Regulation of endothelin-B receptor mRNA expression in human endothelial cells by cytokines and growth factors.
The regulation of endothelin-B receptor (ETB) mRNA expression in human endothelial cells (ECs) by cytokines and growth factors may play an important role in the response of the endothelium to inflammatory and angiogenic stimuli. Using quantitative RT-PCR, we studied ETB expression in human umbilical vein ECs (HUVECs) grown in culture on either plastic or fibrin matrix for 24 h in the presence or absence of tumor necrosis factor-alpha (TNF-alpha, 100 U/ml) or basic fibroblast growth factor (bFGF, 30 ng/ml). In addition, the effect of the nitric oxide (NO) donor S-nitrosyl-acetylpenicillamine (SNAP, 0.4 mM) was examined directly on ETB expression or on the response to bFGF. Under control conditions, ETB mRNA was detected after 35 cycles of amplification as a band of the expected size (553 bp). In the absence of fibrin matrix, ETB was downregulated by bFGF and TNF-alpha and could barely be detected by PCR. Southern analysis of the RT-PCR products after 25 cycles revealed that bFGF reduced ETB mRNA expression by 2.7 +/- 0.4-fold (p < 0.01) and TNF-alpha tended to reduce its expression by 1.8 +/- 0.9-fold of control, although this did not reach statistical significance (p < 0.20). In contrast, on fibrin matrix both bFGF and TNF-alpha increased ETB mRNA expression by 25 +/- 9-fold (p < 0.05) and 68 +/- 19-fold (p < 0.05) of control, respectively, suggesting a role for ETB in the vascular tube formation that occurs under these conditions. Pharmacologic addition of NO mimicked the effect of fibrin, converting the response to bFGF from down- to upregulation of ETB, raising the possibility that NO acts as a molecular switch modulating the response to angiogenic factors.
Smith
P J
PJ
Terrence Donnelly Heart Centre, St. Michael's Hospital, University of Toronto, Ontario, Canada.
Teichert-Kuliszewska
K
K
Monge
J C
JC
Stewart
D J
DJ
D015152:Blotting, Northern; D002460:Cell Line; D016207:Cytokines / Q000494:pharmacology*; D003720:Densitometry; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D006133:Growth Substances / Q000494:pharmacology*; D006801:Humans; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D052250:Nitric Oxide Synthase Type III; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis*; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000096:biosynthesis*; D026423:S-Nitroso-N-Acetylpenicillamine; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016207:Cytokines; D004791:Enzyme Inhibitors; D006133:Growth Substances; D012333:RNA, Messenger; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D014409:Tumor Necrosis Factor-alpha; D016222:Fibroblast Growth Factor 2; D026423:S-Nitroso-N-Acetylpenicillamine; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D010396:Penicillamine