DOI: 10.5281/zenodo.14229565
title: Inhibition of epithelial cell YAP-TEAD/LOX signaling  attenuates pulmonary  fibrosis "

description: Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disease characterized by excessive extracellular matrix (ECM) deposition. Current IPF therapies slow disease progression but do not stop or reverse it. The (myo)fibroblasts are thought to be the main cellular contributors to excessive ECM production in IPF. Here we report that fibrotic AT2 cells regulate production and crosslinking of ECM via the co-transcriptional activator YAP. YAP leads to increase expression of Lysyloxidase (LOX) and subsequent LOX mediated crosslinking by fibrotic AT2 cells. Pharmacological YAP inhibition reverses fibrotic AT2 cell reprogramming and LOX expression in experimental lung fibrosis in vivo and in human fibrotic tissue ex vivo. We thus identify YAP-TEAD/LOX inhibition in AT2 cells as a promising potential new therapy for IPF patients. 

In
description: SnRNAseq analysis of PCLS treated with Verteporfin in fibrotic conditions



Four PCLS slices per control donor (n=2) at day 5 after DMSO, FC, FC+Verteporfin stimulation were washed in cold 1X PBS and snap frozen. Nuclei were extracted using the Nuclei Isolation kit  (CG000505, 10X Genomics,)


Around 20’000 nuclei were loaded on a Chip G with Chromium Single Cell 3′ v3.1 gel beads and reagents (3′ GEX v3.1, 10x Genomics). Final libraries were analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control purposes. cDNA libraries were sequenced on a HiSeq 4000 Illumina platform aiming for 150 million reads per library and a sequencing configuration of 26 base pair (bp) on read1 and 98 bp on read2.). 


Paired reads were filtered if either the cell barcode or unique molecular identifier (UMI) sequence had more than 1 bp with a phred of <20. Reads were aligned with STAR (v2.7.9a) to the human genome reference GRCh38 release 99  from ensemble. Collapsed UMIs with reads that span both exonic and intronic sequences were retained as both separate and combined gene expression assays.
description:  

After preprocessing, analysis of the ex vivo human PCLS snRNA-seq data was conducted using the Seurat package (version 1.8.2). Cells with less than 1000 transcripts profiled were then removed.





To minimize the possible effect of potential batch correction methods, we first processed and annotated each library  separately, before integrating them together and annotating them jointly. To integrate the multiple snRNA-seq datasets, we employed Robust Principal Component Analysis (RPCA). RPCA is a powerful technique for decomposing a data matrix into low-rank and sparse components. Briefly, the low-rank component represents shared biological signals across datasets, while the sparse component captures dataset-specific variations and technical noise. Based on the cellular diversity, we chose to use PCLS treated with DMSO as the reference for the integration. Following the RPCA decomposition, we utilized the low-rank component as the integrated representation of the snRNA-seq datasets. This component captured shared biological signals across conditions while mitigating dataset-specific variations.However,  subsequent analyses, such as clustering and differential expression analysis, were performed on the non integrated  but normalized gene expression values. To validate the effectiveness of the integrated representation, we performed various analyses, including cell-type clustering, identification of marker genes. We also compared the results of these analyses to those obtained from individual datasets to evaluate the improvement gained through the integration process. Marker genes were computed using a Wilcoxon rank-sum test, and genes were considered marker genes if the FDR-corrected p-value was below 0.05 and the log2 fold change was above 0.5. 
description: *genome build/assembly
GRCh38 release 99


*processed data files format and content
Tab-separated values files and matrix files


processed data files format and content
Tab-separated values files and matrix files
description: *library name
*title
*library strategy
*organism
**tissue
**cell line
**cell type
genotype
treatment
batch
*molecule
*single or paired-end
*instrument model
description
processed data file
processed data file
processed data file


Sample1
PCLS,DMSO ,Sample 107
scRNA-seq
Homo sapiens
Lung cell type 
Lung cell type 
Precision Cut Lung Slices
total RNA
DMSO
 
nuclear RNA
paired-end
Illumina HiSeq 4000
10x Genomics
LT107_DMSO_barcodes.tsv
LT107_DMSO_genes.tsv
LT107_DMSO.mtx


Sample2
PCLS,DMSO + Fibrotic Cocktail,Sample 107
scRNA-seq
Homo sapiens
Lung cell type 
Lung cell type 
Precision Cut Lung Slices
total RNA
DMSO-Fibrotic Cocktail 
 
nuclear RNA
paired-end
Illumina HiSeq 4000
10x Genomics
LT107_FC_DMSO_barcodes.tsv
LT107_FC_DMSO_genes.tsv
LT107_FC_DMSO.mtx


Sample3
PCLS,DMSO + Fibrotic Cocktail + Verteporfin Sample 107
scRNA-seq
Homo sapiens
Lung cell type 
Lung cell type 
Precision Cut Lung Slices
total RNA
DMSO-Fibrotic Cocktail-Verteporfin
nuclear RNA
paired-end
Illumina HiSeq 4000
10x Genomics
LT107_VERT_barcodes.tsv
LT107_VERT_genes.tsv
LT107_VERT.mtx


Sample4
PCLS,DMSO Condition,SnRNAseq,Sample 106
scRNA-seq
Homo sapiens
Lung cell type 
Lung cell type 
Precision Cut Lung Slices
total RNA
DMSO
 
nuclear RNA
paired-end
Illumina HiSeq 4000
10x Genomics
LT106_DMSO_barcodes.tsv
LT106_DMSO_genes.tsv
LT106_DMSO.mtx


Sample5
PCLS,DMSO + Fibrotic Cocktail,Sample 106
scRNA-seq
Homo sapiens
Lung cell type 
Lung cell type 
Precision Cut Lung Slices
total RNA
DMSO-Fibrotic Cocktail 
 
nuclear RNA
paired-end
Illumina HiSeq 4000
10x Genomics
LT106_FC_DMSO_barcodes.tsv
LT106_FC_DMSO_genes.tsv
LT106_FC_DMSO.mtx


Sample6
PCLS-DMSO , Fibrotic Cocktail  + Verteporfin ,Sample 106
scRNA-seq
Homo sapiens
Lung cell type 
Lung cell type 
Precision Cut Lung Slices
total RNA
DMSO-Fibrotic Cocktail-Verteporfin
nuclear RNA
paired-end
Illumina HiSeq 4000
10x Genomics
LT106_VERT_barcodes.tsv
LT106_VERT_genes.tsv
LT106_VERT.mtx