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Pharmacological or Genetic Interruption of Focal Adhesion Kinase Improves Endothelial Barrier Dysfunction Resulting from Ischemia/Reperfusion Injury
Author(s) -
Haines Ricci J,
Zhang Shimin,
Eitnier Rebecca A,
Wang Fang,
Yang Clement GY,
Wu Mack H
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.684.18
Subject(s) - focal adhesion , endothelial stem cell , endothelial dysfunction , ischemia , microbiology and biotechnology , reperfusion injury , barrier function , albumin , endothelium , medicine , pharmacology , chemistry , cancer research , phosphorylation , biology , in vitro , biochemistry
Ischemia reperfusion (I/R) injury results from reoxygenation following an initial ischemic insult. Although oxygen levels are restored during reperfusion, the abrupt increase in blood supply results in high levels of reactive oxygen species that leads to endothelial barrier dysfunction and exacerbates the tissue injury originally elicited by the ischemic event. One therapeutic strategy used to enhance the endothelial barrier and prevent further tissue damage is to treat the exacerbated tissue damage caused by reperfusion. Our lab and others have shown that focal adhesion kinase (FAK) is a key player that participates in the signaling events required for endothelial barrier dysfunction. Therefore, we hypothesized that targeting FAK pharmacologically would improve I/R injury in vitro and in vivo . Using murine intestinal endothelial cells, we performed electric cell‐substrate impedance sensing (ECIS) assays with and without FAK inhibitor (PF573228, 1μM) in the presence of H2O2 (250μM). Results showed that FAK inhibition significantly attenuated barrier dysfunction induced by H2O2. Similarly, we also utilized FITC‐albumin flux assays to determine whether targeting FAK improved barrier function resulting from H2O2. These experiments showed that the increase of endothelial albumin flux due to treatment with H2O2 was reduced by approximately 50% when cells were pretreated with FAK inhibitor. Western blots of endothelial cell lysates treated with H2O2 indicated that FAK is activated in as little as 10 minutes and remains elevated for 60 minutes of treatment. Other experiments showed that the use of PF573228 dramatically reduced detection of active FAK (pY397 FAK) as well as total tyrosine phosphorylated proteins despite the presence of inflammatory signals. Using an in vivo model of I/R injury and endothelial specific, tamoxifen inducible, FAK knockout transgenic animals, as well as wild type animals given PF573228, we showed that interruption of FAK activity or expression reduced albumin leakage induced by I/R injury by over two‐fold, while neither mean arterial blood pressure nor diameter was altered by the drug or genetic manipulation. Taken together, these results suggest that targeting FAK may be a novel strategy to improve endothelial barrier dysfunction that occurs as a consequence of I/R injury. Support or Funding Information NIH HL‐120954