[Bezafibrate up-regulate endothelial nitric oxide gene expressions via peroxisome proliferator-activated receptors alpha-dependent and independent pathways in cultured Bovine endothelial cells].
OBJECTIVE
To investigate the effects and related mechanisms of bezafibrate, a ligand of peroxisome proliferator-activated receptors alpha (PPARalpha), on endothelial nitric oxide synthase (eNOS).


METHODS
Firstly, in cultured bovine aorta endothelial cells (BAEC), the effects of bezafibrate on eNOS mRNA and protein levels were investigated by Northern blot and Western blot. The half-life of eNOS mRNA and NO production determined from BAECs treated with bezafibrate were performed by real-time quantitative PCR and NO assay. Next, the effects of bezafibrate on signal pathways in BAEC, through inhibitors of PPARalpha, PI3 kinase and MAPK, were investigated by Western blot. Then luciferase reporter gene driven by human eNOS promoter were cloned and transfected endothelial cells to see the effects of bezafibrate on eNOS promoter-driven luciferase activity.


RESULTS
In cultured BAEC, bezafibrate significantly upregulated eNOS expressions at protein and mRNA levels in a concentration-dependent fashion (50 - 200 micromol/L) (P < 0.05), and increased nitric oxide (NO) production, respectively[control (14.97 +/- 1.29) micromol/L, different concentration groups (25.12 +/- 1.25) micromol/L, (30.12 +/- 1.85) micromol/L, (33.47 +/- 1.22) micromol/L], and enhanced phosphorylation of eNOS-ser-1179 site (P < 0.05), but not eNOS-thr-497 site. Through real-time quantitative PCR, bezafibrate prolonged eNOS mRNA half-life from 3.1 hour to 6.1 hour were demonstrated. Further studies showed that bezafibrate treatments significantly enhanced dose-dependently luciferase activity (relative luciferase activity in 100 micromol/L and 200 micromol/L groups 4429.43 +/- 391.41 and 6187.5 +/- 307.53), compared with untreated luciferase reporter gene group (3361.81 +/- 316.85) (P < 0.05 and P < 0.01, respectively), and induced MAPK phosphorylation expression (P < 0.05 and P < 0.01, respectively). Then these effects of bezafibrate upregulated eNOS expressions could be blocked by PPARalpha antagonist, MAPK and PI3K inhibitor while not affected by PKC and MEK inhibitor (P < 0.01).


CONCLUSIONS
Bezafibrate can upregulate eNOS expression, enhance eNOS-ser-1179 site phosphorylation, increase NO production and stabilize eNOS mRNA through PPARalpha dependent pathway and PPARalpha independent MAPK and PI3K pathways. This mechanism provides additional anti-atherosclerotic and anti-hypertension benefits of bezafibrate in addition of lipid-lowering effects.
Wang
Yan
Y
Cardiovascular Division, Internal Medicine Department, Institute of Hypertension, Tongji Hospital, Huazhong University of Science and Technology, Wuhan 430030, China.
Wang
Yan
Y
Wang
Dao-wen
DW
D000818:Animals; D001629:Bezafibrate / Q000494:pharmacology*; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D004730:Endothelium, Vascular; D005786:Gene Expression Regulation; D020935:MAP Kinase Signaling System; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D047493:PPAR alpha / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism
D047493:PPAR alpha; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D001629:Bezafibrate