Function and signal transduction of thrombospondin gene with respect to cellular growth and apoptosis in vascular system

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We have reported that we tried to introduce the expression vector of thrombospondin (TSP) 1 cDNA into human umbilical vein-endothelial cells (HUVECs) and bovine aortic endothelial cells (BAECs) to establish stable transfected cells, but that we failed it in 1999 during term of the project. In 2000 during term of the project, we therefore reconstituted the project in which we performed establishment of the transient transfection system, testing effect of TSP1 protein on cellular apoptosis and elucidating its signal transduction. To test transient transfection, calcium-phosphate, Lipofectin and Lipofectamine were tried to be used. However, transfection-efficiency were 5% in calcium-phosphate method, 10% in Lipofectin and 10-20% in Lipofectamine. By those transfection methods, DNA fragmentation were induced but quantitative assay of DNA fragmentations did not show significant difference between the wild-type and transfected cells, which is supposed by influence of wild-type cells even in … More the transfectant samples. According to the results above, experiment of transfection should be performed with adenovirus espression vector, and then the expression vector has been being constructed for the time being.On the other hand, we performed experiment whether cell apoptosis were induced by addition of TSP1 in HUVECs to confirm the previous experiment. DNA fragmentation was observed in agarose gel and significantly increased in TSP1-treated cells in dose-dependent manner (maximum induction ; 5 μg/ml (10 nM) of TSP1) by modified Burton's assay. In terms of the signal transduction of the apoptosis by TSP1, we focused on p38 mitogen activator protein kinase (MAPK) which was one of the apoptotic signals in HUVECs induced by oxysterols as we have already found. Five μg/ml of TSP1 activated p38 MAPK with peak activation at 2 hours after the stimulation. This activation was significantly inhibiied by SB203580, a specific inhibitor of p38 MAPK.In addition to the apoptotic signal, p42/44 MAPK and Akt-1 were also estimated as proliferative signals. Both p42/44 MAPK and Akt-1 were not significantly changed in response to 5 μg/ml of TSP1. We also tested the cross-network between p42/44 MAPK and p38 MAPK as reported in 1999 ; SB203580 increased the activation of p42/44 MAPK, suggesting that activation of p38 MAPK suppressed p42/44 MAPK.However, there were no reproducibility about the previous result, and we concluded that there is no relationship between p42/44 MAPK and p38 MAPK.In summary on the basis from the present results, we elucidate that TSP1 induces apoptosis in HUVECs and that activation of p38 MAPK may be in part associated with endothelial cell death as an apoptotic signal transduction. In contrast, p42/44 MAPK and Akt-1 as proliferative signals provide no influence on cell death. It is on study to investigate responsible receptors affecting activation of p38 MAPK by stimulation with TSP1. The candidate may be CD36 or β3 integrin. Less