Cleavage of E-Cadherin Contributes to Defective Barrier Function in Neosquamous Epithelium

Abstract

Background: After ablation of Barrett's esophagus (BE), the esophagus heals with neosquamous epithelium (NSE). Despite normal endoscopic appearance, NSE exhibits defective barrier function with similarities to defects noted in the distal esophageal epithelium in patients with gastroesophageal reflux disease (GERD).

Aim: To determine whether patients with NSE, unlike patients with healthy esophageal epithelium, have C-terminal fragments (CTFs) of e-cad detectable on tissue biopsy. Secondly, to determine whether patients with NSE have elevated levels of N-terminal fragments (NTFs) of e-cad in the serum.

Methods: Fifteen patients with ablated long-segment BE, who had healing with formation of NSE, were enrolled in this pilot study. Western blots for CTFs and NTFs were performed on biopsies of NSE. Venous blood was obtained to assess levels of NTFs. Endoscopic distal esophageal biopsies from patients without esophageal disease served as tissue controls. Control blood samples were obtained from healthy subjects.

Results: Blots of NSE were successful in 14/15 patients, and all 14 (100 %) had a 35-kD CTF of e-cad, while CTFs were absent in healthy control tissues. Despite CTFs in NSE, serum NTFs of e-cad in NSE were similar to controls, p > 0.05. However, unlike healthy controls, blots of NSE also showed NTFs with molecular weights of 70-90 kD.

Conclusions: Cleavage of e-cad, as evidenced by the presence of CTFs and NTFs on biopsy, contributes to defective barrier function in NSE. However, unlike findings reported in GERD patients, serum NTFs are not elevated in NSE patients. This difference may reflect poor absorption with tissue entrapment of NTFs in previously ablated areas with poorly perfused NSE.

Keywords: Barrett’s esophagus; ELISA; Esophageal permeability; Radiofrequency ablation; Western blot.

Fig. 1

a Western blot using antibodies to the C-terminal fragments (CTFs) of e-cadherin for esophageal biopsies illustrated from 3 healthy control subjects and 7 subjects with neosquamous epithelium [specimen of neosquamous epithelium in column 3 was lost in processing]. Note that green bands for intact e-cadherin with molecular weight (MW) 120 kD are present in all 3 controls and all 7 with neosquamous epithelium. Small CTFs of e-cadherin (green bands) with MW 35 kD are shown to be absent in controls and clearly present in 6 of 7 with neosquamous epithelium. The seventh neosquamous epithelium exhibits faint, but positive, staining for CTFs of e-cadherin. Actin staining (red band) is shown at 42 kD. b E-cadherin and c CTF levels were measured by densitometry and normalized to actin. Means of the control group (healthy individuals 1–3) and experiment group (biopsies with neosquamous epithelium 1, 2, 4–8; note that biopsy 2 could not be quantified due to an anomaly in the gel lane) were plotted and compared using the t test. The error bars represent standard error. N.S. not significant

Fig. 2

a Western blot using antibodies to the N-terminal fragments (NTFs) of e-cadherin for esophageal biopsies from 3 healthy control subjects and 7 subjects with neosquamous epithelium [specimen of neosquamous epithelium in column 2 was lost in processing]. Note that green bands for intact e-cadherin with molecular weight (MW) 120 kD are present in all 3 controls and all 7 with neosquamous epithelium. NTFs of e-cadherin with MWs ranging from 70 to 90 kD (green bands) are shown to be absent in controls and clearly present in 6 of 7 with neosquamous epithelium. Actin staining (red bands) are shown at 42 kD. b E-cadherin and c NTF levels were measured by densitometry and normalized to actin. Means of the control group (healthy individuals 1–3) and experiment group (biopsies with neosquamous epithelium 3–7; note that biopsies 1, 2, 8 could not be quantified due to anomalies in the gel lanes) were plotted and compared using the t test. The error bars represent standard error. N.S. not significant

Fig. 3

Boxplot of serum NTF levels from patients with NSE compared to serum NTF levels from healthy controls. The lines within each box represent the median values of each group. Levels obtained using enzyme-linked immunosorbent assay of N-terminal fragments (NTFs) of e-cadherin in sera from patients with neosquamous epithelium (NSE) and healthy controls, p > 0.05. Note: Patient A had a pathologically high acid contact time on esophageal pH monitoring (24.3 %) in association with the high level of NTFs in serum (70.4 pg/mL) and patient B had a normal acid contact time on pH monitoring (1.5 %) in association with a normal level of NTFs (42.7 pg/mL) in serum