Role Of Caveolin-1 In High-glucose-induced Apoptosis Of Lens Epithelial Cells

Purpose:The involvement of caveolin-1 in the regulation of apoptosis has been previously demonstrated in epithelial cells.Lens epithelial cells(LECs) apoptosis is an important factor in determining the early cataract development among the diabetic population.In the first part of current study,LECs were treated with high concentration of glucose,and the evaluation of the cellular apoptosis rate,caveolin-1 mRNA expression,caveolin-1 protein levels,distribution of caveolae,and Immunostaining of caveolin-1 and externalized phosphatidylserine(PS) was performed.The relationship between caveolin-1 and apoptosis of LECs under high glucose(HG) was studied.In the second part of present study,after treated with high glucose,EGF,and simvastatin,an HMG-CoA reductase inhibitor,in Dulbecco's Modified Eagle's Medium(DMEM), LECs were also examined the apoptosis rate,caveolin-1 mRNA expression,caveolin-1 protein levels to further explore the association between caveolin-1 and apoptosis of LECs under high glucose.Furthermore,the possible relationship between caveolin-1 and diabetic cataract are also assessed.Methods:Human lens epithelial cells(HLECs)-B3 were treated with glucose solutions (5-25 mM),mannitol(20 mM) and glucose(5 mM)(as a control to exclude the effect of osmotic pressure).After being grown for 1-48h,induction of apoptosis was measured by flow cytometry and the expression of caveolin-1 was examined by immunofluorescence microscopy,quantitative real-time RT-PCR analysis,and immunoblotting in HLECs treated with glucose solutions and mannitol.Observation of PS externalization was also performed by immunofluorescence microscopy. Transmission electron microscopy was used to examine the distribution of caveolae.In addition,HLECs in different concentrations of glucose(5 and 25 mM) were incubated with simvastatin(10 nM) or EGF(0.1 ng/ml) for 48 h.Flow cytometry,quantitative real-time RT-PCR analysis,Western blot analysis was also performed to measure HLECs apoptosis and the expression of caveolin-1.Results:After 48 h incubation,the increased concentration of glucose(5,10,15,20,25 mM) caused significant enhancement of cell apoptosis(p＜0.05).A significant decrease in caveolin-1 mRNA level was observed in cells in which the concentration of glucose increased from 5 to 25 mM(p＜0.01).The minimal caveolin-1 mRNA level and maximum apoptosis rate appeared in 25 mM glucose-treated cells.Furthermore,the addition ofmannitol as an osmotic control did not influence the percentage of apoptosis or the expression of caveolin-1(p＞0.05).After being incubated in 25 mM glucose from 1 to 48 h,an increase in apoptosis was observed(p＜0.05).However,it was found that longer treatment time resulted in a lower level of caveolin-1 expression,while osmotic control showed no such effects(p＞0.05).In agreement with the RT-PCR data,decreased levels of caveolin-1 protein were detected in cells treated with 10-25 mM glucose for 48 h compared with control.The protein levels of caveolin-1 also decreased in cells treated with 25 mM glucose for 12-48 h.We immunolabeled control and HG-treated cells for caveolin-1 and PS.A significant reduction in caveolin-1 fluorescence intensity was observed in 25 mM glucose-treated cells.The results indicate a decrease in caveolin-1 level in HG-induced HLECs.PS was shown to be translocated from the inner plasma membrane to the exoplasmic membrane region.A significant increase in the rate of PS-stained cells was observed in HG-treated HLECs.Analyzed by transmission electron microscopy,caveolae were downregulated in 25 mM glucose-treated cells.There was no significant change in the caveolin-1 mRNA expression and the percentage of apoptotic cells in 5 mM glucose-treated HLECs between with or without simvastatin and EGF stress(p＞0.05).However,the caveolin-1 mRNA expression in HG-treated HLECs with simvastatin and EGF stimuli increased(p＜0.01),whereas the percentage of apoptotic cells significantly decreased(p＜0.05).Similarly,Western blot analysis also showed that the addition of 0.1 ng/ml EGF or 10 nM simvastatin in 25 mM glucose notably increased caveolin-1 protein expression compared with HG-treated control.Conclusions:1) The expression of cavelolin-1 and caveolus distribution in HG-treated HLECs was examined by immunofluorescence microscopy,quantitative real-time RT-PCR analysis,immunoblotting,and transmission electron microscopy,respectively. When glucose concentration and treatment duration increased,we found that caveolin-1 decreased in HG-treated HLECs,and caveolae were also down-regulated in HG-treated cells.It indicated that the expression of cavelolin-1 and caveolus distribution in HLECs might be effected by different condition of extracellular glucose stimuli.2) We studied the relationship between cavelolin-1 and apoptosis of HLECs under high glucose conditions and found that caveolin-1 expression decreased and apoptosis increased in HLECs when glucose concentration and treatment duration increased.We proposed that high glucose induced apoptosis of lens epithelial cells is associated with decreased caveolin-1 and caveolin-1 presented in lens epithelial cells plays a pivotal role in processes of high glucose induced apoptosis.It will be valuable to the further studies about the relationship of cataract caused by high glucose induced the apoptosis of LECs with the expression of caveolin-1.3) In addition,we suggested the treatment of simvastatin,or EGF increases caveolin-1 in HG-treated HLECs and thus prevents apoptosis.It is meaningful for the further studies about the apoptosis signal regulation in HLECs.