Cell Specific Localization of Altered Gene Expression in Pulmonary Hypertension

描述(申请人提供)：肺动脉高压(PAH)目前是一种不治之症，在晚期被诊断为心脏功能受损。开发逆转这种疾病的治疗方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网络为调查界开发了一种资源，从特发性PAH患者(IPAH)和与其他疾病相关的PAH患者(APAH)以及未使用的供体肺收集和存档组织和血管细胞作为对照。我们的团队作为PHBI肺部采购网站进行了贡献。我们还从IPAH患者和对照肺中获取和培养了CD31阳性(+)(内皮)细胞，并使用包括RNA-Seq在内的“下一代”工具研究了这些细胞中的基因表达。在确定了IPAH与对照组相比CD31+细胞显著上调或下调的转录本后，我们在这项建议中的第一个目标是定位这些转录本在肺组织中的PAEC的表达。CD31+PAECs将通过激光捕获显微解剖从IPAH患者与对照组中发现病变的腺泡内动脉中分离出来，并将通过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引起的差异，但在存在PAH血管病变的血管中表达。然后，我们将确定APAH患者肺组织CD31+细胞中观察到的一些基因表达的变化是否也存在于APAH患者的肺CD31+细胞中。这将使我们将重点放在这些异常的功能意义上，因为它们与IPAH的高级病理有关。在其他研究中，我们使用微流控分析方法分析了新分离的CD31+细胞中基因表达的变化，其中单个细胞被分选并接受48次PCR反应。我们鉴定了IPAH和对照肺中扩增的血管细胞亚群，CD31+和GM-CSFR？+或CD31+和PDGFR？+双阳性。CD31+GM-CSFR？+细胞平均分为主要表达EC标记的细胞和表达单核/巨噬细胞标记的细胞。扩增的CD31+PDGFR？+细胞主要表达单核/巨噬细胞标志。因此，我们的第二个目标是通过共聚焦显微镜定位肺血管系统中的这些亚群，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已鉴定的亚群中哪些正在活跃增殖，以及它们是否选择性地表达在特定目的I的CD31+细胞中鉴定的异常转录。通过鉴定IPAH皮损中异常扩张的细胞亚群，我们可以在体外进一步询问这些细胞，以确定它们是否处于转化或去分化状态，以及它们是否在闭塞性和丛状病变中产生细胞，并可能成为新兴治疗的良好靶点。 公共卫生相关性：我们小组最近的研究发现，与健康(未使用的供体)肺内皮细胞相比，特发性肺动脉高压患者肺内皮细胞培养的基因表达异常。我们现在有机会通过PHBI分析冰冻组织切片中的内皮细胞，以确定病变组织中的细胞是否显示出这些基因在病变血管中表达的变化，而不是反映与培养条件有关的变化。我们已经使用微流体技术来识别IPAH和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管腔的细胞的来源，因此使用新兴的治疗方法可能是重要的靶向治疗。(摘要结束)

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)