Summary. Background: Prostaglandin (PG) E 2 induces expression of matrix metalloproteinases and angiogenic factors, thereby contributing to plaque instability. Objective: To study the influence of cyclooxygenase (COX) and PGE synthase (PGES) isoenzyme expression on PGE 2 and PGI 2 biosynthesis in vascular smooth muscle cells (VSMC) in culture. Methods: Cells were treated with human recombinant IL-1b over different periods of time. Expression of PGI synthase, and COX and PGES isoenzymes was determined by real-time reverse transcriptase polymerase chain reaction and immunoblotting. Biosynthesis of prostanoids from exogenous or endogenous substrate was analyzed by high-performance liquid chromatography or enzyme-immunoassay after incubation of cells with labeled arachidonic acid or thrombin, respectively. Results: Cytosolic PGES and microsomal PGES (mPGES) -1 and -2 were expressed in VSMC. PGES activity was mainly linked to mPGES-1. IL-1b induced COX-2 and mPGES-1 with a different time course. VSMC ability to synthesize PGE 2 and PGI 2 fitted mPGES-1 and COX-2 expression, respectively. The ability of VSMC to produce PGI 2 was downregulated by mPGES-1 expression and was restored when mPGES-1 expression was silenced. Results from COX-1 and COX-2 silencing and selective inhibition showed that both COX-1 and COX-2 were involved in the biosynthesis of PGE 2 and their relative contribution depended on the time of incubation with IL-1b. Conclusions: mPGES-1 is the main PGES responsible for PGE 2 biosynthesis by VSMC and its induction downregulates VSMC ability to produce PGI 2. These results support the concept that under inflammatory conditions VSMC could significantly contribute to plaque instability and that mPGES-1 may be a target for therapeutic intervention in patients with cardiovascular risk.
Human umbilical vein endothelial cells were exposed to free-form and lipid-complexed versions of amphotericin B (alone or in combination with human recombinant interleukin [IL]-1 beta) and to culture medium from the human macrophage cell line THP-1 that had been exposed to amphotericin B. Endothelial cells were then incubated with exogenous-labeled arachidonic acid or were stimulated with histamine. Measurement of the resulting prostanoids indicated that amphotericin B and IL-1 beta acted synergistically to increase the ability of endothelial cells to synthesize prostanoids from endogenous and exogenous substrate and to increase expression of cyclooxygenase-2. This resulted in an increase of the ratio of untransformed prostaglandin (PG) H2 to PGI2 released by endothelial cells. Culture medium from amphotericin B-activated macrophages caused similar effects in endothelial cells. The synergistic effect with IL-1 beta was observed with free-form amphotericin B and, to a lesser extent, with lipid-complexed amphotericin B (Abelcet). Differences between Abelcet and the lipisome carrier (AmBisome) were not significantly different with respect to any of the parameters analyzed.
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