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Background: Esophageal organoids from a variety of pathologies including cancer are grown in Advanced Dulbecco's Modified Eagle Medium-Nutrient Mixture F12 (hereafter ADF). However, the currently available ADF-based formulations are suboptimal for normal human esophageal organoids, limiting the ability to compare normal esophageal organoids with those representing a given disease state. Methods: We have utilized immortalized normal human esophageal epithelial cell (keratinocyte) lines EPC1 and EPC2 and endoscopic normal esophageal biopsies to generate three-dimensional (3D) organoids. To optimize the ADF-based medium, we evaluated the requirement of exogenous epidermal growth factor (EGF) and inhibition of transforming growth factor-(TGF)-β receptor-mediated signaling, both key regulators of the proliferation of human esophageal keratinocytes. We have modeled human esophageal epithelial pathology by stimulating esophageal 3D organoids with interleukin (IL)-13, an inflammatory cytokine, or UAB30, a novel pharmacological activator of retinoic acid signaling. Results: The formation of normal human esophageal 3D organoids was limited by excessive EGF and intrinsic TGFβ-receptor-mediated signaling. Optimized HOME0 improved normal human esophageal organoid formation. In the HOME0-grown organoids, IL-13 and UAB30 induced epithelial changes reminiscent of basal cell hyperplasia, a common histopathologic feature in broad esophageal disease conditions including eosinophilic esophagitis. Conclusions: HOME0 allows modeling of the homeostatic differentiation gradient and perturbation of the human esophageal epithelium while permitting a comparison of organoids from mice and other organs grown in ADF-based media.

Esophageal squamous cell carcinoma (ESCC) is a deadly consequence of radiation exposure to the esophagus. ESCC arises from esophageal epithelial cells that undergo malignant transformation and features a perturbed squamous cell differentiation program. Understanding the dose- and radiation quality-dependence of the esophageal epithelium response to radiation may provide insights into the ability of radiation to promote ESCC. We have explored factors that may play a role in esophageal epithelial radiosensitivity and their potential relationship to ESCC risk. We have utilized a murine three-dimensional (3D) organoid model that recapitulates the morphology and functions of the stratified squamous epithelium of the esophagus to study persistent dose- and radiation quality-dependent changes. Interestingly, although high-linear energy transfer (LET) Fe ion exposure induced a more intense and persistent alteration of squamous differentiation and 53BP1 DNA damage foci levels as compared to Cs, the MAPK/SAPK stress pathway signaling showed similar altered levels for most phospho-proteins with both radiation qualities. In addition, the lower dose of high-LET exposure also revealed nearly the same degree of morphological changes, even though only ~36% of the cells were predicted to be hit at the lower 0.1 Gy dose, suggesting that a bystander effect may be induced. Although p38 and ERK/MAPK revealed the highest levels following high-LET exposure, the findings reveal that even a low dose (0.1 Gy) of both radiation qualities can elicit a persistent stress signaling response that may critically impact the differentiation gradient of the esophageal epithelium, providing novel insights into the pathogenesis of radiation-induced esophageal injury and early stage esophageal carcinogenesis., Competing Interests: Deepa M. Sridharan is from Soley Theraperutics, Inc. The authors declare no conflicts of interest.

During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm-sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury., Competing Interests: The authors have declared that no competing interests exist.

The homeostatic proliferation-differentiation gradient in the esophageal epithelium is perturbed under inflammatory disease conditions such as gastroesophageal reflux disease and eosinophilic esophagitis. Herein we describe the protocols for rapid generation (<14 days) and characterization of single-cell-derived, three-dimensional (3D) esophageal organoids from human subjects and mice with normal esophageal mucosa or inflammatory disease conditions. While 3D organoids recapitulate normal epithelial renewal, proliferation, and differentiation, non-cell autonomous reactive epithelial changes under inflammatory conditions are evaluated in the absence of the inflammatory milieu. Reactive epithelial changes are reconstituted upon exposure to exogenous recombinant cytokines. These changes are modulated pharmacologically or genetically ex vivo. Molecular, structural, and functional changes are characterized by morphology, flow cytometry, biochemistry, and gene expression analyses. Esophageal 3D organoids can be translated for the development of personalized medicine in assessment of individual cytokine sensitivity and molecularly targeted therapeutics in esophagitis patients © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of esophageal organoids from biopsy or murine esophageal epithelial sheets Basic Protocol 2: Propagation and cryopreservation of esophageal organoids Basic Protocol 3: Harvesting of esophageal organoids for RNA isolation, immunohistochemistry, and evaluation of 3D architecture Basic Protocol 4: Modeling of reactive epithelium in esophageal organoids., (© 2020 John Wiley & Sons, Inc.)

