N-乙酰半胱氨酸对TGF-β1诱导的ARPE-19细胞向肌成纤维细胞转分化的影响

背景 视网膜色素上皮(RPE)细胞转分化为肌成纤维细胞是增生性玻璃体视网膜病变发生和发展中的一个核心事件.N-乙酰半胱氨酸(NAC)能够通过抑制转化生长因子-β1(TGF-β1)诱导的细胞内活性氧(ROS)的产生和MAPK蛋白的磷酸化来抑制多种细胞向肌成纤维细胞的转分化,但是NAC对TGF-β1诱导的人RPE细胞系ARPE-19细胞向肌成纤维细胞转分化的影响及其潜在的分子机制尚不清楚. 目的 研究NAC对TGF-β1诱导的ARPE-19细胞向肌成纤维细胞转分化的影响及其潜在的分子机制.方法 体外培养人ARPE-19细胞并分为对照组、TGF-β1处理组、NAC干预组和NAC单独处理组,对照组不做处理,其他3个组分别使用10 ng/ml TGF-β1、10 ng/ml TGF-β1 +5 mmol/L NAC和5 mmol/L NAC处理细胞48 h,相差显微镜下观察细胞的形态学改变.采用实时荧光定量PCR、Western blot、免疫荧光及ELISA法检测转分化标志基因α-平滑肌肌动蛋白(α-SMA)、纤维结合蛋白及Ⅰ型胶原的表达情况.使用非荧光探针DCFH-DA通过流式细胞仪检测在TGF-β1处理后1h,细胞内ROS的产生情况及NAC对细胞内ROS产生的影响.采用Western blot法检测各组细胞内p38MAPK、ERK1/2及SAPK/JNK蛋白磷酸化的影响.采用细胞计数试剂盒-8(CCK-8)法检测不同浓度NAC对ARPE-19细胞生存的影响. 结果 实时荧光定量PCR、Western blot及ELISA实验结果表明,与对照组相比,TGF-β1处理组α-SMA、纤维结合蛋白和Ⅰ型胶原mRNA表达水平均上调,分别是对照组的(2.15±0.29)、(9.54±1.08)和(25.78±0.66)倍,蛋白表达水平分别是对照组的(8.49±0.32)、(2.53±0.69)和(4.45±1.05)倍,差异均有统计学意义(均P＜0.05).NAC干预组α-SMA、纤维结合蛋白和Ⅰ型胶原mRNA的表达水平分别是TGF-β1处理组的(66.70±12.57)％、(66.11±8.35)％和(33.19±6.90)％,蛋白表达水平分别是TGF-β1处理组的(52.30±4.83)％、(55.03±2.58)％和(56.08±3.65)％,差异均有统计学意义(均P＜0.05).流式细胞仪分析结果显示,TGF-β1处理组细胞内ROS的产生水平较对照组增加,是对照组的(2.12±0.20)倍,NAC干预组细胞内ROS水平是TGF-β1处理组的(57.41±9.45)％,差异均有统计学意义(均P＜0.01).Western blot结果显示,与对照组相比,TGF-β1处理组p38MAPK、SAPK/JNK及ERK1/2蛋白磷酸化水平均上调,分别是对照组的(9.18±1.00)、(4.87±0.81)和(4.20±0.69)倍,差异均有统计学意义(均P＜0.05).使用NAC干预处理后,p38MAPK、SAPK/JNK及ERK1/2蛋白磷酸化水平分别是TGF-β1处理组的(48.16±14.82)％、(67.90±2.90)％和(74.52±4.00)％,差异均有统计学意义(均P＜0.05). 结论 NAC可能通过抑制TGF-β1诱导的ROS的产生及p38MAPK、SAPK/JNK及ERK1/2蛋白的磷酸化进而抑制ARPE-19细胞向肌成纤维细胞转分化.     

