Fate of Lung Stem Cells During Pulmonary Disease

描述(由申请人提供)：肺动脉高压(PAH)是血管收缩和血管重塑的结果。我们的目标是了解肺侧群体(SP)祖细胞在低压低氧诱导的PAH中所起的作用。我们已经开发了一种分离/浓缩程序，该程序基于原始祖细胞通过ABCG2转运蛋白将Hoechst染料外流的能力，该转运蛋白对成人干细胞是常见的，称为侧群(SP)。我们将肺SP定位于小鼠肺内的肺泡-毛细血管网络。(具体目标1)干细胞的性质是通过我们的能力反复从肺组织中分离出来，在细胞培养中长期保持，并诱导它们分化为内皮(EC)或肌成纤维细胞系来证明的。研究表明，VE-钙粘附素和血管生成潜力具有诱导分化的作用。肌成纤维细胞以胶原/弹性蛋白合成为特征。(特定目标2)我们实验室的其他研究表明，小鼠肺内SP的增加，结合低压低氧暴露，可以增加心室收缩压。在这些血流动力学研究之后，对肺进行分析以定位肺中的SP细胞。这些外源性给药的肺SP细胞见于与肺相关的淋巴结和实质。(特定目的3)血小板衍生生长因子B(PDGF-BB)是我们的肺SP的体外肌纤维细胞分化所必需的，在我们的低压缺氧诱导的PAH小鼠的肺中，PDGF-BB升高。在PDGF-BB/ABL激酶抑制剂(Gleevec)存在下，我们可以在体外抑制肌成纤维细胞的分化，并在体内降低与肺表面活性物质(SP)增加相关的右室收缩压升高。我们将使用谱系追踪策略来检测体内PAH期间内源性肺SP细胞作为转录因子FoxO1的功能的潜力。我们将通过在体内和体外定量小口径血管的肌化和内皮素-1的SP产生以及使用离体肺灌流分析来检测肺SP细胞的低氧依赖的血管收缩潜力。进一步的研究将确定抑制PDGFR2/ABL激酶信号通路是否减少了低氧诱导的SP细胞的增殖和分化，或者肺SP通过PI3K下游信号通路和FoxO1活性调节对PAH进展的血管收缩作用。 公共卫生相关性：肺高压、慢性阻塞性肺疾病、间质性肺纤维化和其他成人肺部疾病是发病率和死亡率的主要原因。在过去的十年里，由于这些情况而死亡的人数翻了一番。人们越来越重视开发基于细胞的疗法来应对这些疾病，但肺是这些策略的顽固候选者，因为细胞类型和功能多样，而且缺乏对慢性病过程如何影响干细胞分化的了解。因此，在测试基于细胞的治疗之前，最好使用PAH的临床前动物模型来确定疾病发展过程中肺组织的变化如何影响常驻干细胞的分化和功能。

DESCRIPTION (provided by applicant): Pulmonary arterial hypertension (PAH) results from vasoconstriction and vascular remodeling. Our goal is to understand the role lung side population (SP) progenitor cells play in the development of hypobaric hypoxia- induced PAH. We have developed an isolation/enrichment procedure that is based on the ability of primitive progenitor cells to efflux a Hoechst dye via an ABCG2 transporter which is common to adult stem cells, termed the side population (SP). We localized the lung SP as associated with the alveolar-capillary network in mouse lung. (Specific Aim 1) Their stem cell nature was demonstrated by our ability to repeatedly isolate them from lung tissue, maintain them in cell culture for a prolonged period of time and induce them to differentiate into endothelial (EC) or myofibroblast cell lineages. Differentiation was demonstrated by studies that show the induction of VE-cadherin and angiogenic potential. Myofibroblasts were characterized by collagen/elastin synthesis. (Specific Aim 2) Additional studies in our lab demonstrated that augmentation of the lung SP in mice, combined with hypobaric hypoxia exposure, increase ventricular systolic pressure. Following these hemodynamic studies the lungs were analyzed to localize the lung SP cells. These exogenously administered lung SP cells were found in the lung associated lymph nodes and parenchyma. (Specific Aim 3) Platelet- derived growth factor B (PDGF-BB) is required for our in vitro myofibrobalst differentiation of lung SP and is elevated in the lungs of our mice with hypobaric hypoxia induced PAH. In the presence of PDGF-BB/abl kinase inhibitor (Gleevec), we could inhibit myofibroblast differentiation in vitro and decrease elevated right ventricular systolic pressure associated with augmented lung SP in vivo. We will use a lineage tracing strategy to examine the potential of endogenous lung SP cells during PAH in vivo as a function of the transcription factor, FoxO1. We will examine the hypoxia-dependent vasoconstrictive potential of lung SP cells by quantifying muscularization of small caliber vessels and SP production of endothelin-1 in vivo and in vitro and using isolated perfused lung analyses. Additional studies will determine whether inhibition of PDGFR2/abl kinase signaling decreases hypoxia-induced SP cell proliferation and differentiation or the vasoconstrictive effects of lung SP on the progression of PAH thru downstream PI3K signaling and regulation of the FoxO1 activity. PUBLIC HEALTH RELEVANCE: Pulmonary hypertension, chronic obstructive pulmonary disease, interstitial pulmonary fibrosis and other adult lung conditions are a major cause of morbidity and mortality. Deaths due to these conditions have doubled in the last decade. There is an increasing emphasis on the development of cell-based therapies to address these conditions, but the lung is a recalcitrant candidate for these strategies because of the diverse cell types and functions as well as a lack of understanding of how chronic disease processes affect stem cell differentiation. Therefore, prior to testing cell-based therapy, it is desirable to use a pre-clinical animal model of PAH to determine how changes in the lung tissue during the development of disease affect resident stem cell differentiation and function.