Downregulation of tissue factor (TF) by RNA interference induces apoptosis and impairs cell survival of primary endothelium and tumor cells

Abstract

Tissue factor (TF) has been implicated in the thrombotic complications seen during vascular rejection of allografts and may contribute to intimal hyperplasia in chronic allograft vasculopathy. Downregulation of endothelial TF expression post-transplantation could therefore be of therapeutic value. Lentivirus-mediated RNA interference was used in primary endothelial cells (EC) to investigate its effects on TF protein expression and functional activity. Lentivirus-mediated expression of a TF-specific short-interfering (si) RNA with green fluorescent protein as a reporter gene (siRNATF-GFP) resulted in a 42 +/- 3.9% reduction in EC surface-expressed TF as compared with cells expressing a scrambled siRNATF sequence (P = 0.025). The TF content in EC lysates was reduced from 6.85 +/- 1.99 ng to 3.05 +/- 0.82 ng (P = 0.006). Factor X (FX) activation was not impaired on the apical EC surface. The subendothelial matrix of ECs with low TF expression showed significantly reduced TF activity compared with non-transduced cells or with cells harboring the empty vector. ECs expressing siRNATF-GFP exhibited reduced reporter gene (GFP) expression and cell density and an altered morphology. Transfection of control cells with high (J82 cells) or low (MiaPaCa-2 cells) TF expression with siRNATF oligonucleotides caused apoptosis of the J82 but not of the MiaPaCa-2 cells. Thus, lentivirus-mediated RNA interference reduces the TF expression of activated ECs but does not affect FX activation by TF/FVIIa expressed on the apical surface. The downregulation has nevertheless substantial negative effects on the viability of ECs and TF-expressing control cells. These findings imply that certain levels of TF are required for the maintained viability and growth of endothelium and TF-expressing tumor cells.