Balanced progenitor activities are crucial for the development and maintenance of high turn-over organs such as the esophagus. However, the molecular mechanisms regulating these progenitor activities in the esophagus remain to be elucidated. Here, we demonstrated that Yap is required for the proliferation of esophageal progenitor cells (EPCs) in the developing murine esophagus. We found that Yap deficiency reduces EPC proliferation and stratification whereas persistent Yap activation increases cell proliferation and causes aberrant stratification of the developing esophagus. We further demonstrated that the role of YAP signaling is conserved in the developing human esophagus by utilizing 3D human pluripotent stem cell (hPSC)-derived esophageal organoid culture. Taken together, our studies combining loss/gain-of-function murine models and hPSC differentiation support a key role for YAP in the self-renewal of EPCs and stratification of the esophageal epithelium., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Background: Fibrosis and stricture are major comorbidities in patients with eosinophilic esophagitis (EoE). Lysyl oxidase (LOX), a collagen cross-linking enzyme, has not been investigated in the context of EoE., Objective: We investigated regulation of epithelial LOX expression as a novel biomarker and functional effector of fibrostenotic disease conditions associated with EoE., Methods: LOX expression was analyzed by using RNA-sequencing, PCR assays, and immunostaining in patients with EoE; cytokine-stimulated esophageal 3-dimensional organoids; and fibroblast-epithelial cell coculture, the latter coupled with fluorescence-activated cell sorting., Results: Gene ontology and pathway analyses linked TNF-α and LOX expression in patients with EoE, which was validated in independent sets of patients with fibrostenotic conditions. TNF-α-mediated epithelial LOX upregulation was recapitulated in 3-dimensional organoids and coculture experiments. We find that fibroblast-derived TNF-α stimulates epithelial LOX expression through activation of nuclear factor κB and TGF-β-mediated signaling. In patients receiver operating characteristic analyses suggested that LOX upregulation indicates disease complications and fibrostenotic conditions in patients with EoE., Conclusions: There is a novel positive feedback mechanism in epithelial LOX induction through fibroblast-derived TNF-α secretion. Esophageal epithelial LOX might have a role in the development of fibrosis with substantial translational implications., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The esophageal lumen is lined by a stratified squamous epithelium comprised of proliferative basal cells that differentiate while migrating toward the luminal surface and eventually desquamate. Rapid epithelial renewal occurs, but the specific cell of origin that supports this high proliferative demand remains unknown. Herein, we have described a long-lived progenitor cell population in the mouse esophageal epithelium that is characterized by expression of keratin 15 (Krt15). Genetic in vivo lineage tracing revealed that the Krt15 promoter marks a long-lived basal cell population able to self-renew, proliferate, and generate differentiated cells, consistent with a progenitor/stem cell population. Transcriptional profiling demonstrated that Krt15+ basal cells are molecularly distinct from Krt15- basal cells. Depletion of Krt15-derived cells resulted in decreased proliferation, thereby leading to atrophy of the esophageal epithelium. Further, Krt15+ cells were radioresistant and contributed to esophageal epithelial regeneration following radiation-induced injury. These results establish the presence of a long-lived and indispensable Krt15+ progenitor cell population that provides additional perspective on esophageal epithelial biology and the widely prevalent diseases that afflict this epithelium.

Objectives: Eosinophilic esophagitis (EoE) is an immune-mediated allergic disease characterized by progressive esophageal dysmotility and fibrotic stricture associated with chronic esophageal fibroblast activation. It remains unknown how esophageal fibroblasts respond to EoE-relevant matrix stiffness or inflammatory cytokines., Methods: Immunofluorescence was used to evaluate α-smooth muscle actin (α-SMA) expression in endoscopic esophageal biopsies. Primary esophageal fibroblasts from adult and pediatric patients with or without EoE were exposed to transforming growth factor (TGF)β to determine gene expression, collagen-matrix contractility, and cytoskeletal organization. The influence of matrix stiffness upon fibroblast behavior was assessed on the engineered surface of polyacrylamide gels with varying stiffness. Fibroblast traction forces were measured using microfabricated-post-array-detectors., Results: EoE esophageal fibroblasts had enhanced α-SMA expression. TGFβ not only stimulated enhanced fibroblast-specific gene expression but also promoted fibroblast-mediated collagen-matrix contraction, despite disease state or age of patients as the origin of cells. Unlike conventional monolayer cell, culture conditions using plastic surface (1 GPa) that activates fibroblasts constitutively, our engineered platforms recapitulating physiologically relevant stiffness (1-20 kPa) revealed that matrix stiffness defines the extent of α-SMA expression, intracellular collagen fibril organization, SMAD3 phosphorylation, and fibroblast traction force., Conclusions: Matrix stiffness may critically influence TGFβ-mediated gene expression and functions of esophageal fibroblasts ex vivo independent of age and disease conditions. These findings provide a novel insight into the pathogenesis of fibrostenotic disease in EoE.

