自噬在高糖条件下视网膜色素上皮细胞表达血管内皮生长因子促进RF/6A细胞血管生成中的作用
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摘要
目的研究高糖条件下诱导的人视网膜色素上皮细胞株(ARPE-19)自噬对其表达血管内皮生长因子(vascular endothelial growth factor,VEGF),及恒河猴脉络膜视网膜血管内皮细胞(RF/6A)迁移和管腔形成的影响。方法根据不同干预将ARPE-19细胞随机分为对照组、高糖组及3-甲基腺嘌呤(3-MA)+高糖组。对照组细胞在无糖DMEM培养基中常规培养24 h,高糖组细胞在DMEM培养基中加入30 mmol·L~(-1)葡萄糖溶液处理24 h,3-MA+高糖组细胞先用10 mmol·L~(-1) 3-MA处理24 h,然后更换培养基再加入30 mmol·L~(-1)葡萄糖溶液处理24 h。Western blot法检测细胞自噬标志性蛋白微管相关蛋白1轻链3(microtubule-related protein 1 light chain 3,LC3)II型和I型的比值(LC3-II/LC3-I)、Beclin-1及细胞中自噬相关基因3(autophagy-related gene 3,Atg3)的蛋白表达。ELISA法检测ARPE-19细胞上清液中VEGF的蛋白表达。比较三组ARPE-19细胞LC3-II/LC3-I、Beclin-1、Atg3及VEGF的蛋白表达量。然后将以上三组ARPE-19细胞上清液分别加入RF/6A细胞培养基中,将RF/6A细胞也按以上方法分组,分别采用Transwell法及Matrigel胶法检测并比较三组RF/6A细胞迁移数及细胞管腔形成数。结果对照组、高糖组和3-MA+高糖组ARPE-19细胞中LC3-II/LC3-I比值分别为0.405±0.095、0.932±0.024和0.635±0.048;Beclin-1蛋白相对表达量分别为0.205±0.035、0.590±0.120和0.425±0.082;Atg3蛋白相对表达量分别为0.277±0.035、0.539±0.071和0.389±0.019。ARPE-19细胞上清液中VEGF的蛋白表达量三组分别为(44.03±9.08)ng·L~(-1)、(205.70±17.90)ng·L~(-1)和(112.52±21.06)ng·L~(-1);RF/6A细胞迁移数三组分别为(125.60±6.35)个、(153.60±19.20)个和(67.40±7.95)个;细胞管腔形成数三组分别为(12.22±0.84)个、(18.44±1.68)个和(5.44±0.51)个;整体比较差异均有统计学意义(均为P<0.01)。与对照组相比,高糖组ARPE-19细胞的LC3-II/LC3-I比值、Beclin-1及Atg3蛋白相对表达量均明显增加(均为P<0.01),VEGF表达水平均明显升高(P<0.01),RF/6A细胞迁移数和管腔形成数均明显增加(均为P<0.01),3-MA+高糖组ARPE-19细胞中LC3-II/I比值、Beclin-1和Atg3蛋白相对表达量、VEGF表达量、RF/6A细胞迁移数和细胞管腔形成数均较高糖组明显降低(均为P<0.01)。结论高糖激活ARPE-19细胞自噬,进而上调VEGF的表达并促进RF/6A细胞迁移和管腔形成,自噬抑制剂3-MA可抑制ARPE-19细胞自噬,并下调VEGF的表达从而抑制RF/6A细胞的血管形成能力。
Objective To study the effects of autophagy of human retinal pigment epithelial cell line(ARPE-19) induced by high glucose conditions on the expression of vascular endothelial growth factor(VEGF) as well as on the migration and tube formation of rhesus choroid-retinal endothelial cells(RF/6 A).Methods ARPE-19 cells were divided into the control group,high-glucose group and 3-methyladenine(3-MA)+high-glucose group.The cells in the control group were cultured in DMEM medium without glucose for 24 hours.The cells in the high-glucose group were cultured in DMEM medium adding 30 mmol·L~(-1) glucose for 24 hours.The cells in the 3-MA+high-glucose group were pretreated with 10 mmol·L~(-1) 3-MA for 24 hours and then exposed to replaced medium adding 30 mmol·L~(-1) glucose for 24 hours.The ratio of II and I form of microtubule-related protein 1 light chain 3(LC3)(LC3-II/LC3-I),Beclin-1 and autophagy-related gene 3(Atg3) protein expressions in the cells were detected by Western blot,and the expression of VEGF in the cell supernatants was examined by ELISA assay.The expressions of LC3-II/LC3-I,Beclin-1,Atg3 and VEGF among the three groups were compared.Then,the supernatant of ARPE-19 cells in the above three groups was respectively added into RF/6 A cell culture medium and RF/6 A cells were also grouped by the above method.The migration and tube formation of RF/6 A cells were detected by transwell and matrigel assay,respectively.The number of migrated cells and tube formation of the RF/6 A cells were compared.Results In the control group,high-glucose group and 3-MA+high-glucose group,the LC3-II/LC3-I ratio in ARPE-19 cells was 0.405±0.095,0.932±0.024 and 0.635±0.048,respectively;the relative expression of Beclin-1 protein in the