以抑制成纤维细胞MMP9、vimentin、TGFβ/SMAD3等的表达,并且抑制肌成纤维活化标志物α-SMA、col1a1、col1a3表达（p<0.05）。第三部分:一、METTL3在PH患者及SuHxPH动物模型肺组织中表达较对照组均升高（p<0.01）。与对照组相比,PH CD86⁺促炎型巨噬细胞比例明显升高。二、与对照组相比,Su Hx PH组肺动脉血流加速时间/射血时间（PAAT/PAET）比值下降（p<0.05）,右心室游离壁厚度（right ventricular free wall distance,RVFWD）及右心室收缩期内径（right ventricular internal dimension,RVID）增大（p<0.01）,RVSP（Right Ventricular Systolic Pressures,RVSP）显著升高（p<0.05）;与Su Hx PH组比较,Su Hx+STM2457组的PAAT/PAET升高（p<0.01）,RVFWD、RVID和RVSP均下降（p<0.05）。与Mettl3^fl/fl组相比,Mettl3^fl/fl Lyz2^Cre+组RVSP、右心室壁厚度显著下降。三、与对照组相比,Su Hx组的肺组织及心脏组织纤维胶原沉积面积分数增多（p<0.05）;与Su Hx PH组相比,Su Hx+STM2457组肺血管重塑好转,肺组织及心脏组织的胶原沉积面积分数（p<0.01）。与Mettl3^fl/fl组相比,Mettl3^fl/fl Lyz2^Cre+组肺组织及心脏胶原沉积显著下降;肺组织ki67阳性细胞下降。四、与对照组相比,Su Hx组的肺组织中IL-1β、i NOS等促炎相关因子m RNA表达水平上调（p<0.05）;与Su Hx PH组相比,Su Hx+STM2457组的IL-1β、i NOS表达下降（p<0.05）,IL-10、Arg1等抑制炎症相关因子表达升高（p<0.05）。五、与Mettl3^fl/fl组相比,Mettl3^fl/fl Lyz2^Cre+组肺组织CD86⁺和CD206⁺巨噬细胞下降。与对照组相比,Su Hx PH组肺组织中CD86⁺巨噬细胞和CD206⁺巨噬细胞升高（p<0.01和p<0.05）,NFκB-P65和i NOS的荧光强度增高;与Su Hx PH组比较,Su Hx+STM2457组CD86⁺和CD206⁺巨噬细胞在肺组织中比例下降（p<0.01）;NFκB-P65和i NOS的荧光强度下降。结论:一、野百合碱诱导的肺动脉高压大鼠肺组织巨噬细胞METTL3具有差异性表达。二、在低氧/LPS作用下,巨噬细胞METTL3、m6A甲基化水平升高;敲除或抑制METTL3,经典活化巨噬细胞i NOS和IL-1β的表达减少、ROS水平降低、PFKFB3以及乳酸水平下降,并激活平滑肌细胞、成纤维细胞TGFβ/SMAD3通路。三、巨噬细胞敲除或抑制METTL3(METTL3^fl/flLyz2^cre+、STM2457)Su Hx PH,通过NFκB/i NOS信号通路,调节巨噬细胞分化状态,改善PH血流动力学,抑制肺血管和右心重构。更多还原

【Abstract】 Background and Objective:Pulmonary artery hypertension（PH）is characterized by pulmonary vascular resistance increasing,leading to progressive pulmonary vascular remodeling and right ventricular hypertrophy.The adaptive immune system mediating in the pathogenesis and progression of PAH.Moreover,macrophage dysfunction plays a key role in immune system disorders,mediating endothelial dysfunction and vascular remodeling and promoting the occurrence and development of PH.In recent years,studies have shown that N6 methyladenosine（m6A）modification is involved in regulating pulmonary vascular pathological remodeling in PH.However,the potential regulatory mechanisms of m6A regulators in PH have not been elucidated.The aim of this study is to analyze the expression and biological function of m6A regulators at the single-cell level in PH lung tissue and explore the potential mechanism of m6A methylation modification in PH,especially,the role and mechanism of methyltransferase-like protein 3（METTL3）in promoting the progression of PH,incluing pathological vascular remodeling,and right ventricular remodeling,via regulating macrophage function.This study will reveal new mechanisms and therapeutic strategies of PH pathological remodeling.Methods:Firstly,download the whole transcriptome sequencing（RNAseq）microarray data（GSE149713）of lung tissue of monocrotaline（MCT）-induced PAH（MCT-PAH）rat model from GEO database.Next,screened differentially expressed genes（DEGs）and analyzed the expression of m6A regulator.Download MCT-PAH lung tissue single cell sequencing（sc RNA）data.Cell interaction analysis and the differentially expressed m6A regulator in PAEC,PASMC,fibroblasts,macrophages,natural killer cell（NK）,B cells,T cells,and regulatory T cells（Tregs）were conducted.The intersection genes of m6A target genes and macrophage DEGs were functionally annotated using Gene Ontology（GO）functional analysis and Kyoto Encyclopedia of Genes and Genomes（KEGG）approaches to reveal the role of m6A modification in the regulation of macrophage function in PH.Secondly,human myeloid leukemia