Fate of Lung Stem Cells During Pulmonary Disease

描述（由申请方提供）：肺动脉高压（PAH）由血管收缩和血管重塑引起。我们的目标是了解肺侧群体（SP）祖细胞在低压缺氧诱导的PAH发展中的作用。我们已经开发了一种分离/富集程序，该程序基于原始祖细胞通过ABCG 2转运蛋白（称为侧群（SP））流出Hoechst染料的能力，ABCG 2转运蛋白是成体干细胞常见的。我们在小鼠肺中定位了与肺泡毛细血管网络相关的肺SP。（具体目的1）它们的干细胞性质通过我们重复地从肺组织中分离它们、将它们在细胞培养物中维持延长的时间段并诱导它们分化成内皮（EC）或肌成纤维细胞谱系的能力来证明。分化通过显示VE-钙粘蛋白和血管生成潜力的诱导的研究来证明。肌成纤维细胞的特征在于胶原/弹性蛋白合成。（具体目标2）我们实验室的其他研究表明，小鼠肺SP的增加，结合低压缺氧暴露，增加心室收缩压。在这些血液动力学研究之后，分析肺以定位肺SP细胞。在肺相关淋巴结和肺实质中发现这些外源性给予的肺SP细胞。（3）血小板源性生长因子B（PDGF-BB）是肺SP体外成肌纤维细胞分化所必需的，并且在低压缺氧诱导的PAH小鼠肺中升高。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分化，并在体内降低与肺SP增加相关的升高的右心室收缩压。我们将使用谱系追踪策略来检查内源性肺SP细胞在体内PAH期间作为转录因子FoxO 1的功能的潜力。我们将通过定量小口径血管的肌化和内皮素-1在体内和体外的SP产生以及使用离体灌注肺分析来检查肺SP细胞的缺氧依赖性血管收缩潜力。其他研究将确定PDGFR 2/abl激酶信号传导的抑制是否降低缺氧诱导的SP细胞增殖和分化或肺SP通过下游PI 3 K信号传导和FoxO 1活性调节对PAH进展的血管收缩作用。 公共卫生相关性：肺动脉高压、慢性阻塞性肺病、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。在过去十年中，由于这些条件造成的死亡增加了一倍。人们越来越重视开发基于细胞的疗法来解决这些问题，但由于细胞类型和功能的多样性以及缺乏对慢性疾病过程如何影响干细胞分化的了解，肺是这些策略的候选者。因此，在测试基于细胞的疗法之前，需要使用PAH的临床前动物模型来确定疾病发展期间肺组织的变化如何影响驻留干细胞分化和功能。

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.