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Electroporation results in endothelial barrier dysfunction<br />
caused by cytoskeleton disassembly and adherens<br />
junction disruption<br />
Simona Kranjc 1 , Maja ^ema`ar 1 , Gregor Ser{a 1 , Chryso Kanthou 2 ,<br />
Gillian M. Tozer 2<br />
1<br />
Institute of Oncology, Zalo{ka 2, SI-1000 Ljubljana, Slovenia, 2 Academic Unit of Surgical<br />
Oncology, Division of Clinical Sciences (South), University of Sheffield, Royal Hallamshire<br />
Hospital, Sheffield, S10 2JF, UK<br />
Electroporation of cells and tissues obtained by application of short high voltage<br />
electric pulses provides permeabilization of cell membrane, which enables access<br />
of molecules such as antibodies, DNA and drugs to the cytoplasm. In particularly,<br />
enhanced delivery of chemotherapeutic drugs (bleomycin and cisplatin), known as<br />
electrochemotherapy, has shown good antitumour effectiveness in preclinical and<br />
clinical studies. Other application of electroporation, delivery of naked DNA, termed<br />
electrogene therapy, is currently on the preclinical level. Endothelial cells seem<br />
to be potential target of electroporation and electrochemotherapy, as evident by<br />
endothelial cell sensitivity in vitro and significant vascular effects in tissues exposed<br />
to the electric pulses. The aim of this study was to evaluate effects of electroporation<br />
on endothelial cell cytoskeleton and endothelial monolayer permeability.<br />
Human umbilical endothelial cells (HUVEC) were grown on fibronectin coated<br />
polycarbonate membrane transwell inserts. In situ electroporation of adherent<br />
cells was performed using the InSitu electroporation system (EquiBio, UK)<br />
(3 square wave electric pulses, 100 µs, 1 Hz, 20-80 V). Cytoskeletal proteins were<br />
visualised by immunostaining with anti-F-actin, anti-β-tubulin, anti-vimentin and<br />
anti-VE-cadherin antibodies. Cell viability after electroporation was evaluated by<br />
trypan blue exclusion assay. Levels of phosphorylated myosin light chain (MLC),<br />
heat shock protein 70 (HSP70) and cytoskeletal proteins were determined using<br />
western blotting. HUVEC monolayer permeability was determined by diffusion of<br />
FITC-coupled dextran.<br />
90p19<br />
Electroporation induced immediate transient disruption of microtubules and<br />
microfilaments but not intermediate filaments. Induced changes were dependent on<br />
the voltage of electric pulses used and recovered within 1 h after electroporation.<br />
Furthermore, immediate loss and recovery of microfilaments correlated with levels<br />
of phosphorylated MLC. According to unaffected total level of cytoskeletal proteins<br />
and no increase in HSP70 within 16 h after electroporation, we speculate that<br />
electroporation interferes with the organisation of actin and tubulin monomers<br />
into filamentous structures of cytoskeleton without degradation of proteins. Those<br />
cytoskeletal rearrangements were reflected in modified cell morphology. In addition,<br />
electroporation disrupted VE-cadherin in endothelial adherens junctions. Taken<br />
altogether, immediate disruption VE-cadherin endothelial junctions, dissolution<br />
of microfilaments and microtubules resulted in rapid increase in monolayer<br />
permeability.
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