mononuclear（THP-1）macrophages,induced human peripheral blood mononuclear cell（PBMC）macrophages and bone marrow derived macrophages（BMDM）were isolated and extracted from mouse bone marrow were cultured.Detect the m RNA and protein expression levels of METTL3 under hypoxia conditions;the m RNA expression level of inflammatory factor interleukin-1β（Interleukin 1 Beta,IL-1β）,inducible nitric oxide synthase（i NOS）were detect;mito sox fluorescent probes were used to detect the reactive oxygen species（ROS）levels;The m RNA expression levels of glycolytic related genes 6-phosphofructose 2-kinase 3（PFKFB3）and lactate dehydrogenase A（LDHa）were detected.Next,detect the knockdown efficiency of METTL3 m RNA and protein levels by small interfering RNA（si RNA）of METTL3.Alao,the METTL3 inhibitor STM2457 were used to inhibit the function of METTL3;Detect the m RNA expression of inflammatory factor IL-1β,i NOS and the level of IL-1βin supernatant;what’more,the level of ROS were detected in STM2457 groups;the m RNA expression levels of PFKFB3 and LDHa,as well as the release of lactate in cell culture supernatant were detected.Myeloid METTL3conditional gene knockout mice(METTL3^fl/flLyz2^cre+)were constructed using Crispr/cas9 technology in this study,extracted BMDM,and detected i NOS m RNA and protein levels under LPS induction,as well as the m RNA expression of IL-1β;Also,the m RNA and protein expression levels of arginase-1（Arg1）under IL-4 stimulation were detected.In order to explore the effect of macrophages deficient of METTL3 on vascular structural cells,different groups of macrophage supernatants were collected and cocultured with PASMC and fibroblasts.The expression of TGFβ（transforming growth factor beta）,SMAD3,MMP9（matrix metalloprotein 9,MMP9）,vimentin were detected.The fibromuscular related indicators m RNA expression was detected,incluingα-SMA,Col1a1,Col1a3.Transwell were performed to detect PASMC migration under the condition of Recombinant Protein IL-1β.Thirdly,Sugen/Hypoxia（Su Hx）PH mouse model（sugen was intraperitoneally injected with 20mg/kg/week,10%O2）,was divided into two groups,intraperitoneal injection of METTL3 inhibitor STM2457 group and control solvent group.In addition,METTL3^fl/fl and METTL3 ^fl/fl Lyz2^cre+Su Hx PH model were established.Right ventricular systolic pressure（RVSP）,right ventricular free wall distance（RVFWD）and the right ventricular internal diameter were measured;HE staining were performed to observe changes of pulmonary vascular structure and right ventricular remodeling;CD31 andα-Smooth muscle actin（α-smooth muscle Actin,α-SMA）immunofluorescence（IHC）staining were used to evaluate PASMC proliferation and vascular remodeling;pulmonary and ventricular fibrosis were detected;the m RNA expression of IL-1β,i NOS,Arg1,IL-10 and other related genes were investgated of lung tissue;CD86 and CD206 IHC staining were performed to analysis macrophage differentiation.Also,i NOS and nuclear factor NF kappa B p65,（P65-NFκB）IHC Staining were performed.Results:Firstly,the expression of m6A methylation regulators were dysregulated in the lung tissue of MCT-PAH rat model.GO and KEGG analysis of DEGs showed that highly expressed DEGs were significantly enriched in cytokine receptor interactions,ECM（extracellular matrix）receptor interactions,adhesion,p53 signaling pathways,and phosphatidylinositol 3-kinase and AKT/protein kinase b（PI3K Akt）signal pathways.The sc RNA dataset was