• Authors: Okamoto, T., Kawamoto, E., Takagi, Y., Akita, N., Hayashi, T., Park, E. J., Suzuki, K., Shimaoka, M.
  • Year: 2017
  • Journal: Sci Rep 7 6134
  • Applications: in vitro / Protein/Peptide/Antibody / PULSin
  • Cell type: HUVEC
    Description: Human umbilical vein endothelial cells


Briefy, HUVECs were grown to a semi-confuent state with cell-cell contact occurring at the proliferation phase in collagen-coated 60mm tissue culture dishes and were then washed with prewarmed PBS. Mixtures of 200 μL HEPES bufered saline (20 mM HEPES, 150 mM NaCl, pH 7.5), 2 μg of each antibody and 8 μL PULSin were incubated at room temperature for 15 min. HUVECs were incubated with 1800 μL Opti-MEM (Termo Fisher Scientifc) with 200 μL antibody/PULSin mixture at 37 °C for 4 hours.


Endothelial monolayers have shown the ability to signal each other through gap junctions. Gap junction-mediated cell-cell interactions have been implicated in the modulation of endothelial cell functions during vascular inflammation. Inflammatory mediators alter the mechanical properties of endothelial cells, although the exact role of gap junctions in this process remains unclear. Here, we sought to study the role of gap junctions in the regulation of endothelial stiffness, an important physical feature that is associated with many vascular pathologies. The endothelial cellular stiffness of living endothelial cells was determined by using atomic force microscopy. We found that tumor necrosis factor-alpha transiently increased endothelial cellular stiffness, which is regulated by cytoskeletal rearrangement and cell-cell interactions. We explored the role of gap junctions in endothelial cellular stiffening by utilizing gap junction blockers, carbenoxolone, inhibitory anti-connexin 32 antibody or anti-connexin 43 antibody. Blockade of gap junctions induced the cellular stiffening associated with focal adhesion formation and cytoskeletal rearrangement, and prolonged tumor necrosis factor-alpha-induced endothelial cellular stiffening. These results suggest that gap junction-mediated cell-cell interactions play an important role in the regulation of endothelial cellular stiffness.