{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,13]],"date-time":"2026-02-13T06:05:49Z","timestamp":1770962749176,"version":"3.50.1"},"reference-count":31,"publisher":"Public Library of Science (PLoS)","issue":"4","license":[{"start":{"date-parts":[[2021,4,22]],"date-time":"2021-04-22T00:00:00Z","timestamp":1619049600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Innovate Biopharmaceuticals. Inc."}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>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 \u03bcM but not 0.1 \u03bcM or 10 \u03bcM) significantly enhanced transepithelial electrical resistance (TER) above ischemic injured controls and significantly reduced serosal-to-mucosal flux LPS (<jats:italic>P&lt;0<\/jats:italic>.<jats:italic>05<\/jats:italic>). LA (1 \u03bcM) enhanced localization of the sealing tight junction protein claudin-4 in repairing epithelium. To assess for the possibility of fragmentation of LA, an<jats:italic>in vitro<\/jats:italic>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<jats:italic>ex vivo<\/jats:italic>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 \u03bcM 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 \u03bcM) similar in magnitude to that of 1 \u03bcM LA. We conclude that LA stimulates repair of ischemic-injured epithelium at the level of the tight junctions, at an optimal dose of 1 \u03bcM LA. Higher doses were less effective because of inhibition by LA fragments, which could be subverted by chirally-modifying the molecule, or microdosing LA.<\/jats:p>","DOI":"10.1371\/journal.pone.0250165","type":"journal-article","created":{"date-parts":[[2021,4,22]],"date-time":"2021-04-22T19:56:34Z","timestamp":1619121394000},"page":"e0250165","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":12,"title":["Larazotide acetate induces recovery of ischemia-injured porcine jejunum via repair of tight junctions"],"prefix":"10.1371","volume":"16","author":[{"given":"Zachary M.","family":"Slifer","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Liliana","family":"Hernandez","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tiffany A.","family":"Pridgen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexandra R.","family":"Carlson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kristen M.","family":"Messenger","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jay","family":"Madan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"B. Radha","family":"Krishnan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sandeep","family":"Laumas","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0867-7310","authenticated-orcid":true,"given":"Anthony T.","family":"Blikslager","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2021,4,22]]},"reference":[{"issue":"10115","key":"pone.0250165.ref001","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1016\/S0140-6736(17)31796-8","article-title":"Coeliac disease.","volume":"391","author":"B Lebwohl","year":"2018","journal-title":"Lancet"},{"issue":"1","key":"pone.0250165.ref002","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1177\/1756283X15616576","article-title":"The potential utility of tight junction regulation in celiac disease: focus on larazotide acetate","volume":"9","author":"S Khaleghi","year":"2016","journal-title":"Therap Adv Gastroenterol"},{"issue":"2","key":"pone.0250165.ref003","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1016\/j.jcmgh.2016.12.006","article-title":"Celiac Disease: Role of the Epithelial Barrier","volume":"3","author":"M Schumann","year":"2017","journal-title":"Cell Mol Gastroenterol Hepatol"},{"issue":"8","key":"pone.0250165.ref004","doi-asserted-by":"crossref","first-page":"e0200674","DOI":"10.1371\/journal.pone.0200674","article-title":"Epithelial restitution defect in neonatal jejunum is rescued by juvenile mucosal homogenate in a pig model of intestinal ischemic injury and repair","volume":"13","author":"AL Ziegler","year":"2018","journal-title":"PLoS One"},{"issue":"22","key":"pone.0250165.ref005","first-page":"19160","article-title":"Zonula occludens toxin structure-function analysis. Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain","volume":"276","author":"M Di Pierro","year":"2001","journal-title":"J Biol Chem"},{"issue":"3","key":"pone.0250165.ref006","doi-asserted-by":"crossref","first-page":"286","DOI":"10.1001\/archinte.163.3.286","article-title":"Prevalence of celiac disease in at-risk and not-at-risk groups in the United States: a large multicenter study","volume":"163","author":"A Fasano","year":"2003","journal-title":"Arch Intern Med"},{"issue":"Pt 24","key":"pone.0250165.ref007","doi-asserted-by":"crossref","first-page":"4435","DOI":"10.1242\/jcs.113.24.4435","article-title":"Human zonulin, a potential modulator of intestinal tight junctions","volume":"113","author":"W Wang","year":"2000","journal-title":"J Cell Sci"},{"issue":"10","key":"pone.0250165.ref008","doi-asserted-by":"crossref","first-page":"1554","DOI":"10.1038\/ajg.2012.211","article-title":"A randomized, double-blind study of larazotide acetate to prevent the activation of celiac disease during gluten challenge","volume":"107","author":"DA Leffler","year":"2012","journal-title":"Am J Gastroenterol"},{"issue":"2","key":"pone.0250165.ref009","doi-asserted-by":"crossref","first-page":"252","DOI":"10.1111\/apt.12147","article-title":"Larazotide acetate in patients with coeliac disease undergoing a gluten challenge: a randomised placebo-controlled study","volume":"37","author":"CP Kelly","year":"2013","journal-title":"Aliment Pharmacol Ther"},{"issue":"5","key":"pone.0250165.ref010","doi-asserted-by":"crossref","first-page":"757","DOI":"10.1111\/j.1365-2036.2007.03413.x","article-title":"The safety, tolerance, pharmacokinetic and pharmacodynamic effects of single doses of AT-1001 in coeliac disease subjects: a proof of concept study","volume":"26","author":"BM Paterson","year":"2007","journal-title":"Aliment Pharmacol Ther"},{"issue":"1\u20132","key":"pone.0250165.ref011","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/j.ijpharm.2008.08.047","article-title":"Mechanism of action of ZOT-derived peptide AT-1002, a tight junction regulator and absorption enhancer","volume":"365","author":"S Gopalakrishnan","year":"2009","journal-title":"Int J Pharm"},{"issue":"1","key":"pone.0250165.ref012","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1016\/j.peptides.2012.02.015","article-title":"Larazotide acetate regulates epithelial tight junctions in vitro and in vivo","volume":"35","author":"S Gopalakrishnan","year":"2012","journal-title":"Peptides"},{"issue":"1","key":"pone.0250165.ref013","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/j.peptides.2012.02.016","article-title":"Larazotide acetate promotes tight junction assembly in epithelial cells","volume":"35","author":"S Gopalakrishnan","year":"2012","journal-title":"Peptides"},{"issue":"1","key":"pone.0250165.ref014","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1097\/00054725-200102000-00014","article-title":"Mechanisms and modulation of intestinal epithelial repair","volume":"7","author":"AU Dignass","year":"2001","journal-title":"Inflamm Bowel Dis"},{"issue":"5","key":"pone.0250165.ref015","doi-asserted-by":"crossref","first-page":"G885","DOI":"10.1152\/ajpgi.00380.2005","article-title":"Prostaglandin-mediated inhibition of Na+\/H+ exchanger isoform 2 stimulates recovery of barrier function in ischemia-injured intestine","volume":"291","author":"AJ Moeser","year":"2006","journal-title":"Am J Physiol Gastrointest Liver Physiol"},{"issue":"6","key":"pone.0250165.ref016","doi-asserted-by":"crossref","first-page":"C996","DOI":"10.1152\/ajpcell.00113.2016","article-title":"Myosin light chain kinase mediates intestinal barrier dysfunction via occludin endocytosis during anoxia\/reoxygenation injury","volume":"311","author":"Y Jin","year":"2016","journal-title":"Am J Physiol Cell Physiol"},{"issue":"1","key":"pone.0250165.ref017","doi-asserted-by":"crossref","first-page":"G157","DOI":"10.1152\/ajpgi.00005.2001","article-title":"Inducible nitric oxide synthase mediates early epithelial repair of porcine ileum","volume":"283","author":"JL Gookin","year":"2002","journal-title":"Am J Physiol Gastrointest Liver Physiol"},{"issue":"4","key":"pone.0250165.ref018","doi-asserted-by":"crossref","first-page":"353","DOI":"10.2746\/042516408X293574","article-title":"Attenuation of ischaemic injury in the equine jejunum by administration of systemic lidocaine","volume":"40","author":"VL Cook","year":"2008","journal-title":"Equine Vet J"},{"issue":"4","key":"pone.0250165.ref019","doi-asserted-by":"crossref","first-page":"G791","DOI":"10.1152\/ajpgi.00538.2007","article-title":"Mice lacking the Na+\/H+ exchanger 2 have impaired recovery of intestinal barrier function","volume":"295","author":"AJ Moeser","year":"2008","journal-title":"Am J Physiol Gastrointest Liver