Document text
Principal Investigator: Kelly Sabrina Lynn
Organization: EMORY UNIVERSITY
Fiscal Year: 2019
Award: $45,016
Funding agency: National Heart Lung and Blood Institute
ABSTRACT
Acute respiratory distress syndrome (ARDS) is a serious lung condition characterized by airspace flooding and
widespread inflammation. Survival of ARDS is chiefly attributed to the ability to maintain airspace fluid balance.
This involves two complimentary processes: sodium-driven fluid clearance and regulation of fluid diffusion
through the epithelial barrier. The severity and risk of ARDS is magnified with chronic alcohol abuse. In the
alcoholic lung, paracellular diffusion is increased, resulting in fluid leakage into the lung, and thus
compensatory fluid clearance is necessary to maintain fluid balance, leaving the alcoholic lung primed for
ARDS. Currently, our lab is investigating the molecular mechanisms behind increased incidence of ARDS, with
particular interest in the effects on tight junctions. Tight junctions serve as the major functional unit of the
epithelial barrier and are crucial in providing the selective permeability by which both fluid clearance and
diffusion are achieved. Previous work in the Koval lab determined that chronic alcohol ingestion increases
expression of tight junction protein claudin-5 by the alveolar epithelium, which is necessary and sufficient to
decrease alveolar epithelial barrier function. This impairment of the alveolar epithelial barrier correlated to
molecular rearrangement of claudin-18 into spike-like structures perpendicular to the cell junction interface.
These “tight junction spikes” (TJ spikes) appear to be active areas of junction remodeling driven by increased
endocytosis of tight junction proteins. Treatment with the endocytosis inhibitor Dynasore, which targets the
actin-binding protein dynamin, significantly reduces the number of TJ spikes. This suggests a role for clathrin-
mediated, dynamin-dependent endocytosis in TJ spike formation. However, Dynasore is still capable of
inhibiting clathrin-mediated endocytosis in dynamin triple-knockout mouse fibroblasts, suggestive of
Dynasore's off-target effects. Defining roles of one or more dynamin isoforms in TJ spike formation requires a
more detailed molecular analysis. We hypothesize that dynamin induces the rearrangement of claudin-18
into TJ spikes that increase paracellular leak. We plan to test our hypothesis through the following aims. In
Aim 1, we will examine the requirement of dynamin in TJ spike formation by manipulating dynamin expression
in alveolar epithelial cells. It is also possible that TJ spike formation is due to other proteins beyond dynamin. In
order to elucidate the molecular mechanism behind TJ spike formation, more candidates in addition to dynamin
must be explored. In Aim 2, we will identify proteins that interact with claudin-18 that are associated with
promoting TJ spike formation using complementary techniques to assess TJ protein interactions in situ. The
long-term goal is to identify novel therapeutic targets to improve barrier function by redirecting spike-
associated claudin-18 into barrier forming tight junctions.
Terms: <ARDS><Actin-Binding Protein><Acute Respiratory Distress><Acute Respiratory Distress Syndrome><Adult ARDS><Adult RDS><Adult Respiratory Distress Syndrome><Alcohol Chemical Class><Alcohol abuse><Alcoholic><Alcohols><Alveolar><Antibodies><Area><Assay><Bioassay><Biochemical><Biologic Assays><Biological Assay><Boozer><Cell Body><Cell Junctions><Cells><Cessation of life><Chronic><Clathrin><Closure by Ligation><Co-Immunoprecipitations><Common Rat Strains><Critical Illness><Critically Ill><Da Nang Lung><Data><Death><Dependent drinker><Dephosphin><Development><Diffusion><Dynamin><Dysfunction><Endocytosis><Epithelial><Epithelial Cells><Equilibrium><EtOH abuse><Extravasation><Fibroblasts><Flooding><Floods><Fluid Balance><Functional disorder><Goals><Imaging Procedures><Imaging Technics><Imaging Techniques><Immunofluorescence><Immunofluorescence Immunologic><Impairment><In Situ><Incidence><Inflammation><Intercellular Junctions><Ions><Isoforms><KO mice><Knock-out Mice><Knockout Mice><Lead><Leakage><Ligation><Liquid substance><Lung><Lung Alveolar Epithelia><Lung Respiratory System><Measures><Mediating><Methods><Microscopy><Modeling><Molecular><Molecular Analysis><Na element><Null Mouse><Occluding Junctions><Patients><Pb element><Permeability><Physiopathology><Position><Positioning Attribute><Process><Prognosis><Protein Isoforms><Proteins><Publishing><Rat><Rats Mammals><Rattus><Regulation><Research><Resolution><Respiratory Failure><Risk><Role><Severities><Shock Lung><Site><Sodium><Spillage><Staining method><Stains><Stiff lung><Structure><Suggestion><Techniques><Testing><Tight Junctions><Work><Zonula Occludens><alcohol co-abuse><alcohol effect><alcohol problem><alveolar epithelium><balance><balance function><chronic EtOH drinking><chronic alcohol consumption><chronic alcohol drinking><chronic alcohol ingestion><chronic alcohol use><chronic ethanol consumption><chronic ethanol drinking><chronic ethanol ingestion><developmental><ethanol abuse><ethanol effect><experience><fluid><hazardous alcohol use><heavy metal Pb><heavy metal lead><improved><inhibitor><inhibitor/antagonist><interest><knock-down><knockdown><liquid><molecular rearrangement><mortality><new drug target><new druggable target><new pharmacotherapy target><new therapeutic target><new therapy target><novel><novel drug target><novel druggable target><novel pharmacotherapy target><novel therapeutic target><novel therapy target><outcome forecast><pathophysiology><prevent><preventing><problem alcohol use><problem drinker><problem drinking><problematic alcohol consumption><problematic alcohol use><protein complex><pulmonary><pulmonary failure><respiratory insufficiency/failure><response><shRNA><short hairpin RNA><small hairpin RNA><small molecule><social role><therapeutic target><wet lung>