cells was 0.205±0.035,0.590±0.120 and 0.425±0.082,respectively;and the relative expression of Atg3 protein in the cells was 0.277±0.035,0.539±0.071 and 0.389±0.019,respectively.The protein expression of VEGF in the supernatant of ARPE-19 cells was(44.03±9.08)ng·L~(-1),(205.70±17.90)ng·L~(-1) and(112.52±21.06)ng·L~(-1) for the three groups,respectively.The number of migrated RF/6 A cells was 125.60±6.35,153.60±19.20 and 67.40±7.95;the number of tube formation was 12.22±0.84,18.44±1.68 and 5.44±0.51 for the three groups,respectively.There were significant differences among the groups(all P<0.01).Compared with the control group,the ratio of LC3-II/LC3-I and the expression levels of Beclin-1,Atg3 in ARPE-19 cells were significantly increased(all P<0.01) and the expression level of VEGF increased(P<0.01) in the high-glucose group.The number of migrated cells and tube formation of RF/6 A cells were significantly increased in the high-glucose group(all P<0.01) compared with the control group.Compared with the high-glucose group,the ratio of LC3-II/LC3-I and the expression of Beclin-1,Atg3 and VEGF protein in ARPE-19 cells,the number of migrated cells and tube formation of RF/6 A cells were significantly decreased in the 3-MA+high-glucose group(all P<0.01).Conclusion High glucose conditions activate autophagy of ARPE-19 cells,which enhances the expression of VEGF and the migration and tube formation of RF/6 A cells.Autophagy inhibitor 3-MA inhibits the angiogenesis abilities of RF/6 A cells through inhibiting autophagy of ARPE-19 cells and the expression of VEGF.
Objective To study the effects of autophagy of human retinal pigment epithelial cell line(ARPE-19) induced by high glucose conditions on the expression of vascular endothelial growth factor(VEGF) as well as on the migration and tube formation of rhesus choroid-retinal endothelial cells(RF/6 A).Methods ARPE-19 cells were divided into the control group,high-glucose group and 3-methyladenine(3-MA)+high-glucose group.The cells in the control group were cultured in DMEM medium without glucose for 24 hours.The cells in the high-glucose group were cultured in DMEM medium adding 30 mmol·L~(-1) glucose for 24 hours.The cells in the 3-MA+high-glucose group were pretreated with 10 mmol·L~(-1) 3-MA for 24 hours and then exposed to replaced medium adding 30 mmol·L~(-1) glucose for 24 hours.The ratio of II and I form of microtubule-related protein 1 light chain 3(LC3)(LC3-II/LC3-I),Beclin-1 and autophagy-related gene 3(Atg3) protein expressions in the cells were detected by Western blot,and the expression of VEGF in the cell supernatants was examined by ELISA assay.The expressions of LC3-II/LC3-I,Beclin-1,Atg3 and VEGF among the three groups were compared.Then,the supernatant of ARPE-19 cells in the above three groups was respectively added into RF/6 A cell culture medium and RF/6 A cells were also grouped by the above method.The migration and tube formation of RF/6 A cells were detected by transwell and matrigel assay,respectively.The number of migrated cells and tube formation of the RF/6 A cells were compared.Results In the control group,high-glucose group and 3-MA+high-glucose group,the LC3-II/LC3-I ratio in ARPE-19 cells was 0.405±0.095,0.932±0.024 