TGF-β1-induced PAI-1 contributes to a profibrotic network in patients with eosinophilic esophagitis

Abstract

Background: Eosinophilic esophagitis (EoE) is an allergic disease of increasing worldwide incidence. Complications are due to tissue remodeling and involve TGF-β1-mediated fibrosis. Plasminogen activator inhibitor 1 (PAI-1/serpinE1) can be induced by TGF-β1, but its role in EoE is not known.

Objective: We sought to understand the expression and role of PAI-1 in patients with EoE.

Methods: We used esophageal biopsy specimens and plasma samples from control subjects and patients with EoE, primary human esophageal epithelial cells, and fibroblasts from patients with EoE in immunohistochemistry, quantitative PCR, and immunoassay experiments to understand the induction of PAI-1 by TGF-β1, the relationship between PAI-1 and esophageal fibrosis, and the role of PAI-1 in fibrotic gene expression.

Results: PAI-1 expression was significantly increased in epithelial cells of biopsy specimens from patients with active EoE compared with that seen in biopsy specimens from patients with inactive EoE or control subjects (P < .001). Treatment of primary esophageal epithelial cells with recombinant TGF-β1 increased PAI-1 transcription, intracellular protein expression, and secretion. Esophageal PAI-1 expression correlated with basal zone hyperplasia, fibrosis, and markers of esophageal remodeling, including vimentin, TGF-β1, collagen I, fibronectin, and matrix metalloproteases, and plasma PAI-1 levels correlated with plasma TGF-β1 levels. PAI-1 inhibition significantly decreased baseline and TGF-β1-induced fibrotic gene expression.

Conclusions: PAI-1 expression is significantly increased in the epithelium in patients with EoE and reflects fibrosis, and its inhibition decreases TGF-β1-induced gene expression. Epithelial PAI-1 might serve as a marker of EoE severity and form part of a TGF-β1-induced profibrotic network.

Keywords: Eosinophil; SerpinE1; TGF-β1; esophagitis; fibrosis; remodeling.

Figure 1

PAI-1 is expressed in the EoE esophageal epithelium. Representative image of PAI-1 immunohistochemistry on esophageal biopsy specimens from a non-diseased control (A), active EoE (B), and inactive EoE (C). Epithelial staining was quantified as a percent of the total epithelial height in control and active EoE (D) and in paired inactive and active EoE biopsies (E). Lines represent means.

Figure 2

Actively proliferating cells express PAI-1. Representative images of double immunofluorescence for PAI-1 (green, A, B) and cytokeratin 14 (Krt14) (red, A) or nuclear Ki67 (red, B) shows that PAI-1 co-localizes with cytokeratin 14 (yellow, A) and Ki67 positive cells (green cytoplasm, red nucleus, B). Quantification of cells that are KRT14-PAI-1 (C) and Ki67-PAI-1 (D) double positive in the basal (BZ) and non-basal (NBZ) zones of a subset of biopsies. Epithelial PAI-1 expression correlates with the severity of basal zone hyperplasia (E). PAI-1 mRNA expression correlates with Ki67 (F) and cytokeratin 5 (Krt5) (G) in esophageal biopsies from active, inactive, and control subjects.

Figure 3

PAI-1 expression associates with fibrosis and remodeling gene expression. Epithelial expression of PAI-1 correlates with the severity of LP fibrosis (A). PAI-1 mRNA expression correlates with mRNA expression of the pro-fibrotic genes collagen I (B), MMP-14 (C), and MMP-2 (D) and the epithelial mesenchymal transformation marker vimentin (VIM) (E). PAI-1 expression correlates with the numbers of tryptase positive cells (F) and eosinophils (G).

Figure 4

TGFβ1 induces PAI-1 expression in epithelial esophageal cells. Treatment of primary human esophageal epithelial cells with recombinant human TGFβ1 significantly induces PAI-1 gene expression (A), increases intracellular PAI-1 protein expression (B, D), and induces PAI-1 release into the supernatant (C).

Figure 5

PAI-1 expression associates with the degree of TGFβ1 expression. Representative images of epithelial PAI-1 expression (left) and LP TGFβ1 positive cells (right) in control (A) and EoE (B). Epithelial PAI-1 expression correlates with the numbers LP TGFβ1 positive cells (C). mRNA expression of PAI-1 correlates with TGFβ1 in esophageal biopsies (D). Plasma PAI-1 levels correlate with plasma TGFβ1 (E).

Figure 6

PAI-1 inhibition decreases gene expression in EoE fibroblasts. Treatment with the PAI-1 inhibitor TM5275 decreases baseline (A) and TGFβ1-induced (B) expression of pro-fibrotic and myofibroblast genes. Active EoE biopsy (C) and primary human esophageal fibroblasts (D) shows presence of low density lipoprotein like receptor-1.