Guidance of pulmonary fibroblast migration during alveolar septal formation

肺气肿和肺纤维化等破坏性的实质性肺部疾病在很大程度上是不可逆转的， 而诱导肺泡修复和再生的策略是一个重要的优先事项。此应用程序 阐述了引导肺泡成纤维细胞到最佳位置以产生一致的， 机械集成的弹性和胶原纤维网络。了解信令平台如何集成和 肺泡发育过程中来自细胞外环境的状态信号对于改变 成纤维细胞迁移并向肌成纤维细胞(MF)过渡。当它们的神经粘蛋白1(Nrp1)耗尽时， 肺泡间质微循环未见减少，但周围肺泡管增大。初步研究亦包括 显示胶原增强RAS相关的C3肉毒杆菌毒素底物-1(Rac1)的活性，这是 是细胞极化和迁移所必需的。假设：Nrp1和盘状结构域受体-2(DDR2)修饰 血小板衍生生长因子受体α介导的RAC1信号转导肺成纤维细胞迁移和细胞外基质 (ECM)重塑，在肺泡间隔期间。这些信号通路的组件组装在一起 膜脂筏(MLR)，整合素在局部粘连处将细胞外基质连接到细胞肌动蛋白细胞骨架。 在目标1中，携带PDGFRα或Nrp1缺失的小鼠肺中的MF将被用来分析信号转导 从胶原蛋白、β1-整合素和DDR2传递信息以激活RAC1从而 调节片状脂血症的形成。这些研究将(A)研究Nrp1缺失如何改变PDGFRα- 靶向蛋白激酶、适配蛋白和鸟核苷酸交换因子，(B)评估PDGF-A如何 与Nrp1相互作用，以及(C)Nrp1如何调节PDGFFRα的内体运输。目标2将检查缺陷 在PDGFRα、Nrp1或DDR2缺失的小鼠的胶原纤维中，以及这些缺陷如何影响 MF和胶原蛋白纤维。这些研究将显示DDR2和整合素α2β1如何决定成纤维细胞的方式 对纤维样胶原蛋白-1的反应，包括它们对膜脂和膜型基质的极化 迁移过程中金属蛋白酶(MT1-MMPs)的表达。他们还将确定胶原蛋白纤维的硬度 改变rac1活化、局部黏附形成和细胞迁移。AIM 3将调查PDGFRα和 Nrp1与DDR2相互作用，并通过rac1组装足体，其中膜类型1基质 金属蛋白酶(MT1-MMPs)靶向胶原纤维的降解，引导胶原纤维的迁移和定位 Mf.这些研究将探索Podosome和rac1如何使成纤维细胞探测和重塑胶原 纤维沿轴线延伸至远端肺泡隔内。在所有三个目标中，胶原成分和 细胞环境的刚性将被操纵以定义它们如何影响细胞的极性(OF 片状脂体和足体)，rac1激活，向更坚硬的底物迁移(趋杜性)，以及 胶原纤维重塑。了解MLR及其蛋白质成分如何整合和调节信号 细胞外环境对改变成纤维细胞迁移和转化成纤维细胞的方式至关重要。这 这一过程是了解这些疾病以及如何通过肺泡治疗这些疾病的基础 再生。因为目前可用的治疗方法不能阻止或逆转肺泡丢失，形成 肺泡再生的策略可以极大地改善肺气肿的临床预后， 在美国退伍军人中很普遍。

Destructive parenchymal lung diseases such as emphysema and pulmonary fibrosis are largely irreversible, and strategies for eliciting alveolar repair and regeneration are an important priority. This application addresses mechanisms which guide alveolar fibroblasts to optimal locations for generation of a coherent, mechanically integrated elastic and collagen fiber network. Learning how signaling platforms integrate and condition signals from the extracellular environment during alveolar development is critical for modifying how fibroblasts migrate and transition to myofibroblasts (MF). When their neuropilin 1 (Nrp1) was depleted, pulmonary MF did not diminish but the surrounding alveolar ducts were enlarged. Preliminary studies also showed that collagen enhanced Ras-related C3 botulinum toxin substrate-1 (Rac1) activation, which is required for cell polarization and migration. Hypothesis: Nrp1 and discoidin domain receptor-2 (DDR2) modify PDGFRα-mediated signaling through Rac1 to direct lung fibroblast (LF) migration and extracellular matrix (ECM) remodeling, during alveolar septation. Components of these signaling pathways assemble in membrane lipid rafts (MLR) where integrins link the ECM to the cellular actin cytoskeleton at focal adhesions. In Aim 1, MF from the lungs of mice bearing deletions of PDGFRα or Nrp1 will be used to dissect the signaling pathways which transmit information from collagen, β1-integrins, and DDR2 to activate Rac1 and thereby regulate the formation of lamellipodia. These studies will (a) examine how Nrp1-deletion alters PDGFRα- targeted protein kinases, adapter proteins and guanine-nucleotide exchange factors, (b) evaluate how PDGF-A interacts with Nrp1, and (c) how Nrp1 regulates endosomal trafficking of PDGFRα. Aim 2 will examine defects in collagen fibers of PDGFRα, Nrp1, or DDR2-deleted mice and how these defects impact the positioning of MF and collagen fibers. These studies will show how DDR2 and integrin α2β1 determine the way fibroblasts respond to fibrillar collagen-1, including their polarization of lamellipodia and membrane type-1 matrix metalloproteinase (MT1-MMP) during migration. They will also determine how the rigidity of collagen fibers alters Rac1-activation, focal adhesion formation, and cell migration. Aim 3 will investigate how PDGFRα and Nrp1 interact with DDR2 and, via Rac1, assemble podosomes, where membrane type-1 matrix metalloprotease (MT1-MMP) targets collagen fibers for degradation to direct the migration and positioning of MF. These studies will explore how podosomes and Rac1 enable fibroblasts to probe and remodel collagen fibers along the axis extending into the distal alveolar septum. In all three aims the collagen composition and the rigidity of the cellular environment will be manipulated to define how they influence cell polarity (of lamellipodia and podosomes), Rac1 activation, migration towards stiffer substrates (durotaxis), and the remodeling of collagen fibers. Learning how MLR and their protein constituents integrate and condition signals from the extracellular environment is critical to modifying how fibroblasts migrate and transition to MF. This process is fundamental to understanding these diseases and how they may be remediated through alveolar regeneration. Because currently available treatments do not arrest or reverse alveolar loss, developing strategies for alveolar regeneration could greatly improve clinical outcomes for pulmonary emphysema, which is prevalent in the American veteran population.