Targeting CEBPA To Restore Epithelial-Mesenchymal Homeostasis In Lung Fibrosis

项目总结 虽然在了解上皮细胞损伤和成纤维细胞的机制方面取得了巨大的进展 在纤维化中的激活，在充分了解上皮细胞的机制方面仍然存在重大挑战。 间充质串扰在正常肺内稳态以及在损伤、修复和纤维化过程中。和正常肺一样 在发育过程中，肺修复需要上皮细胞和间充质细胞相互协调来调节 细胞的增殖、迁移、分化和凋亡。同样，细胞外基质(ECM)重塑 由大量促纤维化和抗纤维化因子介导，这些因子由上皮细胞和 间质在损伤、修复和消退过程中的作用。因此，异常的上皮间质相互作用可以 导致肺修复中的不可愈合过程，并由肌成纤维细胞导致病理性瘢痕形成 积聚和细胞外基质沉积，最终导致肺纤维化。这样做的长期目标是 建议通过转录调控恢复肺纤维化上皮-间充质平衡 关键的抗纤维化因子。增强内源性抗纤维化或纤维化的靶向机制 疾病进展过程中的分辨信号可能是一种有吸引力的缓解肺部疾病的治疗策略 促进肺纤维化，恢复肺功能。最近，单细胞和批量RNA测序证实了正常 CEBPA对特发性肺间质纤维化上皮细胞特性和异常分化状态的影响 被认为是一个关键的转录调节因子，在IPF中减弱，这是多个独立的 学习。我们的中心假设是肺上皮细胞中CEBPA缺乏促进肺成纤维细胞 活化和纤维化，在修复过程中通常会增加以消除纤维化，而且它可以 使用非基因组编辑CRISPR基因激活治疗恢复，以促进纤维化消退。这 假设将通过三个特定的目标进行检验：第一，我们将评估CEBPA信号的作用 上皮细胞在维持肺上皮-间充质平衡中的作用及其在肺损伤中的保护作用 纤维化症。其次，我们将确定是否能够通过激活CRISPR基因来提高CEBPA的表达 恢复上皮-间充质动态平衡，减轻成纤维细胞活化和纤维化。我们将发展 并使用非基因组编辑CRISPR优化AAV介导的增强CEBPA表达的方法 博莱霉素损伤后表现为肺纤维化未消退的老年小鼠肺中的激活。最后，我们将 检测上皮性CEBPA表达是否由BMP4介导的抗纤维化作用，并进行RNA-seq-DNA序列分析。 基于CEBPA得失功能研究的分选上皮细胞基因表达分析 确定其他新的上皮-间充质动态平衡的候选介体。总而言之， 拟议的研究将揭示关键的抗肝纤维化靶点，以进行旨在恢复 上皮-间充质动态平衡与减轻肺纤维化。

PROJECT SUMMARY While tremendous progress has been made to understand mechanisms of epithelial cell injury and fibroblast activation in fibrosis, significant challenges remain in fully understanding the mechanisms of epithelial– mesenchymal crosstalk in normal lung homeostasis and during injury, repair and fibrosis. As in normal lung development, lung repair requires epithelial cells and mesenchymal cells to coordinate with each other to tune cell proliferation, migration, differentiation and apoptosis. Similarly, extracellular matrix (ECM) remodeling is mediated by a myriad of pro-fibrotic and anti-fibrotic factors that are precisely orchestrated by epithelium and mesenchyme during injury, repair and resolution. Therefore, aberrant epithelial mesenchymal interactions can lead to non-healing processes in lung repair with pathological scar formation due to myofibroblast accumulation and ECM deposition, ultimately contributing to pulmonary fibrosis. The long-term goal of this proposal is to restore epithelial-mesenchymal homeostasis in pulmonary fibrosis with transcriptional regulation of critical anti-fibrotic factors. Targeting mechanisms that augment the endogenous anti-fibrotic or fibrosis resolution signals during disease progression may serve as an attractive therapeutic strategy to alleviate lung fibrosis and restore lung function. Recently, single-cell and bulk RNA sequencing identified loss of normal epithelial cell identities and gain of abnormal indeterminate states of differentiation in IPF, with CEBPA identified as a key transcriptional regulator diminished in IPF that was common to multiple independent studies. Our central hypothesis is that CEBPA deficiency in lung epithelial cells promotes lung fibroblast activation and fibrosis, that it normally increases during repair to resolve fibrosis, and that it can be therapeutically restored using non-genome editing CRISPR gene activation to promote fibrosis resolution. This hypothesis will be tested with three specific aims: First, we will evaluate the role of Cebpa signaling from epithelial cells in maintaining lung epithelial-mesenchymal homeostasis and its protective role in pulmonary fibrosis. Second, we will determine whether increasing Cebpa expression with CRISPR gene activation is able to restore epithelial-mesenchymal homeostasis and attenuate fibroblast activation and fibrosis. We will develop and optimize an AAV-mediated approach to enhance Cebpa expression using non-genome editing CRISPR activation in the lung of aged mice that display non-resolving lung fibrosis after bleomycin injury. Lastly, We will test whether the anti-fibrotic effect of epithelial Cebpa expression is mediated by BMP4 and perform RNA-seq- based gene expression analysis of sorted epithelial cells from Cebpa gain and loss of function studies to identify additional novel candidate mediators of epithelial-mesenchymal homeostasis. Taken together, the proposed research studies will reveal critical anti-fibrotic targets for therapeutic interventions aimed at restoring epithelial-mesenchymal homeostasis and alleviating pulmonary fibrosis.