Investigation On Expression Of Tissue Factor In ECV304 Cell Line Induced By Oxidized High Density Lipoprotein And Its Subfractions And The Underlying Mechanism Involved

OBJECTIVE: To investigate the expression of tissue factor (TF) induced by oxidized high density lipoprotein (oxHDL) and its subfractions (oxHDL2, oxHDL3) in the human umbilical vein cell line ECV304, and the underlying mechanism involved.METHODS: Real time quantitative PCR (RQ-PCR) and western blot were used to detect mRNA and protein expression after the samples were coincubated with thrombin, oxHDL, HDL and its subfractions in different concentrations(oxHDL, HDL and their subfractions: 10, 20, 40, 80, 100, 120mg/L; thrombin : 1, 2, 4, 6, 8, 10IU,after 5 hours stimulation) and different coincubation time (2,3,4,5,6,7,and 8 hours). SP600125(0.1μmol/L), SB203580(0.1μmol/L), PD98059(1μmol/L), and wortmannin(0.1μmol/L) , the specific inhibitors against c-jun terminal NH2 kinase (JNK), p38 MAP kinase (p38), p44/42 MAP kinase (ERK1/2), and phosphatidylinositol 3-kinase (PI3K), respectively, were co-incubated with the cells, and mRNA expression level of TF was analyzed by RQ-PCR.RESULTS:1. The expression of TF mRNA in normal ECV304 was not detected, while thrombin induced TF expression in ECV304 cells in a dose-dependent manner.2. HDL, oxHDL and their subfractions stimulated the expression of TFmRNA significantly both in dose- and time-dependent manner, in which oxHDL3 displayed the strongest effect, followed by oxHDL and oxHDL2.3. Stimulation with oxHDL or oxHDL3 but not oxHDL2, HDL and its subfractions resulted in a detectable TF protein expression.4. Compared with HDL and its subfractions, oxHDL, oxHDL3 induced TF expressions were significantly higher, but to oxHDL2, TF expression were lower than native HDL2.5. SP600125, SB203580, and PD98059 caused a decrease of tissue factor expression induced by oxHDL by 81%, 95%, and 87% respectively (P<0.05).6. SP600125, SB203580, and PD98059 also resulted in a reduction of oxHDL2-stimulated TF mRNA expression by 41%, 64% and 93%, respectively (P<0.05).7. SB203580 and SP600125 induced an increase of oxHDL3-stimulated TF expression by 31% and 34%, respectively (P<0.05), PD98059 resulted in 76% reduction (P<0.05).8. Wortmannin caused a significant augmentation of the TF expression level induced by oxHDL, oxHDL2 and oxHDL3 by 155 %, 93% and 96 % (P<0.05), respectively.CONCLUSIONS:1. TF expression in ECV304 induced by oxHDL and its subfractions could be expressed in dose-, and time-dependent manner. oxHDL3 gets the highest level of TF expression, TF protein level induced by oxHDL3, oxHDL but bot oxHDL2 can be detected by western blot method in our experiment.2. TF expression in ECV304 induced by HDL and its subfractions could be expressed in dose dependent manner.3. Compared with HDL and its subfractions, oxHDL, oxHDL3 induced TF expressions were significantly higher, but to oxHDL2, TF expression was lower than native HDL2.4. MAPKs(ERK1/2,p38MAPK,JNK)and PI3K signal transduction pathway may play a different role in TFmRNA expression of ECV304 cell line.