| IntroductionPeople with diabetes have a two-to four-fold increase in the risk of dying from the complications of cardiovascular disease.Therefore,exploring its mechanisms of development and therapeutic strategies has attracted increasing attention in recent years. The mechanism on the development of cardiovascular diseases mainly relies on atherosclerotic alternation,and endothelial cell senescence has a close relationship with atherosclerosis.Under normal conditions,endothelial cells rarely divide with a turnover rate of approximately once every 3 years,although replication can be initiated through various processes.The ability of endothelial cells to proliferate is limited,and the cells eventually enter a state of irreversible growth arrest known as senescence.Studies have shown that telomere length is shorter in cells isolated from atherosclerotic lesions than from cells in uninvolved areas,and the rate of telomere shortening appears to be higher in vessels with significant hemodynamic stress.Previous studies have shown that endothelial cells in atherosclerotic lesions exhibit the character of cellular senescence including senescence-associatedβ-galactosidase(SA-β-gal) staining and telomere shortening.Importantly,senescent vascular cell behaviors are consistent with the changes observed in atherosclerosis.For example,expressions of inflammatory cytokines and adhesion molecules are up-regulated,and nitric oxide(NO)production is dramatically reduced in senescent endothelial cells.Recent researches show that high glucose may induce senescence of multiple cells including endothelial cells.It seems that high glucose triggers the onset of vascular complications of diabetes by accelerating vascular endothelial cells senescence.The telomere hypothesis is a widely accepted explanation of the occurrence of senescence.Telomeres are repetitive DNA sequences at the ends of eukaryotic chromosomes and are shortened as a linear function of increasing cellular division. When the telomere length is shortened to some extent,the onset of senescence is triggered.Additionally,telomerase is a ribonucleoprotein which can synthesize new telomeric repeats and restore telomere length.In the absence of telomerase or when this enzyme is expressed at very low levels,DNA synthesis through the duration of cell division results in the progressive shortening of telomeric DNA.Several different factors can regulate the length of telomeres and the activity of telomerase during cell division.Asymmetric dimethylarginine(ADMA),an endogenous inhibitor of NO synthase,is one of these factors.ADMA is not only an independent cardiovascular risk factor,but also is able to increase the rate of telomere shortening and accelerate endothelial senescence.These effects may be associated with an increase in reactive oxygen system(ROS) formation and a decrease in NO formation.Furthermore,the clearance of ADMA is mostly performed by dimethylarginine dimethylaminohydrolase (DDAH),an oxidant-sensitive enzyme.Study from Lin et al.has demonstrated ADMA that accumulation occurred in diabetes mellitus and accompanied with the increasing of ROS.In agreement with this,suppression of oxidative stress or application of NO-donors may protect cells from senescence and atherosclerosis.The free-radical theory of aging proposes that degenerative senescence is largely the result of the accumulative effect of ROS,which may promote senescence by shortening telomere through inactivation of the Src kinase family.A number of in vitro and vivo studies have shown that oxidative stress is increased in diabetic patients.Thus premature senescence might be accelerated in diabetic patients.In endothelial cell, excessive ROS can be eliminated by NO.As mentioned earlier,NO production is severely limited in senescent endothelial cells.As a widespread signaling molecule,NO has multiple functions on regulating vascular tone and inhibiting platelet aggregation, apoptosis,smooth muscle cell proliferation and leukocyte adhesion against the initiation and progression of atherosclerosis.NO is generated from L-arginine by endothelial NOS(eNOS).Studies have shown that senescent endothelial cells have significantly lower level of eNOS.Although the underlying mechanism remains unclear,the reduction in NO production appears to be directly related to decreased eNOS expression and NOS activity.Aspirin is one kind of nonsteroidal anti-inflammatory drugs(NSAIDs) popularly for prevention and treatment of cardiovascular diseases,especially for the prevention of heart ischemia.Moreover,recent studies demonstrate that aspirin can also protect endothelial cells from oxidative stress and increase NO production besides its anti-thrombus and anti-inflammation effects.Therefore,aspirin exhibits a potential effect on delaying cellular senescence.In the present study,we investigated(1) whether aspirin delays human endothelial cells senescence exposed to high glucose condition;(2) whether the effect of aspirin on anti-senescence is associated with the increasing of NO and decreasing of ROS.Materials