title: [Tumor necrosis factor-α promote permeability of human umbilical vein endothelial cells via activating RhoA-ERK1/2 pathway].
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  These results indicate that TNF-α-induced EC barrier dysfunction and morphological changes of the F-actin via activating RhoA-ERK/MAPK signal pathway.
authors.affiliation: Department of Cardiology, Shenyang General Hospital, Shenyang 110840, China.
authors.forename: Cheng-Hui
authors.initials: CH
authors.lastname: Yan
authors.forename: Hai-Bo
authors.initials: HB
authors.lastname: Yu
authors.forename: Ming-Fang
authors.initials: MF
authors.lastname: Huang
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.forename: Xiao-Lin
authors.initials: XL
authors.lastname: Zhang
authors.forename: Ya-Ling
authors.initials: YL
authors.lastname: Han
mesh_terms: D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D014409:Tumor Necrosis Factor-alpha; D020928:Mitogen-Activated Protein Kinases; D020742:rhoA GTP-Binding Protein