Reprogramming of type 2 alveolar epithelial cells in idiopathic pulmonary fibrosis and regulation by TGFb1.

特发性肺纤维化中 2 型肺泡上皮细胞的重编程及 TGFb1 的调节。

• 批准号: 10444902
• 负责人: Max Louis Cohen
• 金额: $ 7.62万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 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 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10444902
• 关键词: Abnormal Cell; Abnormal Epithelial Cell; Adult; Affect; Alveolar; Antibodies; Attenuated; Automobile Driving; Basal Cell; Biochemical Markers; Biopsy; Biopsy Specimen; Cell Differentiation process; Cells; Cessation of life; Clinical Trials; Coculture Techniques; Critical Care; Cyst; Data; Dependence; Development; Diagnosis; Diagnostic; Disease; Ectopic Expression; Embryo; Epigallocatechin Gallate; Epithelial; Epithelial Cells; Extracellular Matrix; Fellowship; Fibroblasts; Fibrosis; Gene Expression; Gene Expression Profiling; Genes; Human; Immunohistochemistry; In Vitro; Institutional support resources; Knowledge; Lung; MUC5B gene; Medicine; Mesenchymal; Mesenchyme; Messenger RNA; Modeling; Monitor; Nature; Operative Surgical Procedures; Organoids; Overlapping Genes; Pathogenesis; Pathway Analysis; Pathway interactions; Patients; Phase; Population; RNA; Regulation; Research; Role; Sampling; Schedule; Secretory Cell; Signal Pathway; Signal Transduction; Slice; Source; Structure of parenchyma of lung; Surface; System; Testing; Time; Tissues; Transforming Growth Factor beta; Transforming Growth Factors; Transitional Cell; Type II Epithelial Receptor Cell; Undifferentiated; WNT Signaling Pathway; airway epithelium; alveolar epithelium; base; career; cell type; clinical diagnosis; diagnostic signature; fibrotic interstitial lung disease; fibrotic lung; idiopathic pulmonary fibrosis; in vitro Model; in vivo; inhibitor; insight; lung development; migration; notch protein; repaired; response; self-renewal; surfactant; targeted treatment; tissue culture; transcriptome sequencing; transcriptomics; transdifferentiation

Idiopathic Pulmonary Fibrosis (IPF) is the most common fibrotic interstitial lung disease among adults. The cause of IPF is not fully understood, and it is frequently progressive, often leading to death within several years of diagnosis. In IPF, there is loss of alveolar epithelial cells, including type 1 cells (AEC1s), which line the alveolar airspace surface, and type 2 (AEC2s), which secrete surfactant, self-renew, and give rise to AEC1s and development of honeycomb cysts. These cysts are lined by "bronchiolized" epithelium, so-called because of expression of airway and secretory cell markers such as p63, KRT5, KRT17, and MUC5B. The origin of the cells lining these cysts is not understood, but have generally been thought to be the result of migration of airway epithelial cells (basal and/or club), in a failed attempt at alveolar repair. Recent single-cell RNA (sc-RNA) sequencing studies have uncovered widespread AEC2 and other epithelial cell abnormalities in end-stage IPF tissue, such as intermediate/transitional cell states and ectopic expression of genes associated with airway cells (such as KRT5+ AEC2s). Our lab has recently shown that AEC2s are capable of reprogramming into KRT5+ basal-cell like cells in in vitro organoid cultures. These suggest a new hypothesis that the bronchiolized epithelium lining honeycomb cysts may actually be derived from reprogrammed AEC2s. This study seeks to characterize whether the epithelial abnormalities present in end-stage IPF tissue are also present earlier in the IPF disease course and to determine the role of TGFβ1 in regulating the aforementioned reprogramming. Samples from patients undergoing surgical biopsy for the purpose of clinical diagnosis will be analyzed by sc-RNA sequencing, to characterize the AEC2 and other epithelial cell populations and reconstruct estimated lineages, especially surrounding the induction of the basal-cell differentiation master- regulator Sox2 within AEC2s. These samples will be compared to normal and end-stage IPF tissue, in order to test the hypothesis that AEC2 reprogramming is an early feature of IPF. In addition, diagnostic biopsy samples will be obtained from patients who took epigallocatechin gallate (EGCG) for two weeks prior to biopsy. EGCG is a mesenchyme-specific inhibitor of TGFβ signaling under study in humans and will therefore allow us to examine the hypothesis that AEC2 reprogramming abnormalities seen in diagnostic biopsies can be reversed by TGFβ blockade. Finally, organoid co-cultures and precision-cut lung slices cultures will be used to examine the contribution of important signaling pathways, such as TGFβ, Wnt, and Notch, in driving AEC2 reprograming towards a SPC-/KRT5+ basal-cell like state. Knowledge of the mechanisms driving AEC2 reprograming in IPF may provide fundamental insight into the cause of this disorder and contribute to the development of targeted therapies for this incurable and frequently fatal disease. This proposed project will be performed as part of the research phase of Pulmonary & Critical Care Medicine Fellowship at UCSF and utilizes comprehensive institutional support and resources, in order to prepare the applicant for an independent research career.

