Enhanced prostacyclin synthesis in endothelial cells by retrovirus-mediated transfer of prostaglandin H synthase cDNA.
A retroviral vector (BAG) was used to transfer human prostaglandin H synthase (PGHS-1) gene into a human endothelial cell line for enhancement of PGI2 synthesis. Cells infected with BAG containing PGHS-1 cDNA in the sense orientation relative to the retroviral promoter (PGHS(S)) expressed a 30-fold increase in mRNA but, due to a reading frame shift, did not show an increase in PGHS protein or in PGI2 synthesis, while those with PGHS-1 in reverse orientation relative to the viral promoter (PGHS(R)), produced a > 10-fold increase in PGHS mRNA over the control (169 +/- 22 vs 14.8 +/- 1.2 amol/micrograms RNA) with a concordant increase in PGHS protein (5.82 +/- 1.07 vs 0.23 +/- 0.04 ng/mg protein) and enzyme activity. Primer extension analysis of PGHS(R) revealed two transcription start sites located in the SV40 late promoter region adjacent to PGHS-1 cDNA. PGHS(R) cells produced a high basal PGI2 level which was increased by several-fold in response to stimulation by ionophore, arachidonic acid, and thrombin. Kinetic analysis revealed the PGI2 synthetic rate to be 14 ng/min-1 per million cells and t1/2 of PGI2 synthesis, 13.3 min. These findings indicate that transfer of PGHS-1 gene into vascular cells enhances PGI2 synthesis and may be a useful strategy for restoring thromboprotective property of damaged blood vessels.
Xu
X M
XM
Department of Internal Medicine, University of Texas Health Science Center, Houston.
Ohashi
K
K
Sanduja
S K
SK
Ruan
K H
KH
Wang
L H
LH
Wu
K K
KK
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D011464:Epoprostenol / Q000096:biosynthesis*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D012190:Retroviridae / Q000502:physiology*; D014162:Transfection / Q000502:physiology
D004247:DNA; D011464:Epoprostenol; D011451:Prostaglandin-Endoperoxide Synthases