Eosinophilic esophagitis (EoE) is a chronic Th2 and food antigen-mediated disease characterized by esophageal eosinophilic infiltration. Thymic stromal lymphopoetin (TSLP), an epithelial derived cytokine which bridges innate and Th2-type adaptive immune responses in other allergic conditions, is overexpressed in esophageal biopsies of EoE subjects. However, the triggers of TSLP expression in the esophageal epithelium are unknown. The objective of the current study was to characterize TSLP expression in human esophageal epithelium in EoE in vivo and to determine the role of food antigens upon epithelial TSLP expression in vitro. Using immunohistochemistry (IHC), we localized TSLP in esophageal biopsies of active EoE (≥15 eos/hpf), inactive EoE (<15 eos/hpf) and non-EoE control subjects, and found that TSLP expression was restricted to the differentiated suprabasal layer of the epithelium in actively inflamed EoE biopsies. Consistent with these results in vivo, inducible TSLP protein secretion was higher in CaCl2 differentiated telomerase-immortalized esophageal epithelial cells (EPC2-hTERT) compared to undifferentiated cells of the basal phenotype, following stimulation with the TLR3 ligand poly(I:C). To determine whether food antigens could directly induce epithelial TSLP secretion, differentiated and undifferentiated primary esophageal epithelial cells from EoE and non-EoE subjects were challenged with food antigens clinically relevant to EoE: Chicken egg ovalbumin (OVA), wheat, and milk proteins beta-lactoglobulin (blg) and beta-casein. Food antigens failed to induce TSLP secretion by undifferentiated cells; in contrast, only OVA induced TSLP secretion in differentiated epithelial cells from both EoE and control cell lines, an effect abolished by budesonide and NF-κb inhibition. Together, our study shows that specific food antigens can trigger innate immune mediated esophageal TSLP secretion, suggesting that esophageal epithelial cells at the barrier surface may play a significant role in the pathogenesis of EoE by regulating TSLP expression.

Acetaldehyde is an ethanol-derived definite carcinogen that causes oesophageal squamous cell carcinoma (ESCC). Aldehyde dehydrogenase 2 (ALDH2) is a key enzyme that eliminates acetaldehyde, and impairment of ALDH2 increases the risk of ESCC. ALDH2 is produced in various tissues including the liver, heart, and kidney, but the generation and functional roles of ALDH2 in the oesophagus remain elusive. Here, we report that ethanol drinking increased ALDH2 production in the oesophagus of wild-type mice. Notably, levels of acetaldehyde-derived DNA damage represented by N(2)-ethylidene-2'-deoxyguanosine were higher in the oesophagus of Aldh2-knockout mice than in wild-type mice upon ethanol consumption. In vitro experiments revealed that acetaldehyde induced ALDH2 production in both mouse and human oesophageal keratinocytes. Furthermore, the N(2)-ethylidene-2'-deoxyguanosine levels increased in both Aldh2-knockout mouse keratinocytes and ALDH2-knockdown human keratinocytes treated with acetaldehyde. Conversely, forced production of ALDH2 sharply diminished the N(2)-ethylidene-2'-deoxyguanosine levels. Our findings provide new insight into the preventive role of oesophageal ALDH2 against acetaldehyde-derived DNA damage.

Esophageal cells overexpressing epidermal growth factor receptor (EGFR) and TP53 mutation can invade into the extracellular matrix when grown in 3D-organotypic cultures (OTC) and mimic early invasion in esophageal squamous cell carcinoma (ESCC). We have performed laser capture microdissection with RNA microarray analysis on the invasive and non-invasive tumor cells of p53(R175H)-overexpressing OTC samples to determine candidate genes facilitating tumor invasion. WNT10A was found to be >4-fold upregulated in the invasive front. Since WNT10A is also prominently upregulated during placode promotion in hair follicle development, a process that requires epithelial cells to thicken and elongate, in order to allow downward growth, we hypothesized that WNT10A may be important in mediating a similar mechanism of tumor cell invasion in ESCC. We have found that WNT10A expression is significantly upregulated in human ESCC, when compared with normal adjacent tissue. Furthermore, high WNT10A expression levels correlate with poor survival. Interestingly, we observe that WNT10A is expressed early in embryogenesis, but is reduced dramatically postnatally. We demonstrate that overexpression of WNT10a promotes migration and invasion, and proliferation of transformed esophageal cells. Lastly, we show that WNT10A overexpression induces a greater CD44(High)/CD24(Low) population, which are putative markers of cancer stem cells, and increases self-renewal capability. Taken together, we propose that WNT10A acts as an oncofetal factor that is highly expressed and may promote proper development of the esophagus. During tumorigenesis, it is aberrantly overexpressed in order to promote ESCC migration and invasion, and may be linked to self-renewal of a subset of ESCC cells., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Tissue homeostasis requires balanced self-renewal and differentiation of stem/progenitor cells, especially in tissues that are constantly replenished like the esophagus. Disruption of this balance is associated with pathological conditions, including eosinophilic esophagitis (EoE), in which basal progenitor cells become hyperplastic upon proinflammatory stimulation. However, how basal cells respond to the inflammatory environment at the molecular level remains undetermined. We previously reported that the bone morphogenetic protein (BMP) signaling pathway is critical for epithelial morphogenesis in the embryonic esophagus. Here, we address how this pathway regulates tissue homeostasis and EoE development in the adult esophagus. BMP signaling was specifically activated in differentiated squamous epithelium, but not in basal progenitor cells, which express the BMP antagonist follistatin. Previous reports indicate that increased BMP activity promotes Barrett's intestinal differentiation; however, in mice, basal progenitor cell-specific expression of constitutively active BMP promoted squamous differentiation. Moreover, BMP activation increased intracellular ROS levels, initiating an NRF2-mediated oxidative response during basal progenitor cell differentiation. In both a mouse EoE model and human biopsies, reduced squamous differentiation was associated with high levels of follistatin and disrupted BMP/NRF2 pathways. We therefore propose a model in which normal squamous differentiation of basal progenitor cells is mediated by BMP-driven NRF2 activation and basal cell hyperplasia is promoted by disruption of BMP signaling in EoE.

