Human Esophagus Research Articles

Rat Reflux Model of Esophageal Cancer and Its Implication in Human Disease

The epidemiologic shift in esophageal cancer from squamous cell carcinoma to esophageal adenocarcinoma coincided with popularization of proton pump inhibitors and has focused attention on gastroesophageal reflux disease as a causative factor in this shift. The aim of this study is to review the literature on the rat reflux model in an effort to elucidate this phenomenon. An extensive online literature review (PubMed) was carried out to identify all seminal contributions to the study of esophageal adenocarcinoma using the rat reflux model. The rat reflux model is a validated reproducible model for the development of Barrett's esophagus and esophageal adenocarcinoma. Esophageal reflux of an admixture of gastric acid and duodenal juice induces Barrett's esophagus followed by adenocarcinoma. A high-pH environment created by surgical gastrectomy or proton pump inhibitor therapy in combination with a high-fat diet seems to potentiate the development of Barrett's esophagus and adenocarcinoma. Early surgical intervention to prevent reflux reduces the progression toward esophageal adenocarcinoma. Anti-inflammatory, antioxidant, and nitrate-trapping agents reduce the incidence of tumorigenesis. As in the rat so also in humans, reflux of an admixture of gastric acid and duodenal juice in a high-pH environment induces the development of Barrett's esophagus followed by esophageal adenocarcinoma. This has led to the hypothesis that to prevent Barrett's esophagus and subsequent esophageal adenocarcinoma in humans, the reflux of an admixture of acid and bile must be controlled before the development of Barrett's esophagus by methods other than acid-suppression therapy.

Black raspberries demethylate Sfrp4, a WNT pathway antagonist, in rat esophageal squamous cell papilloma.

Aberrant methylation of DNA is a common event in the development of cancers, including squamous cell carcinoma (SCC) of the human esophagus. In the present study, we determined: (a) whether aberrant DNA methylation also occurs in the development of N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis in the rat esophagus, a model of human esophageal SCC; and (b) if so, whether dietary black raspberries (BRBs) are capable of preventing this aberrant DNA methylation. A diet containing 5% BRBs inhibited the development of NMBA-induced tumors in the rat esophagus. This inhibition was associated with reduced mRNA levels of the DNA methyltransferases, Dnmt1 and Dnmt3b, in both dysplastic lesions and in papillomas of the esophagus. In addition, promoter methylation of Sfrp4, a WNT pathway antagonist, was significantly reduced by the berry diet, and this was associated with decreased nuclear localization of β-CATENIN and reduced expression of c-MYC protein in NMBA-treated esophagi. Decreased promoter methylation of Sfrp4 correlated with decreased expression of Dmnt3b and, ultimately, with increased Sfrp4 mRNA expression. This suggests that epigenetic alterations in NMBA-induced rat esophageal tumorigenesis recapitulate epigenetic events in human esophageal SCC, and that BRBs could be useful in preventing the aberrant DNA methylation involved in the development of human esophageal SCC. © 2015 Wiley Periodicals, Inc.

Cytoskeletal changes induced by allosteric modulators of calcium-sensing receptor in esophageal epithelial cells.

The calcium-sensing receptor (CaSR), a G-protein-coupled receptor, plays a role in glandular and fluid secretion in the gastrointestinal tract, and regulates differentiation and proliferation of epithelial cells. We examined the expression of CaSR in normal and pathological conditions of human esophagus and investigated the effect of a CaSR agonist, cinacalcet (CCT), and antagonist, calhex (CHX), on cell growth and cell–cell junctional proteins in primary cultures of porcine stratified squamous esophageal epithelium. We used immunohistochemistry and Western analysis to monitor expression of CaSR and cell–cell adhesion molecules, and MTT assay to monitor cell proliferation in cultured esophageal cells. CCT treatment significantly reduced proliferation, changed the cell shape from polygonal to spindle-like, and caused redistribution of E-cadherin and β-catenin from the cell membrane to the cytoplasm. Furthermore, it reduced expression of β-catenin by 35% (P < 0.02) and increased expression of a proteolysis cleavage fragment of E-cadherin, Ecad/CFT2, by 2.3 folds (P < 0.01). On the other hand, CHX treatment enhanced cell proliferation by 27% (P < 0.01), increased the expression of p120-catenin by 24% (P < 0.04), and of Rho, a GTPase involved in cytoskeleton remodeling, by 18% (P < 0.03). In conclusion, CaSR is expressed in normal esophagus as well as in Barrett’s, esophageal adenocarcinoma, squamous cell carcinoma, and eosinophilic esophagitis. Long-term activation of CaSR with CCT disrupted the cadherin–catenin complex, induced cytoskeletal remodeling, actin fiber formation, and redistribution of CaSR to the nuclear area. These changes indicate a significant and complex role of CaSR in epithelial remodeling and barrier function of esophageal cells.

