The Role of dNp63 in Maladaptive Regeneration and Repair Following Severe Pulmonary Injury

dNp63 在严重肺损伤后适应不良再生和修复中的作用

• 批准号: 10541255
• 负责人: Aaron Weiner
• 金额: $ 1.29万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2023-05-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10541255
• 关键词: Adhesions; Affect; Alveolar; Alveolar Cell; Alveolus; Basal Cell; Basement membrane; Biological Assay; COVID-19 pandemic; CRISPR/Cas technology; Cause of Death; Cell Cycle; Cell Maintenance; Cell physiology; Cells; ChIP-seq; Clustered Regularly Interspaced Short Palindromic Repeats; Cyst; Data; Defect; Distal; Emergency Situation; Epithelial Cells; Epithelium; Equilibrium; Exhibits; Failure; Flow Cytometry; Functional Regeneration; Gases; Genes; Genetic Transcription; Histologic; Hospitalization; Image; Immunohistochemistry; Impairment; In Vitro; Individual; Infiltration; Influenza; Injury; Interstitial Lung Diseases; Knock-out; Left; Lung; Maintenance; Mediating; Migration Assay; Molecular; Mus; Natural regeneration; Organ; Outcome; Oxygen; Persons; Physiological; Play; Process; Proliferating; Prostate; Protein Isoforms; Pseudostratified Epithelium; Pulmonary function tests; Regenerative pathway; Regenerative response; Regulation; Respiratory Disease; Respiratory Tract Infections; Role; Skin; Slice; Survivors; Tissues; Trachea; United States; Up-Regulation; Viral Pneumonia; airway epithelium; alveolar epithelium; cell motility; cell type; directed differentiation; experimental study; follow-up; hospitalization rates; in vivo regeneration; influenza infection; injured; insight; lung injury; lung regeneration; mammary; migration; mortality; novel; overexpression; progenitor; programs; pulmonary function; repaired; response; response to injury; self-renewal; severe injury; stem; stem cells; surfactant; transcriptome sequencing

Abstract Unlike many mammalian vital organs, the lung exhibits a robust regenerative response to severe injuries such as influenza infection, which primarily targets epithelial cells airways and alveoli. Quiescent lung-resident epithelial progenitors enter the cell cycle, proliferate, and differentiate following lung injury, participating in two distinct regenerative pathways: functionally beneficial regeneration and maladaptive tissue remodeling. Intralobular airway-resident distal p63+ progenitors are one such progenitor cell type that migrates into the alveoli, adheres to the denuded alveolar basement membrane, and rapidly proliferates to generate ectopic bronchiolar- like tissue, forming honeycomb-like cysts that fail to resolve after injury and that do not participate in gas exchange. Though ultimately a maladaptive injury response, this ectopic bronchiolization does appear to benefit individuals with severe alveolar injuries by providing an “emergency” epithelial barrier. Distal p63+ progenitors are the only cells in the distal lung that express the master epithelial regulator Trp63, specifically the ΔN isoform (ΔNp63). ΔNp63 is highly active in the proliferative basal stem cells of other epithelial tissues such as the skin, mammary, prostate, and trachea, in which it confers basal cells with their stem-like identity and transcriptionally regulates the cellular processes of migration, adhesion, and proliferation. In my own preliminary data, I have found that influenza-injured mice with broad ΔNp63 deletion in the airway epithelium display a completely abrogated maladaptive alveolar remodeling response. Besides this data, there have been no studies directly investigating the role of ΔNp63 in maladaptive remodeling and the mechanisms by which it promotes this regenerative pathway. Aim 1 of this proposal will utilize conditional deletion of ΔNp63 in and lineage-tracing of distal p63+ progenitors to investigate if loss of ΔNp63 causes a cell identity change in distal p63+ progenitors following pulmonary injury. Intracellular flow cytometry, immunohistochemistry, and qPCR will be used to assess the fate decisions of ΔNp63-/- distal p63+ progenitors upon deletion both prior to and following influenza injury; pulmonary function tests will additionally be used to evaluate the physiological consequences of ΔNp63 knockout. Aim 2 will employ CRISPRa-mediated overexpression of p63 in tandem with in vitro and ex vivo migration assays to determine if cell motility is affected by ΔNp63 overexpression in distal p63+ progenitors as it is in other ΔNp63-expressing cell and tissue types. Finally, upon confirmation/identification of known and/or previously unidentified ΔNp63 migration targets in distal p63+ progenitors, CRISPR-mediated knockout of these targets followed by in vitro and ex vivo migration assays will be utilized to evaluate the importance of the ΔNp63- driven migration program in injury-activated distal p63+ progenitor motility. These experiments will investigate the role of ΔNp63 in distal p63+ progenitor fate choice and activation following injury, in turn yielding insight into the mechanistic underpinnings of lung regeneration pathways following severe pulmonary injury.

摘要 与许多哺乳动物的重要器官不同，肺对严重损伤表现出强大的再生反应， 如流感感染，主要针对气道和肺泡上皮细胞。静止性肺驻留 上皮祖细胞在肺损伤后进入细胞周期、增殖和分化，参与两个 不同的再生途径：功能上有益的再生和适应不良的组织重塑。 小叶内气道驻留的远端p63+祖细胞是一种迁移到肺泡中的祖细胞类型， 粘附在裸露的肺泡基底膜上，并迅速增殖，产生异位细支气管- 像组织一样，形成蜂窝状囊肿，在受伤后无法解决，并且不参与气体 交易所虽然最终是一种适应不良的损伤反应，但这种异位细支气管化似乎确实有益于 通过提供“紧急”上皮屏障来治疗严重肺泡损伤的个体。远端p63+祖细胞 是远端肺中唯一表达主要上皮调节因子Trp 63的细胞，特别是ΔN亚型 （ΔNp63）。Δ Np 63在其他上皮组织如皮肤的增殖基底干细胞中高度活跃， 乳腺，前列腺和气管，其中它赋予基底细胞干细胞样身份和转录 调节迁移、粘附和增殖的细胞过程。在我自己的初步数据中， 研究发现，在气道上皮中广泛缺失Δ Np 63的流感损伤小鼠显示出完全的 消除适应不良的肺泡重塑反应。除了这些数据，还没有直接的研究 研究Δ Np 63在适应不良重塑中的作用及其促进机制， 再生途径该提议的目的1将利用Δ Np 63的条件性缺失和Δ Np 63的谱系追踪。 远端p63+祖细胞，以研究Δ Np 63的缺失是否导致远端p63+祖细胞中的细胞身份改变 肺损伤后。将使用细胞内流式细胞术、免疫组织化学和qPCR评估 流感损伤前后Δ Np 63-/-远端p63+祖细胞在缺失后的命运决定; 肺功能测试将额外用于评估Δ Np 63的生理后果 漂亮目的2将采用CRISPRa介导的p63过表达，与体外和离体方法串联。 迁移试验，以确定细胞运动性是否受到远端p63+祖细胞中Δ Np 63过表达的影响， 在其他表达Δ Np 63的细胞和组织类型中。最后，在确认/识别已知和/或 先前未鉴定的Δ Np 63迁移靶点在远端p63+祖细胞中，CRISPR介导的这些敲除 靶点，然后进行体外和离体迁移试验，将用于评估Δ Np 63- 在损伤激活的远端p63+祖细胞运动中的驱动迁移程序。这些实验将研究 Δ Np 63在损伤后远端p63+祖细胞命运选择和激活中的作用，进而深入了解 严重肺损伤后肺再生途径的机制基础。