Barrett's esophagus (BE) is defined as an incomplete intestinal metaplasia characterized generally by the presence of columnar and goblet cells in the formerly stratified squamous epithelium of the esophagus. BE is known as a precursor for esophageal adenocarcinoma. Currently, the cell of origin for human BE has yet to be clearly identified. Therefore, we investigated the role of Notch signaling in the initiation of BE metaplasia. Affymetrix gene expression microarray revealed that BE samples express decreased levels of Notch receptors (NOTCH2 and NOTCH3) and one of the the ligands (JAG1). Furthermore, BE tissue microarray showed decreased expression of NOTCH1 and its downstream target HES1. Therefore, Notch signaling was inhibited in human esophageal epithelial cells by expression of dominant-negative-Mastermind-like (dnMAML), in concert with MYC and CDX1 overexpression. Cell transdifferentiation was then assessed by 3D organotypic culture and evaluation of BE-lineage specific gene expression. Notch inhibition promoted transdifferentiation of esophageal epithelial cells toward columnar-like cells as demonstrated by increased expression of columnar keratins (K8, K18, K19, K20) and glandular mucins (MUC2, MUC3B, MUC5B, MUC17) and decreased expression of squamous keratins (K5, K13, K14). In 3D culture, elongated cells were observed in the basal layer of the epithelium with Notch inhibition. Furthermore, we observed increased expression of KLF4, a potential driver of the changes observed by Notch inhibition. Interestingly, knockdown of KLF4 reversed the effects of Notch inhibition on BE-like metaplasia. Overall, Notch signaling inhibition promotes transdifferentiation of esophageal cells toward BE-like metaplasia in part via upregulation of KLF4. These results support a novel mechanism through which esophageal epithelial transdifferentiation promotes the evolution of BE.

This protocol describes the isolation and characterization of mouse and human esophageal epithelial cells and the application of 3D organotypic culture (OTC), a form of tissue engineering. This model system permits the interrogation of mechanisms underlying epithelial-stromal interactions. We provide guidelines for isolating and cultivating several sources of epithelial cells and fibroblasts, as well as genetic manipulation of these cell types, as a prelude to their integration into OTC. The protocol includes a number of important applications, including histology, immunohistochemistry/immunofluorescence, genetic modification of epithelial cells and fibroblasts with retroviral and lentiviral vectors for overexpression of genes or RNA interference strategies, confocal imaging, laser capture microdissection, RNA microarrays of individual cellular compartments and protein-based assays. The OTC (3D) culture protocol takes 15 d to perform.

Background & Aims: The Notch receptor family regulates cell fate through cell-cell communication. CSL (CBF-1/RBP-jκ, Su(H), Lag-1) drives canonical Notch-mediated gene transcription during cell lineage specification, differentiation, and proliferation in the hematopoietic system, the intestine, the pancreas, and the skin. However, the functional roles of Notch in esophageal squamous epithelial biology are unknown., Methods: Normal esophageal keratinocytes were stimulated with calcium chloride to induce terminal differentiation. The squamous epithelia were reconstituted in organotypic 3-dimensional culture, a form of human tissue engineering. Notch was inhibited in culture with a γ-secretase inhibitor or dominant negative mastermind-like 1 (DNMAML1). The roles of Notch receptors were evaluated by in vitro gain-of-function and loss-of-function experiments. Additionally, DNMAML1 was targeted to the mouse esophagus by cytokeratin K14 promoter-driven Cre (K14Cre) recombination of Lox-STOP-Lox-DNMAML1. Notch-regulated gene expression was determined by reporter transfection, chromatin immunoprecipitation assays, quantitative reverse-transcription polymerase chain reaction, Western blotting, immunofluorescence, and immunohistochemistry., Results: NOTCH1 (N1) was activated at the onset of squamous differentiation in the esophagus. Intracellular domain of N1 (ICN1) directly activated NOTCH3 (N3) transcription, inducing HES5 and early differentiation markers such as involucrin (IVL) and cytokeratin CK13 in a CSL-dependent fashion. N3 enhanced ICN1 activity and was required for squamous differentiation. Loss of Notch signaling in K14Cre;DNMAML1 mice perturbed esophageal squamous differentiation and resulted in N3 loss and basal cell hyperplasia., Conclusions: Notch signaling is important for esophageal epithelial homeostasis. In particular, the cross talk of N3 with N1 during differentiation provides novel, mechanistic insights into Notch signaling and squamous epithelial biology., (Copyright © 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Insulin-like growth factor-binding protein (IGFBP)-3 is overexpressed frequently in esophageal squamous cell carcinoma. Yet, the role of IGFBP3 in esophageal tumor biology remains to be elucidated. We find that IGFBP3 facilitates transforming growth factor (TGF)-beta1-mediated epithelial-to-mesenchymal transition (EMT) in transformed human esophageal epithelial cells, EPC2-hTERT-EGFR-p53(R175H). In organotypic 3D culture, a form of human tissue engineering, laser-capture microdissection revealed concurrent upregulation of TGF-beta target genes, IGFBP3 and EMT-related genes in the cells invading into the stromal compartment. IGFBP3 enhanced TGF-beta1-mediated EMT as well as transcription factors essential in EMT by allowing persistent SMAD2 and SMAD3 phosphorylation. TGF-beta1-mediated EMT and cell invasion were enhanced by ectopically expressed IGFBP3 and suppressed by RNA interference directed against IGFBP3. The IGFBP3 knockdown effect was rescued by IGFBP3(I56G/L80G/L81G), a mutant IGFBP3 lacking an insulin-like growth factor (IGF)-binding capacity. Thus, IGFBP3 can regulate TGF-beta1-mediated EMT and cell invasion in an IGF or insulin-like growth factor 1 receptor-independent manner. IGFBP3(I56G/L80G/L81G) also promoted EMT in vivo in a Ras-transformed human esophageal cell line T-TeRas upon xenograft transplantation in nude mice. In aggregate, IGFBP3 may have a novel IGF-binding independent biological function in regulation of TGF-beta1-mediated EMT and cell invasion.

