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: 8335473
• 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/8335473
• 关键词: 7-(N-(3-aminopropyl)amino)heptan-2-one; Address; Antibodies; Archives; Arteries; Biological Assay; Blood Vessels; Cardiac; Cell Culture Techniques; 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; Sorting - Cell Movement; Source; Structure of parenchyma of lung; TNF gene; Technology; Tissue Sample; Tissues; Transcript; Vascular remodeling; 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

ABSTRACT 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) cels 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+ cels 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 cels 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.

摘要 肺动脉高压（PAH）目前是一种无法治愈的疾病，在晚期诊断， 心脏功能经常受损。在发展治疗方法以逆转 这种疾病一直缺乏肺组织和细胞，可以研究从病人。PHBI网络 因此，通过采集和存档组织，为调查界开发了一种资源， 来自特发性PAH（IPAH）患者和与其他医学疾病相关的PAH患者的血管细胞 （APAH），以及来自未使用的供体肺，作为对照。我们的团队作为PHBI肺采购 绝佳的价钱我们还从IPAH患者中收获并培养了CD 31阳性（+）（内皮）细胞， 我们使用“下一代”工具研究这些细胞中的基因表达，包括RNA- Seq.已经鉴定了在来自IPAH的CD 31 + T细胞中显著上调或下调的转录物， 控制肺，我们在这个建议中的第一个目标是将这些转录本的表达定位于肺中的PAEC。 肺组织将通过激光捕获显微切割从腺泡内动脉中分离CD 31 + PAEC 其中在IPAH患者与对照中发现病变，并将通过RNA提取和qRT进行分析。 PCR法这些研究将使我们能够验证这些变化是由文化差异诱导的，但 在存在PAH血管病变的血管中表达。然后我们将确定一些 在组织中的IPAH CD 31+细胞中观察到的基因表达变化也存在于 APAH患者的肺部。这将使我们关注这些异常的功能意义， 它们与IPAH的晚期病理学有关。在其他的研究中，我们分析了基因表达的变化， 新鲜分离的CD 31+细胞，使用微流体分析，其中单个细胞被分选并进行48次 PCR反应。我们在IPAH和对照肺中鉴定了血管细胞的扩增亚群， CD 31+和GM-CSFR <$+或CD 31+和PDGFR <$+双阳性。这些CD 31 +GM-CSFR+细胞是 同样细分为主要表达EC标志物的细胞和表达单核细胞/巨噬细胞的细胞 标记。来自IPAH患者肺的扩增的CD 31 +PDGFR <$+细胞主要表达 单核细胞/巨噬细胞标志物。因此，我们的第二个目标是将这些亚群定位在肺中 通过共聚焦显微镜观察血管，并确定它们是否与特定血管的血管相关。 大小，或具有特定病变。然后我们可以通过微流体和免疫荧光进一步评估， 的已鉴定亚群正在积极增殖，以及它们是否选择性地表达异常的 在特异性目的I中在CD 31+细胞中鉴定的转录物。通过识别异常扩张的亚群 在IPAH病变的细胞，我们可以进一步询问这些细胞离体，以确定他们是否处于一种状态， 的转变或去分化，以及他们是否引起了在消灭和 丛状病变，可能是新兴疗法的良好靶点。