Hydrogen sulfide synthesis enzymes reduced in lower esophageal sphincter of patients with achalasia.

The etiology of achalasia remains largely unknown. Considerable evidence reveals that the lower esophageal sphincter dysfunction is due to the lack of inhibitory neurotransmitter, secondary to esophageal neuronal inflammation or loss. Recent studies suggest hydrogen sulfide may act as an inhibitory transmitter in gastrointestinal tract, but study about hydrogen sulfide in human esophagus still lack. The aim of the study was to investigate if hydrogen sulfide synthesis enzymes could be detected in human esophagus and if the synthesis of the endogenous hydrogen sulfide could be affected in achalasia patients. Tissue samples in cardia, lower esophageal sphincter, 2 cm and 4 cm above lower esophageal sphincter were obtained from achalasia patients undergoing peroral endoscopic myotomy. Control tissues in lower esophageal sphincter were obtained from esophageal carcinoma patients. Expression of cystathionine-β-synthase and cystathionine-γ-lyase in lower esophageal sphincter of achalasia patients and control were detected by immunohistochemical staining. In addition, expression of cystathionine-β-synthase and cystathionine-γ-lyase were compared among different parts of esophagus in achalasia patients. Compared with control, the expression of cystathionine-β-synthase and cystathionine-γ-lyase in lower esophageal sphincter of achalasia patients was significantly reduced (χ2 = 11.429, P = 0.010). The expression of cystathionine-β-synthase and cystathionine-γ-lyase were lower in lower esophageal sphincter than that in 2 cm and 4 cm above lower esophageal sphincter, respectively (all P < 0.05). In conclusion, the expression of hydrogen sulfide synthesis enzymes, cystathionine-β-synthase and cystathionine-γ-lyase, can be detected in human esophagus and is reduced in patients with achalasia, which implicates the involvement of the two hydrogen sulfide synthesis enzymes in the pathophysiology of achalasia.

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett’s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett's esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

Characterization of oncocytes in deep esophageal glands.

Deep esophageal glands play a vital role in the protection and regeneration of the esophageal mucosa. Conditions such as gastroesophageal reflux disease and Barrett's esophagus have been associated with a change in the usual glands by oncocytic metaplasia. However, little is known regarding the function of oncocytes or the relevance of this metaplastic change in the human esophagus. We hypothesized that oncocytes of deep esophageal glands also express markers characteristic of a ductal epithelial phenotype because similar oncocytes have been described as part of large ductal epithelial cells in salivary glands. We used immunohistochemical stains to define structural, functional, proliferative, and potential stem/progenitor characteristics of oncocytes. Oncocytes did not express mucins or lysozyme C, two molecules found in mucous cells and used for antimicrobial defense. Oncocytes did not express CK5, a cytokeratin found in myoepithelial cells and basal epithelial cells, but expressed CK7, a cytokeratin found in intralobular ductal epithelial cells and luminal epithelial cells of the main duct. Oncocytes expressed cystic fibrosis transmembrane conductance regulator and sodium/potassium ATPase, ion channels that play a role in bicarbonate secretion. Membrane-bound beta-catenin was detected in oncocytes, but these cells did not express the proliferative marker Ki67. Approximately, a third of oncocytes expressed SOX9 and p63, transcription factors expressed in epithelial progenitor cells in multiple organs. Moreover, oncocytes expressed CD44, a transmembrane Glycoprotein expressed in cancer stem cells. Taken together, our data show that oncocytes express markers of intralobular ductal epithelial cells and luminal epithelial cells of the main duct. Additionally, our observations suggest that oncocytes act as epithelial progenitor cells and play a role in bicarbonate secretion. Since oncocytic metaplasia is associated with conditions of chronic acid injury, it is possible that oncocytes replace the mucous cells in deep esophageal glands (dEG) as an adaptive change to counteract injury from acid reflux. The marker characterization suggests that oncocytes may originate from transdifferentiation of myoepithelial and mucous cells. This transdifferentiation might lead to an overall decrease of mucins production and secretion by the dEG and a subsequent reduction of the protection conferred by the viscoelastic mucous layer.

