Cell Specific Localization of Altered Gene Expression in Pulmonary Hypertension

肺动脉高压中基因表达改变的细胞特异性定位

• 批准号: 8211919
• 负责人: Marlene Rabinovitch
• 金额: $ 7.9万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-23 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8211919
• 关键词: 7-(N-(3-aminopropyl)amino)heptan-2-one; Address; Antibodies; Archives; Arteries; Biological Assay; Blood Vessels; Cardiac; Cell Culture Techniques; Cell Separation; Cells; Colony-Stimulating Factor Receptors; Communities; Companions; Confocal Microscopy; Cultured Cells; Development; Disease; Dissection; Endothelial Cells; Fluorescence Microscopy; Freezing; Functional disorder; Gene Expression; Genes; Goals; Harvest; Immunofluorescence Immunologic; Immunohistochemistry; Inflammation; Inflammatory; Infusion procedures; Inherited; Lasers; Lesion; Location; Lung; Medical; Microfluidic Analytical Techniques; Microfluidics; Modification; Mus; Mutation; Nature; PECAM1 gene; Pathology; Patients; Penetrance; Platelet-Derived Growth Factor Receptor; Population; Positioning Attribute; Proliferating; Proteins; Pulmonary Hypertension; Pulmonary artery structure; RNA; Reaction; Resources; Sample Size; Scanning; Site; Source; Structure of parenchyma of lung; TNF gene; Technology; Tissue Sample; Tissues; Transcript; Vascular remodeling; abstracting; bone morphogenetic protein receptor type II; cohort; cytokine; disease diagnosis; granulocyte; interest; laser capture microdissection; macrophage; monocyte; monocyte colony stimulating factor; next generation; protein expression; pulmonary arterial hypertension; pulmonary artery endothelial cell; response; selective expression; therapy development; tool

DESCRIPTION (provided by applicant): Pulmonary arterial hypertension (PAH) is currently an incurable disease diagnosed at an advanced state and when there is often compromise of cardiac function. The major drawback in developing therapies to reverse this disease has been the lack of lung tissue and cells that could be studied from patients. The PHBI Network has therefore developed a resource for the investigative community by harvesting and archiving tissue and vascular cells from patients with idiopathic PAH (IPAH) and from PAH associated with other medical conditions (APAH), as well as from unused donor lungs, as controls. Our group contributes as a PHBI lung procurement site. We have also harvested and cultured CD31 positive(+) (endothelial) cells from patients with IPAH and from control lungs, and we study gene expression in these cells using 'next generation' tools including RNA-Seq. Having identified transcripts that are significantly up- or downregulated in CD31+ cells from IPAH vs. control lungs, our first aim in this proposal is to localize expression of these transcripts to the PAECs in the lung tissue. The CD31+ PAECs will be isolated by laser-capture microdissection from intra-acinar arteries where lesions are found in the IPAH patients vs. controls, and will be analyzed by RNA extraction and qRT-PCR. These studies will allow us to verify that the changes are differentially induced by culture but are expressed in vessels where the vascular lesions of PAH are present. We will then determine whether some of the changes in gene expression observed in IPAH CD31+ cells in tissue are also present in CD31+ cells from lungs in APAH patients. This will position us to focus on the functional significance of these abnormalities as they relate to the advanced pathology of IPAH. In other studies, we analyzed changes in gene expression in freshly isolated CD31+ cells using microfluidics assays, in which single cells are sorted and subjected to 48 PCR reactions. We identified expanded subpopulations of vascular cells in IPAH vs control lungs that are double positive for CD31+ and GM-CSFR?+, or CD31+ and PDGFR?+. Those CD31+GM-CSFR?+ cells are equally subdivided into cells that express primarily EC markers, and those that express monocyte/macrophage markers. The expanded CD31+PDGFR?+ cells from IPAH patient lungs express primarily monocyte/macrophage markers. Our second aim is therefore to localize these subpopulations in the lung vasculature by confocal microscopy, and to determine whether they are associated with a vessel of a particular size, or with a specific lesion. We can then further assess, by microfluidics and by immunofluorescence, which of the identified subpopulations are actively proliferating, and whether they selectively express the abnormal transcripts identified in CD31+ cells in Specific Aim I. By identifying the abnormally expanded sub-populations of cells in IPAH lesions, we can further interrogate these cells ex-vivo to determine whether they are in a state of transformation or de-differentiation, and whether they are giving rise to the cels in the obliterative and plexiform lesions, and could be good targets for emerging therapies. PUBLIC HEALTH RELEVANCE: Recent studies from our group identified aberrant gene expression in endothelial cells cultured from pulmonary arteries from lungs of patients with idiopathic pulmonary arterial hypertension, compared to those from healthy (unused donor) lungs. We now have the opportunity, through the PHBI, to analyze endothelial cells from frozen tissue sections to determine whether the cells in the diseased tissue show the altered expression of these genes in vessels with lesions and do not reflect a change related to the condition of culture. We have used microfluidic technology to identify expanded sub-populations of endothelial cells in IPAH vs. control lungs, and will now use confocal microscopy to determine the location of these cells, and which of the sub- populations might be the source of the cells that occlude the vessel lumen, and might therefore be important to target using emerging therapies. (End of Abstract)

