The Effects of Hypoxia and Inflammation on Claudin‐1 in CaCo‐2 Cells as a Model Intestinal Epithelium.

Abstract

BackgroundThe use of hydrocortisone in association with indomethacin is a known risk factor for spontaneous intestinal perforation in extremely low birthweight infants. Hypoxia, inflammation and non‐steroidal anti‐inflammatory drugs independently alter apical junctional complexes of epithelial cells. We are interested in understanding the combined effects of hypoxia, inflammation and non‐steroidal anti‐inflammatory drugs on the apical junctional complex and barrier integrity in a gastrointestinal epithelial cell model.MethodsThrough the use of CaCo‐2 BBe monolayers, an established in vitro model of the intestinal epithelium, we analyzed the effects of the pro‐inflammatory agents (TNF and IFN‐gamma) and anti‐inflammatory agents (indomethacin and hydrocortisone) on junctional protein expression and localization, cell proliferation using Edu labeling, cellular ATP concentration and barrier function.ResultsProtein expression analyzed by Western blotting techniques demonstrated an alteration of claudin‐1, a tight junction sealing protein. The soluble and insoluble fractions of the cells were analyzed in order to determine the location of these proteins. We determined inflammatory conditions produced a higher concentration in the soluble fraction indicating impaired function. Confocal analysis after 24‐hour exposure to hypoxia showed that the combined use of hypoxia and inflammatory agents elicited no significant change in cell proliferation levels. Cellular ATP levels were analyzed to corroborate proliferation studies. Confocal imaging under the same experimental conditions was also conducted in order to probe for claudin‐1 localization within the cells.ConclusionsThere was no observed significant difference in the cell proliferation rate or marked changes in ATP levels in our model. Therefore, we hypothesize that disturbance in barrier function may be related to specific junctional mechanisms. We found claudin‐1 protein expression and localization to be sensitive to both hypoxia and inflammatory cytokines contributing to impaired barrier integrity. These cellular studies suggest claudin‐1 stability may be a potential target in the progression of spontaneous intestinal perforation.Disclosure: The view(s) expressed herein are those of the author(s) and do not reflect the official policy or position of Brooke Army Medical Center, the U.S. Army Medical Department, the U.S. Army Office of the Surgeon General, the Department of the Air Force and Army, Department of Defense or the U.S. Government.Support or Funding InformationSan Antonio Medical Foundation Grant, Trinity University, US Air Force 59th Medical Wing Clinical Research WingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.