【作者】 陈德伟；

【导师】 高钰琪；

【作者基本信息】 第三军医大学 ， 高原医学， 2014， 博士

【摘要】 研究背景：低氧性肺动脉高压(Hypoxia Pulmonary Hypertension，HPH)是由低氧引起的以持续肺动脉压力增高、肺血管结构改建，并伴有右心室肥大的慢性进行性疾病，是高原心脏病和肺源性心脏病发病的中心环节。HPH的形成是一个复杂的病理生理过程，已有研究表明，炎症免疫反应和血管活性物质分泌失调是其中的两个重要机制。炎症免疫细胞在血管壁的黏附聚集是炎症发生的起始环节，而血管内皮细胞中ICAM-1、VCAM-1和E-selectin等细胞黏附分子(cell adhesion molecules，CAMs)是其中的关键作用分子。低氧时内皮细胞中CAMs基因转录激活，促进循环免疫细胞的黏附聚集和血管周围炎症微环境的形成，引起肺动脉高压和肺血管结构改建。内皮素1(Endothelin1，ET-1)是内皮细胞分泌的缩血管作用最强的活性多肽，低氧可诱导ET-1表达上调，参与HPH的发生。目前已知，CAMs和ET-1基因表达受核因子kB(Nuclear factor kappaB，NF-kB)、低氧诱导因子1α(Hypoxia inducible factor1α，HIF-1α)和巨核细胞性白血病因子1(Megakaryocytic leukemia1，MKL1)等转录因子调节。低氧时上述转录因子进入核内增多，与启动子序列特异结合，启动基因转录和表达。真核细胞的DNA由组蛋白包裹形成核小体乃至染色体，基因转录时首先需要打开核小体结构，暴露启动子序列，即染色质重构。Brahma-related gene1(Brg1)和brahma(Brm)是哺乳动物染色质重构复合物SWI(mating-type switching)/SNF(sucrosefermentation)的核心ATP酶亚单位，两者结构相似、功能互补。Brg1和Brm可以水解ATP释放能量实现对核小体的重排和置换，从而调节靶基因的转录。研究表明，Brg1和Brm在胚胎发育过程中的血管平衡维持和应激相关疾病发生中都具有重要作用。最新研究发现，HIF-1α可以招募Brg1和Brm至其靶基因，参与细胞低氧反应，但Brg1和Brm是否及如何参与低氧诱导的CAMs及ET-1转录调控，进而促进HPH的发生和发展，迄今未见报道。因此，本研究在明确低氧诱导肺血管及内皮细胞中Brg1和Brm基因表达的基础上，研究其对内皮细胞CAMs和ET-1转录调控的作用及机制；特异干扰内皮细胞中的Brg1和Brm，进一步探讨Brg1和Brm在肺血管结构改建和HPH形成中的作用和机制。为干预或预防HPH提供新的思路和有效靶点。方法：1.模拟海拔5000m高原28d复制雄性SD大鼠HPH模型，分离肺动脉；将人肺动脉内皮细胞(Human pulmonary arterial endothelial cell，HPEC)和人脐静脉内皮细胞(Human umbilical vein endothelial cell，HUVEC)低氧(1%O2)处理24h；RT-qPCR及WB法检测肺动脉及内皮细胞中Brg1和Brm基因的mRNA及蛋白表达。2.通过外源性质粒转染内皮细胞(HPEC、HUVEC)过表达或siRNA转染干扰内源性的Brg1和Brm基因，采用报告基因法检测CAMs启动子转录活性；采用RT-qPCR及WB方法检测CAMs基因的mRNA及蛋白表达；采用细胞黏附实验检测内皮细胞的黏附功能。为了进一步探讨低氧时Brg1和Brm激活CAMs转录的机制，采用染色质免疫共沉淀(Chromatin immunoprecipitation，CHIP)方法检测低氧后Brg1和Brm与内皮细胞中CAMs启动子的结合水平；siRNA转染干扰Brg1和Brm后，进一步运用CHIP方法检测NF-kB/p65与CAMs启动子的结合水平，同时运用CHIP方法检测CAMs启动子周围AcH3和H3K4M3等组蛋白修饰变化。3.内皮细胞(HPEC、HUVEC)中外源性质粒转染过表达或siRNA干扰内源性的Brg1和Brm基因，采用报告基因法检测ET-1启动子的转录活性；采用RT-qPCR及ELISA法检测ET-1的mRNA表达；采用CHIP方法检测低氧后特异结合在内皮细胞(HUVEC) ET-1启动子上的Brg1和Brm以及MKL1蛋白水平；siRNA转染干扰Brg1和Brm后，采用CHIP方法检测ET-1启动子周围AcH3和H3K4M3等组蛋白修饰变化。4.将雄性C57/BL6小鼠随机分为常氧组、低氧组、低氧对照病毒组和低氧Brg1/Brm干扰病毒组。常氧组和低氧组给予生理盐水，低氧对照病毒组给予对照病毒液，低氧Brg1/Brm干扰病毒组尾静脉注射内皮细胞特异性Brg1/Brm干扰质粒慢病毒敲减Brg1和Brm基因表达；低氧各组置于低压氧舱内模拟海拔5000m低氧处理28d复制小鼠HPH模型。采用导管法检测右心室血流动力学指标；采用HE染色及免疫组化(Immunohistochemisty， IHC)染色检测肺血管改建，采用免疫荧光(Immunofluorescence，IF)法检测肺血管内皮细胞中Brg1和Brm的表达以及肺血管周围炎症细胞浸润；采用RT-qPCR法检测肺组织中CAMs的表达；采用ELISA法检测肺组织匀浆IL-1β、IL-6、MCP-1、TNF-α等炎症因子的表达；采用CHIP法检测低氧后肺组织中Brg1和Brm与ICAM-1启动子的结合水平。结果：1.低氧可显著上调SD大鼠的肺动脉(5000m，28d)和内皮细胞(HPEC、HUVEC，1%O2，24h) Brg1和Brm的mRNA和蛋白表达。2.内皮细胞(HPEC、HUVEC)中过表达Brg1和Brm能显著增强低氧(1%O2，24h)诱导的CAMs启动子转录活性，且该作用依赖于其酶催化活性；过表达Brg1和Brm显著上调内皮细胞中内源性CAMs的mRNA和蛋白表达；敲减Brg1和Brm基因可显著抑制低氧诱导的CAMs启动子转录活性增强和内皮细胞内源性CAMs表达，且内皮细胞与白细胞的黏附作用降低。CHIP实验显示低氧能显著增加Brg1和Brm在内皮细胞CAMs启动子上p65的结合区域的结合；干扰内皮细胞中的Brg1和Brm能显著抑制p65与CAMs启动子的结合，CAMs启动子周围AcH3和H3K4Me3等具有转录活化作用的组蛋白也显著减少，Pol II的招募也显著减少。3.