Molecular Biology Reports

, Volume 40, Issue 6, pp 3891–3900

Cellular repressor of E1A stimulated genes enhances endothelial monolayer integrity

  • Yan Duan
  • Shaowei Liu
  • Jie Tao
  • Yang You
  • Guitang Yang
  • Chenghui Yan
  • Yaling Han
Article

DOI: 10.1007/s11033-012-2373-6

Cite this article as:
Duan, Y., Liu, S., Tao, J. et al. Mol Biol Rep (2013) 40: 3891. doi:10.1007/s11033-012-2373-6

Abstract

Cellular repressor of E1A stimulated genes (CREG) is a novel modulator that maintains the homeostasis of vascular cells. The present study aimed to investigate the effects of CREG on tumor necrosis factor (TNF)-α-mediated inflammatory injury of vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were cultured and CREG overexpressing (VC), knockdown (VS) and mock-transfected (VE) HUVECs were challenged with TNF-α. We demonstrated that TNF-α prompted robust intercellular filamentous actin (F-actin) stress fiber formation as examined by rhodamin-phalloidin staining. Transwell assay and rhodamine B isothiocyanate–dextran staining indicated that TNF-α induced intercellular hyperpermeability of the HUVEC monolayers. These effects were attenuated in VC cells with forced CREG overexpression but significantly potentiated in VS cells with CREG silencing. After TNF-α stimulation, interleukin (IL)-6 and IL-8 secretions in VE cells were markedly increased and inducible nitric oxidase (iNOS) expression substantially elevated, whereas these effects were pronouncedly damped in VC cells. Conversely, in VS cells, the increase in inflammatory markers was substantially potentiated. Immunofluorescence staining demonstrated that nuclear factor κB (NF-κB) slowly and transiently translocated into the nuclei of VC cells upon TNF-α stimulation. However, a more swift and sustained nuclear translocation was observed in VS as compared to VE cells. Corresponding changes in the pattern of its protein expression was also observed. These data suggested that CREG can inhibit NF-κB activation, TNF-α-induced inflammatory responses and the hyperpermeability of endothelial cells, and may therefore represent a potential therapeutic target for pathological vascular injury.

Keywords

Cellular repressor of E1A stimulated genesVascular endothelial cellsInflammatory injuryNuclear factor κB

Abbreviations

CREG

Cellular repressor of E1A stimulated genes

TNF-α

Tumor necrosis factor-α

HUVEC

Human umbilical vein endothelial cell

VC

CREG-overexpressing HUVECs

VS

CREG-knockdown HUVECs

VE

Mock-transfected HUVECs

F-actin

Filamentous actin

IL

Interleukin

iNOS

Inducible nitric oxidase

NF-κB

Nuclear factor κB

EC

Endothelial cell

IκB

Inhibitor of κB

p-IκB

Phosphorylated form of IκB

shRNAs

Short hairpin RNAs

ECL

Enhanced chemiluminescence

RITC

Rhodamine B isothiocyanate

PBS

Phosphate-buffered saline

DAPI

4′,6-diamidino-2-phenylindole

BCA

Bicinchoninic acid

DTT

Dithiothreitol

PMSF

Phenylmethylsulfonyl fluoride

ELISA

Enzyme linked immunosorbent assay

SD

Standard deviation

PDTC

Pyrrolidine dithiocarbamate

MAPK

Mitogen-activated protein kinase

ERK

Extracellular signal-regulated kinase

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Yan Duan
    • 1
  • Shaowei Liu
    • 1
  • Jie Tao
    • 1
  • Yang You
    • 1
  • Guitang Yang
    • 1
  • Chenghui Yan
    • 1
  • Yaling Han
    • 1
  1. 1.Department of Cardiology, Shenyang Northern HospitalCardiovascular Research InstituteShenyangChina