Mechano-niche in Lung Repair after Injury

肺的正常结构和功能保持在动态平衡，并在下列情况下修复/再生 由区域定义的干细胞/祖细胞造成的不同损伤。干细胞驻留在独特的组织中 微环境，称为干细胞的“生态位”，它构成干细胞后代，其他生态位的支持 细胞包括间充质细胞(MC)和周围的细胞外基质(ECM)。干细胞的利基 为干细胞的自我更新和分化提供了有益的线索。纤维化的肺经历了实质性的 组织生物力学特性的变化，表现为细胞外基质僵硬。驻留在干细胞中的细胞 细胞小生境感知并对微环境中的僵硬变化做出反应，突出基质 僵硬是干细胞壁龛的一个重要机械组成部分。在初步研究中，我们有 描述了与PDGFRα肺微血管细胞相关的肺泡2型上皮干细胞(AT2)生态位的僵硬。 在新开发的可调节硬度的3D水凝胶中进行肺泡有机样培养证明了基质 僵硬构成了AT2利基。我们最近发现α6-整合素是一种机械敏感性整合素 亚基；刚性基质诱导的α6表达，主要是具有较短细胞质结构域的α6亚型(α6S)， 介导肺成纤维细胞侵入基底膜。新的初步数据现在显示，在 除了α6的表达外，基质刚性还调节α6前α肺MC的选择性剪接。 导致不同的α6亚型与较长的细胞质结构域(α6L)在Soft下的差异表达 僵硬/纤维化的基质条件和从α6L到α6S的优势转换 条件。我们发现α-6L的表达促进了肺巨噬细胞的成脂分化，并赋予AT2- 利基功能，促进肺内稳态的恢复。相反，α6S的表达损害了AT2- 并促进肺巨噬细胞的纤维化/侵袭性分化，从而促进肺纤维化。在这 建议，我们假设α6-整合素的基质刚性依赖的选择性剪接调节修复 通过控制肺间充质细胞的肺泡营养性分化和纤维性分化来治疗受损肺。特定的 本研究的目的是：(1)确定基质刚度的调节机制。 α6-整合素的选择性剪接；(2)不同α6-整合素机制的测定 胞质变异体介导肺间充质细胞的肺泡营养性分化和纤维性分化；以及(3) 检测靶向基质刚性依赖的α6-整合素选择性剪接逆转肝癌的可能性 持续的小鼠肺纤维化。了解肺上皮干细胞的作用机制 与其在正常与病理情况下对受损肺的修复相互作用将提供新的治疗方法 预防、治疗和潜在逆转肺纤维化的方法。

Normal structure and function of the lung is maintained in homeostasis and repaired/regenerated following diverse injuries by regionally defined stem/progenitor cells. Stem cells reside in unique tissue microenvironments, known as the stem cell “niche”, which constitutes stem cell progeny, other niche-support cells including mesenchymal cells (MCs), and the surrounding extracellular matrix (ECM). The stem cell niche provides instructive cues for stem cell self-renewal and differentiation. Fibrotic lungs undergo substantial changes in the tissue biomechanical properties, manifested by stiffening of the ECM. Cells residing in the stem cell niche sense and respond to alterations in the stiffness of the microenvironment, highlighting matrix stiffness as an important mechanical component of the stem cell niche. In preliminary studies, we have characterized the stiffness of alveolar type 2 epithelial stem cell (AT2) niche associated with Pdgfrα+ lung MCs. Alveolar organoid culture in newly developed, stiffness-tunable 3D hydrogels demonstrated that matrix stiffness constitutes an AT2 niche. We recently identified that α6-integrin is a mechanosensitive integrin subunit; stiff matrix-induced α6 expression, primarily an α6 isoform with a shorter cytoplasmic domain (α6S), mediates lung fibroblast invasion into the basement membrane. New preliminary data now show that in addition to α6 expression, matrix stiffness regulates alternative splicing of α6 pre-mRNA in Pdgfrα+ lung MCs, resulting in differential expression of a distinct α6 isoform with a longer cytoplasmic domain (α6L) under soft /homeostatic matrix conditions and a switch from α6L to α6S predominance under stiff/fibrotic matrix conditions. We found that α6L expression promotes lipogenic differentiation of lung MCs and confers the AT2- niche function, facilitating reinstatement of lung homeostasis. In contrast, α6S expression impairs the AT2- niche function and promotes fibrogenic/invasive differentiation of lung MCs, contributing to lung fibrosis. In this proposal, we hypothesize that matrix stiffness-dependent alternative splicing of α6-integrin regulates the repair of injured lungs by controlling alveolotrophic vs. fibrogenic differentiation of lung mesenchymal cells. Specific aims in the proposed study are: (1) determination of the mechanisms by which matrix stiffness regulates alternative splicing of α6-integrin; (2) determination of the mechanisms by which distinct α6-integrin cytoplasmic variants mediate alveolotrophic vs. fibrogenic differentiation of lung mesenchymal cells; and (3) testing the potential of targeting matrix stiffness-dependent alternative splicing of α6-integrin for the reversal of sustained pulmonary fibrosis in mice. Understanding the mechanisms by which lung epithelial stem cells interact with their niches in normal vs. pathological repair of the injured lung will provide novel therapeutic approaches to prevent, treat, and potentially reverse pulmonary fibrosis.