内皮细胞(HUVEC)中过表达Brg1和Brm能显著增强低氧诱导的ET-1启动子转录活性，酶失活的Brg1和Brm并不增强ET-1启动子转录活性；干扰内皮细胞(HPEC、HUVEC)中的Brg1和Brm能显著抑制ET-1启动子的转录活性以及ET-1的表达和分泌；CHIP实验发现低氧能显著增加Brg1和Brm以及MKL1在内皮细胞(HUVEC) ET-1启动子上的结合；干扰Brg1和Brm后，ET-1启动子周围AcH3和H3K4Me3等具有转录活化作用的组蛋白也显著减少。4.成功构建HPH小鼠模型，IF结果显示干扰病毒组小鼠肺血管内皮细胞中Brg1和Brm的表达显著降低；伴随着肺血管内皮细胞中Brg1和Brm的表达抑制，肺组织中CAMs基因的mRNA表达也显著降低，小鼠右心室收缩压和肥厚指数也显著降低，肺血管厚度显著变薄，肺血管重构显著改善，肺血管周围炎症细胞浸润显著减少，肺组织匀浆中IL-1β、IL-6、MCP-1的分泌显著降低。CHIP结果显示HPH小鼠肺组织中的Brg1和Brm被更多的招募到ICAM-1的启动子上。结论：1. HPH大鼠肺血管中Brg1和Brm基因表达增多，内皮细胞可能是其中一种重要效应细胞；2. Brg1和Brm介导低氧内皮细胞中CAMs转录和表达，参与调节低氧内皮细胞与白细胞的黏附作用，其作用机制依赖与NF-kB/p65的相互作用，以及对CAMs启动子周围的组蛋白修饰的影响；3. Brg1和Brm参与调控低氧内皮细胞中ET-1转录和表达，其作用机制依赖与MKL1的相互作用及其对ET-1启动子周围组蛋白修饰的调节作用；4.特异干扰HPH小鼠内皮细胞中Brg1和Brm基因，可显著降低右心室收缩压、抑制肺血管重构和右心室肥厚；其机制与CAMs及ET-1转录抑制并表达降低、肺血管周围炎症细胞浸润减少和炎症细胞因子表达降低密切相关；5.肺血管内皮细胞中的Brg1和Brm基因在HPH的形成中发挥重要作用，有望成为干预或防治HPH的有效靶点。更多还原

【Abstract】 Background：Hypoxia Pulmonary Hypertension is a chronic progressive disease which caused bylow oxygen, characterized by elevated pulmonary hypertrophy and pulmonary vascularremodeling and associated with right ventricular hypertrophy. The formation of HPH is acomplex pathophysiological process. Pulmonary vasoconstriction, inflammation, cellproliferation and transformation, all of them were involved in HPH. Hypoxic pulmonaryvasoconstrictions mediated by endothelin, which secrete from vascular endothelial cells,play an important role in the development of HPH. In addition, inflammation may play animportant role in this process. Elevated inflammation is invariably initiated by theaggregation and adhesion of immune cells on the vessel wall. Pivotal to this process is theadhesion of leukocytes to the vessel wall facilitated by the up-regulation of cell adhesionmolecules (CAMs) in vascular endothelial cells. Therefore, accelerated transcription ofCAMs genes in hypoxic endothelial cells represents a key event that foretells enhancedleukocyte adhesion, perpetuated vascular inflammation, and eventually the development ofHPH. As we know，CAMs and ET-1transactivation were regulated by nuclear factor kappaB (NF-kB), hypoxia inducible factor1α (HIF-1α) and megakaryocytic leukemia1(MKL1).These transcription factors combined with specific promoter sequence under hypoxia, tostart CAMs and ET-1transcription and expression. But the epigenetic switch that dictatesCAMs and ET-1transactivation in response to hypoxia in endothelial cells leading up toHPH is not fully appreciated.Unlike the prokaryotic organisms, eukaryotic genes are wrapped by histones intoindividual nucleosomes to form chromatin that needs to be unfolded in order for the basictranscription machinery to gain access. Brahma-related gene1(Brg1) and brahma (Brm)are the catalytic components of the mammalian chromatin remodelling complex playingimportant roles in maintaining vascular homoeostasis in