Nicotinamide suppresses bevacizumab-induced epithelial-mesenchymal transition of ARPE-19 cells by attenuating oxidative stress

AIM:To investigate the effects of nicotinamide(NAM)on bevacizumab(BEV)-induced epithelial-mesenchymal transition(EMT)of human retinal pigment epithelial cells(ARPE-19)and the underling mechanisms.METHODS:ARPE-19 cells were treated with BEV for 24,48,and 72 h,and the variation degrees of EMTrelated markers(fibronectin,α-SMA,vimentin,and ZO-1)were assessed by Western blotting to select the optimal treatment time point which exhibited the most obvious changes of EMT-related markers for the subsequent experiments.Furthermore,NAM was added to the medium,the m RNA and protein levels of the EMT-related markers were then measured.The accumulation of reactive oxygen species(ROS)and H₂O₂ and the total antioxidant capacity(TAC)of the cells were also measured to evaluate the level of oxidative stress.RESULTS:After being treated with BEV for 72 h,the protein expression levels of EMT-related markers in ARPE-19 cells showed significant changes.Meanwhile the levels of ROS and H₂O₂ were obviously increased,and the TAC of ARPE-19 cells was decreased.Totally 72 h was chosen to be the optimal treatment time point in subsequentexperiments.Furthermore,NAM inhibited BEV-induced EMT by downregulating fibronectin,α-SMA,and vimentin and upregulating ZO-1,decreased the accumulation of ROS and H₂O₂,and enhanced TAC in BEV-treated ARPE-19 cells.CONCLUSION:This study demonstrates that NAM suppressed BEV-induced EMT in ARPE-19 cells by attenuating oxidative stress.Hence,NAM may be a potential therapeutic agent for alleviating neovascular fibrosis of the ocular fundus after anti-vascular endothelial growth factor therapy.     

ALK5抑制剂对转化生长因子-β1（TGF-β1）致视网膜色素上皮细胞纤维化的干预作用

目的　探讨转化生长因子-β1（transforming growth factor-beta1，TGF-β1）对人视网膜色素上皮（retinal pigment epithelial，RPE）细胞向成纤维细胞转化的影响，及活化素受体样激酶5（activated receptor kinase 5，ALK5）抑制剂（SB-431542）对这一影响的干预作用。方法　体外培养人RPE细胞，随机分为对照组、TGF-β1处理组、TGF-β1+SB-431542处理组、SB-431542处理组。对照组不做任何处理，其余三组分别用10 μg·L^-1 TGF-β1、10 μg·L^-1 TGF-β1+30 μmol·L^-1 SB-431542、30 μmol·L^-1 SB-431542处理细胞24 h。MTT法检测各组RPE细胞增殖率；划痕实验观察各组处理后0 h、12 h、24 h RPE细胞迁移情况；免疫荧光法检测各组细胞ALK5蛋白的分布和表达；Western blot法检测各组ALK5、血管内皮生长因子（vascular endothelial growth factor，VEGF）、α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、Ⅰ型胶原（collagen typeⅠ，ColⅠ）以及纤维粘连素（fibronectin，FN）蛋白的表达。结果　与对照组相比，TGF-β1作用于RPE细胞24 h后，可改变RPE细胞表型，使其呈成纤维细胞样外观，并明显促进RPE细胞增殖和迁移。10.0 g·L^-1 TGF-β1作用24 h后RPE细胞增殖率为对照组的（1.33±0.08）倍，30 μmol·L^-1 SB-431542作用24 h后RPE细胞增殖率为10.0 g·L^-1 TGF-β1处理组的（67.77±6.78）%。经TGF-β1作用24 h后RPE细胞内ALK5、VEGF、α-SMA、ColⅠ及FN蛋白的表达显著上调，分别是对照组的（3.69±0.37）倍、（1.76±0.05）倍、（2.58±0.18）倍、（1.86±0.11）倍、（1.74±0.08）倍；SB-431542可逆转TGF-β1诱导的ALK5、VEGF、α-SMA、ColⅠ及FN蛋白表达上调，分别是TGF-β1处理组的（67.73±5.15）%、（71.71±3.50）%、（79.87±0.05）%、（63.59±3.16）%、（83.07±2.31）%，差异均有统计学意义（均为P＜0.05）。结论　TGF-β1可促进RPE细胞增殖、迁移，向成纤维细胞转化，并能显著上调RPE细胞内ALK5、VEGF、α-SMA、ColⅠ及FN蛋白的表达；SB-431542可通过抑制TGF-β1的作用而抑制RPE细胞的纤维化。     