描述（由申请人提供）： 肺动脉高压（PAH）目前是一种无法治愈的疾病，诊断时已处于晚期并且心脏功能经常受到损害。开发逆转这种疾病的疗法的主要缺点是缺乏可以从患者身上进行研究的肺组织和细胞。因此，PHBI 网络通过从特发性肺动脉高压 (IPAH) 患者和与其他医疗状况相关的肺动脉高压 (APAH) 患者以及未使用的供体肺中采集和存档组织和血管细胞作为对照，为研究界开发了一种资源。我们的团队作为 PHBI 肺采购网站做出了贡献。我们还从 IPAH 患者和对照肺中收获并培养了 CD31 阳性（+）（内皮）细胞，并使用包括 RNA-Seq 在内的“下一代”工具研究这些细胞中的基因表达。在鉴定出 IPAH 与对照肺的 CD31+ 细胞中显着上调或下调的转录本后，我们本提案的首要目标是将这些转录本的表达定位于肺组织中的 PAEC。将通过激光捕获显微切割从 IPAH 患者与对照中发现病变的腺泡内动脉中分离 CD31+ PAEC，并通过 RNA 提取和 qRT-PCR 进行分析。这些研究将使我们能够验证这些变化是由培养物差异诱导的，但在存在 PAH 血管病变的血管中表达。然后，我们将确定在组织中的 IPAH CD31+ 细胞中观察到的一些基因表达变化是否也存在于 APAH 患者肺部的 CD31+ 细胞中。这将使我们能够关注这些异常的功能意义，因为它们与 IPAH 的高级病理学相关。在其他研究中，我们使用微流体分析分析了新鲜分离的 CD31+ 细胞中基因表达的变化，其中对单个细胞进行分选并进行 48 次 PCR 反应。我们发现，与对照肺相比，IPAH 中的血管细胞亚群增多，这些细胞亚群呈 CD31+ 和 GM-CSFR?+ 或 CD31+ 和 PDGFR?+ 双阳性。这些CD31+GM-CSFR？+细胞同样细分为主要表达EC标记物的细胞和表达单核细胞/巨噬细胞标记物的细胞。来自 IPAH 患者肺部的扩增 CD31+PDGFR?+ 细胞主要表达单核细胞/巨噬细胞标记。因此，我们的第二个目标是通过共聚焦显微镜将这些亚群定位在肺脉管系统中，并确定它们是否与特定大小的血管或特定病变相关。然后，我们可以通过微流体和免疫荧光进一步评估哪些已识别的亚群正在活跃增殖，以及它们是否选择性表达在特定目标 I 中 CD31+ 细胞中识别出的异常转录本。通过识别 IPAH 病变中异常扩增的细胞亚群，我们可以进一步在体外询问这些细胞，以确定它们是否处于转化状态或 去分化，以及它们是否在闭塞性和丛状病变中产生细胞，并且可能是新兴疗法的良好靶标。 公共健康相关性：我们小组最近的研究发现，与来自健康（未使用的供体）肺部的肺动脉相比，从特发性肺动脉高压患者的肺动脉培养的内皮细胞中存在异常基因表达。我们现在有机会通过 PHBI 分析来自冷冻组织切片的内皮细胞，以确定病变组织中的细胞是否在病变血管中显示出这些基因表达的改变，并且不反映与培养条件相关的变化。我们已经使用微流体技术来识别IPAH与对照肺中扩大的内皮细胞亚群，现在将使用共聚焦显微镜来确定这些细胞的位置，以及哪些亚群可能是阻塞血管腔的细胞的来源，因此对于使用新兴疗法可能很重要。 （摘要完）