Selective induction of cyclo‐oxygenase‐2 activity in the permanent human endothelial cell line HUV‐EC‐C: biochemical and pharmacological characterization

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Abstract
Cyclo‐oxygenase (COX), the enzyme responsible for the conversion of arachidonic acid (AA) to prostaglandin H2 (PGH2), exists in two forms, termed COX‐1 and COX‐2 which are encoded by different genes. COX‐1 is expressed constitutively and is known to be the site of action of aspirin and other non‐steroidal anti‐inflammatory drugs. COX‐2 may be induced by a series of pro‐inflammatory stimuli and its role in the development of inflammation has been claimed. Endothelial cells are an important physiological source of prostanoids and the selective induction of COX‐2 activity has been described for finite cultures of endothelial cells, but not for permanent endothelial cell lines. The HUV‐EC‐C line is a permanent endothelial cell line of human origin. We have determined the COX activity of these cells under basal conditions and after its exposure to two different stimuli, phorbol 12‐myristate 13‐acetate (PMA) and interleukin‐1β (IL‐1β). Both PMA and IL‐1β produced dose‐ and time‐dependent increases of the synthesis of the COX‐derived eicosanoids. These increases were maximal after the treatment with 10 nM PMA for 6 to 9 h. Under these conditions, the main eicosanoid produced by the cells was PGE2. The increase of COX activity by PMA or IL‐1β correlated with an increase of the enzyme's apparent Vmax, whilst the affinity for the substrate, measured as apparent Km, remained unaffected. Treatment of the cells with PMA induced a time‐dependent increase in the expression of both COX‐1 and COX‐2 mRNAs. Nevertheless, this increase was reflected only as an increase of the COX‐2 isoenzyme at the protein level. The enzymatic activity of the PMA‐induced COX was measured in the presence of a panel of enzyme inhibitors, and the IC50 values obtained were compared with those obtained for the inhibition of human platelet COX activity, a COX‐1 selective assay. Classical non‐steroidal anti‐inflammatory drugs (NSAIDs) inhibited both enzymes with varying potencies but only the three compounds previously shown to be selective COX‐2 inhibitors (SC‐58125, NS‐398 and nimesulide) showed higher potency towards the COX of PMA‐treated HUV‐EC‐C. Overall, it appears that the stimulation of the HUV‐EC‐C line with PMA selectively induces the COX‐2 isoenzyme. This appears to be a suitable model for the study of the physiology and pharmacology of this important isoenzyme, with a permanent endothelial cell line of human origin. British Journal of Pharmacology (1997) 121, 171–180; doi:10.1038/sj.bjp.0701112