Guidance of pulmonary fibroblast migration during alveolar septal formation

肺气肿和肺纤维化等破坏性肺实质疾病在很大程度上是不可逆的， 并且引发肺泡修复和再生的策略是重要的优先事项。本申请 提出了将肺泡成纤维细胞引导到最佳位置以产生连贯的， 机械整合的弹性和胶原纤维网络。了解信令平台如何集成， 在肺泡发育过程中，来自细胞外环境的条件信号对于改变 成纤维细胞迁移并转变为肌成纤维细胞（MF）。当它们的神经纤毛蛋白1（Nrp 1）耗尽时， 肺MF没有减少，但周围的肺泡管扩大。初步研究还 显示胶原蛋白增强Ras相关的C3肉毒杆菌毒素底物-1（Rac 1）活化， 细胞极化和迁移所需的。假设：Nrp 1和盘状结构域受体-2（DDR2）修饰 PDGFRα介导的信号传导通过Rac 1指导肺成纤维细胞（LF）迁移和细胞外基质 (ECM)在牙槽分隔过程中的重塑。这些信号通路的组成部分组装在 膜脂筏（MLR），其中整合素将ECM连接至粘着斑处的细胞肌动蛋白细胞骨架。 在目的1中，来自携带PDGFRα或Nrp 1缺失的小鼠的肺的MF将用于剖析信号传导。 传递来自胶原蛋白、β1-整合素和DDR2的信息以激活Rac 1，从而 调节板状伪足的形成。这些研究将（a）检查Nrp 1缺失如何改变PDGFRα- 靶向蛋白激酶、衔接蛋白和鸟嘌呤-核苷酸交换因子，（B）评估PDGF-A 与Nrp 1相互作用，以及（c）Nrp 1如何调节PDGFRα的内体运输。目标2将检查缺陷 在PDGFRα、Nrp 1或DDR2缺失小鼠的胶原纤维中，以及这些缺陷如何影响 MF和胶原纤维。这些研究将显示DDR2和整合素α2β1如何决定成纤维细胞 对纤维胶原蛋白-1的反应，包括它们的板状伪足和膜1型基质的极化 金属蛋白酶（MT 1-MMP）。他们还将确定胶原纤维的硬度 改变Rac 1活化、粘着斑形成和细胞迁移。目的3将研究PDGFRα和 Nrp 1与DDR2相互作用，并通过Rac 1组装podosomes，其中膜1型基质 金属蛋白酶（MT 1-MMP）靶向胶原纤维进行降解，以引导胶原纤维的迁移和定位。 MF。这些研究将探索如何podosomes和Rac 1使成纤维细胞探测和重塑胶原蛋白 纤维沿着轴延伸到远端牙槽隔。在所有三个目标中，胶原蛋白组成和 细胞环境的刚性将被操纵以定义它们如何影响细胞极性（ 板状伪足和足体），Rac 1活化，向更硬的基质迁移（硬旋），以及 胶原纤维的重塑。了解MLR及其蛋白质成分如何整合和调节信号 从细胞外环境中释放对改变成纤维细胞迁移和转化为MF的方式至关重要。这 这一过程对于理解这些疾病以及如何通过肺泡 再生因为目前可用的治疗不能阻止或逆转肺泡损失， 肺泡再生的策略可以大大改善肺气肿的临床结果， 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.