The Effect of Tetomilast (OPC-6535) on Cultured Human Vascular Endothelial Cells

Abstract

Purpose: Tetomilast is currently in clinical development for the treatment of inflammatory bowel disease and chronic obstructive pulmonary disease. Leukocyte adhesion to endothelium and transmigration into the damaged tissue are essential features of the inflammatory process; endothelial permeability and adhesion molecule expression are increased with inflammation. Although the mechanism of action for tetomilast has not been fully elucidated, tetomilast is known to inhibit phosphodiesterase 4 (PDE4). To investigate the mechanisms involved in tetomilast's anti-inflammatory action, we used primary vascular endothelial cells (EC) to study its effect on cell monolayer permeability and adhesion molecule expression, as compared with that of PDE4-selective inhibitors. Methods: Human umbilical vein EC (HUVEC) were cultured on a membrane insert. The permeability increase was induced by incubation with thrombin (0.2 unit/mL) or histamine (10 μM) at 37°C for 1 hr and quantified by the detection of diffused FITC-dextran through the HUVEC monolayer. For the study of adhesion molecule expression, a cell-based ELISA was used to measure VCAM-1, ICAM-1, and E-selectin expression in HUVEC and human lung microvessel EC (HLMVEC) following TNF-αα stimulation. Results: Tetomilast (20 μM) inhibited thrombin- or histamine-induced permeability increase by 29.2% and 58.8%, respectively, whereas the PDE4 inhibitors cilomilast (30 μM) and roflumilast (0.3 μM) did not. In both cell types, a concentration-dependent inhibition of E-selectin expression by tetomilast (5 to 30 μM) was observed with both 30-min and overnight (16-hr) pretreatments, whereas inhibition of ICAM-1 and VCAM-1 was clearly seen only with overnight pretreatment. In contrast, cilomilast, rolipram (both up to 30 μM), and roflumilast (up to 0.3 μM) had no such effect on expression of any of the adhesion molecules. cDNA microarray data showed that preincubation with tetomilast for 6 hr downregulated interferon regulatory factor (IRF)-1 expression. Using a luciferase-based IRF-1 pathway-specific reporter assay in HLMVEC, the inhibitory effect of tetomilast on IRF-1 pathway was observed over the same concentration range. Conclusions: Tetomilast may mediate its anti-inflammatory effect partially through the inhibition of vascular permeability increase and adhesion molecule expression, independent of PDE4 inhibition. Our data indicate that tetomilast inhibits VCAM-1 and ICAM-1 expression on EC, likely by inhibiting IRF-1 pathway.