[Relationship between activation of Rho kinase signal pathway and permeability of hypoxic vascular endothelial cells].
OBJECTIVE
To 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).


CONCLUSIONS
Activation of ROCK signal pathway may be involved in the pathogenesis of vascular endothelial cell hyperpermeability induced by hypoxia.
Institute of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burns and Combined Injury, the Third Military Medical University, Chongqing 400038, China.
Chen
C
Liu
Pei
P
Wang
Feng-jun
FJ
Wang
D015687:Cell Hypoxia; D002460:Cell Line; D002463:Cell Membrane Permeability; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D015398:Signal Transduction; D054460:rho-Associated Kinases / Q000378:metabolism*
D054460:rho-Associated Kinases