Fast detection of tissue factor and tissue factor pathway inhibitor messenger RNA in endothelial cells and monocytes by sensitive reverse transcription polymerase chain reaction

We developed fast and sensitive reverse transcription-polymerase chain reac tion (RT-PCR) procedures to study the expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI-1) mRNA in human endothelial cells an d monocytes. The sensitivity of the technique was checked by performing RT- PCR with limited numbers of cells. Cells were stimulated either with tumor necrosis factor (TNF-alpha) or endotoxin to induce TF mRNA expression or wi th phorbol ester to increase TFPI-1 mRNA expression. Thus, RT-PCR specific for TF mRNA provided detection from as few as 10(3) TNF-alpha stimulated en dothelial cells and 5.10(2) monocytes stimulated by endotoxin. TF mRNA expr ession was increased by TNF-alpha in endothelial cells and in monocytes sti mulated by endotoxin. Elevated expression of TF mRNA in monocytes without s timulation by endotoxin was mainly related to cell adhesion. TFPI-1 mRNA wa s constitutively expressed in endothelial cells and was detected in only 5. 10(2) unstimulated cells and 10(2) phorbol ester-stimulated cells. Expressi on was increased upon stimulation with phorbol ester. With this technique, TFPI-1 mRNA in monocytes was rather low even when cells were stimulated wit h phorbol ester or after adhesion. (C) 1999 Elsevier Science Ltd. All right s reserved.