Molecular Profile of Barrett’s Esophagus and Gastroesophageal Reflux Disease in the Development of Translational Physiological and Pharmacological Studies

Abstract

Barrett’s esophagus (BE) is a premalignant condition caused by gastroesophageal reflux disease (GERD), where physiological squamous epithelium is replaced by columnar epithelium. Several in vivo and in vitro BE models were developed with questionable translational relevance when implemented separately. Therefore, we aimed to screen Gene Expression Omnibus 2R (GEO2R) databases to establish whether clinical BE molecular profile was comparable with animal and optimized human esophageal squamous cell lines-based in vitro models. The GEO2R tool and selected databases were used to establish human BE molecular profile. BE-specific mRNAs in human esophageal cell lines (Het-1A and EPC2) were determined after one, three and/or six-day treatment with acidified medium (pH 5.0) and/or 50 and 100 µM bile mixture (BM). Wistar rats underwent microsurgical procedures to generate esophagogastroduodenal anastomosis (EGDA) leading to BE. BE-specific genes (keratin (KRT)1, KRT4, KRT5, KRT6A, KRT13, KRT14, KRT15, KRT16, KRT23, KRT24, KRT7, KRT8, KRT18, KRT20, trefoil factor (TFF)1, TFF2, TFF3, villin (VIL)1, mucin (MUC)2, MUC3A/B, MUC5B, MUC6 and MUC13) mRNA expression was assessed by real-time PCR. Pro/anti-inflammatory factors (interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, tumor necrosis factor α, interferon γ, granulocyte-macrophage colony-stimulating factor) serum concentration was assessed by a Luminex assay. Expression profile in vivo reflected about 45% of clinical BE with accompanied inflammatory response. Six-day treatment with 100 µM BM (pH 5.0) altered gene expression in vitro reflecting in 73% human BE profile and making this the most reliable in vitro tool taking into account two tested cell lines. Our optimized and established combined in vitro and in vivo BE models can improve further physiological and pharmacological studies testing pathomechanisms and novel therapeutic targets of this disorder.

Barrett’s Metaplasia Progression towards Esophageal Adenocarcinoma: An Attempt to Select a Panel of Molecular Sensors and to Reflect Clinical Alterations by Experimental Models

“…This could partially be explained by the fact that lowering pH towards pKa values of unconjugated (pKa 5.2–6.2; e.g., deoxycholic acid) or glycine-conjugated (pKa 3.8–4.8; e.g., sodium glycocholate) may unionize bile salts, which makes them more lipophilic and able to enter the epithelial cells by cell membrane rupture and influence intracellular pathways [ 24 ]. This is also in accordance with our previous study, where we observed that acidified BM is more cytotoxic for esophageal HET-1A and EPC2 cells [ 21 ].…”

“…However, highly expressed KRT15 protein may contribute to esophageal carcinoma progression [ 59 ], whereas overexpression of KRT18 in colorectal cancer exerts an oncogenic role [ 60 ]. Moreover, our previously published data confirmed that these markers reflect clinical alterations within an in vitro model of BE [ 21 ]. The expression of COXs (COX1 and COX2, encoded by PTGS1 and PTGS2 ) was linked to the development of GI tumors and early BE-derived neoplastic transformation [ 61 , 62 ].…”

“…The gene expression of IL1R1 did not fulfill the criteria ( Table 2 ). The mRNA expression of KRT4, KRT15 was significantly downregulated in BE samples, whereas mRNA expression of KRT8, KRT18 was significantly upregulated in BE samples compared to normal epithelium and based on the analysis published elsewhere [ 21 ].…”

“…Deeply evaluated molecular mechanisms underlying the generation of bile mixture (BM)-induced esophageal lesions may contribute to the development of innovative pro-drugs and pharmacological treatment methods. Nonetheless, recent studies have undertaken an approach to set up an in vitro model of BE since the available translational animal models are relatively difficult to handle and require appropriate surgical skills or specific infrastructure [ 21 , 22 , 23 ]. Various cell cultures have been studied to investigate bile and acid reflux and its role in stimulation and/or inhibition of BE development [ 24 ].…”

“…Our previous work showed that the treatment of a primary immortalized human esophageal epithelial squamous cell line (EPC2) and human normal esophageal epithelial cell line (HET-1A) with acidified medium (pH 5.0) and BM altered gene expression, reflecting a clinical human BE-specific gene expression profile [ 21 ]. For example, an analysis of the expression profile of keratins (KRTs), a major constituent of the esophageal epithelium, revealed significant changes from squamous epithelia specific towards those expressed in columnar epithelium, as observed within the analysis of clinical samples [ 21 ]. However, it is unclear whether the molecular changes in BE- or EAC-derived cell lines and the previously implemented experimental design will also reflect the dysplastic progression of BE to adenocarcinoma.…”