Fate of Lung Stem Cells During Pulmonary Disease

描述（由申请人提供）：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（SP）祖细胞在低压缺氧诱导的多环芳烃的发展中所起的作用。我们开发了一种基于原始祖细胞通过ABCG2转运体排出Hoechst染料的能力的分离/富集程序，这种转运体在成体干细胞中很常见，称为侧群（SP）。我们在小鼠肺中定位了与肺泡-毛细血管网络相关的肺SP。（具体目标1）我们能够从肺组织中反复分离出它们，在细胞培养中长时间维持它们，并诱导它们分化成内皮细胞（EC）或肌成纤维细胞系，从而证明了它们的干细胞性质。分化被研究证实，显示ve -钙粘蛋白和血管生成潜能的诱导。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SP的增加，加上低压缺氧暴露，会增加心室收缩压。在这些血流动力学研究之后，对肺进行分析以确定肺SP细胞的位置。这些外源性给药的肺SP细胞在肺相关淋巴结和实质中发现。（特异性目的3）血小板衍生生长因子B （PDGF-BB）是肺SP体外肌纤维母细胞分化所必需的，在低压缺氧诱导的PAH小鼠的肺中升高。在PDGF-BB/abl激酶抑制剂（Gleevec）存在的情况下，我们可以在体外抑制肌成纤维细胞分化，并降低体内与肺SP增强相关的右心室收缩压升高。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fox01的功能的潜力。我们将通过量化体内和体外小口径血管的肌肉化和内皮素-1的SP生成，并使用分离的灌注肺分析，来检查肺SP细胞的缺氧依赖性血管收缩电位。进一步的研究将确定PDGFR2/abl激酶信号的抑制是否会降低缺氧诱导的SP细胞增殖和分化，或者肺部SP通过下游PI3K信号传导和fox01活性调节对PAH进展的血管收缩作用。

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.