Simvastatin Increase The Activity Of ENOS Via Phosphorylation

Background:The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or Statins, were developed as lipid-lowering drugs, and they have been shown in numerous clinical trials to promote the primary and secondary prevention of cardiovascular morbidity and mortality in patients with hypercholesterolemia. The clinical benefit was initially believed to be solely attributable to its low-density lipoprotein (LDL)-lowering effect. There is increasing evidence, however, that Statins yield profound benefits beyond their lipid-lowering action. A number of large clinical trials have demonstrated their clinical usefulness for preventing cardiovascular events, such as myocardial infarction, stroke, and sudden cardiac death. Such pleiotropic effects on the vessel wall have been suggested to contribute to antioxidative, antiinflammatory, and improved endothelial functions through enhanced eNOS expression in endothelial cells. It is well recognized that statins protect the vascular endothelium by stabilizing eNOS mRNA and causing an increase in circulating endothelial progenitor cells. Laufs and colleagues showed that Simvastatin and Lovastatin improved the stability of the messenger RNA for eNOS and increased the half-life of the messenger RNA for eNOS from 13 to 38 hrs, with a consequent enhanced generation of NO from the endothelium. The endothelium synthesizes a variety of autocrine and paracrine factors, including nitric oxide (NO), which is synthesized by endothelial nitric oxide synthase (eNOS). NO regulate vascular tone, maintain blood cell/vessel wall interaction, prevent thrombosis, and limit smooth muscle cell proliferation. Acting as a messenger molecule, NO mediates the majority of endothelium-dependent responses in the brain. There is increasing evidence that Statins are known to enhance vascular expression of endothelial (eNOS). However, a trifle of study has ever addressed the regulation on the activity of eNOS. Thus, the present study was designed to establishe G418-resistant human fetal kidney cell line and to investigate whether Statins could regulate the activity of eNOS, moreover elucidate the potential mechanisms.Methods: The plasmid pcDNA3.l- eNOS /neo containing neo gene was purified and transfected into human fetal kidney cell with Lipofectin. The transfected cells would be survived in culture medium containing G418 antibiotic because the neo gene could express a G418 resistant product. The identification of the G418-resistant human fetal kidneycell line was conducted by using Western blot. G418-resistant human fetal kidney cells were coincubated with Simvastatin for 2 hours. The activity of eNOS is determined by the convertion of radiolabelled 3H-L-arginine to 3H-L-citrulline. The levels of phospho-eNOS, phospho-Akt and phospho-Ampk were measured by Western blot.Results: The transfected cells could express fluorescin after 48h. The concentration gradient of G418 was set from 100μg/ml to 1000μg/ml. The minimum lethal dose concentration of G418 was 800μg/ml. Under the condition of G418 (800μg/ml), G418-resistant human fetal kidney cell clones appeard after 12 to18 days but the negative control all died after 14 days. The positive clones wered cultivate and then they were detected the expression of eNOS protein by Western blot. The results of Western blot showed the transformants of 20% could express eNOS protein. The activity of eNOS is determined by the convertion of radiolabelled 3H-L-arginine to 3H-L-citrulline and the eNOS expression levels were measured by Western blotting method. We found the activity of eNOS had a significant increase when G418-resistant human fetal kidney 293 cell were incubated with Simvastatin (10μmol/L) for 2 hours. Besides, the levels of phospho-eNOS, phospho-Akt and phospho-Ampk had a significant increase, but the total expression of eNOS had no marked change.Conclusion: The eNOS stably transfected human embryonic kidney 293 cell line was established by Lipofectin, which may be useful to investigate eNOS. Besides, it's investigated that Simvastatin could stimulate the activity of eNOS via phosphorylation, which provides new explanation for statin application to cardiovascular diseases.