藤黄酸通过CYLD/NF-κB信号通路抑制高糖环境下RPE细胞上皮-间充质转化的作用

目的 基于去泛素化酶圆柱瘤蛋白/核因子κB(CYLD/NF-κB)通路,研究藤黄酸(GA)对高糖环境下视网膜色素上皮(RPE)细胞上皮-间充质转化(EMT)的作用。方法 体外培养ARPE-19细胞,高糖诱导,实验分为NC组(含5.5 mmol·L^-1葡萄糖),HG组(含30 mmol·L^-1葡萄糖),HG+不同剂量GA组(2μmol·L^-1、4μmol·L^-1、8μmol·L^-1GA分别预处理RPE细胞1 h,加30 mmol·L^-1葡萄糖)。CCK-8检测各组RPE细胞增殖活力,免疫荧光染色检测RPE细胞中α平滑肌肌动蛋白(α-SMA)、去泛素化酶圆柱瘤蛋白(CYLD)表达;Western blot检测RPE细胞中α-SMA、CYLD、磷酸化核转录因子κB(p-NF-κB)蛋白表达。结果 与NC组相比,HG组RPE细胞出现明显增殖,RPE细胞中α-SMA、p-NF-κB蛋白表达均增加,CYLD蛋白表达减少(均为P<0.01);与HG组相比,HG+不...     

杞黄颗粒对H2O2诱导的人视网膜色素上皮细胞炎症损伤的保护作用

目的　探讨杞黄颗粒（QHG）对H₂O₂诱导的人视网膜色素上皮（RPE）细胞系ARPE-19炎症损伤的保护作用。方法　常规培养ARPE-19细胞,随机分为对照组、H₂O₂损伤组(200 μmol?L^-1 H₂O₂处理细胞）、QHG低剂量组（1 g?L^-1 QHG作用24 h后再加入H₂O₂处理细胞）和QHG高剂量组（2 g?L^-1QHG作用24 h后再加入H₂O₂处理细胞）。使用相差显微镜观察各组ARPE-19细胞形态;流式细胞仪检测各组ARPE-19细胞凋亡率,Real-time PCR检测各组ARPE-19细胞β淀粉样蛋白（Aβ)mRNA表达,ELISA检测各组ARPE-19细胞Aβ以及攻膜复合物（MAC）的蛋白表达水平。结果　相差显微镜下可见,QHG低剂量组、QHG高剂量组ARPE-19细胞形态改变均较H₂O₂损伤组轻。对照组、H₂O₂损伤组、QHG低剂量组、QHG高剂量组ARPE-19细胞凋亡率分别为（6.85±0.14）%、（43.60±1.80）%、（23.23±1.43）%、（17.90±0.78）%,H₂O₂损伤组细胞凋亡率较对照组明显增加,QHG低剂量组及QHG高剂量组细胞凋亡率均较H₂O₂损伤组明显降低,且QHG高剂量组较QHG低剂量组降低更明显,差异均有统计学意义（均为P<0.05）。与对照组相比,H₂O₂损伤组ARPE-19细胞Aβ mRNA及Aβ、MAC蛋白表达水平均升高,差异均有统计学意义（均为P<0.01）。与H₂O₂损伤组相比,QHG低剂量组及QHG高剂量组均可明显降低ARPE-19细胞Aβ mRNA 相对表达量及Aβ、MAC蛋白的表达水平,差异均有统计学意义（均为P<0.01）。结论　QHG能有效抑制H₂O₂诱导的人RPE细胞凋亡,减少细胞Aβ及MAC表达,从而起到保护人RPE细胞的作用。     

