Influence of Stretch-induced Thrombomodulin (TM) Expression on Endothelial Cell Migration and Regulation of TM Stability by Cytokine
Date Issued
2009
Date
2009
Author(s)
Liu, Fang-Hsuan
Abstract
Atherosclerosis is a chronic inflammatory disease. Inflammation and coagulation play an important role in this disease. Thrombomodulin (TM) is a highly glycosylated transmembrane protein in vascular endothelial cells, and has anti-inflammation and anti-coagulation properties. In recent study, it can also regulate angiogenesis, cell proliferation and migration. Vascular endothelial cells (ECs) are exposed to stretch generated by the blood pressure. Therefore, stretch is an important factor in regulation of EC functions. In this study, bovine aortic endothelial cells (BAECs) were utilized to investigate the influence of stretch-induced TM expression on cell migration and the regulatory mechanism of TNF-a-mediated TM protein stabilityo investigate the effect of TM expression in endothelial cell wound healing ability, BAECs were transfected with TM plasmid containing C-terminal FLAG tag (TM-flag) and the wound healing assay was carried out for 7 hours with low serum (2% FBS) or high serum (10% FBS)-containing culture medium. The results showed that, cultured with low serum-containing culture medium, the degree of healing of BAECs transfected with TM-flag was facilitated about 10% compared with that transfected with empty vector. But when cells cultured with high serum-containing culture medium, there was no significant difference between cells transfected with empty-vector and TM-flag.e also used TM knock-down experiment to investigate its effect on the wound healing. Under low serum culture condition, there was no significant difference in wound healing between TM knock-down cells and scramble control. But when cells cultured with high serum-containing culture medium, the degree of healing of TM knock-down cells was about 10% less than scramble control in a 7-hr wound healing assay.hen endothelial cells were exposed to stretch with 16% strain for 7 hours, TM protein expression was up-regulated by 2.5 to 3.5-fold compared with static control. But weather the scar was perpendicular or parallel to the stretch direction, wound healing ability of BAECs after stretch stimulation for 7 hours was about 20% less than the static control. However, when the same stretch force stimulated cells for 15 hours, TM protein expression was suppressed to about 0.7-fold of static control. The results suggested that stretch-induced TM expression did not substantially regulate endothelial cell migration.o study TM protein stability, we added 10 ng/ml TNF-a to cultured BAECs for 6 hours. The results showed that the TM protein expression was down-regulated to 0.6-fold in comparison with control. When BAECs were transfected with TM-flag, TM-Y/A, TM-T/A or TM-S/A mutants and treated TNF-a for 6 hours, the results suggested that the de-phosphorylation of C-terminal Tyr (Y534) and Ser (S550) residues of TM may be related to TM stability and protect it against TNF-a-induced TM down-regulation. To invesgate the mechanism of TNF-a-induced TM down-regulation, we pre-treared BAECs with 10 uM MG132 for 1 hour followed by 6 hours TNF-a treatment. We found that MG132 protected TM from TNF-a-induced down-regulation, but in the immumoprecipitation experiment, no significant ubiquitination occurred suggesting that the down-regulation of TM by TNF-a is not mainly through ubiquitinaiton pathway.n summary, increased TM expression facilitated endothelial cell wound healing ability. Stretch-induced TM expression was not involved in cell migration. In TM protein stability investigation, its stability was down-regulated by TNF-a. The mutant of C-terminal Tyr (Y534) and Ser (S550) residues of TM increased its stability and protected it from TNF-a-induced down-regulation.
Subjects
thrombomodulin
stretch
migration
TNF-{alpha}
Type
thesis
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