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Abstract: Objective: Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, is a novel cardiovascular risk factor produced by endothelial cells. ADMA levels are mainly regulated by the activity of dimethylarginine dimethylaminohydrolases (DDAH). Endothelial release of ADMA is increased in the presence of oxidized LDL cholesterol (oxLDL), whereas estrogens stimulate NO production by endothelial cells by increasing both expression and activity of NO synthase and by reducing ADMA levels. Thus, the aim of the present study was to evaluate the estradiol effects on the DDAH/ADMA/NO pathway in cultured human umbilical vein endothelial cells (HUVEC) exposed to LDL. Methods: After 24 h of exposure to various treatments, culture medium was collected to measure NO production by using an amperometric sensor specific for NO, and to measure dimethylarginines by high-performance liquid chromatography (HPLC). DDAH-I and II mRNA expression and protein content were quantified by real-time PCR assay and immunoblotting, respectively. Results: Exposure of HUVEC to 100 μg/mL oxLDL, but not to 100 μg/mL of native LDL (nLDL), reduced DDAH-II expression at both the mRNA as well as the protein levels, which in turn increased ADMA levels and reduced NO production. Estradiol (1 nM) alone increased DDAH-II mRNA and protein expression, which reduced ADMA levels and increased NO production. In cells exposed to estradiol in combination with either nLDL or oxLDL, levels of DDAH-II, ADMA, and NO were the same as those for estradiol alone. Conclusion: Estradiol completely reverses the effects induced by oxLDL on the DDAH/ADMA/NO pathway. [Copyright &y& Elsevier]

Objective: To examine the effects of raloxifene on prostacyclin production by human umbilical vein endothelial cells (HUVEC) and to shed light on the molecular details of that action. Design: Cell culture for 4, 8, 16, 24, and 48 hours. Setting: University research laboratory. Patient(s): Source of HUVEC. Intervention(s): Measurement of prostacyclin production and of protein levels and mRNA expression of cyclooxygenase (COX)-1 and -2. Main Outcome Measure(s): Prostacyclin production was measured by enzyme immunoassay, the mRNA expression of COX-1 was measured by quantitative real time-polymerase chain reaction, and the protein levels of COX-1 and -2 were measured by immunoblotting. Result(s): Raloxifene significantly increased prostacyclin release in a time- and dose-dependent manner, being higher than control after 24 hours. Raloxifene, at 0.1–10 nM, increased the mRNA expression of COX-1 and the protein content of both COX-1 as well as COX-2. All of these effects were independent of the classical pathway for estrogen receptor (ER) activation because the treatment of cells with the ER antagonist ICI 182780 did not eliminate any of the effects. Although treatment with either the selective COX-1 inhibitor SC-560 or the selective COX-2 inhibitor NS-398 significantly diminished prostacyclin release (20% ± 5% and 24% ± 7%, respectively), co-treatment with raloxifene and either SC-560 or NS-398 was followed by a smaller increase than that achieved by raloxifene alone. The nonselective COX inhibitor indomethacin, however, reduced prostacyclin production to 37% ± 11% of control values. Conclusion(s): Raloxifene increased HUVEC prostacyclin release through a mechanism possibly distinct from the classical ER pathway and involving enhanced COX-1 and COX-2 expression and activity. [Copyright &y& Elsevier]