identified by cell classification using key marker genes,including PAEC,PASMC,fibroblasts,macrophages,T cells,NK cells,neutrophils,dendritic cells（DC）,and other subpopulations.By analyzing the DEGs at the single-cell level in PH lung tissue,the results revealed that METTL3 showed differential expression in macrophages,while the m6A methylated reading protein HNRNPC was upregulated in macrophages,YTHDC1,ZC3H13,and YTHDF2 were downregulated in macrophages.Cellular communication analysis revealed interactions between macrophages and PASMC,PAEC,fibroblasts,and T cells.Compared with the control group,the interaction between macrophages and PASMC in MCT-PAH was associated with an increase in the COL1A1/CD44,COL4A1/CD44,FN1/CD44,LAMC1/CD44,and COL6A2/CD44 signaling pathways.The transcriptome DEGs of macrophages were intersected with target genes of m6A methylation regulator,and biological functional analysis was performed.It was found that m6A methylation participated in important biological functions of macrophages,including oxidative stress,tumor necrosis factor（TNF）signal pathway,apoptosis,oxidative phosphorylation,IL-17 signal pathway,mitogen activated protein kinase（MAPK）signal pathway,etc.Secondly,pro-inflammatory factor IL-1β,i NOS significantly increased in macrophage under Hypo/LPS conditions（p<0.01）.The expression levels of METTL3m RNA and protein in THP-1 macrophages and BMDM stimulated by hypoxia were significantly increased compared to the control group（p<0.01）;meanwhile,the m6A methylation level of total RNA was upregulated（p<0.01）.IL-1βelevated in PBMC macrophages under hypoxia conditon（p<0.01）and STM2457 group significantly inhibited IL-1βExpression（p<0.01）;Knockdown of METTL3 or inhibit METTL3using STM2457 on THP-1 macrophages（p<0.01）,both of which inhibit the inflammatory factor IL-1βExpression（p<0.01）.In addition,the expression of IL-1βwas significantly reduced compared to the Mettl3^fl/flLyz2^cre–group under LPS stimulation（p<0.01）.STM2457 inhibits the expression of induced inflammatory factor IL-1βin BMDM under Hypo/LPS（p<0.01）,what’s more,STM2457 inhibits i NOS m RNA and protein levels in Hypo/LPS.Mettl3^fl/flLyz2^Cre+BMDM showed a decrease in i NOS m RNA levels（p<0.05）,and significantly inhibited i NOS protein expression under LPS stimulation.Also,Mettl3^fl/flLyz2^Cre+BMDM significantly increased the m RNA and protein expression of Arg1 under IL-4 induction（p<0.01）.Increasing of mitochondrial ROS levels in macrophages under hypoxia and STM2457 inhibit the resease of mitochondrial ROS.Single cell sequencing data analysis found that the expression of a variety of key glycolytic enzymes in macrophages of PH lung tissue was disordered.Among them,the expression of hexokinase 2（HK2）,PFKFB3,and LDHa in macrophages was up-regulated in the lung sample of PH（p<0.05）,and HK2,PFKFB3,and LDHa in BMDM were highy expressed under hypoxia（p<0.05）.Compared to the normoxia group,hypoxia promoted the production and release of lactate levels in BMDM（p<0.01）,while STM2457 inhibited the release of lactate under hypoxia（p<0.05）.Thirdly,the expression of TGFβand SMAD3 of PASMC were increasing cultured with Hypo/LPS macrophage supernatant,additionly,the level of TGFβand SMAD3 of STM2457 group were decreasing compared with Hypo/LPS group.The macrophage supernatant of STM2457 group exhibited decreasing expressed MMP9,vimentin,and TGFβ/SMAD3 in fibroblasts and downregulating myofibroblast activation