OBJECTIVETo investigate the relationship between activation of Rho kinase (ROCK) signal pathway and permeability of hypoxic vascular endothelial cells.

METHODS(1) Human vascular endothelial cell line VE cells were planted onto 6-well plates Transwell and divided into control group (without hypoxia treatment) and hypoxia for 1, 2, 3, 6, 12 h groups (exposed to 1%O2, 5%CO2, and 94%N2 for corresponding time) according to the random number table, with 5 wells in each group. The expression levels of ROCKI, ROCKII, myosin light chain phosphatase target subunit 1 (MYPT1) and phosphorylated MYPT1 (p-MYPT1), myosin light chain (MLC), p-MLC in cells were detected by Western blotting. The ratios of p-MYPT1/MYPT1 and p-MLC/MLC were calculated. (2) VE cells were planted onto 24-well plates Transwell, and the monolayer cells were divided into control group (without hypoxia treatment) and hypoxia for 6 h group (exposed to 1% O(2), 5% CO(2), and 94% N(2) for 6 h) according to the random number table, with 5 wells in each group. Permeability of monolayer cells was determined by fluorescence spectrophotometer. Data were processed with one-way analysis of variance or t test, and Newman-Keuls method was used in paired comparison among groups.

RESULTS(1) ROCKI protein expression in control and hypoxia for 1, 2, 3, 6, 12 h groups was obviously 0.63 ± 0.14 and 0.36 ± 0.08, 1.25 ± 0.21, 1.98 ± 0.16, 1.49 ± 0.38, 0.79 ± 0.24 (F = 36.52, P < 0.01). ROCKI protein expression in hypoxia for 2, 3, 6 h groups were obviously higher than that in control group (with P values all below 0.01). (2) There was significant statistical difference among all groups in ROCKII protein expression (F = 17.84, P < 0.01). ROCKII protein expression in hypoxia for 2 h group (1.33 ± 0.17) was significantly higher than that in control group (1.05 ± 0.04, P < 0.01). (3) p-MYPT1/MYPT1 ratio in control and hypoxia for 1, 2, 3, 6, 12 h groups was respectively 0.62 ± 0.13 and 0.62 ± 0.11, 0.65 ± 0.10, 1.06 ± 0.23, 1.37 ± 0.16, 1.91 ± 0.32 (F = 37.41, P < 0.01). p-MYPT1/MYPT1 ratio in hypoxia for 3, 6, 12 h groups were obviously higher than that in control group (with P values all below 0.01). (4) p-MLC/MLC ratio in control and hypoxia for 1, 2, 3, 6, 12 h groups was respectively 0.72 ± 0.19 and 0.83 ± 0.17, 0.91 ± 0.15, 1.39 ± 0.16, 2.02 ± 0.15, 0.90 ± 0.25 (F = 36.92, P < 0.01). p-MLC/MLC ratio in hypoxia for 3, 6 h groups were obviously higher than that in control group (with P values all below 0.01). (5) Permeability of VE monolayer in hypoxia for 6 h group (36.1 ± 8.0) was obviously higher than that in control group (9.1 ± 2.1, t = 7.30, P < 0.01).

CONCLUSIONSActivation of ROCK signal pathway may be involved in the pathogenesis of vascular endothelial cell hyperpermeability induced by hypoxia.