缓激肽对转化生长因子-β1（TGF-β1）诱导视网膜色素上皮细胞发生上皮间充质转化的影响

目的　制作增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)的上皮间充质转化（epithelial-mesenchymal transformation,EMT）细胞模型,研究缓激肽（bradykinin,BK）对视网膜色素上皮（retinal pigment epithelium,RPE）细胞发生EMT的影响,并探讨BK对PVR的影响机制。方法　体外培养人RPE细胞株ARPE-19细胞,采用不同浓度转化生长因子-β1（transforming growth factor- β1,TGF- β1）分别作用于ARPE-19细胞24 h、48 h,于倒置显微镜下观察细胞形态变化;采用CCK-8检测细胞增殖情况,确定TGF-β1浓度及作用时间;利用Western blot和细胞免疫荧光检测EMT标志蛋白E-钙黏素、α-平滑肌肌动蛋白（α-smooth muscle actin,α-SMA）和波形蛋白（Viminten）表达情况;细胞划痕实验、Transwell实验检测细胞迁移能力。同时采用Western blot检测TGF/Smad通路下游pSmad3和Smad7的表达。结果　TGF- β1刺激ARPE-19细胞后可以成功诱导EMT体外细胞模型。当TGF-β1浓度为10 μg·L^-1、作用时间为48 h时,细胞增殖最明显。BK在TGF- β1诱导的EMT中可以降低α-SMA、Viminten的表达,升高E-钙黏素的表达并且降低细胞迁移能力。这些影响能被BK-2受体拮抗剂HOE-140逆转。TGF-β1诱导ARPE-19细胞发生EMT时,pSmad3表达量升高;TGF-β1刺激前给予BK刺激,pSmad3表达量减少;加入BK前预敷HOE-140,然后给予TGF-β1刺激,BK作用减弱,pSmad3表达量升高。Smad7表达趋势与pSmad3表达趋势相反。结论　10 μg·L^-1 TGF-β1可导致ARPE-19细胞发生EMT。BK通过TGF-/Smad信号通路上调Smad 7的表达、下调pSmad 3的表达,从而逆转TGF-β1诱导的EMT。提示BK可能是一种新的、有效的治疗PVR的方法。     

miR-21对H2O2诱导的视网膜色素上皮细胞凋亡及PTEN/AKT通路的影响

目的　探讨miR-21对H₂O₂诱导的视网膜色素上皮细胞凋亡以及PTEN/AKT通路的影响。方法　体外培养人视网膜色素上皮细胞系ARPE-19,实验分为四组:空白对照组、阴性对照组、H₂O₂组、H₂O₂+miR-21mimics组,使用q-RT-PCR法检测各组ARPE-19细胞中miR-21表达,流式细胞术检测各组细胞凋亡率,Western blot检测凋亡相关蛋白Bax、Bcl-2、Caspase-3以及人第10号染色体缺失的磷酸酶（phosphatase and tensin homolog deleted on chromosome ten,PTEN）、磷酸化AKT蛋白表达。结果　与空白对照组和阴性对照组相比,H₂O₂组和H₂O₂+miR-21 mimics组miR-21表达量（1.14±0.23、2.18±0.44）、SOD水平［（5.49±1.10） U·L^-1、（14.28±2.86） U·L^-1］、Bcl-2蛋白含量（0.34±0.07、0.62±0.12）、p-PI3K表达水平（0.46±0.09、0.68±0.13）、p-AKT水平（0.53±0.11、1.16±0.13）均显著降低,差异均有统计学意义（均为P＜0.05）,而活性氧自由基（reactive oxygen species,ROS）水平（30.58±7.96、23.25±5.67）、MDA水平［（3.95±0.79）mol·L^-1、（2.13±0.43）mol·L^-1］、凋亡率［（25.48±5.10）%、（17.63±3.52）%］、PTEN蛋白表达（0.59±0.12、0.43±0.11）、Bax表达（0.73±0.15、0.49±0.10）、Caspase-3蛋白表达（0.85±0.17、0.42±0.08）均显著升高,差异均有统计学意义（均为P＜0.05）。与H₂O₂组相比,H₂O₂+miR-21 mimics组miR-21表达量、SOD水平、Bcl-2蛋白表达、p-PI3K及p-AKT蛋白表达均显著升高（均为P＜0.05）,ROS水平、MDA水平、细胞凋亡率、PTEN蛋白表达、Bax及Caspase-3蛋白表达均显著降低（均为P＜0.05）。结论　上调miR-21可能通过激活PTEN/AKT信号通路抑制H₂O₂诱导的视网膜色素上皮细胞细胞凋亡。     