Background: Left atrial-esophageal fistula is a serious and poorly understood complication of catheter ablation of atrial fibrillation. The purpose of this study was to (1) develop a canine model of esophageal injury and left atrial-esophageal fistula after applications of forward-firing high-intensity focused ultrasound (HIFU) and side-firing unfocused ultrasound (SFU); (2) examine the relationship to esophageal temperature (Eso-temp); and (3) study the evolution of injury/healing., Methods and Results: Twenty dogs were studied. After transeptal puncture, HIFU catheter (ProRhythm Inc; 13 dogs) was positioned close to the esophagus, either outside (n=6) or inside (n=7) the inferior pulmonary vein (PV). In 7 other dogs, an SFU catheter was placed deep inside the PV, close to the esophagus. A balloon (20- to 25-mm diameter) with 7 thermocouples (2-mm separation) was positioned in the esophagus (Eso-balloon). Variable air filling of the Eso-balloon controlled the distance from the esophagus to the sonication source, pressing the esophagus against left atrium/PV. One to 9 (median, 5) HIFU (35 W) and 5 to 7 (median, 5) SFU (40 W) sonications were delivered for 40 seconds. Maximum luminal Eso-temp was closely related to HIFU Eso-balloon distance. For HIFU outside PV, Eso-temp >or=50 degrees C occurred only for HIFU Eso-balloon distance or=50 degrees C, with HIFU Eso-balloon distance up to 6.8 mm. Endoscopy identified esophageal ulcer immediately after ablation in 11 of 13 HIFU dogs and 7 of 7 SFU dogs, all with Eso-temp >or=50 degrees C. Endoscopy at 2 weeks showed ulcer healing in 5 of 11 chronic dogs and ulcer size progression with relaxation of the lower esophageal sphincter and esophagitis in 6 dogs. Two dogs developed left atrial-esophageal fistula and died at 2 weeks., Conclusions: This model produces esophageal ulcer when Eso-temp is >or=50 degrees C. Eso-temp is higher with HIFU/SFU applications closer to the esophagus and with HIFU/SFU applications inside the PV. Ulcer progression and left atrial-esophageal fistula were associated with reflux esophagitis.

Barrett's esophagus (BE) is the replacement of normal squamous esophageal mucosa with an intestinalized columnar epithelium. The molecular mechanisms underlying its development are not understood. Cdx2 is an intestine-specific transcription factor that is ectopically expressed in BE, but its role in this process is unclear. Herein, we describe a novel cell culture model for BE. Retroviral-mediated Cdx2 expression in immortalized human esophageal keratinocytes [EPC-human telomerase reverse transcriptase (hTERT)] could transiently be established but not maintained and was associated with a reduction in cell proliferation. Coexpression of cyclin D1, but not a dominant-negative p53, rescued proliferation in the Cdx2-expressing cells. Cdx2 expression in the EPC-hTERT.D1 cells decreased cell proliferation but did not induce intestinalization. We investigated for other treatments to enhance intestinalization and found that acidic culture conditions uniformly killed EPC-hTERT.D1.Cdx2 cells. However, treatment with 5-aza-2-deoxycytidine (5-AzaC) to demethylate epigenetically silenced genes did appear to be tolerated. Multiple Cdx2 target genes, markers of intestinal differentiation and markers of BE, were induced by this 5-AzaC treatment. More interestingly, the expression level of several of these genes was enhanced only in the EPC-hTERT.D1-Cdx2 cells treated with 5-AzaC. Two of these, SLC26a3/DRA (downregulated in adenoma) and Na+/H+ exchanger 2 (NHE2), were not previously known to be elevated in BE; however, we confirmed their elevation in BE tissue samples. 5-AzaC treatment also induced cell senescence, even at low doses. We conclude that ectopic proliferation signals, alterations in epigenetic gene regulation and the inhibition of tumor suppressor mechanisms are required for Cdx2-mediated intestinalization of human esophageal keratinocytes in BE.