A stochastic model for soft tissue failure using acoustic emission data

The strength of soft tissues is due mainly to collagen fibers. In most collagenous tissues, the arrangement of the fibers is random, but has preferred directions. The random arrangement makes it difficult to make deterministic predictions about the starting process of fiber breaking under tension. When subjected to tensile stress the fibers are progressively straighten out and then start to be stretched. At the beginning of fiber breaking, some of the fibers reach their maximum tensile strength and break down while some others remain unstressed (this latter fibers will assume then bigger stress until they eventually arrive to their failure point). In this study, a sample of human esophagi was subjected to a tensile breaking of fibers, up to the complete failure of the specimen. An experimental setup using Acoustic Emission to detect the elastic energy released is used during the test to detect the location of the emissions and the number of micro-failures per time unit. The data were statistically analyzed in order to be compared to a stochastic model which relates the level of stress in the tissue and the probability of breaking given the number of previously broken fibers (i.e. the deterioration in the tissue). The probability of a fiber breaking as the stretch increases in the tissue can be represented by a non-homogeneous Markov process which is the basis of the stochastic model proposed. This paper shows that a two-parameter model can account for the fiber breaking and the expected distribution for ultimate stress is a Fréchet distribution.

Interspecies Differences in Electromechanical and Histological Characteristics of Human and Swine Esophagus1

Interspecies Differences in Electromechanical and Histological Characteristics of Human and Swine Esophagus1

PTH-166 Generation of a human oesophageal tissue culture model from forceps biopsies

<h3>Introduction</h3> Barrett’s oesophagus is the condition whereby the normal stratified squamous epithelium at the lower oesophagus is replaced with simple columnar epithelium in the context of gastro-oesophageal reflux disease. This increases the risk of oesophageal adenocarcinoma (OA) ten-fold, and the majority of people diagnosed with OA do not survive for 12 months. Our understanding of the molecular and cellular mechanisms underlying Barrett’s oesophagus is hindered due to lack of at a suitable model. We set out to develop a model from explant tissue biopsies of adult human oesophagus, as opposed to murine models (mice have a keratinised oesophageal epithelium and do not get Barrett’s oesophagus) or the use of cell lines (which do not recapitulate the normal tissue). <h3>Method</h3> We devised a study protocol and secured ethical approval (REC reference number 13/YH/0197) to obtain forceps biopsy specimens from consenting adults attending for routine diagnostic gastroscopies at the Royal United Hospital Bath. Specimens were then transported to the University of Bath where we have tested multiple culture conditions. These include choice of culture media, substrate (eg glass, plastic – coated or uncoated, semi-permeable membrane at air/liquid interface), cell adhesion factors (eg collagen I, collagen IV, fibronectin, Matrigel®), choice of tissue preparation (enzyme treatment with dispase in the absence or presence of trypsin, dissection with scissors or minced with a blade), plating density, media volume and use of a cell feeder layer. <h3>Results</h3> We have recruited 42 participants to date and attempted tissue culture on 36 of these. (Biopsies from 6 patients have been processed for RNA analysis.) Over time we have gradually optimised culture conditions. Of the most recent 6 participants, 5 have cultured successfully with cell survival for at least 14 days. Explant colony growth had been particularly vigorous (&gt;12 mm) when plated on Matrigel® and a feeder layer cell line (mitomycin C-inactivated mouse embryonic fibroblast (iMEF) cell line 3T3-Swiss albino, ATCC® CCL-92™) on plastic coverslips. Explants have also adhered and cultured successfully on collagen IV-coated (sc-29010, Santa Cruz Biotech) plastic coverslips without a feeder layer. Explant colonies were fixed/permeabalised before immunofluorescent staining was performed for cytokeratin 14 (K14), smooth muscle actin (SMA), p63, E-cadherin and SLUG antigens. iMEFs are clearly delineated with SMA staining and the explant outgrowths are positive for K14, p63, E-cadherin and SLUG. <h3>Conclusion</h3> It is possible to culture adult human oesophageal tissue <i>in vitro</i>from standard pinch-biopsy forceps specimens at gastroscopy and we are in the final stages of optimising conditions, confirming characterisation by repeating experiments and devising a protocol. <h3>Disclosure of interest</h3> None Declared.