4-苯基丁酸对高糖诱导的人晶状体上皮细胞发生上皮间质转化的影响

目的　探讨4-苯基丁酸（4-PBA）对高糖诱导的人晶状体上皮细胞发生上皮间质转化（EMT）的影响。方法　体外培养人晶状体上皮细胞系HLE-B3细胞,将培养的HLE-B3细胞分为正常对照组、高糖组和4-PBA预处理+高糖组。利用流式细胞术检测各组细胞内活性氧（ROS）的含量;利用Western blot检测各组细胞中内质网应激（ERS）相关通路分子的蛋白表达;采用免疫荧光染色观察各组细胞中上皮因子E-cadherin和间质因子α-SMA的表达;分别采用实时荧光定量PCR和Western blot检测各组细胞中EMT标志分子E-cadherin、α-SMA、Snail的 mRNA及蛋白表达。结果　高糖组细胞呈长梭形改变,丢失上皮细胞特性,类似纤维细胞的表型;4-PBA预处理+高糖组细胞形态不规则,相对于高糖组细胞,长梭形细胞数量较少。流式细胞术检测结果显示,高糖组细胞内ROS含量高于正常对照组,4-PBA预处理后ROS含量降低,差异均有统计学意义（均为P<0.01）。Western blot检测结果显示,高糖组细胞中ERS相关通路分子GRP78、CHOP、p-eIF2α的蛋白相对表达水平均高于正常对照组（均为P<0.05）;4-PBA预处理+高糖组细胞中GRP78、CHOP、p-eIF2α的蛋白相对表达水平均明显低于高糖组（均为P<0.05）。实时荧光定量PCR和Western blot检测结果显示,与正常对照组相比,高糖组细胞中E-cadherin mRNA及蛋白表达下调,α-SMA和Snail的mRNA及蛋白表达均上调（均为P<0.05）;与高糖组相比,4-PBA预处理+高糖组细胞中E-cadherin mRNA及蛋白的相对表达量均明显上调,α-SMA和Snail的mRNA及蛋白的相对表达量则与之相反（均为 P<0.05）。免疫荧光染色结果显示,高糖组细胞中E-cadherin荧光表达明显弱于正常对照组,4-PBA 预处理后荧光增强;α-SMA荧光表达则与之相反。结论　4-PBA通过抑制ERS相关通路分子的表达、减少ROS产生来抑制高糖诱导的晶状体上皮细胞发生EMT,从而对晶状体上皮细胞起到一定的保护作用。     

LRG1 promotes epithelial-mesenchymal transition of retinal pigment epithelium cells by activating NOX4

AIM:To investigate the effect of leucine-rich-alpha-2-glycoprotein 1(LRG1)on epithelial-mesenchymal transition(EMT)in retinal pigment epithelium(RPE)cells,and to explore the role of NADPH oxidase 4(NOX4).METHODS:RPE cells(ARPE-19 cell line)were treated with transforming growth factor-β1(TGF-β1)to induce EMT.Changes of the m RNA and protein expression levels of LRG1 were tested in the TGF-β1 treated cells.The recombinant human LRG1 protein(r LRG1)and si RNA of LRG1 were used to establish accumulation of exogenous LRG1 model and the down-regulation of LRG1 model in ARPE-19 cells respectively,and to detect EMT-related markers including fibronectin,α-smooth muscle actin(α-SMA)and zonula occludens-1(ZO-1).The m RNA and protein expression level of NOX4 were measured according to the above treatments.VAS2870 was used as a NOX4 inhibitor in r LRG1-treated cells.EMT-related markers were detected to verify the effect of NOX4 in the process of EMT.RESULTS:TGF-β1 promoted the expression of LRG1 at both the m RNA and protein levels during the process of EMT which showed the up-regulation of fibronectin andα-SMA,as well as the down-regulation of ZO-1.Furthermore,the r LRG1 promoted EMT of ARPE-19 cells,which manifested high levels of fibronectin andα-SMA and low level of ZO-1,whereas knockdown of LRG1 prevented EMT by decreasing the expressions of fibronectin andα-SMA and increasing the expression of ZO-1 in ARPE-19 cells.Besides,the r LRG1 activated and LRG1 si RNA suppressed NOX4 expression.EMT was inhibited when VAS2870 was used in the r LRG1-treated cells.CONCLUSION:These results for the first time demonstrate that LRG1 promotes EMT of RPE cells by activating NOX4,which may provide a novel direction to explore the mechanisms of subretinal fibrosis.     

