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.

Public Health Relevance

The current study seeks to delineate the homeostatic and fibrosis-resolving roles of anti-fibrotic factor CEBPA in lung epithelial cells and to develop a new regenerative therapeutic strategy to treat lung fibrosis in experimental murine models using non-genome editing CRISPR gene activation to promote fibrosis resolution and restore epithelial-mesenchymal homeostasis. The outcomes from proposed studies are expected to have an important positive impact on human chronic lung diseases since identification and application of novel anti- fibrotic regulators will help to overcome current limitations of treating pulmonary fibrosis in human patients.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL153026-01
Application #
10026419
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Vuga, Louis J
Project Start
2020-07-15
Project End
2025-06-30
Budget Start
2020-07-15
Budget End
2021-06-30
Support Year
1
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905