The esophageal epithelium is a prototypical stratified squamous epithelium that exhibits an exquisite equilibrium between proliferation and differentiation. After basal cells proliferate, they migrate outward toward the luminal surface, undergo differentiation, and eventually slough due to apoptosis. The identification and characterization of stem cells responsible for the maintenance of the esophageal epithelium remains elusive. Here, we employed Hoechst dye extrusion and BrdU label-retaining assays to identify in mice a potential esophageal stem cell population that localizes to the basal cell compartment. The self-renewing capacity of this population was characterized using a clonogenic assay and a 3D organotypic culture model. The putative esophageal stem cells were also capable of epithelial reconstitution in vivo in direct esophageal epithelial injury models. In both the 3D organotypic culture and direct mucosal injury models, the putative stem cells gave rise to undifferentiated and differentiated cells. These studies therefore provide a basis for understanding the regenerative capacity and biology of the esophageal epithelium when it is faced with injurious insults.

Esophageal cancer is a prototypic squamous cell cancer that carries a poor prognosis, primarily due to presentation at advanced stages. We used human esophageal epithelial cells as a platform to recapitulate esophageal squamous cell cancer, thereby providing insights into the molecular pathogenesis of squamous cell cancers in general. This was achieved through the retroviral-mediated transduction into normal, primary human esophageal epithelial cells of epidermal growth factor receptor (EGFR), the catalytic subunit of human telomerase (hTERT), and p53(R175H), genes that are frequently altered in human esophageal squamous cell cancer. These cells demonstrated increased migration and invasion when compared with control cells. When these genetically altered cells were placed within the in vivo-like context of an organotypic three-dimensional (3D) culture system, the cells formed a high-grade dysplastic epithelium with malignant cells invading into the stromal extracellular matrix (ECM). The invasive phenotype was in part modulated by the activation of matrix metalloproteinase-9 (MMP-9). Using pharmacological and genetic approaches to decrease MMP-9, invasion into the underlying ECM could be suppressed partially. In addition, tumor differentiation was influenced by the type of fibroblasts within the stromal ECM. To that end, fetal esophageal fibroblasts fostered a microenvironment conducive to poorly differentiated invading tumor cells, whereas fetal skin fibroblasts supported a well-differentiated tumor as illustrated by keratin "pearl" formation, a hallmark feature of well-differentiated squamous cell cancers. When inducible AKT was introduced into fetal skin esophageal fibroblasts, a more invasive, less-differentiated esophageal cancer phenotype was achieved. Invasion into the stromal ECM was attenuated by genetic knockdown of AKT1 as well as AKT2. Taken together, alterations in key oncogenes and tumor suppressor genes in esophageal epithelial cells, the composition and activation of fibroblasts, and the components of the ECM conspire to regulate the physical and biological properties of the stroma.

Heparanase is an endo-beta-glucuronidase that specifically cleaves heparan sulfate (HS) chains. Heparanase is involved in the process of metastasis and angiogenesis through the degradation of HS chains of the extracellular matrix and cell surface. Recently, we demonstrated that heparanase was localized in the cell nucleus of normal esophageal epithelium and esophageal cancer, and that its expression was correlated with cell differentiation. However, the nuclear function of heparanase remains unknown. To elucidate the role of heparanase in esophageal epithelial differentiation, primary human esophageal cells were grown in monolayer as well as organotypic cultures, and cell differentiation was induced. Expression of heparanase, HS, involucrin, and p27 was determined by immunostaining and Western blotting. SF4, a novel pharmacological inhibitor, was used to specifically inhibit heparanase activity. Upon esophageal cell differentiation, heparanase was translocated from the cytoplasm to the nucleus. Such translocation of heparanase appeared to be associated with the degradation of HS chains in the nucleus and changes in the expression of keratinocyte differentiation markers such as p27 and involucrin, whose induction was inhibited by SF4. Furthermore, these in vitro observations agreed with the expression pattern of heparanase, HS, involucrin, cytokeratin 13, and p27 in normal esophageal epithelium. Nuclear translocation of heparanase and its catalytic cleavage of HS may play a critical role in the differentiation of esophageal epithelial cells. Our study provides a novel insight into the role of heparanase in an essential differentiation process.

IGF and EGF regulate various physiological and pathological processes. IGF binding protein (IGFBP)-3 regulates cell proliferation in IGF-dependent and -independent fashions. Recently, we identified IGFBP-3 as a novel EGF receptor (EGFR) downstream target molecule in primary and immortalized human esophageal epithelial cells, suggesting an interplay between the EGF and IGF signaling pathways. However, the regulatory mechanisms for IGFBP-3 expression and its functional role in esophageal cell proliferation remain to be elucidated. Herein, we report that IGFBP-3 mRNA and protein were induced upon growth factor deprivation in primary and immortalized human esophageal cells through mechanisms requiring p53-independent de novo mRNA transcription and protein synthesis. This occurred in the face of the activated phosphatidylinositol 3-OH-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway. Secreted IGFBP-3 neutralized IGFs and prevented IGF-I receptor (IGF-IR) activation. In contrast, EGF suppressed IGFBP-3 mRNA and protein expression through activation of MAPK in an EGFR-tyrosine kinase-dependent manner to restore the cellular response to IGF-I. When stably overexpressed, wild-type IGFBP-3 but not I56G/L80G/L81G (GGG) mutant IGFBP-3, which has a reduced affinity to IGFs, prevented IGF-I from activating IGF-IR and Akt as well as stimulating cell proliferation. However, unlike other cell types where IGFBP-3 exerts antiproliferative effects, neither wild-type nor GGG mutant IGFBP-3 alone affected cell proliferation or EGFR activity. These results indicate that IGF signaling is subject to negative regulation through IGFBP-3 and positive regulation by EGF, the latter of which suppresses IGFBP-3. This provides a platform for understanding the novel cross talk between EGF- and IGF-mediated pathways.

