Investigating pulmonary complications due to abnormal collagen/ER stress in Osteogenesis Imperfecta

项目摘要 成骨不全(OI)是一种以骨脆性增加为特征的遗传性异质性疾病 导致骨折，主要是由于结构和/或分泌量的I型 胶原蛋白。虽然骨脆性是OI发病的主要原因，但肺损害是主要原因。 死亡的威胁。我们和其他人发现在OI中存在异常的骨细胞外基质(ECM)结构 I型胶原表达细胞表现为内质网应激。肺间充质来源的细胞也非常丰富 表达I型胶原蛋白。我们假设OI通过两种机制对肺产生负面影响：分泌 细胞外基质异常和慢性内质网应激。使用代表主要形式的OI的两个OI小鼠模型 (I型胶原的错义/功能缺失突变)，这一建议将解决以下假设 I型胶原分泌和内质网应激导致肺形态异常，影响肺细胞 分化，损害肺损伤恢复，改变ECM和内质网应激，对信号产生负面影响 小路。我们通过三个目标解决这些假说：1.确定I型胶原突变对 肺的生后动态平衡与细胞分化/通讯。假设：I型胶原基因突变 基因导致OI肺形态和细胞分化的改变。策略：使用AGa2和Col1a1+/- 小鼠模型，将通过组织学/免疫组织化学研究肺在多个发育阶段 (IHC)用于分化和ECM组成。利用体外上皮细胞/成纤维细胞共培养类有机物 实验我们将确定异常的ECM分泌对肺细胞分化、增殖、 和细胞凋亡。2.确定I型胶原突变所致内质网应激对肺细胞分化的影响。 组织动态平衡，以及对损伤的反应。假设：内质网应激对肺I型胶原表达的影响 细胞影响肺细胞的分化和功能。策略：使用目标1中的模型，我们将确定ER 肺细胞应激水平及4-PBA对体内内质网应激的调节作用 影响细胞分化和动态平衡。利用有机化合物实验，我们将定义 慢性内质网应激对肺细胞分化、增殖和凋亡的影响。为了研究OI肺损伤易感性， 我们将用博莱霉素对WT、AGa2和Col1a1+/-小鼠进行体内治疗，观察其对细胞的影响 4-PBA治疗对OI肺组织的损伤；3.确定肺细胞群的变化 细胞外基质和内质网异常应激背景下的分布和基因表达。假设：肺部有 细胞外基质和内质网应激的改变会影响细胞分化和功能中重要的信号通路。战略： 利用AGa2和Col1a1+/-小鼠模型，来自肺的单细胞RNA-seq细胞/组织将被 用于确定与信号级联相关的细胞发育和基因表达的变化 定位于特定的肺细胞群。这些目标的完成将揭示致因机制，同时 介绍OI肺功能障碍的新治疗方法，OI肺功能障碍是OI死亡的主要原因。

Project Summary Osteogenesis imperfecta (OI) is a genetically heterogenous disorder characterized by increased bone fragility leading to fractures and primarily results from defects in the structure and/or the amount of secreted type I collagen. While bone fragility is the primary cause of morbidity in OI, pulmonary compromise is the leading cause of mortality. We and others showed that in OI there is an abnormal bone extracellular matrix (ECM) structure and type I collagen expressing cells manifest ER stress. Pulmonary mesenchymal derived cells also abundantly express type I collagen. We hypothesize that OI negatively impacts the lung through two mechanisms; secretion of an abnormal ECM and chronic ER stress. Using two OI mouse models representing major forms of OI (missense/loss of function mutations in type I collagen), this proposal will address the hypotheses that mutant type I collagen secretion and ER stress produces abnormal pulmonary morphology, affects lung cell differentiation, impairs lung damage recovery, and that altered ECM and ER stress negatively impact signaling pathways. We address these hypotheses via three aims: 1. Determine the effect of type I collagen mutations on lung postnatal homeostasis and cell differentiation/communication. Hypothesis: Mutations in type I collagen genes lead to alterations in OI lung morphology and cell differentiation. Strategy: Using Aga2 and Col1a1+/- mouse models, the lung will be studied at multiple stages of development via histological/immunohistochemical (IHC) for differentiation and ECM composition. Using in vitro epithelial cell/fibroblast co-culture organoid experiments we will define the contribution of abnormal ECM secretion to lung cell differentiation, proliferation, and apoptosis. 2. Determine the effect of ER stress due to type I collagen mutations on lung cell differentiation, tissue homeostasis, and reaction to damage. Hypothesis: ER stress in pulmonary type I collagen expressing cells affect lung cell differentiation and function. Strategy: Using the models from Aim 1, we will determine ER stress levels in pulmonary cells and whether modulating ER stress in vivo with the chaperone 4-PBA can influence cell differentiation and homeostasis. Using organoid experiments, we will define the contribution of chronic ER stress to lung cell differentiation, proliferation, and apoptosis. To study OI lung damage susceptibility, we will perform in vivo treatment of WT, Aga2, and Col1a1+/- mice with bleomycin to observe the effects of cellular damage on OI lung tissue in conjunction with the 4-PBA treatment; 3. Identify changes in lung cell population distribution and gene expression in the context of an abnormal ECM and ER stress. Hypothesis: Lungs with altered ECM and ER stress affect signaling pathways important in cell differentiation and function. Strategy: Using the Aga2 and Col1a1+/- mouse models, single-cell RNA-seq cells/tissues derived from lung will be performed to identify changes in cell development and gene expression correlated with signaling cascades localized to specific lung cell populations. Completion of these aims will reveal causative mechanisms while introducing novel treatment methods of OI pulmonary dysfunction, the major cause of mortality in OI.