Electroporation results in endothelial barrier dysfunction caused by cytoskeleton disassembly and adherens junction disruption Simona Kranjc 1 , Maja ^ema`ar 1 , Gregor Ser{a 1 , Chryso Kanthou 2 , Gillian M. Tozer 2 1 Institute of Oncology, Zalo{ka 2, SI-1000 Ljubljana, Slovenia, 2 Academic Unit of Surgical Oncology, Division of Clinical Sciences (South), University of Sheffield, Royal Hallamshire Hospital, Sheffield, S10 2JF, UK Electroporation of cells and tissues obtained by application of short high voltage electric pulses provides permeabilization of cell membrane, which enables access of molecules such as antibodies, DNA and drugs to the cytoplasm. In particularly, enhanced delivery of chemotherapeutic drugs (bleomycin and cisplatin), known as electrochemotherapy, has shown good antitumour effectiveness in preclinical and clinical studies. Other application of electroporation, delivery of naked DNA, termed electrogene therapy, is currently on the preclinical level. Endothelial cells seem to be potential target of electroporation and electrochemotherapy, as evident by endothelial cell sensitivity in vitro and significant vascular effects in tissues exposed to the electric pulses. The aim of this study was to evaluate effects of electroporation on endothelial cell cytoskeleton and endothelial monolayer permeability. Human umbilical endothelial cells (HUVEC) were grown on fibronectin coated polycarbonate membrane transwell inserts. In situ electroporation of adherent cells was performed using the InSitu electroporation system (EquiBio, UK) (3 square wave electric pulses, 100 µs, 1 Hz, 20-80 V). Cytoskeletal proteins were visualised by immunostaining with anti-F-actin, anti-β-tubulin, anti-vimentin and anti-VE-cadherin antibodies. Cell viability after electroporation was evaluated by trypan blue exclusion assay. Levels of phosphorylated myosin light chain (MLC), heat shock protein 70 (HSP70) and cytoskeletal proteins were determined using western blotting. HUVEC monolayer permeability was determined by diffusion of FITC-coupled dextran. 90p19 Electroporation induced immediate transient disruption of microtubules and microfilaments but not intermediate filaments. Induced changes were dependent on the voltage of electric pulses used and recovered within 1 h after electroporation. Furthermore, immediate loss and recovery of microfilaments correlated with levels of phosphorylated MLC. According to unaffected total level of cytoskeletal proteins and no increase in HSP70 within 16 h after electroporation, we speculate that electroporation interferes with the organisation of actin and tubulin monomers into filamentous structures of cytoskeleton without degradation of proteins. Those cytoskeletal rearrangements were reflected in modified cell morphology. In addition, electroporation disrupted VE-cadherin in endothelial adherens junctions. Taken altogether, immediate disruption VE-cadherin endothelial junctions, dissolution of microfilaments and microtubules resulted in rapid increase in monolayer permeability.
In conclusion, this study shows that electroporation, using electrical parameters that do not affect cell viability, induced reversible effects on actin, tubulin, but not on vimentin intracellular arrangement and disrupted VE-cadherin endothelial junctions. These changes resulted in an increase in permeability of endothelial monolayer that could potentially contribute toward transient impairment of the normal function of the microvasculature, which was observed in vivo after application of electric pulses to tumours and normal tissues. 91