The epidermal growth factor receptor (EGFR) activates several signaling cascades in response to epidermal growth factor stimulation. One of these signaling events involves tyrosine phosphorylation of signal transducer and activator of transcription (STAT), whereas another involves activation of the phosphatidylinositol 3-OH kinase pathway. Two possibilities for STAT activation exist: a janus kinase (JAK)-dependent and a JAK-independent mechanism. Herein, we demonstrate that EGFR overexpression in primary esophageal keratinocytes activates STAT in a JAK-dependent fashion with the functional consequence of enhanced cell migration, which can be abolished by use of a JAK-specific inhibitor, AG-490. We determined the mechanisms underlying the signal transduction pathway responsible for increased cell migration. Stimulation of EGFR induces Tyr701 phosphorylation of STAT1 and initiates complex formation of STAT1 and STAT3 with JAK1 and JAK2. Thereafter, the STATs translocate to the nucleus within 15 min. In addition, we found that activation of this signaling pathway results in matrix metalloproteinase-1 (MMP-1) activity. By contrast, Akt activation does not impact the EGFR-STATs-JAKs complex formation and nuclear translocation of the STATs with subsequent MMP-1 activity, although Akt activation may contribute to cell migration through an independent mechanism. Taken together, we find that the recruitment of the STAT-JAK complex by EGFR is responsible for keratinocyte migration that, in turn, might be mediated by MMP-1 activation.

Epidermal growth factor receptor (EGFR) overexpression is observed in a number of malignancies, especially those of esophageal squamous cell origin. However, little is known about the biological functions of EGFR in primary esophageal squamous epithelial cells. Using newly established primary human esophageal squamous epithelial cells as a platform, we overexpressed EGFR through retroviral transduction and established novel three-dimensional organotypic cultures. Additionally, EGFR was targeted in a cell type- and tissue-specific fashion to the esophageal epithelium in transgenic mice. EGFR overexpression in primary esophageal keratinocytes resulted in the biochemical activation of Akt and STAT pathways and induced enhanced cell migration and cell aggregation. When established in organotypic culture, EGFR-overexpressing cells had evidence of epithelial cell hyperproliferation and hyperplasia. These effects were also observed in EGFR-overexpressing transgenic mice and the esophageal cell lines established thereof. In particular, EGFR-induced effects upon aggregation appear to be mediated through the relocalization of p120 from the cytoplasm to the membrane and increased interaction with E-cadherin. EGFR modulates cell migration through the up-regulation of matrix metalloproteinase 1. Taken together, the functional effects of EGFR overexpression help to explain its role in the initiating steps of esophageal squamous carcinogenesis.

Background: Eosinophilic esophagitis (EoE) is a chronic allergic disease associated with type 2 inflammation and epithelial barrier dysfunction. The etiology is unknown, however, genetic heritability studies suggest environmental factors play a key role in pathogenesis. Detergents, such as sodium dodecyl sulfate (SDS), are common ingredients in household products such as dish soap and toothpaste. We hypothesized detergent exposure decreases epithelial barrier function and induces esophageal inflammation. Methods: Immortalized esophageal epithelial cells (EPC2) were cultured in air‐liquid interface (ALI) and exposed to SDS. Barrier function/activity was assessed by transepithelial electrical resistance (TEER), FITC‐dextran flux, and RT‐PCR. Additionally, SDS‐treated mouse esophageal organoids were evaluated for morphology. To investigate the effects of SDS in vivo, mice were treated with 0.5% SDS in drinking water for 14 days. Esophagi were assessed by gross morphology, histopathology, protein expression, and bulk RNA sequencing. Results: When EPC2 cells were exposed to SDS (5 μg/ml) for 96 h, TEER decreased (p = 0.03), and FITC‐dextran flux increased (p = 0.0002). mRNA expression of IL‐33 increased 4.5‐fold (p = 0.02) at 6 h and DSG1 decreased (p < 0.0001) by 72 h. Disrupted epithelial integrity was noted in SDS‐treated esophageal organoids. When mice were exposed to SDS, they showed increased esophageal width, chemokine, and metalloprotease levels. Mice treated with SDS also showed increased IL‐33 protein expression, basal zone hyperplasia, CD4+ cell infiltration, and esophageal eosinophilia. RNA sequencing revealed upregulation of immune response pathway genes. Conclusion: Exposure to SDS decreases esophageal barrier integrity, stimulates IL‐33 production, and promotes epithelial hyperplasia and tissue eosinophilia. Detergents may be a key environmental trigger in EoE pathogenesis. [ABSTRACT FROM AUTHOR]

