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Larazotide acetate induces recovery of ischemia-injured porcine jejunum via repair of tight junctions
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-...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8061941/ https://www.ncbi.nlm.nih.gov/pubmed/33886649 http://dx.doi.org/10.1371/journal.pone.0250165 |
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author | Slifer, Zachary M. Hernandez, Liliana Pridgen, Tiffany A. Carlson, Alexandra R. Messenger, Kristen M. Madan, Jay Krishnan, B. Radha Laumas, Sandeep Blikslager, Anthony T. |
author_facet | Slifer, Zachary M. Hernandez, Liliana Pridgen, Tiffany A. Carlson, Alexandra R. Messenger, Kristen M. Madan, Jay Krishnan, B. Radha Laumas, Sandeep Blikslager, Anthony T. |
author_sort | Slifer, Zachary M. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8061941 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-80619412021-05-04 Larazotide acetate induces recovery of ischemia-injured porcine jejunum via repair of tight junctions Slifer, Zachary M. Hernandez, Liliana Pridgen, Tiffany A. Carlson, Alexandra R. Messenger, Kristen M. Madan, Jay Krishnan, B. Radha Laumas, Sandeep Blikslager, Anthony T. PLoS One Research Article 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. Public Library of Science 2021-04-22 /pmc/articles/PMC8061941/ /pubmed/33886649 http://dx.doi.org/10.1371/journal.pone.0250165 Text en © 2021 Slifer et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Slifer, Zachary M. Hernandez, Liliana Pridgen, Tiffany A. Carlson, Alexandra R. Messenger, Kristen M. Madan, Jay Krishnan, B. Radha Laumas, Sandeep Blikslager, Anthony T. Larazotide acetate induces recovery of ischemia-injured porcine jejunum via repair of tight junctions |
title | Larazotide acetate induces recovery of ischemia-injured porcine jejunum via repair of tight junctions |
title_full | Larazotide acetate induces recovery of ischemia-injured porcine jejunum via repair of tight junctions |
title_fullStr | Larazotide acetate induces recovery of ischemia-injured porcine jejunum via repair of tight junctions |
title_full_unstemmed | Larazotide acetate induces recovery of ischemia-injured porcine jejunum via repair of tight junctions |
title_short | Larazotide acetate induces recovery of ischemia-injured porcine jejunum via repair of tight junctions |
title_sort | larazotide acetate induces recovery of ischemia-injured porcine jejunum via repair of tight junctions |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8061941/ https://www.ncbi.nlm.nih.gov/pubmed/33886649 http://dx.doi.org/10.1371/journal.pone.0250165 |
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