Abstract
Intestinal ischemia results in mucosal injury, including paracellular barrier loss due to disruption of tight junctions. Larazotide acetate (LA), a small peptide studied in Phase III clinical trials for treatment of celiac disease, regulates tight junctions (TJs). We hypothesized that LA would dose-dependently hasten recovery of intestinal ischemic injury via modulation of TJs. Ischemia-injured tissue from 6-8-week-old pigs was recovered in Ussing chambers for 240-minutes in the presence of LA. LA (1 μM but not 0.1 μM or 10 μM) significantly enhanced transepithelial electrical resistance (TER) above ischemic injured controls and significantly reduced serosal-to-mucosal flux LPS (P<0.05). LA (1 μM) enhanced localization of the sealing tight junction protein claudin-4 in repairing epithelium. To assess for the possibility of fragmentation of LA, an in vitro enzyme degradation assay using the brush border enzyme aminopeptidase M, revealed generation of peptide fragments. Western blot analysis of total protein isolated from uninjured and ischemia-injured porcine intestine showed aminopeptidase M enzyme presence in both tissue types, and mass spectrometry analysis of samples collected during ex vivo analysis confirmed formation of LA fragments. Treatment of tissues with LA fragments had no effect alone, but treatment with a fragment missing both amino-terminus glycines inhibited barrier recovery stimulated by 1 μM LA. To reduce potential LA inhibition by fragments, a D-amino acid analog of larazotide Analog #6, resulted in a significant recovery response at a 10-fold lower dose (0.1 μM) similar in magnitude to that of 1 μM LA. We conclude that LA stimulates repair of ischemic-injured epithelium at the level of the tight junctions, at an optimal dose of 1 μM LA. Higher doses were less effective because of inhibition by LA fragments, which could be subverted by chirally-modifying the molecule, or microdosing LA.
Highlights
Intestinal epithelium exists as a monolayer that forms both transcellular and paracellular barriers to separate luminal contents from the subsurface interstitium
Celiac disease (CeD), a disease triggered by the ingestion and subsequent digestion of gluten with a worldwide prevalence of about 1% [1], includes pathophysiology consisting of compromised tight junction (TJ) proteins, which may result in the loss of paracellular barrier function
Paracellular barrier loss can occur after intestinal ischemic injury, which induces the loss of paracellular barrier function via disruption of TJs
Summary
Intestinal epithelium exists as a monolayer that forms both transcellular and paracellular barriers to separate luminal contents from the subsurface interstitium. Celiac disease (CeD), a disease triggered by the ingestion and subsequent digestion of gluten with a worldwide prevalence of about 1% [1], includes pathophysiology consisting of compromised TJ proteins, which may result in the loss of paracellular barrier function. This loss of barrier function can lead to the exposure of luminal gliadin and antigenic interaction with immune cells within the lamina propria, furthering the dysregulation of TJ proteins and paracellular barrier dysfunction [2, 3]. This disruption of TJs following injury can result in the onset of sepsis and death in the host [4]
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