markers,incluingα-SMA,col1a1,and col1a3（p<0.05）.Also,Recombinant Cell Factor IL-1βpromoted the migration of PASMC.The expression of METTL3 in lung tissue of PH patients and rodent PH animal models was higher than that of the control group（p<0.01）.Furthermore,the proportion of PH CD86⁺pro-inflammatory macrophages was significantly increased compared with the control group.The results showed that the ratio of PAAT/PAET in the Su Hx PH group significantly decreased compared with the control group（p<0.05）,and the Su Hx+STM2457 group was significantly higher than that in the Su Hx PH group（p<0.01）.The RVFWD and RVID of the Su Hx PH group increased compared to the control group（p<0.01）,and the RVFWD and RVID of the Su Hx+STM2457 group decreased compared to the Su Hx PH group（p<0.05）.The RVSP of the Su Hx PH group was significantly higher than that of the control group,while the RVSP of the Su Hx+STM2457 group was lower than that of the Su Hx PH group（p<0.05）.Compared with Mettl3^fl/fl group,the thickness of RVSP and Right ventricle wall in Mettl3^fl/fl Lyz2^Cre+group decreased significantly.HE staining of lung tissue andα-SMA fluorescence showed pulmonary vascular remodeling alleviated in the STM2457 group compared to the Su Hx PH group.The collagen deposition area fraction of lung and heart tissue in the Su Hx group increased（p<0.05）,while the collagen deposition area fraction of lung and heart tissue in the Su Hx+STM2457 group was decrfeasing in the Su Hx PH group（p<0.01）.Compared with the Mettl3^fl/flgroup,the Mettl3^fl/fl Lyz2^Cre+group showed a significant decrease in collagen deposition in the lung tissue and heart.Upregulation of m RNA expression levels of pro-inflammatory factors IL-1β,i NOS（p<0.05）,and The expression of IL-1β,i NOS decreased in Su Hx+STM2457 group compared to the Su Hx PH group（p<0.05）,while the Su Hx+STM2457 group showed an increase in the expression of inflammatory related factors such as IL-10 and Arg1 compared to the Su Hx PH group（p<0.05）.Compared with the Mettl3^fl/fl group,CD86+and CD206+macrophages showed a decrease in lung tissue in the Mettl3^fl/flLyz2^Cre+group.CD86⁺macrophages and CD206⁺macrophages in the Su Hx PH group increased in lung tissue（p<0.01）,while the proportion of CD86⁺macrophages in the Su Hx+STM2457 group decreased compared to the Su Hx group（p<0.01）;meanwhile,the CD206⁺macrophages in the Su Hx+STM2457 group decreased in lung tissue compared to the Su Hx PH group（p<0.01）.The fluorescence intensity of NFκB-P65 and i NOS in lung tissues of Su Hx PH was significantly higher than that of the control group;while Su Hx STM2457 group showed decreasing fluorescence intensity compared to Su Hx PH group.Conclusion:Firstly,disorder of m6A regulators was observed in MCT-PAH lung samples and METTL3 was differentially expressed in macrophages.Next,Under the condition of hypoxia/LPS,the methylation levels of m6A and the expression of METTL3 in macrophages were increased;Knockout or inhibition of METTL3,the expression of i NOS and IL-1β,ROS level,PFKFB3 and lactic acid level was decreased in classical activation of macrophages,which inhibited TGFβ/SMAD3 pathway in smooth muscle cells and fibroblasts.Thirdly,Macrophage knockout or inhibition of METTL3（METTL3fl/fl Lyz2cre+,STM2457）Su Hx PH regulates macrophage differentiation status,improves PH hemodynamics,and inhibits pulmonary vascular and right heart remodeling by NFκB/i NOS signaling pathway更多还原