Simvastatin combined with nifedipine enhances endothelial cell protection by inhibiting ROS generation and activating Akt phosphorylation.
OBJECTIVE
To investigate the protective effects of simvastatin (Sim) combined with nifedipine (Nif) on endothelial cells and elucidate the action mechanism.


METHODS
Human umbilical vein endothelial cells (HUVEC) were used. mRNA and protein levels were measured by using reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. Intracellular calcium and reactive oxygen species (ROS) were detected using confocal microscopy. The Griess assay was used to evaluate nitric oxide (NO) release.


RESULTS
Treatment of HUVEC with H(2)O(2) 100 micromol/L for 30 min inhibited the mRNA and protein expression of endothelial nitric oxide synthase (eNOS). With increased concentrations of Nif, eNOS mRNA and protein levels increased (P<0.05). Combined treatment with Sim 1.0 micromol/L and Nif 1.0 micromol/L significantly increased the mRNA and protein expression of eNOS and NO release compared with Sim or Nif alone (P<0.05). The combination significantly lowered the intracellular ROS level (P<0.05), which was correlated with the increase in eNOS and NO, but there was no visible change in intracellular calcium (P>0.05). Compared with individual drug treatment, Akt phosphorylation and the ratio of p-eNOS/eNOS were up-regulated in the combination group, and this effect was inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002.


CONCLUSIONS
The Sim-Nif combination effectively protects HUVEC against H(2)O(2) injury by inhibiting intracellular ROS generation, increasing the ratio of p-eNOS/eNOS and up-regulating Akt phosphorylation.
Chen
Xiao-niao
XN
Nankai University, Tianjin, China.
Xu
Jun
J
Feng
Zhe
Z
Fan
Ming
M
Han
Jing-yao
JY
Yang
Zhuo
Z
D015153:Blotting, Western; D002121:Calcium Channel Blockers / Q000494:pharmacology; D004347:Drug Interactions; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology*; D052250:Nitric Oxide Synthase Type III / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019821:Simvastatin / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects
D002121:Calcium Channel Blockers; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D019821:Simvastatin; D006861:Hydrogen Peroxide; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt; D009543:Nifedipine