embryogenesis and promoting a pathogenic agenda under stress conditions. This remodelling complex utilizes ATP tomobilize nucleosomes and alter transcription. Brg1and Brm playing important roles inmaintaining vascular homeostasis in embryogenesis and promoting a pathogenic agendaunder stress conditions. Brg1and Brm were also involved in cellular hypoxia reactionwhich was mediated by HIF-1alpha. However, whether Brg1/Brm is upregulated byhypoxia stress and accelerated the CAMs and ET-1genes in hypoxic endothelial cells. Andthe transcription mechanism of CAMs and ET-1involved in HPH development is not fullyunderstood.Therefore, this study was undertaken to observe the expression levels of Brg1and Brmin isolated pulmonary arteries of HPH rats and cultured human endothelial cells. Andobserve the regulation of transactivation of CAMs and ET-1genes in endothelial cells inresponse to hypoxic stress. In addition, we constructed the endothelial-specific vector thatencodes shRNA targeting both Brg1and Brm, to test the role of Brg1and Brm in thehypoxia-induced vascular inflammation and pulmonary vascular remodeling. As such,targeting Brg1/Brm in endothelial cells may yield promising strategies in the interventionand/or prevention of HPH.Methods：1. SD rats were housed in a hypobaric hypoxia chamber (Simulated altitude:5000m)for4weeks to induce hypoxic pulmonary hypertension. Pulmonary arteries were thenisolated to measure the expression of Brg1and Brm by RT-qPCR and Western blot. HPEC、HUVEC were exposed to1%O2, the expressions of Brg1and Brm were also detected.2. Over-expression of exogenous Brg1and Brm or knocked down the expression ofendogenous Brg1and Brm using siRNA in endothelial cells. The promoter activities ofCAM genes induced by hypoxia were detected by reporter assays. The endogenous CAMsmessages induced by hypoxia were also detected by RT-qPCR and Western blot. In vitro,adhesion assay was used to detect the interaction between endothelial cells and leukocytes.To further explore the interplay between p65and Brg1/Brm in hypoxia-inducedtransactivation of CAMs genes, we knocked down the expression of p65or Brg1/Brm withsiRNA in endothelial cells. ChIP assay to detect the combination of NF-kB/p65orBrg1/Brm with CAMs promoter. We also detect the histone modifications surrounding theCAMs promoters by ChIP assay 3. Over-expression of exogenous Brg1and Brm or knocked down the expression ofendogenous Brg1and Brm using siRNA in endothelial cells. The promoter activitie of ET-1gene induced by hypoxia was detected by reporter assays. The endogenous ET-1messagesinduced by hypoxia were also detected by RT-qPCR and ELISA. ChIP assay was used todetect the combination of Brg1/Brm with ET-1promoter. The histone modificationssurrounding the ET-1promoter