Dose-Dependent Effect of Nocodazole on Endothelial Cell Cytoskeleton

摘要

The endothelium lining the inner surface of blood vessels fulfils an important barrier function andspecifically, it controls vascular membrane permeability as well as nutrient and metabolite exchange in circu-lating blood and tissue fluids. Disturbances in vascular endothelium barrier function (vascular endothelium dys-function) are coupled to cytoskeleton rearrangements, actomyosin contractility, and as a consequence, forma-tion of paracellular gaps between endothelial cells. Microtubules constitute the first effector link in the reactioncascade resulting in vascular endothelium dysfunction. Increased vascular permeability associated with manyhuman diseases is also manifested as a side effect in anticancer mitosis-blocking therapy. The aim of this studywas to examine the possibility of preventing side effects of mitostatic drugs in patients with vascular endothe-lium dysfunction and to establish effective doses able to disrupt the microtubular network without interferingwith the endothelial barrier function. Previously, it was found that the population of endothelial cell microtu-bules is heterogeneous. Along with dynamic microtubules, cell cytoplasm contains a certain amount of post-translationally modified microtubules that are less active and less susceptible to external influences thandynamic microtubules. We have shown that the area occupied with stable microtubules is relatively large(approx. one third of the total cell area). We assume that it can account for a higher resistance of the endothelialmonolayer to factors responsible for vascular endothelium dysfunction. This hypothesis was validated in thisstudy, in which nocodazole was used to induce vascular endothelium dysfunction in lung endothelial cells. Theeffect of nocodazole on endothelial cell cytoskeleton was found to be dose-dependent. Nocodazole in micro-molar concentrations not only irreversibly changed the barrier function, but also upset the viability of endothe-lial cells and induced their death. Nanomolar concentrations of nocodazole also increased the permeability ofthe endothelial monolayer; this effect was reversible at the drug concentration ranging from 100 to 200 nM. At100 nM, nocodazole induced partial disruption of the microtubule network near the cell margin without anyappreciable effect on acetylated microtubules and actin filaments. At 200 nM, nocodazole exerted a pronouncedeffect on the system of dynamic (but not acetylated) microtubules and increased the population of actin fila-ments in the central region of the cell. Our data suggest that disruption of peripheral microtubules triggers acascade of reactions culminating in endothelial barrier dysfunction; however, the existence of a large populationof microtubules resistant to nanomolar concentrations of the drug provides higher viability of endothelial cellsand restores their functional activity.