and methods1.Morphology inspection,β-gal stain,human telomerase activity by PCR-ELISA method and cell cycle analysis by flow cytometry were used to evaluate cell senescence.2.Griess reaction was used to assay total NO of endothelial cells by evaluating NO2-/NO3- level.3.To determine NOS activity,3-amino-4-aminomethyl-2',7'- difluorescein, diacetate(DAF-FM DA,Molecular Probes)was used.4.Total eNOS protein and eNOS(Ser-1177) protein expression of endothelial cell were analyzed by Western blotting.5.eNOS mRNA expression of endothelial cell was analyzed by RT-PCR.6.Intracellular ROS levels were determined using cell permeable 2',7'-dichlorofluorescein diacetate(DCFH-DA)by flow cytometry.7.Time of flight mass spectrometer was used to measure ADMA concentrations and DDAH activity of endothelial cells.Results1.Endothelial cells exhibited the characteristic of senescence after exposure to high glucose(11-33 mM),e.g.,they increased in size,displayed a more flat morphology and increased the number ofβ-gal positive cells.All these phenomena were reversed by low dose aspirin(0.01-1 mM) in a dose-dependent manner and reached the peak at 48 h(P＜0.05).Furthermore,low dose aspirin increased telomerase activity dose-dependently(P＜0.05) and promote cell proliferation(P＜0.05).However, there was no significant difference between 3 mM aspirin group and high glucose group(33 mM) as morphology inspection,β-gal stain and telomerase activity show (P＞0.05).The effect of aspirin on anti-senescence was completely abolished by the NOS inhibitor,L-NAME.2.The level of NO in endothelial cell was significantly decreased under high glucose(33 mM)condition in a time-dependent manner(P＜0.05) and it was reversed by 1 mM aspirin in the same manner(P＜0.05).Furthermore,0.01-1 mM aspirin increased the NO level in a dose-dependent manner after 48 h co-incubation(P＜0.05). However,3 mM aspirin had no significant effect on increasing NO level compared with high glucose(P＞0.05).L-NAME,completely abolished the effect of aspirin on increasing NO level(P＜0.05).3.The expressions of eNOS mRNA and eNOS total protein of endothelial cells had no significant changes among normal group,high glucose group and 1 mM aspirin group after 48 h exposure(P＞0.05).4.The NOS activity of endothelial cell was significantly decreased in high glucose group in a time-dependent manner(P＜0.05) and it was reversed by 1 mM aspirin in the same manner(P＜0.05).Furthermore,0.01-1 mM aspirin increased the NOS activity in a dose-denpendent manner after 48 h co-incubation(P＜0.05).However,3 mM aspirin had no significant effect on increasing NOS activity compared with high glucose (P＞0.05).L-NAME,completely abolished the effect of aspirin on increasing NOS activity(P＜0.05).5.After exposure to normal glucose,high glucose and high glucose containing 0.01-3 mM aspirin for 48 h,the eNOS phosphorylation at Ser-1177 of endothelial cells was analyzed by western blotting.High glucose significantly downregulated the phosphorylation of eNOS,which was prevented by 0.01-1 mM aspirin in a dose-dependent manner(P＜0.05).However,3 mM aspirin had no effect on enhancing the phosphorylation of eNOS.6.The level of ROS was significantly increased under high glucose condition in a time-dependent manner(P＜0.05) and it was reversed by 1 mM aspirin in the same manner(P＜0.05).Furthermore,0.01-1 mM aspirin decreased the ROS in a dose-dependent manner after 48 h co-incubation(P＜0.05).However,3 mM aspirin had no significant effect on decreasing ROS level compared with high glucose(P＞0.05). L-NAME,completely abolished the effect of aspirin on decreasing ROS(P＜0.05).7.After exposure to normal glucose,high glucose and high glucose containing 0.01-3 mM aspirin for 48 h,DDAH activity and ADMA formation were detected.High glucose significantly decreased DDAH activity and increased ADMA accumulation, which was prevented by 0.01-1 mM aspirin in a dose-dependent manner.However,3 mM aspirin had no effect on regulating DDAH-ADMA system under high glucose condition.Conclusion1.The senescence of endothelial cells are accelerated by high glucose in both dose-dependent and time-dependent manners.2.Low dose aspirin(0.01-1 mM) delay endothelial cell senescence exposed to high glucose in a dose-dependent manner and the effect reaches the peak at 48 h.3.Low dose aspirin delay endothelial cell senescence exposed to high glucose by increasing NO and decreasing ROS.4.The effect of low-dose aspirin on increasing NO level of endothelial cells under glucose condition is associated with the enhancing of NOS activity,but without changing the expressions of eNOS mRNA and eNOS total protein.5.The effect of low dose aspirin on up-regulating eNOS phosphorylation at Ser-1177 may account for the enhancement of NOS activity.6.Low dose aspirin can increase DDAH activity and decrease ADMA accumulation,which may further strengthen the effect of aspirin on anti-senescence and anti-atherosclerosis.7.3 mM aspirin has no effect on delaying endothelial cell senescence exposed to high glucose.
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