Regulation of fibroblast polarity during pulmonary alveolar septal formation

美国退伍军人通常会患上坚持不懈的肺部疾病，其中肺泡 组成气体交换区的气体被摧毁，导致频繁住院。减少 吸烟并没有伴随着与此相关的医疗费用和死亡人数的减少 疾病，强调需要治疗以减轻进一步的破坏和修复受损的肺泡。至 开发这种治疗方法很重要的一点是确定在发育过程中活跃的因素，并可能 被操纵来修复成人的肺泡。申请者主要研究肺间质成纤维细胞(LF)。 在肺泡形成过程中，现在建议进行研究，以阐明LF是如何被引导离开的 它们在喷发时的初始轨迹在次级隔膜期间从初级隔膜进入空域。 假设：血小板衍生生长因子受体-α(PDGFRA)和Sonic Hedgehog(Shh) 通过培养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次级间隔轴线定向 伸长(极化)以及PDGFRA和Shh对极化的影响。具体目标2：本地化 Lf迁移过程中PDGF-A和Shh细胞内信号通路中的共同信号分子 并确定了它们的激活与初级纤毛、中心粒和 重塑微管。初步研究表明，表达PDGFRA的LF优先定位 在肺泡间隔的最远端，更丰富地显示了初级纤毛，并定位了它们的 中心粒朝向肺泡进入环(AER)。PDGFRA调节Akt，促进增殖，减少 表达该受体的LF中的细胞凋亡。Shh信号通过一条非正则途径涉及G- 蛋白偶联受体特性的平滑。PDGF-A和Shh协同增强靶向性 LF的迁移和共享重要的信号中间产物改变微管(MT)和直接细胞- 两极。建议进行研究以实现这些目标，包括对来自 内源性PDGFRA启动子调节区的一个等位基因驱动绿色基因表达的小鼠 荧光蛋白(GFP)标签。这将启用该LF填充的本地化和枚举，并且 识别这些细胞的极性与其他肺泡细胞在PDGF-A和Shh方面的不同 发信号。PDGF-A和Shh信号将被条件删除的PDGFRA或 平滑，或通过抑制PDGFRA-激酶活性。这样的扰动将确定这些路径是如何 通过在迁徙过程中调节其极性，确保低频的适当和定向的移动。活细胞， 将使用时间推移成像来量化微流控设备中LF迁移的速度和持久性 以了解PDGF-A、Shh和细胞形状的改变是如何改变这些参数的。信令 事件和MT重塑将使用免疫荧光显微镜进行定位：通过 免疫组织化学和追踪一个LF细胞系表达的荧光标记蛋白。注意力将会 关注维持极性的途径(磷脂酰肌醇3-激酶、PI3K和Akt/蛋白激酶-B)。 调节前缘微管的稳定性。这些拟议的研究将阐明LF如何达到 它们的最佳位置，并沿着适当方向的肺泡间隔延长轴持续迁移。 了解LF如何定向到其最佳位置可能有助于理解弹性纤维是如何 以机械优化的模式存放。来自拟议研究的信息也将作为 为未来研究呼吸引起的机械应变如何影响LF定位奠定了基础。理解 需要基本的结构和细胞通路来识别和开发促进 肺气肿的肺泡修复。

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.