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: 8634274
• 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/8634274
• 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; Erinaceidae; 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; 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; Relative (related person); Resistance; Respiration; Signal Pathway; Signal Transduction; Signaling Molecule; Speed; Stroke; Structural Protein; Structure; Surface; Symptoms; System; Therapeutic; Time; Vascular Diseases; Veterans; Weight-Bearing state; base; capillary; cigarette smoking; extracellular; human SMO protein; interstitial; migration; platelet-derived growth factor A; 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有肺泡的肺部疾病 包括气体交换区域的销毁，导致住院频繁。减少 吸烟尚未伴随着与这些相关的医疗保健费用和死亡的降低 疾病，强调需要治疗进一步破坏并修复受损的肺泡。到 开发这种治疗方法很重要的是要确定在开发过程中活跃的因素，并且可能是 操纵以修复成人的肺泡。申请人集中于间质肺成纤维细胞（LF） 在肺泡形成期间，现在提议进行调查，以阐明如何指示LF离开 在次要分隔期间，它们在从原发隔片中爆发到空域的初始轨迹。 假设：血小板衍生的生长因子受体-Alpha（PDGFRA）和声波刺猬（SHH） 通过促进成纤维细胞极化和方向性来调节次级肺泡伸长 迁移。特定目的1：研究肺成纤维细胞如何沿继发间隔的轴轴心 伸长（极化）以及极化如何受PDGFRA和SHH的影响。特定目标2：本地化 LF迁移期间PDGF-A和SHH细胞内信号通路中的共享信号分子 体外，并定义其激活与主要纤毛，中心和 重塑微管。初步研究表明，表达PDGFRA的LF优先定位 朝着肺泡隔膜的最远端部分，更丰富地显示原发性纤毛，并将其定向 朝向肺泡入口环（AER）的中心元组。 PDGFRA调节AKT，增强增殖并减少 表达该受体的LF中的凋亡。 SHH通过涉及g-的非规范途径信号 平滑的蛋白偶联受体特性。 PDGF-A和SHH合作增强了方向性 LF的迁移并共享重要的信号转导中间体，以修改微管（MT）和直接细胞 极性。提出了研究来实现这些目标，包括对肺的立体分析 内源性PDGFRA促启动器调节区域的一个等位基因驱动绿色表达的小鼠 荧光蛋白（GFP）标签。这将使该LF人群的本地化和枚举，以及 识别这些细胞的极性与其他肺泡细胞相对于PDGF-A和SHH的差异如何 信号。 PDGF-A和SHH信号将使用PDGFRA的条件缺失或 平滑，或通过抑制PDGFRA-激酶活性。这样的扰动将确定这些途径如何 通过调节其在迁移过程中的极性，确保LF的适当和定向运动。活细胞， 延时成像将用于量化微流体设备中LF迁移的速度和持久性 为了了解这些参数如何通过PDGF-A，SHH和细胞形状的变化来修改这些参数。信号 事件和MT造型将使用免疫荧光显微镜进行定位： 免疫组织化学并通过跟踪LF细胞系表示的荧光标记的蛋白质。关注会 专注于途径（磷酸肌醇3-激酶，PI3K和AKT/蛋白激酶-B），它们通过 调节前缘的微管稳定。这些提出的研究将阐明LF如何达到 它们的最佳位置，并沿着正确定向的肺泡伸长伸长轴持续迁移。 知道如何将LF定向到其最佳位置可能有助于了解弹性纤维的含量 以机械优化的模式沉积。拟议研究的信息也将作为 将来研究呼吸引起的机械应变如何影响LF定位的基础。理解 需要基本的结构和细胞途径来识别和开发药理学剂以促进 肺气肿的牙槽修复。