Regulation of fibroblast polarity during pulmonary alveolar septal formation

肺泡间隔形成过程中成纤维细胞极性的调节

• 批准号: 8974249
• 负责人: STEPHEN E MCGOWAN
• 金额: --
• 依托单位: IOWA CITY VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8974249
• 关键词: Address; Adult; Air Sacs; Alleles; Alveolar; Alveolar Cell; Alveolar Duct; Alveolus; American; Apoptosis; Area; Attention; Birth; Blood capillaries; Blood-Air Barrier; Cell Line; Cell Polarity; Cell Shape; Cells; Centrioles; Cessation of life; Chronic Kidney Insufficiency; Chronic Obstructive Airway Disease; Cilia; Complex; Connective Tissue; Deposition; Development; Diabetes Mellitus; Disease; Disease Progression; Distal; Elastic Fiber; Elastin; Ensure; Epithelial; Event; Exposure to; Fibroblasts; Fostering; Future; G-Protein-Coupled Receptors; Gases; Goals; Green Fluorescent Proteins; Health Care Costs; Hospitalization; Image; Immigration; Immunofluorescence Microscopy; Immunohistochemistry; In Vitro; Investigation; Label; Learning; Life; Location; Lung; Lung diseases; Mechanics; Mediating; Membrane; Mesenchymal; Microfluidic Microchips; Microtubule Stabilization; Microtubule-Organizing Center; Microtubules; Military Personnel; Molecular Structure; Movement; Mus; Natural regeneration; Nucleic Acid Regulatory Sequences; PDGFA gene; Pathway interactions; Pattern; Phosphotransferases; Platelet-Derived Growth Factor; Platelet-Derived Growth Factor alpha Receptor; Population; Positioning Attribute; Process; Property; Proteins; Proto-Oncogene Proteins c-akt; Pulmonary Emphysema; Regulation; Resistance; Respiration; SHH gene; Signal Pathway; Signal Transduction; Signaling Molecule; Speed; Stroke; Structural Genes; Structure; Surface; Symptoms; System; Therapeutic; Time; Vascular Diseases; Veterans; Weight-Bearing state; base; capillary; cigarette smoking; extracellular; interstitial; migration; promoter; receptor; repaired; response; smoking prevalence; smoothened signaling pathway

American military veterans commonly develop unremitting pulmonary diseases in which the alveoli comprising the gas-exchange region are destroyed leading to frequent hospitalizations. Reductions in cigarette smoking have not been accompanied by reductions in health care costs and deaths related to these diseases, highlighting the need for treatments to allay further destruction and repair damaged alveoli. To develop such treatments it is important to identify factors which are active during development, and could be manipulated to repair alveoli in adults. The applicant has concentrated on interstitial lung fibroblasts (LF) during alveolar formation and now proposes investigations to elucidate how LF are directed to move away from their initial locus at eruptions from the primary septa into the airspace during secondary septation. Hypothesis: Platelet-derived growth factor receptor-alpha (PDGFRa) and sonic hedgehog (Shh) cooperatively regulate secondary alveolar septal elongation by fostering fibroblast polarization and directional migration. Specific Aim 1: To investigate how lung fibroblasts orient along the axis of the secondary septal elongation (polarize) and how polarization is influenced by PDGFRa and Shh. Specific Aim 2: To localize shared signaling molecules in the PDGF-A and Shh intracellular signaling pathways during LF migration in vitro, and define the topography of their activation in relationship to the primary cilium, centriole, and remodeling microtubules. Preliminary studies have shown that LF expressing PDGFRa preferentially localize towards the most distal portions of the alveolar septum, more abundantly display primary cilia, and orient their centrioles towards the alveolar entry ring (AER). PDGFRa regulates Akt, enhances proliferation, and reduces apoptosis in LF which express this receptor. Shh signals through a non-canonical pathway involving the G- protein coupled receptor properties of Smoothened. PDGF-A and Shh cooperatively enhance directional migration of LF and share important signaling intermediates that modify microtubules (MT) and direct cell- polarity. Studies are proposed to accomplish these objectives, including stereological analysis of lungs from mice in which one allele of the endogenous PDGFRa-promoter regulatory region drives expression of a green fluorescent protein (GFP) tag. This will enable localization and enumeration of this LF-population, and recognition of how the polarity of these cells differs from other alveolar cells, with respect to PDGF-A and Shh signaling. PDGF-A and Shh signaling will be perturbed using conditional deletions of PDGFRa or smoothened, or by inhibiting PDGFRa-kinase activity. Such perturbations will identify how these pathways ensure adequate and directed movement of LF, through regulation of their polarity during migration. Live cell, time-lapse imaging will be used to quantify the speed and persistence of LF migration in microfluidic devices in order to learn how these parameters are modified by PDGF-A, Shh and alterations in cell shape. Signaling events and MT-remodeling will be localized using immunofluorescence microscopy: both through immunohistochemistry and by tracking fluorescently labeled proteins expressed by a LF-cell line. Attention will be focused on pathways (phosphoinositol 3-kinase, PI3K and Akt/protein kinase-B) which maintain polarity by regulating microtubule stabilization at the leading edge. These proposed studies will illuminate how LF reach their optimal locations and persistently migrate along a properly oriented axis of alveolar septal elongation. Knowing how LF are directed to their optimal location may facilitate understanding how elastic fibers are deposited in a mechanically optimized pattern. Information from the proposed studies will also serve as a basis for future studies of how mechanical strain induced by respiration impacts LF positioning. Understanding basic structural and cellular pathways is required to identify and develop pharmacologic agents to promote alveolar repair in emphysema.

