{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,31]],"date-time":"2026-01-31T19:09:46Z","timestamp":1769886586349,"version":"3.49.0"},"reference-count":59,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,1,29]],"date-time":"2026-01-29T00:00:00Z","timestamp":1769644800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000050","name":"NHLBI","doi-asserted-by":"crossref","award":["R01 HL141387"],"award-info":[{"award-number":["R01 HL141387"]}],"id":[{"id":"10.13039\/100000050","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000050","name":"NHLBI","doi-asserted-by":"crossref","award":["P01 HL126609"],"award-info":[{"award-number":["P01 HL126609"]}],"id":[{"id":"10.13039\/100000050","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Endothelial cell (EC) barrier integrity is tightly regulated by the activity of the non-muscle myosin light chain kinase (nmMLCK) under diverse pathological inflammatory conditions (pneumonia, sepsis) and exposure to mechanical stress. Inflammatory stimuli, including lipopolysaccharide (LPS), cytokines, and damage-associated molecular patterns (DAMPs), increase EC permeability through nmMLCK-dependent EC paracellular gap formation. However, the exact mechanisms by which nmMLCK regulates vascular barrier dysfunction in acute lung injury (ALI) remain incompletely understood. We hypothesized that inflammation-induced ROS results in the peroxynitrite-mediated nitration of nmMLCK that contributes to EC barrier disruption. Human lung EC exposure to either the peroxynitrite donor, SIN-1, or to LPS, triggered significant nmMLCK nitration, which was abolished by the oxidant scavenger, MnTMPyP. Mass spectrometry of SIN-1-treated nmMLCK identified multiple nitrated tyrosines. Nitration of Y1410 proved a critical PTM as site-directed substitution with alanine (Y1410A) abolished both SIN-1- and LPS-induced nmMLCK nitration. nmMLCK nitration disrupts wild-type nmMLCK interaction with Kindlin-2, a cytoskeletal regulator of vascular barrier stability, whereas EC transfected with the Y1410A nmMLCK mutant exhibited preserved Kindlin-2 binding, reflected by alterations in trans-EC electrical resistance (TEER). Consistent with these observations, LPS-challenged murine lungs displayed enhanced nmMLCK nitration and diminished nmMLCK-Kindlin-2 association. Functionally, SIN-1 markedly impaired EC barrier integrity (TEER), which was not observed in ECs expressing the Y1410A mutant. Together, these findings suggest that nmMLCK nitration at Y1410 is a critical molecular mechanism contributing to vascular leakage, highlighting this modification as a potential therapeutic target to reduce inflammation-induced vascular permeability. Given nmMLCK\u2019s established role in barrier regulation, we hypothesized that LPS-induced peroxynitrite formation may promote the nitration of nmMLCK tyrosine residues: a PTM that potentially contribute to nmMLCK\u2019s regulation of EC barrier integrity.<\/jats:p>","DOI":"10.3390\/cells15030261","type":"journal-article","created":{"date-parts":[[2026,1,29]],"date-time":"2026-01-29T14:52:36Z","timestamp":1769698356000},"page":"261","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Involvement of Non-Muscle Myosin Light Chain Kinase Nitration in Molecular Regulation of Inflammation-Induced Endothelial Cell Barrier Dysfunction"],"prefix":"10.3390","volume":"15","author":[{"given":"Haifei","family":"Xu","sequence":"first","affiliation":[{"name":"Center for Inflammation Science and Systems Medicine, University of Florida Scripps Research Institute, Jupiter, FL 33458, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0123-2909","authenticated-orcid":false,"given":"Jin H.","family":"Song","sequence":"additional","affiliation":[{"name":"Center for Inflammation Science and Systems Medicine, University of Florida Scripps Research Institute, Jupiter, FL 33458, USA"},{"name":"Department of Molecular Medicine, University of Florida Scripps Research Institute, Jupiter, FL 33458, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joseph B.","family":"Mascarenhas","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of Arizona Health Sciences, Tucson, AZ 85721, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0654-0644","authenticated-orcid":false,"given":"Libia A.","family":"Garcia","sequence":"additional","affiliation":[{"name":"Center for Inflammation Science and Systems Medicine, University of Florida Scripps Research Institute, Jupiter, FL 33458, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Susannah","family":"Patarroyo-White","sequence":"additional","affiliation":[{"name":"Center for Inflammation Science and Systems Medicine, University of Florida Scripps Research Institute, Jupiter, FL 33458, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Annie","family":"Hernandez","sequence":"additional","affiliation":[{"name":"Center for Inflammation Science and Systems Medicine, University of Florida Scripps Research Institute, Jupiter, FL 33458, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carrie L.","family":"Kempf","sequence":"additional","affiliation":[{"name":"Center for Inflammation Science and Systems Medicine, University of Florida Scripps Research Institute, Jupiter, FL 33458, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1929-737X","authenticated-orcid":false,"given":"Paul R.","family":"Langlais","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of Arizona Health Sciences, Tucson, AZ 85721, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Donna D.","family":"Zhang","sequence":"additional","affiliation":[{"name":"Center for Inflammation Science and Systems Medicine, University of Florida Scripps Research Institute, Jupiter, FL 33458, USA"},{"name":"Department of Molecular Medicine, University of Florida Scripps Research Institute, Jupiter, FL 33458, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6934-0420","authenticated-orcid":false,"given":"Joe G. N.","family":"Garcia","sequence":"additional","affiliation":[{"name":"Center for Inflammation Science and Systems Medicine, University of Florida Scripps Research Institute, Jupiter, FL 33458, USA"},{"name":"Department of Molecular Medicine, University of Florida Scripps Research Institute, Jupiter, FL 33458, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1146\/annurev-physiol-042222-030731","article-title":"A Razor\u2019s Edge: Vascular Responses to Acute Inflammatory Lung Injury\/Acute Respiratory Distress Syndrome","volume":"86","author":"Price","year":"2024","journal-title":"Annu. Rev. Physiol."},{"key":"ref_2","first-page":"L989","article-title":"Regulation of endothelial cell myosin light chain kinase by Rho, cortactin, and p60src","volume":"276","author":"Garcia","year":"1999","journal-title":"Am. J. 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