Introduction  1 2 3 7 4 6 12 12 13 15 4 12 16 20 4 12 16 19 1 21 22 Fig. 1 3 7 dashed arrow 2 19  Materials and methods Cell culture 23 2 l Simulation of I/R 24 25 2 4  2 2 4  2 2  2 Fig. 2 Schematic representation of the experimental protocol. Cells were pre-treated for 1 h with Y-27632, cytochalasinD, latrunculinA, jasplakinolide or wortmannin. One hour of simulated ischemia was followed by 1–24 h of simulated reperfusion. No reperfusion for ATP measurement and F-actin staining, 1 h for ATP measurement, F-actin staining and analysis of Akt activity by western blotting and 24 h for quantification of apoptosis. The drugs were also present during the reperfusion phase  ATP measurement We measured ATP after ischemia and I/R (1 h of reperfusion) in order to confirm ischemic substrate deprivation. Cells were grown in a gelatin-coated 12-well chamber and treated according to the simulated I/R protocol. ATP was measured using the ENLITEN ATP Assay System Bioluminiscence Detection Kit for ATP (Promega Corporation, Madison, Wisconsin, USA). Proteins in lysate were measured with BCA Protein Assay Kit (Pierce, Rockford, Illinois, USA) in order to calculate nmol ATP/μg protein. Quantification of apoptosis ® TM TM F-actin cytoskeleton staining ® Analysis of Akt phosphorylation 3 4 Statistical analysis t t R 2 P Results ATP levels and apoptosis following simulated ischemia and I/R 2 P n P n n P 3 Fig. 3 n n n n P n P n # P n  The effect of Rho-kinase inhibition on apoptosis 3 P P Akt activity in Y-27632-mediated protection against apoptosis 4 4 Fig. 4 n P n P n # P n  The F-actin cytoskeleton in Y-27632-mediated protection against apoptosis n 5 Fig. 5 Control cells Ischemic cells I/R cells  5 6 P P Fig. 6 n n P n P n # P n  7 Fig. 7 n P n P n # P n  8 Fig. 8 R 2    P  Discussion This study shows that (1) inhibition of Rho-kinase attenuates I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity and (2) the effects of Rho-kinase inhibition on PI3-kinase/Akt and apoptosis are facilitated by prevention of F-actin rearrangement. 1 5 13 26 4 12 16 20 22 27 22 27 22 27 22 28 29 30 31 32 32 12 16 25 25 33 34 13 26 17 18 4 5 8 12 35 Conclusion Prevention of F-actin rearrangement or F-actin bundle formation by Rho-kinase inhibition or by treatment with actin depolymerizator cytochalasinD, independent of Rho-kinase, attenuated I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity. The present finding suggests that the changes in the F-actin cytoskeleton play a pivotal role in the negative regulation of PI3-kinase and Akt by Rho-kinase. Further studies are required to conclude on the mechanisms by which F-actin rearrangement facilitates the effects of Rho-kinase on PI3-kinase/Akt and apoptosis.