Cellular and molecular delineation of pathologic fibroblasts in pulmonary fibrosis

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Principal Investigator: Tatsuya  Tsukui
Organization: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
Fiscal Year: 2024
Award: $248,947
Funding agency: National Heart Lung and Blood Institute

PROJECT SUMMARY
Pulmonary fibrosis is a chronic and intractable disease with a 5-year survival rate comparable to pancreatic or
lung cancers. Deterioration of respiratory function in pulmonary fibrosis is caused by progressive replacement
of normal tissue for gas exchange to dense fibrotic scar with fibrillar collagens. Pathologic fibroblasts
accumulate at the sites of fibrogenesis and work as effector cells for excessive collagen deposition.
Development of therapeutic strategies for targeting pathologic fibroblasts is hindered by the lack of
understanding to cellular lineage and molecular detail of pathologic fibroblasts. In our previous study, we
performed single-cell RNA-sequencing of normal and fibrotic lungs of mouse and human with a specialized
protocol to identify all collagen-producing cells. We identified several fibroblast subsets that localize in different
compartments of the lung. One of the fibroblast subsets emerge in fibrotic lungs of both mouse and human and
show the highest levels of collagen gene expression and enhanced migratory capacity. These fibroblasts are
characterized by specific expression of Cthrc1 (collagen triple helix repeat containing 1) and localized within
fibroblastic foci of idiopathic pulmonary fibrosis, suggesting their pathologic role in pulmonary fibrosis. We
recently generated and validated a novel mouse strain, Cthrc1-CreER, which allows us to specifically
manipulate the pathologic fibroblast population in pulmonary fibrosis. The goal of this K99/R00 proposal is to
elucidate the role and transcriptional regulations of pathologic fibroblasts in pulmonary fibrosis by using our
innovative murine tools. Aim 1 (K99 phase) will reveal the role and fate of pathologic fibroblasts by ablating
Cthrc1+ cells or lineage-tracing Cthrc1-CreER-labeled cells over the course of bleomycin-induced pulmonary
fibrosis. Aim 2 (K99 phase) will reveal the transcriptional and epigenetic landscape of pathologic fibroblasts by
performing RNA-seq, ChIP-seq, and ATAC-seq of purified Cthrc1-CreER-labeled cells at multiple time points of
pulmonary fibrosis to seek master regulators for activation and deactivation. We will also seek the
transcriptional regulations of CTHRC1+ cells in human pulmonary fibrosis. Aim 3 (R00 phase) will demonstrate
the role of genes regulating pathologic fibroblasts by using intratracheal adoptive transfer of fibroblasts with
lentiviral gene modifications and by conditionally knocking out candidate genes in fibroblasts in pulmonary
fibrosis. These studies using the murine genetic tool highly specific for pathologic fibroblasts will shed light on
cellular function and transcriptional regulations of pathologic fibroblasts in pulmonary fibrosis. This proposal is
also designed to provide the candidate with training opportunity to obtain skill sets for murine genetic approach
in search of therapeutic targets and functional genomics approach integrating RNA-seq, ChIP-seq, and ATAC-
seq. The success of this project will enable the candidate to establish his expertise in the field of pulmonary
fibrosis and lead to the candidate’s transition to scientific independence over the course of award period.