Eosinophilic esophagitis (EoE) is a food allergen-induced inflammatory disorder. EoE is increasingly recognized as a cause of swallowing dysfunction, food impaction and esophageal stricture. Inflammation of the esophageal mucosa involves immune cell infiltrate, reactive epithelial changes and fibroblast activation, culminating in robust tissue remodeling toward esophageal fibrosis characterized by excess collagen deposition in the subepithelial lamina propria. Fibrosis contributes to a unique mechanical property of the EoE-affected esophagus that is substantially stiffer than the normal esophagus. There is a great need to better understand the processes behind esophageal fibrosis in order to foster improved diagnostic tools and novel therapeutics for EoE-related esophageal fibrosis. In this review, we discuss the role of esophageal inflammatory microenvironment that promotes esophageal fibrosis, with specific emphasis upon cytokines-mediated functional epithelial-stromal interplays, recruitment and activation of a variety of effector cells, and tissue stiffness. We then explore the current state of clinical methodologies to detect and treat the EoE-related esophageal stricture. [ABSTRACT FROM AUTHOR]

Abstract: Minimally invasive central corpectomy (MICC) for cervical segmental ossified posterior longitudinal ligament (OPLL) is described. The procedure of MICC includes upper- or lower-half central corpectomy of the involved cervical spine, transdiscal decompression of the adjacent disc level, dissection and partial removal of the OPLL, removal of the OPLL behind the vertebral body via these windows, and fusion with cylindrical titanium cages. Anterior plate fixation is not necessary. From January 2008 to December 2009 we surgically treated three patients with cervical OPLL by MICC. All three patients showed remarkable improvement of their symptoms within a few days after the operation. No neurological or radiological complication was observed during that period. MICC is beneficial in avoiding complete corpectomy and long fusion, usage of an anterior plate, and usage of a large external orthosis. MICC also reduces the risk of postoperative esophageal perforation due to a screw backing out of the plate. [ABSTRACT FROM AUTHOR]

Oncogenic Ras induces premature senescence in primary cells. Such an oncogene-induced senescence involves activation of tumor suppressor genes that provide a checkpoint mechanism against malignant transformation. In mouse, the ARF-p53 pathway mediates Ha-RasG12V-induced senescence, and p19ARF-/- and p53-/- cells undergo transformation upon Ras activation. In addition, mouse cells, unlike human cells, express constitutively active telomerase and have long telomeres. However, it is unclear how Ras activation affects human cells of epithelial origin with p53 mutation and/or telomerase activation. In order to address this question, Ha-RasG12V was expressed ectopically in primary as well as hTERT-immortalized human esophageal keratinocytes stably expressing dominant-negative p53 mutants. In human esophageal keratinocytes, we found that Ha-RasG12V induced senescence regardless of p53 status and telomerase activation. Ras activation resulted in changes of cellular morphology, activation of senescence-associatedß-galactosidase, and suppression of cell proliferation, all coupled with reduction in the hyperphosphorylated form of the retinoblastoma protein (pRb). Furthermore, Ha-RasG12V upregulated p16INK4a and downregulated cyclin-dependent kinase Cdk4 in human esophageal keratinocytes. Thus, Ras-mediated senescence may involve distinct mechanisms between human and mouse cells. Inactivation of the pRb pathway may be necessary for Ras to overcome senescence and transform human esophageal epithelial cells.Oncogene (2004) 23, 6760-6768. doi:10.1038/sj.onc.1207923 Published online 26 July 2004 [ABSTRACT FROM AUTHOR]

Although eosinophil count is the standard used to monitor disease activity in patients with eosinophilic esophagitis (EoE), there are often disparities between patient-reported symptoms and eosinophil counts. We examined the prevalence of epithelial alterations, namely basal cell hyperplasia (BCH) and spongiosis, among patients with inactive EoE (eosinophil counts below 15 following therapy) and aimed to determine whether maintenance of these changes in epithelial morphology are associated with persistent clinical findings. Esophageal biopsies of 243 patients (mean age, 16.9 years) undergoing routine endoscopy at the University of Pennsylvania were evaluated for epithelial BCH and spongiosis. Univariable analysis was used to calculate the association between epithelial changes and symptoms as well as endoscopic findings and peak eosinophil count. We validated our findings using data from a cohort of patients at the University of North Carolina. The discovery and validation cohorts each included patients with inactive EoE, based on histologic factors, but ongoing BCH and spongiosis. Ongoing BCH, but not spongiosis, in patients with inactive EoE was associated with symptoms (odds ratio, 2.14; 95% CI, 1.03–4.42; P =.041) and endoscopic findings (odds ratio, 7.10; 95% CI, 3.12–16.18; P <.001). In patients with EoE, the presence of BCH might indicate ongoing disease activity, independent of eosinophil count. This might account for the persistent symptoms in patients who are considered to be in remission based on histologic factors. [ABSTRACT FROM AUTHOR]