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 reaction (RT-PCR) procedures to study the expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI-1) mRNA in human endothelial cells and 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 with 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 endothelial cells and 5 x 10(2) monocytes stimulated by endotoxin. TF mRNA expression was increased by TNF-alpha in endothelial cells and in monocytes stimulated by endotoxin. Elevated expression of TF mRNA in monocytes without stimulation by endotoxin was mainly related to cell adhesion. TFPI-1 mRNA was constitutively expressed in endothelial cells and was detected in only 5 x 10(2) unstimulated cells and 10(2) phorbol ester-stimulated cells. Expression was increased upon stimulation with phorbol ester. With this technique, TFPI-1 mRNA in monocytes was rather low even when cells were stimulated with phorbol ester or after adhesion.
D001773:Blood Cells / Q000737:chemistry / Q000378:metabolism; D002448:Cell Adhesion; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000166:cytology*; D015870:Gene Expression; D006801:Humans; D007231:Infant, Newborn; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000378:metabolism; D008074:Lipoproteins / Q000097:blood* / Q000235:genetics*; D009000:Monocytes / Q000737:chemistry* / Q000166:cytology; D012333:RNA, Messenger / Q000097:blood; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013925:Thromboplastin / Q000032:analysis*