美国退伍军人通常患上持续性肺部疾病， 包括气体交换区域的气体被破坏，导致频繁的住院治疗。减少 吸烟并没有伴随着健康护理费用的减少和与此相关的死亡。 这些疾病突出表明需要治疗以减轻进一步的破坏和修复受损的肺泡。到 开发这种治疗方法，重要的是要确定在发育过程中活跃的因素， 用来修复成人的肺泡申请人专注于间质性肺成纤维细胞（LF） 在肺泡形成过程中，现在提出研究，以阐明LF是如何被引导远离 它们的最初所在地是在次生分隔期间从初级隔膜喷发到空气中。 假设：血小板衍生生长因子受体α（PDGFRa）和音刺猬（Shh） 通过促进成纤维细胞极化和定向协同调节继发性肺泡间隔延长 迁移具体目标1：研究肺成纤维细胞如何沿着次级间隔轴定向 伸长率（Tg）以及偏振如何受PDGFRa和Shh影响。具体目标2：本地化 在LF迁移过程中PDGF-A和Shh细胞内信号传导途径中共享信号分子， 体外，并确定其激活的地形关系的初级纤毛，中心粒， 重塑微管初步研究表明，表达PDGFRa的LF优先定位于 朝向肺泡隔的最远端部分，更丰富地显示初级纤毛，并且定向它们的 中心粒朝向肺泡入口环（AER）。PDGFRa调节Akt，增强增殖，并减少Akt的表达。 表达该受体的LF细胞凋亡。Shh信号通过涉及G- Smoothened的蛋白偶联受体特性。PDGF-A和Shh协同增强方向性 LF的迁移和共享重要的信号中间体，修饰微管（MT）和直接细胞， 极性提出了实现这些目标的研究，包括对肺的体视学分析， 小鼠，其中内源性PDGFRa启动子调控区的一个等位基因驱动绿色 荧光蛋白（GFP）标签。这将使得能够定位和枚举该LF群体，并且 认识到这些细胞的极性如何不同于其他肺泡细胞，关于PDGF-A和Shh 发信号。PDGF-A和Shh信号传导将使用PDGFRa或Sh的条件性缺失来干扰。 平滑的，或通过抑制PDGFRa-激酶活性。这种扰动将确定这些途径如何 通过调节迁移过程中LF的极性，确保LF的充分和定向运动。活细胞， 延时成像将用于量化LF迁移在微流体装置中的速度和持久性， 为了了解这些参数是如何被PDGF-A，Shh和细胞形状的改变所改变的。信令 事件和MT重塑将使用免疫荧光显微镜进行定位： 通过免疫组织化学和通过追踪由LF细胞系表达的荧光标记的蛋白质来检测。注意力将 重点关注通过以下途径维持极性的途径（磷酸肌醇3-激酶、PI 3 K和Akt/蛋白激酶-B）： 调节微管在前缘的稳定性。这些拟议的研究将阐明LF如何达到 它们的最佳位置，并持续地沿着肺泡间隔伸长的适当定向的轴沿着迁移。 了解LF如何被引导到它们的最佳位置可以有助于理解弹性纤维是如何被引导到它们的最佳位置的。 以机械优化的图案沉积。拟议研究的资料也将作为 呼吸引起的机械应变如何影响LF定位的未来研究的基础。理解 需要基本的结构和细胞途径来鉴定和开发药理学试剂，以促进 肺气肿的肺泡修复