and 0.635±0.048,respectively;the relative expression of Beclin-1 protein in the cells was 0.205±0.035,0.590±0.120 and 0.425±0.082,respectively;and the relative expression of Atg3 protein in the cells was 0.277±0.035,0.539±0.071 and 0.389±0.019,respectively.The protein expression of VEGF in the supernatant of ARPE-19 cells was(44.03±9.08)ng·L~(-1),(205.70±17.90)ng·L~(-1) and(112.52±21.06)ng·L~(-1) for the three groups,respectively.The number of migrated RF/6 A cells was 125.60±6.35,153.60±19.20 and 67.40±7.95;the number of tube formation was 12.22±0.84,18.44±1.68 and 5.44±0.51 for the three groups,respectively.There were significant differences among the groups(all P<0.01).Compared with the control group,the ratio of LC3-II/LC3-I and the expression levels of Beclin-1,Atg3 in ARPE-19 cells were significantly increased(all P<0.01) and the expression level of VEGF increased(P<0.01) in the high-glucose group.The number of migrated cells and tube formation of RF/6 A cells were significantly increased in the high-glucose group(all P<0.01) compared with the control group.Compared with the high-glucose group,the ratio of LC3-II/LC3-I and the expression of Beclin-1,Atg3 and VEGF protein in ARPE-19 cells,the number of migrated cells and tube formation of RF/6 A cells were significantly decreased in the 3-MA+high-glucose group(all P<0.01).Conclusion High glucose conditions activate autophagy of ARPE-19 cells,which enhances the expression of VEGF and the migration and tube formation of RF/6 A cells.Autophagy inhibitor 3-MA inhibits the angiogenesis abilities of RF/6 A cells through inhibiting autophagy of ARPE-19 cells and the expression of VEGF.
引文
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[15] MATSUNAGA N,CHIKARAISHI Y,IZUTA H,OGATA N,SHIMAZAWA M,Matsumura M,et al.Role of soluble vascular endothelial growth factor receptor-1 in the vitreous in proliferative diabetic retinopathy[J].Ophthalmology,2008,115(11):1916-1922.
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[17] MURROW L,DEBNATH J.ATG12-ATG3 connects basal autophagy and late endosome function[J].Autophagy,2015,11(6):961-962.
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[19] MAIURI M C,LE TOUMELIN G,CRIOLLO A,RAIN J C,GAUTIER F,JUIN P,et al.Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1[J].EMBO J,2007,26(10):2527-2539.
[20] LI R,TIAN J,DU J,ZHAO L,YAO Y,YU Z,et al.Manipulation of autophagy:a novelly potential therapeutic strategy for retinal neovascularization[J].BMC Ophthalmol,2018,18(1):110.
[21] SZATMáRI-TóTH M,KRISTóF E,VERéB Z,AKHTAR S,FACSKó A,FéSüS L,et al.Clearance of autophagy-associated dying retinal pigment epithelial cells-a possible source for inflammation in age-related macular degeneration[J].Cell Death Dis,2016,7(9):e2367.
[2] LI Y Y,YANG X F,GU H,SNELLINGEN T,LIU X P,LIU N P.The relationship between insulin resistance/β-cell dysfunction and diabetic retinopathy in Chinese patients with type 2 diabetes mellitus:the Desheng Diabetic Eye Study[J].Int J Ophthalmol,2018,11(3):493-500.
[3] CHEUNG N,MITCHELL P,WONG T Y.Diabetic retinopathy[J].Lancet,2010,376 (9735):124-136.
[4] CARMELIET P,COLLEN D.Molecular analysis of blood vessel formation and disease[J].Am J Physiol,1997,273(5 Pt 2):H2091-2104.