was also detectd by ChIP assay.4. Male C57/BL6mice were randomly divided into normoxia group, hypoxia group,control virus group and interfere virus group. The last three group were housed in ahypobaric hypoxia chamber (Simulated altitude:5000m) for4weeks to induce hypoxicpulmonary hypertension. In the meantime, these mice were injected via tail veinphysiological saline or lentiviral particles carrying a SCR or endothelial-specific vector thatencodes shRNA targeting both Brg1and Brm. Hemodynamic and pulmonarypathomorphology data were gathered. The hypertrophy of right ventricle was evaluated bythe ratio of weight of the right ventricle (RV) to the weight of the left ventricle (LV) plusventricular septal (VS) and RV weight over body weight. The remodelling of pulmonaryvasculature, as measured by vessel wall thickness, was detected by HE or IHC. Therecruitment of immune cells to the vessels was detected by IF. The activities of cytokineIL-1β、IL-6、MCP-1and TNF-α in lung tissue homogenate extracts was detected by ELISA.The combination of Brg1/Brm with ICAM-1promoter in lung tissue was also detectd byChIP assayResults：1. The levels of Brg1and Brm were significantly elevated in HPH rats as opposed toage-and sex-matched control rats. Exposure to1%O2caused an up-regulation in themessage levels of Brg1and Brm in both HUVEC and HPECs.2. Brg1and Brm were able to enhance the activation of CAMs promoters by hypoxia.Point mutations (ED, enzyme dead) that interfere with the catalytic domain deprived Brg1and Brm of their ability to activate CAMs promoters under hypoxic conditions. Moreimportantly, over-expression of Brg1and Brm led to a marked increase in the levels ofendogenous adhesion molecules. When knocked down the expression of endogenous Brg1and Brm using siRNA, induction of CAMs promoter activities by hypoxia was severelycrippled. Furthermore, the induction of endogenous CAMs messages by hypoxia was also limited in both HUVEC and HPEC. In vitro adhesion assay also revealed that theinteraction between endothelial cells and leukocytes was weakened by the elimination ofBrg1and Brm. ChIP assay indicates there was a dramatic increase in the occupancies ofBrg1and Brm on the promoter region of the CAMs genes spanning the NF-kB site underhypoxic conditions. Consistent with these observations, re-ChIP assays demonstrate thathypoxia promoted the interaction between Brg1/Brm and NF-kB/p65on the CAMspromoters. Reciprocally, depletion of Brg1or Brm negatively affected the binding of p65on all three CAMs promoters. Ablation of Brg1and Brm greatly reduced the levels of AcH3and H3K4Me3, all of which herald transcriptional activation, on the CAMs promoterregions. As a result, the recruitment of RNA polymerase II (Pol II) was hampered probablyleading to a