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Research Articles: Therapeutics, Targets, and Development

The endothelial cytoskeleton as a target of electroporation-based therapies

¹ Cancer Research UK Tumour Microcirculation Group, Academic Unit of Surgical Oncology, Royal Hallamshire Hospital, University of Sheffield, Sheffield, United Kingdom; ² Department of Experimental Oncology, Institute of Oncology Ljubljana; and ³ Faculty of Electrical Engineering, Laboratory of Biocybernetics, University of Ljubljana, Ljubljana, Slovenia

Requests for reprints: Maja Cemazar, Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia. E-mail: mcemazar{at}onko-i.si or Chryso Kanthou, Cancer Research UK Tumour Microcirculation Group, Academic Unit of Surgical Oncology, University of Sheffield, Floor K, Royal Hallamshire Hospital, Glossop Road, Sheffield S10 2JF, United Kingdom. Phone: 44-114-271-1725. E-mail: C.Kanthou{at}sheffield.ac.uk

Abstract

Electroporation-based therapies, such as electrochemotherapy and electrogene therapy, result in the disruption of blood vessel networks in vivo and cause changes in blood flow and vascular permeability. The effects of electroporation on the cytoskeleton of cultured primary endothelial cells and on endothelial monolayer permeability were investigated to elucidate possible mechanisms involved. Human umbilical vein endothelial cells (HUVECs) were electroporated in situ and then immunofluorescence staining for filamentous actin, ß-tubulin, vimentin, and VE-cadherin as well as Western blotting analysis of levels of phosphorylated myosin light chain and cytoskeletal proteins were performed. Endothelial permeability was determined by monitoring the passage of FITC-coupled dextran through endothelial monolayers. Exposure of endothelial cells to electric pulses resulted in a profound disruption of microfilament and microtubule cytoskeletal networks, loss of contractility, and loss of vascular endothelial cadherin from cell-to-cell junctions immediately after electroporation. These effects were voltage dependent and reversible because cytoskeletal structures recovered within 60 min of electroporation with up to 40 V, without any significant loss of cell viability. The cytoskeletal effects of electroporation were paralleled by a rapid increase in endothelial monolayer permeability. These results suggest that the remodeling of the endothelial cytoskeleton and changes in endothelial barrier function could contribute to the vascular disrupting actions of electroporation-based therapies and provide an insight into putative mechanisms responsible for the observed increase in permeability and cessation of blood flow in vivo. [Mol Cancer Ther 2006;5(12):3145–52]

Grant support: Slovenian Research Agency grants P3-0003 and J3-7044 and Cancer Research UK Programme grant C1276/A3307.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/14/06; revised 9/10/06; accepted 10/13/06.