OBJECTIVETo investigate the changes in endothelial cytoskeletal reorganization and the role of Rho in the signal transduction pathway.

METHODSECV304 cells were cultured and randomly divided into following groups: i.e. sham (with normal rat serum treatment), burn (with burn rat serum treatment), Y (with 30 micromol/L Rho kinase inhibitor Y-27632 treatment), burn plus Y (pretreatment of cells with burn serum before treated with 30 micromol/L Y-27632), Y plus burn (pretreatment of cells with Y-27632 for 1 hour before treated with burn serum), LPA (with normal rat serum and 13 micromol/L LPA), and LPA plus Y (pretreatment of cells with LPA before treated with Y-27632) groups. The indices were examined at 6, 7 and 8 posttreatment hours (PTH) in all groups except in Y group. The endothelial morphology was observed with HE staining. Endothelial cytoskeleton was observed by dual-fluorescence labeling of filamenta (F) with Rhodamine-phalloidin and monomer (G) with oregon green labeled DNAase. The actin content in the cells in all groups was measured with flow cytometry.

RESULTSIn sham and control group, the cells were in fusiform or polygonal shape, with satisfactory growth; filamentous actin (F-actin) was mainly distributed in the peripheral site of the cytoplasm and formed peripheral filamental band. The cells became confluent to form a single layer with reticular structure. Globular actin (G-actin) was concentrated in the nucleus and per nucleus. In burn group, after 6 hours of burn serum treatment, the ability of cells to adhere to vessel wall was weakened, and a striking reorganization of the actin cytoskeleton and the formation of the stress fibers were found. Furthermore, the fluorescent intensity of the peripheral filament bands was weakened, and dispersed actin monomers were seen in the cytoplasm. This reaction was enhanced along with elapse of stimulation time. In burn plus Y or Y plus burn group, the cells grew and adhered well to the wall of culture vessel. The distribution of the filamentous actin was the same as the sham group, while the stress fiber decreased in amount obviously. The structure of globular actin was condensed with little G-actin in the cytoplasm. The changes in actin cytoskeleton in LPA group was similar to that in burn group. The effects of LPA on actin reorganization could also be reversed by Y-27632. The content of F-actin in burn group at 6 PTH (0.63 +/- 0.07) was lower than that in sham group (0.75 +/- 0.08), while the content of G-actin in burn group (1.28 +/- 0.27) was higher than that in sham group (1.16 +/- 0.16, P > 0.05).

CONCLUSIONBurn serum induces vascular endothelial actin cytoskeleton reorganization in endothelial cells via the Rho-dependent signal pathway. Similar to the effect of LPA, this effect could be reversed by Y-27632.