Effect of aspirin on high glucose-induced senescence of endothelial cells.
BACKGROUND
Endothelial cell senescence is accelerated under high glucose condition, which may contribute to the vascular complications in the diabetics. It has been proved that aspirin has multiple cytoprotective effects. This study aimed to investigate the effect of aspirin on high glucose-induced endothelial cell senescence and its possible mechanism.


METHODS
Human umbilical venous endothelial cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with different treatments including the normal glucose (5.5 mmol/L), high glucose (33 mmol/L) and aspirin (0.01 - 1.00 mmol/L) with high glucose. And 300 micromol/L L-NAME was added to the culture medium when needed. After 48 hours, SA-beta-gal staining was used to evaluate the senescence. Total nitric oxide (NO) production and NO synthase (NOS) activity were measured using Griess reaction and molecular probes of 3-amino-4-aminomethyl-2', 7'-difluorescein, diacetate. The level of intracellular reactive oxygen species was monitored by flow cytometry using 2', 7'-dichlorofluorescein diacetate. Endothelial NOS (eNOS), caveolin-1 protein expressions and caveolin-1/eNOS interaction were analyzed by immunoblotting and immunoprecipitation respectively. Asymmetric dimethylarginine (ADMA) concentration was determined by high-performance liquid chromatography.


RESULTS
Exposure to 33 mmol/L glucose for 48 hours significantly increased the number of SA-beta-gal positive cells. Co-incubation with aspirin markedly inhibited SA-beta-gal activity dose-dependently. Aspirin increased NOS activity with eNOS protein expression unchanged and increased NO levels and alleviated oxidative stress. Consistent with these findings, caveolin-1 expression, caveolin-1/eNOS interaction and ADMA accumulation were also decreased. All the inhibitory effects of aspirin on senescence were completely obliterated by L-NAME, the NOS inhibitor.


CONCLUSIONS
The anti-senescent effects of aspirin are fulfilled by increasing NO production via the up-regulation of NOS activity and preventing caveolin-1 expression, caveolin-1/eNOS interaction and ADMA accumulation.
Department of Endocrinology and Metabolism, First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, China.
Tong-Ning
TN
Yi
Hong-Yu
HY
Zhao
Jing-Sheng
JS
Zhang
Hai-Yan
HY
Shan
Xin
X
Meng
Jin
J
Zhang
D000873:Anthracenes / Q000378:metabolism; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D001241:Aspirin / Q000494:pharmacology*; D051242:Caveolin 1 / Q000378:metabolism; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects*; D002851:Chromatography, High Pressure Liquid; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D011407:Propane / Q000031:analogs & derivatives / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
D000873:Anthracenes; D000894:Anti-Inflammatory Agents, Non-Steroidal; D051242:Caveolin 1; D017382:Reactive Oxygen Species; C018524:N,N-dimethylarginine; D001120:Arginine; D052250:Nitric Oxide Synthase Type III; D005947:Glucose; D001241:Aspirin; D011407:Propane; D019331:NG-Nitroarginine Methyl Ester