Basic Research
Section of Gastroenterology & Hepatology has two major research grants funded by NIH:
Esophageal Cytoprotection – Agents & Mechanisms: The long term goal of this proposal is to understand the pathogenesis of the human disease, reflux esophagitis (RE), and through this means, to open new avenues for its cure or prevention. The goal is approached mechanistically and from the relatively uncommon vantage point of the target tissue, the esophageal epithelium (EE). In this Merit Award proposal, we focus on the barrier properties of intercellular junctions (ICJs) in EE, and especially on the role of E-cadherin which plays a major role in ICJ regulation of shunt permeability. The importance of the ICJs is that experimental models show them to be the initial target for attack by luminal acid or acid-pepsin - with the resulting damage increasing shunt permeability and setting in motion a sequence of events that culminate in esophagitis. Also clinically the presence of dilated intercellular spaces (DIS) provides circumstantial evidence that defects in ICJ barrier function are present in patients with nonerosive reflux esophagitis (NERD) and reflux esophagitis (RE). Therefore, the goals of the present proposal are to: a) further characterize ICJ structure and function in healthy and acid or acid-pepsin damaged EE; b) identify and characterize the associated proteins and mechanisms by which E-cadherin regulates ICJ barrier function in EE; and c) document and characterize the defects in ICJ barrier function in patients with NERD and RE – the latter by mounting endoscopic biopsies in mini-Ussing chambers for electrical measurements basally and in response to Na-free and Cl-free solutions and for performance of circuit analysis. Morphology of human biopsy material is also done for correlation of electrical changes with the presence of DIS or changes in E-cadherin quantity or pattern of distribution within the ICJs. Other tools used in this investigation include standard Ussing chamber technology, primary cell culture, protein purification and separation techniques, amino acid analysis, immunohistochemistry, transmission electron microscopy, and freeze fracture.
Mechanisms of Acid Resistance in Barrett’s Esophagus -- Barrett's esophagus denotes the presence of specialized columnar epithelium (SCE) in distal esophagus. It develops in subjects with acid reflux disease as replacement for destroyed esophageal stratified squamous epithelium (ESSE). In ~90%, it remains stable for life and irrespective of treatment since most - presumably because of its acid resistance - are (reflux) symptom-free. In one sense, then, Barrett’s is a successful adaptation for esophageal protection. Barrett’s, however, has a high cell turnover rate which establishes it as a premalignant lesion that increases esophageal cancer risk 40-fold over the general population. Though clinically important, little is known about the biology of SCE, and specifically about the mechanisms for acid resistance that are at the heart of its very existence. Therefore the goals of this proposal are: a) to define the preepithelial and epithelial mechanisms for SCE’s survival under acidic stress: b) compare them to ESSE in Barrett’s in order to test the hypothesis that SCE develops in GERD because of intrinsic mechanisms that make it more acid resistant than ESSE, and c) since alcohol and cigarette smoking increase the risk of malignancy in SCE, test the hypothesis that this risk reflects the ability of these agents to shorten cell survival (which increases cell turnover) by impairing SCE’s ability to protect itself under an acidic stress. The specific aims are to determine in SCE and ESSE in Barrett’s: 1) the magnitude of the lumen-to-surface pH gradient and role of mucus and HCO3, 2) the permeability of the apical membrane-junctional complex to H+, 3) the total cell buffer capacity and role of carbonic anhydrase, 4) the nature, location and activity of transporters for pHi regulation, 5) the effect of alcohol and smoking on cell survival under an acid stress, and to: 6) compare the results to ESSE in those without esophageal disease. Studies are performed using esophageal biopsies for cell isolation, culture and mounting in mini-Ussing chambers. Techniques include: pH microelectrodes, immunohistochemistry, impedance analysis and fluorescence microscopy. Achievement of the proposed goals will yield information of fundamental importance to understanding the pathogenesis and malignant potential of SCE in reflux subjects and provide insights into possible novel strategies for its prevention, stabilization or selective eradication for reduction of cancer risk.
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