TNF-α decreases expression of Thrombomudulin and endothelial protein C receptor in human endothelial cells

Abstract

Introduction. Thrombomodulin (TM) and endothelial protein C receptor (EPCR) are intrinsic anticoagulant factors of normal endothelial cells. The objective of this study was to determine the effects of TNF-α on TM and EPCR gene expression in human endothelial cells as well as the effect of a spice and coloring food compound curcumin as a potential therapeutic agent. Methods. Human umbilical vein endothelial cells (HUVECs) were cultured without or with TNF-α (2.0 ng/ml) for different times. The expression of TM and EPCR was determined by real-time RT-PCR, Western blot, and ELISA. Dose-dependent study of TNF-α in 6 h culture was also performed. Blocking effect of anti-inflammatory agent curcumin (5, 10, and 25 μM) on TNF-α was determined. Results. HUVECs treated with TNF-α (2.0 ng/ml) reduced TM mRNA levels by 80, 97, 94, and 97% at 3, 6, 12, and 24 h, respectively ( P < 0.01). Dose-dependent study showed that TM mRNA levels of HUVECs were decreased by 86, 89, 92, 91, and 94% after treatment of TNF-α (0, 0.25, 0.5, 1, 2, and 4 ng/ml) for 6 h, respectively ( P < 0.01). TM protein levels were significantly reduced by 69% in TNF-α-treated cells as compared to controls ( P < 0.01). Secreted protein and activity of TM in the supernatant of HUVEC cultures were also significantly reduced after TNF-α treatment. In addition, EPCR mRNA and protein levels were significantly reduced in TNF-α-treated group as compared to controls ( P < 0.05). Furthermore, these effects were observed in other types of endothelial cells from human coronary arteries, lungs, and skins. Curcumin effectively blocked these effects of TNF-α on down regulation of TM and EPCR. Conclusions. These data demonstrate that TNF-α significantly decreases expression of TM and EPCR at both mRNA and protein levels in several human endothelial cells. Curcumin can effectively block TNF-α-induced endothelial dysfunction. This study suggests a new molecular mechanism of inflammation associated thrombosis and a new strategy to prevent this problem.