全反式视黄酸诱导人视网膜色素上皮细胞凋亡的实验研究

目的　探讨梯度浓度全反式视黄酸（all-trans retinoic acid,ATRA）诱导人视网膜色素上皮细胞凋亡的作用。方法　采用流式细胞术检测视网膜色素上皮细胞凋亡、活性氧（ROS）活性。采用实时定量聚合酶链反应和Western blot，从蛋白水平检测梯度浓度ATRA对ARPE-19细胞内质网应激（endoplasmic reticulum stress，ERS）标记蛋白表达的影响。观察抗氧化剂NAC及ERS抑制剂Salubrinal抑制ARPE-19细胞诱导ROS和ERS的作用。结果　CCK-8检测结果显示:ATRA处理ARPE-19细胞24 h和48 h后的半抑制浓度（IC₅₀）分别为13.88 μmol?L^-1和11.99 μmol?L^-1。流式细胞术检测结果显示，10.0 μmol?L^-1、15.0 μmol?L^-1、20.0 μmol?L^-1ATRA处理后细胞凋亡水平均较对照组显著上升，差异均有统计学意义（均为P<0.001）；2.5 μmol?L^-1、5.0 μmol?L^-1、10.0 μmol?L^-1、20.0 μmol?L^-1ATRA处理后细胞的ROS水平均较对照组显著增加，差异均有统计学意义（均为P<0.001）。Western blot检测结果显示，ERS标记蛋白C/EBP同源蛋白（C/EBPhomologous protein,CHOP）、结合免疫球蛋白（binding immunoglobulin protein，BIP）的蛋白表达水平与对照组差异均有统计学意义（均为P<0.05）；经ATRA处理的模型组较对照组的血管内皮生长因子A(VEGF-A)、CHOP蛋白表达水平均显著上升，NAC-ARTA处理组、Salubrinal-ARTA处理组及NAC-Salubrinal-ARTA联合处理组较模型组的VEGF-A、CHOP蛋白表达水平均显著下降，差异均有统计学意义（均为P<0.05）。结论　ATRA通过激活ROS和ERS信号通路诱导ARPE-19细胞凋亡。     

miR-155在转化生长因子β2诱导的人视网膜色素上皮细胞上皮-间质转化中的促进作用

目的:探讨微小RNA-155(miR-155)在转化生长因子β2(TGF-β2)诱导的人视网膜色素上皮细胞上皮-间质转化过程中的作用及其机制。方法:取视网膜色素上皮细胞ARPE-19细胞系分为对照组和TGF-β2组,分别用DMEM培养液和含10 ng/ml TGF-β2的DMEM培养液培养,另取ARPE-19细胞系分为...     

The role of gremlin, a BMP antagonist, and epithelial-to-mesenchymal transition in proliferative vitreoretinopathy

