Effects of different statins on endothelial nitric oxide synthase and AKT phosphorylation in endothelial cells☆

Abstract 

Background

In the present study, we examined effects of pitavastatin and cerivastatin on NO production and their mechanisms in EC.

Methods

HUVEC cells (1×10⁴ cells/well) were seeded into 96-well plates in 100 μl of culture medium for overnight, and then treated with various concentrations of pitavastatin or cerivastatin for 48 h. The cytotoxicity was evaluated using a WST-8 assay; The cells were cultured for 6 h in 200 μl of fresh medium containing increasing doses of pitavastatin or cerivastatin at 37 °C for 6 h, the NO production was detected by diaminofluoresceins (DAFs) assay; Simultaneously, The cells (1×10⁵ cells/well) were seeded into 96-well plates in medium for overnight, and then treated with reagents at 37 °C for 30 min, cGMP level was measured by enzyme-immunoassay. The cells were cultured in 2 ml of fresh medium containing increasing doses of pitavastatin or cerivastatin at 37 °C for 30 min, the phosphorylations of eNOS and Akt were detected by Western blotting.

Results

We found that pitavastatin not only induced NO production, but also increased cGMP level in HUVECs. Furthermore, EC were incubated with pitavastatin or cerivastatin for 30 min, Western blot analysis showed that pitavastatin (0.1 μM) significantly upregulated the phosphorylation of eNOS and Akt about 1.4-fold or 1.3-fold compared with control, however, cerivastatin (0.1 μM) did not have any effects on them.

Conclusion

Low dose of pitavastatin (0.1 μM) involves Akt pathway, activates eNOS activity, increases cGMP level and produces NO in EC, which is higher than that of cerivastatin.

Keywords: Nitric oxide synthase, Vascular endothelial cell, Akt, Statin