Modulation of Cox-2 Expressed in LPS-Treated Endothelial Cells by Cox-Metabolites

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Pravit Akarasereenont
Kitirat Techatraisak
Sirikul Chotewuttakorn
Athiwat Thaworn

Abstract

Here, we have investigated the effects of COX-metabolites (PGI2, PGE2, PGF2α and
TXA2) on COX-2 expressed in human umbilical vein endothelial cells (HUVEC) treated with lipopolysaccharide (LPS). Human umbilical vein endothelial cells (HUVEC) were obtained from babies born to normal pregnant women (HUVEC) and cultured in 96-well/6-well plates as standard techniques. Cells were grown to confluent and replaced with fresh medium containing no addition, LPS alone, COX-metabolites alone and LPS plus COX-metabolites (0.001, 0.01, 0.1 or 1 μg/ml) for 24h. Then, the medium was removed and replaced with fresh medium containing arachidonic acid (10 μM for 10 min) after which time the medium was collected to

measured COX activity by the production of 6-keto-PGF1α (stable metabolites of PGI2) using enzyme immunoassay. The remained cells were extracted and detected COX isoform expression by using immunoblotting. PGI2, PGE2, PGF2α or TXA2, did not affect on basal COX activity in untreated HUVEC (24h incubation). Untreated HUVEC contained COX-1 protein but not COX-2 protein. When HUVEC were treated with LPS (1 μg/ml for 24h), COX activity and COX-2 protein was increased in a dose dependent manner. The increased COX activity in LPS (1 μg/ml) treated HUVEC was inhibited with PGE2, but not PGI2, PGF2α or TXA2 in a dose dependent manner. Similary, COX-2 protein expression in LPS treated HUVEC was also inhibited with PGE2, but not PGI2, PGF2α or TXA2 in a dose dependent manner. These results suggested that PGE2, but not PGI2, PGF2α or TXA2 is a key in feedback regulation of COX-metabolites produced in HUVEC.

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Section
2001 Annual Meeting Abstracts/Lectures