[5] WU Y,ZHANG Q,ANN D K,AKHONDZADEH A,DUONG H S,MESSADI D V,et al.Increased vascular endothelial growth factor may account for elevated level of plasminogen activator inhibitor-1 via activating ERK1/2 in keloid fibroblasts[J].Am J Physiol Cell Physiol,2004,286(4):C905-912.
[6] BAHRAMI B,HONG T,GILLES M C,CHANG A.Anti-VEGF therapy for diabetic eye diseases[J].Asia Pac J Ophthalmol (Phila),2017,6(6):535-545.
[7] THOMAS A A,FENG B,CHAKRABARTI S.ANRIL:A regulator of VEGF in diabetic retinopathy[J].Invest Ophthalmol Vis Sci,2017,58(1):470-480.
[8] MEHRPOUR M,ESCLATINE A,BEAU I,CODOGNO P.Overview of macroautophagy regulation in mammalian cells[J].Cell Res,2010,20(7):748-762.
[9] LI R,DU J,CHANG Y.Role of autophagy in hypoxia-induced angiogenesis of RF/6A cells in vitro[J].Curr Eye Res,2016,41(12):1566-1570.
[10] DU J H,LI X,LI R,CHENG B X,KUERBANJIANG M,MA L.Role of autophagy in angiogenesis induced by high glucose condition in RF/6A cells[J].Ophthalmologica,2017,237(2):85-95.
[11] GOLESTANEH N,CHU Y,XIAO Y Y,STOLERU G L,THEOS A C.Dysfunctional autophagy in RPE,a contributing factor in age-related macular degeneration[J].Cell Death Dis,2017,8(1):e2537.
[12] SIMóR I,VILLARROEL M,CORRALIZA L,HERNáNDEZ C,GARCIA-RAMíREZ M.The retinal pigment epithelium:something more than a constituent of the blood-retinal barrier--implications for the pathogenesis of diabetic retinopathy[J].J Biomed Biotechnol,2010,2010:190724.
[13] SHIMADA K,BATA T,NEUGEBAUER S,ONOZAKI A,YAMADA D,MIDORIKAWA S,et al.Plasma vascular endothelial growth factor in Japanese type 2 diabetic patients with and without nephropathy[J].J Diabetes Complications,2002,16(6):386-390.
[14] KOCH S,TUGUES S,LI X,GUALANDI L,CLAESSON-WELSH L.Signal transduction by vascular endothelial growth factor receptors[J].Biochem J,2011,437(2):169-183.
[15] MATSUNAGA N,CHIKARAISHI Y,IZUTA H,OGATA N,SHIMAZAWA M,Matsumura M,et al.Role of soluble vascular endothelial growth factor receptor-1 in the vitreous in proliferative diabetic retinopathy[J].Ophthalmology,2008,115(11):1916-1922.
[16] ABOUNIT K,SCARABELLI T M,MCCAULEY R B.Autophagy in mammalian cells[J].World J Biol Chem,2012,3(1):1-6.
[17] MURROW L,DEBNATH J.ATG12-ATG3 connects basal autophagy and late endosome function[J].Autophagy,2015,11(6):961-962.
[18] MIZUSHIMA N,YOSHIMORI T,LEVINE B.Methods in mammalian autophagy research[J].Cell,2010,140(3):313-326.
[19] MAIURI M C,LE TOUMELIN G,CRIOLLO A,RAIN J C,GAUTIER F,JUIN P,et al.Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1[J].EMBO J,2007,26(10):2527-2539.
[20] LI R,TIAN J,DU J,ZHAO L,YAO Y,YU Z,et al.Manipulation of autophagy:a novelly potential therapeutic strategy for retinal neovascularization[J].BMC Ophthalmol,2018,18(1):110.
[21] SZATMáRI-TóTH M,KRISTóF E,VERéB Z,AKHTAR S,FACSKó A,FéSüS L,et al.Clearance of autophagy-associated dying retinal pigment epithelial cells-a possible source for inflammation in age-related macular degeneration[J].Cell Death Dis,2016,7(9):e2367.