Tumor necrosis factor-α promote permeability of human umbilical vein endothelial cells via activating RhoA-ERK1/2 pathway / 中华心血管病杂志

ABSTRACT

<p><b>OBJECTIVE</b>Tumor necrosis factor-α (TNF-α) is known to induce changes in endothelial cell morphology and permeability. The aim of this study is to determine the underlying signaling mechanisms involved in these responses.</p><p><b>METHODS</b>Cultured human umbilical vein endothelial cells (HUVECs) were exposed to TNF-α, and HUVEC cytoskeletal changes were evaluated by observing fluorescence of F-actin following ligation with labeled antibodies. Endothelial permeability was detected by measuring the flux of horseradish peroxidase (HRP)-albumin across the EC monolayers. To explore the signaling pathways behind TNF-α-induced changes in HUVEC morphology and permeability, HUVECs were treated with either the Rho GTPase inhibitor Y27632 or the mitogen-activated protein kinases (MAPK) inhibitors PD98059 and SB203580 before TNF-α administration. To further elucidate possible involvement of the RhoA and ERK pathways in TNF-α-induced HUVEC changes, retrovirus-carried recombinant dominant-negative forms and constitutive-activative forms of RhoA, namely T19NRhoA and Q63LRhoA, were pre-infected into HUVECs prior to TNF-α exposure.</p><p><b>RESULTS</b>TNF-α induced F-actin cytoskeleton rearrangement and increased HUVEC permeability in a dose and time-dependent manner. The maximal increase in the HRP-BSA flux (40 ng/ml) was seen in cells exposed to TNF-α at 100 ng/ml after 2 h. Preconditioning of HUVEC monolayer with Y27632 or PD98059 significantly reduced TNF-α induced permeability increase (HRP concentration from 40 ng/ml decreased to 12.5 ng/ml, P < 0.05) and F-actin cytoskeleton rearrangement, HUVEC pre-infection with activated forms of Q63LRhoA increased HUVEC permeability and upregulated pERK compared to GFP infection, while HUVEC pre-infection with inhibited forms of T19NRhoA attenuated the effects of TNF-α on HUVEC permeability.</p><p><b>CONCLUSION</b>These results indicate that TNF-α-induced EC barrier dysfunction and morphological changes of the F-actin via activating RhoA-ERK/MAPK signal pathway.</p>