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett's Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa. A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa. This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett's Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

Identification and genetic manipulation of human and mouse oesophageal stem cells

ObjectiveHuman oesophageal stem cell research is hampered by the lack of an optimal assay system to study self-renewal and differentiation. We aimed to identify and characterise human and mouse oesophageal...

Tu1426 Differences in the Control of Distension-Induced Secondary Peristalsis in Human Esophagus: the Role of 5-HT4 Receptor Versus TRPV1 Receptor

Tu1426 Differences in the Control of Distension-Induced Secondary Peristalsis in Human Esophagus: the Role of 5-HT4 Receptor Versus TRPV1 Receptor

Expression of neuron-specific enolase and synaptophysin in esophageal development of human embryos

To investigate the expression of neuron-specific enolase (NSE) and synaptophysin(SYN) proteins in different developmental stages of human embryonic esophagus. Immunohistochemistry was used to detect the expressions of NSE and SYN proteins in embryonic esophagus tissues of fetuses of 2, 3 and 4 month gestational age (n=16). One-way ANOVA and LSD-t test were employed to compare the staining intensity and number of positive expression cells in embryonic esophageal tissues of different gestational age. In fetuses with 2, 3 and 4 months of gestation, the number of NSE-positive nerve cells in the myenteric nerve plexus and submucosa of human embryonic esophageal tissues were 18.38 ± 8.37, 25.00 ± 11.54 and 38.00 ± 15.09, respectively; the staining intensity of NSE-positive nerve cells and nerve fibers in myenteric nerve plexus and submucosa of embryonic esophageal tissues were 74.38 ± 14.93, 62.25 ± 18.59 and 56.44 ± 14.70, respectively. NSE-positive cells were detected in the esophageal epithelium only at the third month. In the fetuses at 2, 3 and 4 months of gestation, SYN in all layers of esophageal tissue were positively or strong positively expressed, especially in the myenteric plexus and submucosal plexus. The staining intensity of SYN-positive cells in embryonic esophagus tissues of 2, 3 and 4 month gestation were 54.69 ± 9.34, 51.84 ± 6.10 and 46.41 ± 6.44, respectively. SYN and NSE may be involved in the regulation of nerve system of esophageal tissues during the human embryonic development.

Isolation of myofibroblasts from mouse and human esophagus.

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express α-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

Isolation of Myofibroblasts from Mouse and Human Esophagus

Isolation of Myofibroblasts from Mouse and Human Esophagus

Myanmar: An Endemic Country for Oral Cancer.

Myanmar: An Endemic Country for Oral Cancer.

Blood vessels in ganglia in human esophagus might explain the higher frequency of megaesophagus compared with megacolon.

