Prone to rupture atherosclerotic plaques (AT) show significant angiogenesis. The mechanism of neovessel formation in the growing AT is still unknown. We have previously shown that tissue factor (TF) and its signaling mediators have a significant role in mature neovessel formation.Objective
To define the signaling pathways involved in TF-induced angiogenesis.Methods and Results
We analyzed the Akt, a downstream effector of the TF-induced signaling via activation of PI3K. The following results were obtained: a) TF-siRNA inhibition of TF in the human endothelial cells (HMEC-1) resulted in a highly significant inhibition of endothelial-tube-likeformation (ETub) with stable phenotype in 3D cultures (2500 ± 25 μm random-siRNA versus 260 ± 45 μm TF-siRNA). The inhibition in ETub was associated with a down-regulation of Akt expression (2x) and with increased Raf-phosphorylation at Ser259 (1x up) resulting in a reduced Raf kinase expression and a reduction of ERK1/2 phosphorylation (1x down) and Ets-1 transcription factor inhibition (2x down); b) Overexpression of TF resulted in an increase in ETub (3250 ± 15 μm versus 2500 ± 25 μm) and in the up-regulation of Akt protein; c) Immunoprecipitation of Akt revealed that TF is directly associated with Akt; d) The effect of silencing TF was only reversed by PAR2 agonist (H-5042) to a 65% of control ETub (1630 ± 25 μm versus 260 ± 45 μm); e) Enforced expression of Akt by pcDNA3-MyrHA-Akt1 plasmid in TF-silenced endothelial cells rescued ETub formation (2250 ± 45 μm versus 260 ± 45 μm) and induced Ets-1 phosphorylation.Conclusion
TF and Akt1 form a complex that regulates Etub formation signaling through Raf/ERK/Ets-1. The triggering effect of TF on the formation of endothelial-tube-like formation may have a significant impact not only in atherosclerotic plaque neovessel formation but in cancer metastasis and other processes cursing with neovascularization.