Tissue factor-Akt signaling triggers microvessel formation.

Summary.  Background:  Tissue factor (TF) and its signaling mediators play a crucial role in angiogenesis. We have previously shown that TF-induced endothelial cell (EC) CCL2 release contributes to neovessel formation. Objective:  In this study, we have investigated the signaling pathways involved in TF-induced EC tube formation. Methods:  The human microvascular endothelial cell line (HMEC-1) cultured onto basement membrane-like gel (Matrigel) was used to study TF signaling pathways during neovessels formation. Results:  Inhibition of endogenous TF expression in ECs using siRNA resulted in inhibition of a stable tube-like structure formation in three-dimensional cultures, associated with a down-regulation of Akt activation, an increased phosphorylation of Raf at Ser259 with a subsequent reduction of Raf kinase and a reduction of ERK1/2 phosphorylation ending up in Ets-1 transcription factor inhibition. Conversely, overexpression of TF resulted in an increase in tube formation and up-regulation of Akt protein. Moreover, immunoprecipitation of Akt and western blotting of the immunoprecipitates with anti-TF antibody revealed a direct interaction between TF and Akt. The effects of silencing TF were partially reversed by a PAR2 agonist that rescued tube formation, indicating that the TF-Akt pathway induces PAR2-independent effector signaling. Finally, enforced expression of Akt in TF-silenced ECs rescued tube formation in a Matrigel assay and induced Ets-1 phosphorylation. Conclusions:  In EC, TF forms a complex with Akt activating Raf/ERK and Ets-1 signaling induces microvessel formation.