This study aimed to determine the existence of blood vessels within ganglia of the myenteric plexus of the human esophagus and colon. At necropsy, 15 stillborns, newborns and children up to two years of age, with no gastrointestinal disorders, were examined. Rings of the esophagus and colon were analyzed and then fixed in formalin and processed for paraffin. Histological sections were stained by hematoxylin-eosin, Giemsa and immunohistochemistry for the characterization of endothelial cells, using antibodies for anti-factor VIII and CD31. Blood vessels were identified within the ganglia of the myenteric plexus of the esophagus, and no blood vessels were found in any ganglia of the colon. It was concluded that the ganglia of the myenteric plexus of the esophagus are vascularized, while the ganglia of the colon are avascular. Vascularization within the esophageal ganglia could facilitate the entrance of infectious agents, as well as the development of inflammatory responses (ganglionitis) and denervation, as found in Chagas disease and idiopathic achalasia. This could explain the higher frequency of megaesophagus compared with megacolon.

Murine and human tissue-engineered esophagus form from sufficient stem/progenitor cells and do not require microdesigned biomaterials.

Tissue-engineered esophagus (TEE) may serve as a therapeutic replacement for absent foregut. Most prior esophagus studies have favored microdesigned biomaterials and yielded epithelial growth alone. None have generated human TEE with mesenchymal components. We hypothesized that sufficient progenitor cells might only require basic support for successful generation of murine and human TEE. Esophageal organoid units (EOUs) were isolated from murine or human esophagi and implanted on a polyglycolic acid/poly-l-lactic acid collagen-coated scaffold in adult allogeneic or immune-deficient mice. Alternatively, EOU were cultured for 10 days in vitro prior to implantation. TEE recapitulated all key components of native esophagus with an epithelium and subjacent muscularis. Differentiated suprabasal and proliferative basal layers of esophageal epithelium, muscle, and nerve were identified. Lineage tracing demonstrated that multiple EOU could contribute to the epithelium and mesenchyme of a single TEE. Cultured murine EOU grew as an expanding sphere of proliferative basal cells on a neuromuscular network that demonstrated spontaneous peristalsis in culture. Subsequently, cultured EOU generated TEE. TEE forms after transplantation of mouse and human organ-specific stem/progenitor cells in vivo on a relatively simple biodegradable scaffold. This is a first step toward future human therapies.

Evidence for up-regulation of purinergic receptor genes associating with TRPV1 receptors and neurotrophic factors in the inflamed human esophagus.

Purinergic receptors are implicated in nociceptive signaling in small primary afferents via activation of adenosine triphosphate (ATP). ATP appears to mediate HCl-induced transient receptor potential vanilloid receptor 1 (TRPV1) activation in esophageal mucosa. Up-regulation of TRPV1 expression in gastroesophageal reflux disease (GERD) is associated with increased nerve growth factor (NGF) and glial derived neurotrophic factor (GDNF). This study aims to genetically determine the expression of purinergic receptors in severe inflamed human esophagus. Distal esophageal biopsies from the subjects with erosive GERD, asymptomatic patients (AP) and healthy ones were examined. Using real-time qPCR for detecting purinergic receptors (P2X2, P2X3, P2X7, P2Y1, P2Y2, P2Y4, P2Y6 and P2Y12), TRPV1, TRPV4, NGF, and GDNF was done in this study. Both P2X3 and P2X7 mRNA expressions in GERD patients significantly increased than those in healthy controls (P < 0.001) and AP (P < 0.001), but P2X2, P2Y1, P2Y2, P2Y4, P2Y6, P2Y12 or P2Y12 had no difference within the control, AP or GERD subjects. The well correlated expression in P2X3 gene with TRPV1 (r = 0.46, P = 0.002), NGF (r = 0.54, P = 0.0002), and GDNF (r = 0.64, P = 0.0001) was found. The P2X7 gene expressions also well correlated with TRPV1 (r = 0.47, P = 0.002), NGF (r = 0.32, P = 0.037), and GDNF (r = 0.42, P = 0.005). These results suggest that chronic esophagitis increases mRNA expressions of P2X3 and P2X7 receptors accompanied by up-regulation of TRPV1 and neurotrophic factors (NGF and GDNF). These genetical alterations in esophageal mucosa might mediate sensitization of inflamed human esophagus.