Effects of H₂O₂ on Microfilaments of Cultured Endothelial Cells

Abstract

Hydrogen peroxide (H₂O₂) is an important granulocyte derived mediator of endothelial cell injury. Alterations of the microfilaments system (especially of actin) in endothelial cells may be relevant for the pathogenesis of vascular leakage. In the study presented effects of H₂O₂ on actin monomers (G-actin) and filamentous actin (F-actin) were examined in cultured pulmonary artery endothelial cells. Phalloidin which blocks actin depolymerization by inhibiting actin monomer dissociation and C. Botulinum C2 toxin which ADP-ribosylates G-actin thereby inhibiting actin polymerization were used as tools for the study of H₂O₂–related actin alterations. Exposure of cells to 2 mM H₂O₂ resulted in a biphasic change of F-actin with an early decrease (15 min) and a subsequent doubling (120 min) paralleled by an inverse G-actin pattern. In endothelial cells with a 20% reduction of F-actin-brought about by preincubation with C2-toxin for 150 min- H₂O₂-related actin polymerization was unimpaired. In cells with completely dissolved F-actin (Bot. C2-toxin for 210 min) no actin polymerization occurred upon H₂O₂ application. Phalloidin blocked the early H₂O₂-induced F-actin decrease and slowed down late actin polymerization. Effects of H₂O₂ on endothelial actin were abolished in the presence of scavengers of oxygen metabolites (catalase) and by the poly (ADP) ribose-polymerase inhibitor aminobenzamid. The data presented are compatible with the concept that H₂O₂ stimulates actin turnover and actin nucleation which—in the long run—result in H₂O₂ related formation of new actin filaments. This process was blocked by oxygen metabolite scavengers and by inhibition of DNA strand break repair mechanisms.