Terms: <ATAC sequencing><ATAC-seq><ATACseq><Ablation><Adoptive Transfer><Alveolar><Apoptosis><Apoptosis Pathway><Assay for Transposase-Accessible Chromatin using sequencing><Award><Basal Transcription Factor><Basal transcription factor genes><Binding><Bleo><Bleomycin><Candidate Disease Gene><Candidate Gene><Cell Body><Cell Count><Cell Cycle Kinetics><Cell Function><Cell Kinetics><Cell Number><Cell Physiology><Cell Process><Cell-Extracellular Matrix><Cells><Cellular Function><Cellular Physiology><Cellular Process><ChIP Sequencing><ChIP-seq><ChIPseq><Chromatin><Chronic><Cicatrix><Collagen><Collagen Gene><Computer Analysis><Data><Deposit><Deposition><Deterioration><Development><Disease><Disorder><ECM><Effector Cell><Epigenetic><Epigenetic Change><Epigenetic Mechanism><Epigenetic Process><Extracellular Matrix><Fibrillar Collagen><Fibroblasts><Fibrosing Alveolitis><Fibrosis><Fibrotic lesions in lung><Gases><Gene Expression><Gene Modified><Gene Transcription><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic><Genetic Transcription><Genome><Genomic approach><Goals><Human><Impairment><In Situ Hybridization><Inflammation><Injury><Knowledge><Label><Lung><Lung Respiratory System><Lung Tissue Fibrosis><Lung scar><Lung tissue scar><Malignant Pancreatic Neoplasm><Malignant Tumor of the Lung><Malignant neoplasm of lung><Malignant neoplasm of pancreas><Methodology><Mice><Mice Mammals><Modeling><Modern Man><Molecular><Molecular Interaction><Morphology><Mouse Strains><Murine><Mus><Non-Polyadenylated RNA><Normal Tissue><Normal tissue morphology><Pancreas Cancer><Pancreatic Cancer><Pathologic><Patients><Phase><Phenotype><Play><Population><Process><Programmed Cell Death><Proliferating><Proteins><Protocol><Protocols documentation><Pulmonary Cancer><Pulmonary Fibrosis><Pulmonary Scar><Pulmonary Tissue fibrosis><Pulmonary malignant Neoplasm><RNA><RNA Expression><RNA Gene Products><RNA Seq><RNA sequencing><RNAseq><Regulator Genes><Reporter><Resolution><Respiratory physiology><Ribonucleic Acid><Role><Scarring at the lung><Scarring in the lung><Scars><Site><Subcellular Process><Survival Rate><Therapeutic><Time><Transcription><Transcription Factor Proto-Oncogene><Transcription Regulation><Transcription factor genes><Transcriptional Control><Transcriptional Regulation><Transcriptional Regulatory Elements><Work><Wound Repair><assay for transposase accessible chromatin followed by sequencing><assay for transposase accessible chromatin seq><assay for transposase accessible chromatin sequencing><assay for transposase-accessible chromatin with sequencing><candidate identification><chromatin immunoprecipitation-sequencing><computational analyses><computational analysis><computer analyses><conditional knock-out><conditional knockout><design><designing><developmental><diffuse interstitial pulmonary fibrosis><epigenetic regulation><epigenetically><experiment><experimental research><experimental study><experiments><fibrogenesis><fibrosis in the lung><fibrotic lung><functional genomics><gene modification><genetic approach><genetic strategy><genetically modified><genomic effort><genomic strategy><idiopathic pulmonary fibrosis><in situ Hybridization Genetics><in situ Hybridization Staining Method><in vitro Assay><in vivo><injured><injuries><innovate><innovation><innovative><knock-down><knockdown><knockout gene><lung cancer><lung development><lung fibrosis><migration><mouse development><novel><overexpress><overexpression><pancreatic malignancy><programs><pulmonary><regulatory gene><resolutions><respiratory function><scATAC sequencing><scATAC-seq><scRNA-seq><single cell ATAC-seq><single cell ATAC-sequencing><single cell Assay for Transposase Accessible Chromatin sequencing><single cell RNA-seq><single cell RNAseq><single cell expression profiling><single cell sequencing assay for transposase accessible chromatin><single cell transcriptomic profiling><single-cell Assay for Transposase-Accessible Chromatin with sequencing><single-cell RNA sequencing><single-cell assay for transposase-accessible chromatin using sequencing><single-cell assay for transposase-accessible chromatin-seq><skills><social role><success><therapeutic agent development><therapeutic development><therapeutic target><tool><training opportunity><trans acting element><transcription factor><transcriptome sequencing><transcriptomic sequencing><triple helix><wound healing><wound recovery><wound resolution>