The endothelial cytoskeleton as a target of electroporation-based therapies

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, B-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.

Vascular disrupting action of electroporation and electrochemotherapy with bleomycin in murine sarcoma

“…Laser Doppler flowmetry demonstrated that vascular shutdown occurred very rapidly, which cannot be attributed to the death of endothelial cells and thus confirm the results of our in vitro study on adherent endothelial cell monolayer (Kanthou et al, 2006). Specifically, we demonstrated that the electroporation-induced vascular effects can be attributed to the rapid disruption of microfilament and microtubule cytoskeletal networks that paralleled an increase in endothelial monolayer permeability (Kanthou et al, 2006). This may lead to increased liquid extravasation and, as a consequence of the increased interstitial fluid pressure, also to the collapse in blood vessel structures.…”

“…Therefore, this technique could be used for planning of combined treatment approaches where antivascular therapies are combined with other therapies targeting hypoxic cells or therapies that should be applied at the time of maximal reperfusion after the first treatment (Shaked and Kerbel, 2007). Laser Doppler flowmetry demonstrated that vascular shutdown occurred very rapidly, which cannot be attributed to the death of endothelial cells and thus confirm the results of our in vitro study on adherent endothelial cell monolayer (Kanthou et al, 2006). Specifically, we demonstrated that the electroporation-induced vascular effects can be attributed to the rapid disruption of microfilament and microtubule cytoskeletal networks that paralleled an increase in endothelial monolayer permeability (Kanthou et al, 2006).…”

“…This phenomenon was well documented by different techniques demonstrating reduced blood flow and oxygenation in the tumours (Sersa et al, 1999b. In addition, we demonstrated that electroporation of adherent endothelial monolayer induced disruption of actin filaments and microtubule network cytoskeleton as well as loss of cell -cell contact junctions, which altogether increased endothelial monolayer permeability (Kanthou et al, 2006). This suggests that in addition to the direct effect of electric pulses applied to the tumour, there may also be an indirect effect on tumour via the effect of electric pulses on tumour blood vessels (vascular disrupting effect).…”

“…In the case of electroporation, actin as well as microtubule cytoskeleton is disrupted as a consequence of the applied electric field; however, disruption by electroporation appears to uncouple Rho and inactivate Rho kinases. It is likely that electroporation inactivates Rho by preventing its association with the cell membrane (Kanthou et al, 2006).…”

Electroporation in Biological Cell and Tissue: An Overview

“…Consequently, the tissue oxygenation level is reduced by electroporation resulting in enhanced tumor cell death (Serša et al 1999;Čemaar et al 2001;Serša et al 2002;Gehl et al 2002;Kanthou et al 2006). …”