Physiol"},{"issue":"4","key":"pone.0250165.ref020","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1136\/jcp.43.4.307","article-title":"Brush border enzymes in coeliac disease: histochemical evaluation","volume":"43","author":"J Mercer","year":"1990","journal-title":"J Clin Pathol"},{"issue":"4","key":"pone.0250165.ref021","doi-asserted-by":"crossref","first-page":"478","DOI":"10.1001\/archsurg.1970.01340280030009","article-title":"Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal.","volume":"101","author":"CJ Chiu","year":"1970","journal-title":"Arch Surg"},{"issue":"1","key":"pone.0250165.ref022","doi-asserted-by":"crossref","first-page":"346","DOI":"10.1053\/j.gastro.2007.11.049","article-title":"Life in the gut without oxygen: adaptive mechanisms and inflammatory bowel disease","volume":"134","author":"AT Blikslager","year":"2008","journal-title":"Gastroenterology"},{"issue":"3","key":"pone.0250165.ref023","doi-asserted-by":"crossref","DOI":"10.3390\/ijms21030972","article-title":"The Integral Role of Tight Junction Proteins in the Repair of Injured Intestinal Epithelium","volume":"21","author":"ZM Slifer","year":"2020","journal-title":"Int J Mol Sci"},{"issue":"3","key":"pone.0250165.ref024","doi-asserted-by":"crossref","first-page":"e24978","DOI":"10.4161\/tisb.24978","article-title":"Claudins in intestines: Distribution and functional significance in health and diseases","volume":"1","author":"Z Lu","year":"2013","journal-title":"Tissue Barriers"},{"issue":"2","key":"pone.0250165.ref025","doi-asserted-by":"crossref","first-page":"G63","DOI":"10.1152\/ajpgi.00112.2013","article-title":"Animal models of ischemia-reperfusion-induced intestinal injury: progress and promise for translational research","volume":"308","author":"LM Gonzalez","year":"2015","journal-title":"Am J Physiol Gastrointest Liver Physiol"},{"issue":"10","key":"pone.0250165.ref026","article-title":"The Regulation of Intestinal Mucosal Barrier by Myosin Light Chain Kinase\/Rho Kinases","volume":"21","author":"Y Jin","year":"2020","journal-title":"Int J Mol Sci"},{"issue":"6","key":"pone.0250165.ref027","doi-asserted-by":"crossref","first-page":"S-487","DOI":"10.1016\/S0016-5085(19)38078-3","article-title":"Abstract\u2014Su1019 Larazotide Protects the Intestinal Tight Junction Barrier During Anoxia\/Reoxygenation Injury Via Inhibition of Myosin Light Chain Kinase","volume":"156","author":"C. P. Jin YP","year":"2019","journal-title":"Gastroenterology"},{"issue":"1","key":"pone.0250165.ref028","doi-asserted-by":"crossref","first-page":"194","DOI":"10.1053\/j.gastro.2008.03.023","article-title":"Gliadin induces an increase in intestinal permeability and zonulin release by binding to the chemokine receptor CXCR3","volume":"135","author":"KM Lammers","year":"2008","journal-title":"Gastroenterology"},{"issue":"2","key":"pone.0250165.ref029","doi-asserted-by":"crossref","first-page":"G441","DOI":"10.1152\/ajpgi.00303.2007","article-title":"EGF receptor transactivation and MAP kinase mediate proteinase-activated receptor-2-induced chloride secretion in intestinal epithelial cells","volume":"294","author":"JQ van der Merwe","year":"2008","journal-title":"Am J Physiol Gastrointest Liver Physiol"},{"issue":"4","key":"pone.0250165.ref030","doi-asserted-by":"crossref","first-page":"e1251384","DOI":"10.1080\/21688370.2016.1251384","article-title":"Zonulin, a regulator of epithelial and endothelial barrier functions, and its involvement in chronic inflammatory diseases","volume":"4","author":"C Sturgeon","year":"2016","journal-title":"Tissue Barriers"},{"issue":"7","key":"pone.0250165.ref031","doi-asserted-by":"crossref","first-page":"1098","DOI":"10.1007\/s13361-013-0808-5","article-title":"Understanding gas phase modifier interactions in rapid analysis by differential mobility-tandem mass spectrometry","volume":"25","author":"A Kafle","year":"2014","journal-title":"J Am Soc Mass Spectrom"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0250165","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,5,2]],"date-time":"2021-05-02T07:54:23Z","timestamp":1619942063000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0250165"}},"subtitle":[],"editor":[{"given":"Tomohito","family":"Higashi","sequence":"first","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2021,4,22]]},"references-count":31,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2021,4,22]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0250165","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,4,22]]}}}