Human esophageal myofibroblasts increase squamous epithelial thickness via paracrine mechanisms in an in vitro model of gastroesophageal reflux disease.

Anisa Shaker,Meng Li,Chunying Zhang,Kevin Yang,Nicholas Clemons,Liping Hu

doi:10.1371/journal.pone.0238852

Abstract

The pathogenesis of esophageal injury in gastroesophageal reflux disease (GERD) is incompletely understood. We modeled exposure of human esophageal myofibroblasts (HEMFs) to gastroesophageal reflux by repeated treatment with pH 4.5 and pH 4.5 bile salts and determined the effects on the epithelium in a 3D organotypic-like air-liquid interface model. Total, basal and supra-basal thickness of the epithelium were measured and immunostaining for p63, for basal (CK 14) and supra-basal (CK 4) squamous differentiation markers, and for cell proliferation (PCNA) were performed. Epithelial cell proliferation in response to HEMF conditioned media was also assessed in 2D culture. In the 3D organotypic model, total epithelial thickness increased similarly with pH 4.5 and pH 4.5 bile salt treated versus untreated and bile salt treated HEMF conditioned media. Epithelial p63 immunostaining was increased and multilayered. There was expansion of the CK14+ basal and CK4+ supra-basal layers in the epithelium established with conditioned media from pH 4.5 and pH 4.5 bile salt treated HEMFs versus untreated HEMF conditioned media. PCNA + cells per μm of tissue were unchanged in the basal layer across all treatment conditions while PCNA + cells per total DAPI + cells were decreased. In 2D culture, basal epithelial proliferation decreased with conditioned media from pH 4.5 and pH 4.5 bile salt treated HEMFs compared to conditioned media from untreated HEMF conditioned media. Secreted factors from HEMFs treated with acidic stimuli encountered in GERD increase epithelial thickness compared to secreted factors from untreated HEMFs and expand both basal and supra-basal layers. Our findings demonstrate for the first time paracrine regulation of the squamous epithelium from acid stimulated HEMFs. The effects of secreted factors from acid treated HEMFs on basal cell proliferation in this model and the mechanism mediating the increase in epithelial thickness merit further investigation.

Highlights

Squamous epithelium in non-erosive and erosive gastro-esophageal reflux disease (GERD) is characterized by an increase in epithelial thickness and dilated intercellular spaces [1, 2]
Total epithelial thickness increases in 3D OTC -air-liquid interface (ALI) established with conditioned media from pH 4.5 and pH 4.5 bile salt treated human esophageal myofibroblasts (HEMFs) compared to untreated HEMF conditioned media or HEMFs treated with bile salts alone (Fig 1)
Because we did not observe an effect with conditioned media from bile salt treated HEMFs, additional studies were not performed with this condition

Summary

Introduction

Squamous epithelium in non-erosive and erosive gastro-esophageal reflux disease (GERD) is characterized by an increase in epithelial thickness and dilated intercellular spaces [1, 2]. The effect of noxious luminal factors on the sub-epithelial non-immune cellular components has not been rigorously investigated. We previously described human esophageal myofibroblasts (HEMFs) subjacent to the basal layer of the squamous epithelium in normal human esophagus [6]. We observed an increase in this population in de-identified biopsies from patients with histology typical of GERD (basal intracellular edema, intraepithelial squamous infiltration by neutrophils, lymphocytes and eosinophils, basal cell hyperplasia, and elongation of vascular papillae) as well as NF-B activation in sub-epithelial HEMFs [6]. We have previously shown that growth factors in conditioned media from untreated HEMFs increase epithelial proliferation and thickness in a 3D organotypic-like air-liquid interface model (3D OTC-ALI), with expansion limited to the basal layer [8]

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