特发性肺纤维化(IPF)是最常见的纤维性间质性肺疾病 成年人。IPF的病因尚未完全了解，而且往往是渐进性的，往往是领先的 在确诊后的几年内死亡。在IPF中，肺泡上皮细胞丢失， 包括排列在肺泡表面的1型细胞(AEC1s)和2型(AEC2s)，后者 分泌表面活性物质，自我更新，促进AEC1和蜂窝囊的发育。这些 囊内衬有“细支气管化”的上皮，所谓“细支气管化”是因为它表达了呼吸道和 分泌细胞标记，如p63、Krt5、Krt17和MUC5B。排列在这些区域的细胞的来源 目前尚不清楚，但通常被认为是呼吸道移行的结果。 上皮细胞(基细胞和/或杆状细胞)，试图修复肺泡失败。最近的单电池 核糖核酸(sc-rna)测序研究发现了广泛存在的AEC2和其他上皮细胞 终末期IPF组织异常，如中间/过渡细胞状态和异位 与呼吸道细胞相关的基因(如Krt5+AEC2)的表达。我们的实验室最近发现 AEC2能够在体外器官样细胞中重编程为Krt5+基底细胞样细胞 文化。这提示了一种新的假设，即蜂窝囊内的毛细支气管化上皮 可能实际上源自重新编程的AEC2。这项研究试图表征 在终末期IPF组织中出现的上皮异常也在IPF的早期出现 并确定转化生长因子β1在调节上述重编程中的作用。 以临床诊断为目的而接受手术活检的患者的样本将 通过sc-RNA测序分析，以表征AEC2和其他上皮细胞群和 重建估计的谱系，特别是围绕基底细胞的诱导 AEC2s中的分化主调控因子Sox2。这些样本将与正常和 终末期IPF组织，以验证AEC2重新编程是早期特征的假设 对IPF的影响。此外，诊断性活组织检查样本将从服用了 在活检前两周应用表没食子儿茶素没食子酸酯(EGCG)。EGCG是一种间充质细胞特异性 转化生长因子β信号的抑制剂正在研究中，因此将使我们能够检查 诊断活检中出现的AEC2重编程异常可以通过以下方法逆转的假说 转化生长因子β拮抗剂。最后，有机共培养和精切肺切片培养将用于 研究重要的信号通路，如转化生长因子β、Wnt和Notch在驾驶中的作用 AEC2重编程进入SPC-/Krt5+类基底细胞状态。关于驱动机制的知识 IPF中AEC2的重新编程可能为这种疾病的原因提供根本的洞察，并 为这种不治之症和经常致命的疾病开发有针对性的治疗方法 疾病。这项拟议的项目将作为研究阶段的一部分进行。 加州大学旧金山分校的重症监护医学奖学金，并利用全面的机构支持和 资源，以便为申请人的独立研究生涯做好准备。