Blockade of geranylgeranylation by rosuvastatin upregulates eNOS expression in human venous endothelial cells

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Abstract

Endothelial dysfunction is associated with a reduction in nitric oxide (NO) bioavailability. Positive effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) on the improvement of endothelial dysfunction have been shown. We investigated the effects of rosuvastatin and isoprenoid metabolites on endothelial NO synthase (eNOS) mRNA and protein expression in human umbilical venous endothelial cells after exposure to 10−8–10−5 mol/l rosuvastatin for 8 and 12 h. Cell viability was not significantly altered after exposure to the statin for 12 h. In a concentration-dependent manner, rosuvastatin upregulated eNOS mRNA and protein expression. The effects on eNOS expression mediated through rosuvastatin could be reversed by treatment with mevalonate indicating inhibition of HMG-CoA reductase as the underlying mechanism. Treatment with geranylgeranylpyrophosphate, but not farnesylpyrophosphate, reversed the increase of eNOS expression induced by rosuvastatin. Rosuvastatin may have beneficial effects on endothelial dysfunction associated with cardiovascular diseases beyond its effects on lowering cholesterol.

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Materials and methods

Cell culture. Human venous endothelial cells (HUVEC) were isolated from umbilical cords and cultured according to standard procedures. The endothelial phenotype was confirmed using phase-contrast microscopy and staining for the endothelial-specific von Willebrand factor. Confluent cell layers were exposed to rosuvastatin concentrations ranging from 10−8 to 10−5 mol/l for 8 or 12 h. In some experiments, the media were supplemented with mevalonic acid (MEV, 10−4 mol/l), geranylgeranylpyrophosphate

Effects of rosuvastatin on cell viability of HUVEC

As presented in Fig. 1, after 12 h exposure to rosuvastatin (10−8–10−5 mol/l) viability of human endothelial cells was only marginally affected. As this was not concentration-related it may reflect variability in the assay. Addition of the isoprenoid intermediates mevalonic acid, GGPP or FPP in the presence of the statin did not change cell viability.

Rosuvastatin increases eNOS mRNA and protein expression in a concentration-dependent manner

Treatment of endothelial cells with rosuvastatin for 8 h increased eNOS mRNA and protein expression in a concentration-dependent manner. As shown in

Discussion

The presented data show that rosuvastatin, which is a hydrophilic statin, has no relevant effect on the viability of human endothelial cells. This is in accordance with a previously published study by Kaneta et al. [19]. The authors were able to show that the hydrophilic pravastatin had no effect on cell viability after a 24-h incubation period in murine endothelial cells. As hydrophobic statins can enter endothelial cells by penetrating the lipid bilayers of the cell membranes, they are able

Acknowledgments

The authors thank the Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe, Ernst-Moritz-Arndt-Universität, Greifswald, Germany, for the generous supply of human umbilical cords. The excellent technical assistance of R. Dressler and M. Wendt is gratefully acknowledged. We are grateful to Dr. J. Sonnemann, Peter Holtz Research Centre of Pharmacology and Experimental Therapeutics, for his initial help with the Alamar Blue assay. This research was supported by a grant from AstraZeneca to

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