Mechano-niche in Lung Repair after Injury

肺的正常结构和功能维持在稳态中，并在以下情况下修复/再生 区域性干/祖细胞造成的不同损伤。干细胞存在于独特的 微环境，被称为干细胞"生态位"，它构成干细胞后代，其他生态位支持 细胞，包括间充质细胞（MC）和周围的细胞外基质（ECM）。干细胞龛 为干细胞的自我更新和分化提供了有益的线索。纤维化肺经历了实质性的 组织生物力学性质的变化，表现为ECM的硬化。干细胞 细胞龛的感觉和反应的变化，在刚度的微环境，突出矩阵 刚度作为干细胞龛的重要机械组成部分。在初步研究中， 表征了与Pdgfr α +肺MC相关的肺泡2型上皮干细胞（AT 2）龛的硬度。 在新开发的刚度可调的3D水凝胶中进行的肺泡类器官培养表明，基质 刚度构成了AT2生态位。我们最近发现α 6-整合素是一种机械敏感的整合素 亚基;刚性基质诱导的α 6表达，主要是具有较短胞质结构域（α 6S）的α 6同种型， 介导肺成纤维细胞侵入基底膜。新的初步数据显示， 除了α 6表达外，基质硬度还调节Pdgfr α +肺MC中α 6前mRNA的选择性剪接， 导致不同的α 6亚型在软组织中的差异表达，具有较长的胞质结构域（α 6L）， /稳态基质条件和在僵硬/纤维化基质下从α 6L到α 6S优势的转换 条件我们发现α 6L表达促进肺MC的脂肪生成分化，并赋予AT2- 生态位功能，促进肺内稳态的恢复。与此相反，α 6S表达损害了AT2- 生态位功能，并促进肺MC的纤维化/侵袭性分化，从而导致肺纤维化。在这 因此，我们假设α 6整合素的基质硬度依赖性选择性剪接调节了修复， 通过控制肺间充质细胞的肺泡营养分化与纤维化分化来治疗受损肺。具体 研究的目的是：（1）确定基质刚度调节的机制 α 6-整合素的选择性剪接;（2）确定不同α 6-整合素 细胞质变体介导肺间充质细胞的肺泡营养分化与纤维化分化;和（3） 测试靶向α 6-整联蛋白的基质硬度依赖性可变剪接逆转 持续性肺纤维化。了解肺上皮干细胞 在损伤肺的正常与病理修复中与它们的小生境相互作用， 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.