Purpose: Tissue factor (TF)-mediated signaling regulates gene expression and protein synthesis leading to alterations in cell function. Recently we have demonstrated in microvascular endothelial cells (mECs) that TF induces ETS1, regulates CCL2 expression and stimulates smooth muscle cell recruitment stabilizing the newly formed microvessels. However, TF may signal through more than one transcription factor to regulate gene expression in motile mECs. We have investigated whether additional transcription factors are involved in mature microvessel formation.
Methods: Human mECs were cultured in three dimensional basement membrane matrices. Cells were bioengineered to either abrogate or overexpress the TF gene. At 4 or 24 hours of culture RNA was obtained and real-time PCR gene arrays performed. Bioinformatic and systems biology analysis were applied to obtain the most probable regulated gene neural networks and targets. Targets genes were further validated.
Results: TF activate different transcription factors as ETS1, KFL2, SMAD3, STAT3 and PPARγ to induce downstream expression of genes encoding molecules implicated in angiogenesis. SMAD3 through protein-protein interaction regulates ETS1 function. Whereas the ability of TF overexpressing cells to induce gene expression through ETS1 is dependent of AKT activation, in presence of SMAD3 ETS1 does not require AKT activation. SMAD3 bypasses the requirement of ETS1 for AKT activation and signals through ERK1/2 to activate target gene promoter activity.
Conclusions: In mECs TF can trigger the expression of CCL2 through transcription factor synergism in a complex regulatory network with ETS1 as a main transcription factor to ensure the recruitment of perycites to stabilize newly formed microvessels.