Background/Aims:
Oesophageal stricture is a clinically important problem in inflammatory diseases such as Crohn's disease and Behçet's disease, in oesophageal cancer itself and in post-operative settings following open surgery or endoscopic submucosal dissection (ESD). After oesophageal ESD procedure, massive epithelial removal and thermal damage caused by electrocautery are supposed to contribute to fibroblast (FB) activation. Indeed, mucosal defects of more than three-quarters of the periluminal lumen are a risk of post-endoscopic stricture, especially in the oesophagus compared to the small and large intestine (ref.1). However, pathogenesis of oesophageal stricture is not yet fully understood due to the lack of suitable animal models that mimic post-ESD stricture and a global understanding of the uniqueness of oesophageal FBs. The aim of this study is to develop a clinically useful models for investigating post-ESD stricture and to unveil the unique characteristics of oesophageal FBs and find potential therapeutic targets on oesophageal strictures.

Methods:
A reproducible post-ESD stenosis model was developed by thermally stimulating the oesophagus of mice under anaesthesia with a pre-heated micro-iron stick. To investigate the comprehensive functions of FBs in the oesophagus, we prepared RNA-sequencing (RNA-seq) libraries from FBs of the oesophagus (Oeso), small intestine (SI), large intestine (LI), and cecum (Ce), and compared global gene expression of FBs across organ. Differentially expressed genes (DEGs) in oesophageal FBs were subjected to gene ontology analysis performed by Metascape (ref.2). Finally, highly expressed genes were confirmed by single cell RNA-seq analysis.

Results:
We successfully developed a post-ESD stenosis model with significant increase in fibrotic areas (Fig.1). The characters of oesophageal FBs were significantly different from other gastrointestinal tracts (Fig.2a). We identified 401 DEGs highly expressed in the oesophageal FBs, where genes related to positive regulation of cell migration were significantly enriched (Fig.2b). Among these DEGs, Csf2 (GM-CSF), Ccl2 and Ccl7, important for macrophage differentiation and chemoattraction, as well as Thbs4, Col8a1 and Col6a6, ECM proteins related to fibrosis, were highly and ubiquitously expressed in oesohpageal FBs (Fig.2c,d).

Conclusions:
Global gene expression analysis of FBs across gastrointestinal tracts revealed unique properties of oesophageal FBs, which have a high capacity to recruit and mature macrophages and to produce excess extracellular matrix. These data can contribute to future prophylaxis for post operative strictures as well as inflammation and cancer in the oesophagus.

Reference:
Kanehara, et al. Guidelines for Diagnosis and Treatment of Carcinoma of the Esophagus. 4th ed.
Zhou, et al. Nat Commun. 2019 Apr 3;10(1):1523.

Fig.1 Representative haematoxylin-eosin (H&E) staining of a colon section (top; scale bar 100 µm) and Sirius red staining of the oesophageal fibrosis model (bottom) of pre- and post-treatment.

Fig.2 Global gene expression of fibroblasts in the gastrointestinal tract.<br /> 2a. Heatmap of the correlation matrix generated by Spearman rank correlation between samples of osesophagus (Oeso), small intestine (SI), large intestine (LI), and cecum (Ce).<br /> 2b. Gene ontology analysis showing biological pathways enriched in 401 DEGs highly expressed in the oesophageal FBs using Metascape. Bar plot of -log10 P-value of each enriched term. DEGs were defined as FDR q-val<0.05, fold change >2.<br /> 2c. Representative individual plot of DEGs of oesophagus, small intestine, large intestine and cecum.<br /> 2d. Representative genes expression of single cell analysis of <i>Thbs4 </i>and<i> Ccl7</i> at a single cell level.

Fig.2 Global gene expression of fibroblasts in the gastrointestinal tract.
2a. Heatmap of the correlation matrix generated by Spearman rank correlation between samples of osesophagus (Oeso), small intestine (SI), large intestine (LI), and cecum (Ce).
2b. Gene ontology analysis showing biological pathways enriched in 401 DEGs highly expressed in the oesophageal FBs using Metascape. Bar plot of -log10 P-value of each enriched term. DEGs were defined as FDR q-val<0.05, fold change >2.
2c. Representative individual plot of DEGs of oesophagus, small intestine, large intestine and cecum.
2d. Representative genes expression of single cell analysis of Thbs4 and Ccl7 at a single cell level.

Yohei Mikami

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