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Connexin43 Modulates Endothelial Wound Healing, Cell Proliferation and Locomotion
Author(s) -
Chen ChengHung,
Mayo Jamie N.,
Bearden Shawn E.
Publication year - 2013
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.27.1_supplement.685.9
Subject(s) - gene knockdown , wound healing , transfection , microbiology and biotechnology , cell growth , endothelial stem cell , small interfering rna , connexin , chemistry , gap junction , biology , intracellular , immunology , apoptosis , gene , biochemistry , in vitro
We previously reported that homocysteine (Hcy) impairs wound healing and increases connexin 43 (Cx43) levels in endothelial cells by activating metabotropic glutamate receptor 5 (mGluR5). Here, we tested the hypothesis that Cx43 regulates wound closure, cell proliferation, and locomotion in cerebral endothelium. We confirmed that Hcy increases Cx43 levels via an mGluR5‐dependent mechanism by Western blotting and specific pharmacology. We then used siRNA and DNA transfection to knockdown and overexpress Cx43 levels in cerebral microvascular endothelial cells. Cells were studied by time lapse confocal microscopy in a scrape wound model and in an open field migration/proliferation assay. Increasing expression of Cx43 significantly impaired wound closure rate (% of closure at 3 days: siRNA 100±0 vs. Control 86.7±4.2 vs. DNA 50±4.5, n=6, p<0.05 all pairwise). Knockdown of Cx43 expression increased cell proliferation (siRNA 116.6±10.3 vs. Control 33.4±1.7 & DNA 16.5±2.1 cells/mm 2 , n=6, p<0.05), locomotion (siRNA 10.6±0.6 vs. Control 6.6±0.4 & DNA 5.9±0.5 μm/hr, n=10, p<0.05), and net speed (siRNA 4.9±0.3 vs. Control 2.5±0.3 & DNA 1.6±0.4 μm/hr, n=10, p<0.05). Cx43 regulated the cycling and number of cellular protrusions/retractions and the efficiency of migration in a dose dependent manner. Cx43 is a key regulator of migratory dynamics and growth in cerebral endothelium. Support: HL106548 ; S11–28U.