decelerated transcription rate.3. Brg1and Brm were able to enhance the activation of ET-1promoters by hypoxia,which require the enzymatic activity. When knocked down the expression of endogenousBrg1and Brm using siRNA, induction of ET-1promoter activities by hypoxia was severelycrippled. Furthermore, the induction of endogenous ET-1messages was also limited inendothelial cells. ChIP assay indicates there was a dramatic increase in the occupancies ofBrg1and Brm on the promoter region of the ET-1gene under hypoxic conditions. Ablationof Brg1and Brm greatly reduced the levels of AcH3, H3K4Me2and H3K4Me3around theET-1promoter.4. In the mice HPH model, Immunofluorescence staining revealed that the expressionof Brg1and Brm was effectively reduced in the pulmonary endothelium by the viralparticles which contains endothelial-specific vector that encodes shRNA targeting bothBrg1and Brm. Correction of HPH by Brg1/Brm depletion was accompanied by thedown-regulation of all three CAM molecules in pulmonary arteries. As a result, elevation ofright ventricular pressure (RVSP) was significantly relieved by endothelial-specificBrg1/Brm silencing, as was as the hypertrophy of right ventricle. And, there was decreasedrecruitment of immune cells to the vessels. Furthermore, remodelling of pulmonaryvasculature, as measured by vessel wall thickness, was suppressed in mice injected withendo-siBrg1/siBrm compared with mice injected with control shRNA. Also the activities ofIL-1β, IL-6and MCP-1were inhibited remarkably. More importantly, hypoxia recruitedmore Brg1and Brm to the ICAM-1promoter in pulmonary arteries isolated from HPH mice when compared with the control mice.Conclusion：1. The expression of Brg1and Brm fluctuates in HPH rats and endothelial cells inresponse to oxygen tension indicative of a potential role for Brg1/Brm in endothelialmalfunction and pathogenesis of HPH.2. Brg1and Brm are both sufficient and necessary for hypoxia-induced CAMstransactivation and leukocyte adhesionin vitro. Brg1/Brm/p65complex forms on the CAMspromoters in response to hypoxia to activate transcription in endothelial cells. Brg1andBrm can form a crosstalk with the histone modification machinery to influencehypoxia-induced CAMs transactivation.3. Brg1and Brm are indispensable for hypoxia-induced transactivation of the ET-1gene. They can form a crosstalk with the histone modification machinery to influencehypoxia-induced ET-1transactivation.4. Endothelial-specific Brg1/Brm silencing stalled remodelling of pulmonaryvasculature and development of HPH, likely owing to the normalization of endothelialfunction and pulmonary inflammation in vivo.5. As such, targeting Brg1and Brm in endothelial cells may yield promising strategiesin the intervention and/or prevention of HPH.更多还原