PURPOSE: Proliferative vitreoretinopathy (PVR), a major reason for failure of retinal detachment surgery, is characterized by the formation of scarlike tissue that contains transdifferentiated retinal pigment epithelial (RPE) cells. The scar tissue occurs in response to growth factors such as transforming growth factor (TGF)-beta and epidermal growth factor (EGF). The authors postulate that transdifferentiation of RPE cells may arise via epithelial-to-mesenchymal transition (EMT). Bone morphogenetic proteins (BMPs) are expressed in the retina and have an antiproliferative role. Gremlin is expressed in the outer retina and is a BMP antagonist. The study was conducted to establish a model of PVR by inducing EMT in the human RPE cell line ARPE-19, using TGF-beta and EGF and to establish the contribution of gremlin to EMT. METHODS: ARPE-19 cells were cultured and stimulated with TGF-beta1, EGF, and gremlin. The expression of alpha-smooth muscle actin (alpha-SMA), vimentin, and zona occludens (ZO)-1 were examined via PCR, Western blot analysis, and immunofluorescence. Zymography was performed for matrix metalloproteinase (MMP) activity. Scratch assays were performed to assess migration. RESULTS: A model of EMT was established in the ARPE-19 cell line. The characteristics of EMT include gain of alpha-SMA, loss of ZO-1, upregulation of MMP activity and enhanced migration. Gremlin plays an important role in this process, contributing to the gain of alpha-SMA, loss of ZO-1, and upregulation of MMP activity. CONCLUSIONS: EMT occurs in vitro in the ARPE-19 cell line in response to the growth factors TGF-beta1 and EGF. EMT is also induced by Gremlin.     

Bevacizumab modulates retinal pigment epithelial-to-mesenchymal transition via regulating Notch signaling

AIM: To investigate the effect of bevacizumab treatment on Notch signaling and the induction of epithelial-of-mesenchymal transition (EMT) in human retinal pigment epithelial cells (ARPE-19) in vitro. METHODS: In vitro cultivated ARPE-19 cells were treated with 0.25 mg/mL bevacizumab for 12, 24, and 48h. Cell morphology changes were observed under an inverted microscope. The expression of zonula occludens-1 (ZO-1), vimentin and Notch-1 intracellular domain (NICD) was examined by immunofluorescence. The mRNA levels of ZO-1, α-SMA, Notch-1, Notch-2, Notch-4, Dll4, Jagged-1, RBP-Jk and Hes-1 expression were evaluated with quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of α-SMA, NICD, Hes-1 and Dll-4 expression were examined with Western blot. RESULTS: Bevacizumab stimulation increased the expression of α-SMA and vimentin in ARPE-19 cells which changed into spindle-shaped fibroblast-like cells. Meanwhile, the mRNA expression of Hes-1 increased and the protein expression of Hes-1 and NICD also increased, which Notch signaling was activated. The mRNA expression of Notch-1, Jagged-1 and RBP-Jk increased at 48h, and while Dll4 mRNA and protein expression did not change after bevacizumab treatment. CONCLUSION: Jagged-1/Notch-1 signaling may play a critical role in bevacizumab-induced EMT in ARPE-19 cells, which provides a novel insight into the pathogenesis of intravitreal bevacizumab-associated complication.     

Zn2+-induced cell death is mediated by the induction of intracellular ROS in ARPE-19 cells

PURPOSE: Recent studies have shown that Zn2+ induced cell death in retinal pigment epithelial cells. Here we sought to investigate the mode of Zn2+-induced cell death and the role of reactive oxygen species (ROS) in human retinal pigment epithelial cell line, ARPE-19 cells. METHODS: Cell viability was measured by MTT assay. Cell death of ARPE-19 cells was measured by annexin V-fluorescein isothiocyanate (FITC) binding assay, TUNEL assay. The formation of intracellular ROS was measured using 2',7'-dichlorofluorescein diacetate (DCFH-DA). The activation of mitogen-activated protein kinase (MAPK) was examined by Western blot analysis. RESULTS: This study demonstrated that Zn2+ treatment induced both necrosis and apoptosis in ARPE-19 cells. Exposure of ARPE-19 cells to Zn2+ led to the activation of ERK1/2, JNK1/2/3, and p38 MAPKs. The activation of these MAPKs was blocked by treatment with the antioxidant, N-acetylcystein (NAC). More importantly, inhibition of ROS production by NAC completely prevented Zn2+-induced cell death in RPE cells. CONCLUSIONS: This study suggests that Zn2+ induces both apoptosis and necrosis in ARPE-19 cells and that its cytotoxicity may depend on the induction of intracellular ROS.     

Cadmium-induced apoptotic death of human retinal pigment epithelial cells is mediated by MAPK pathway

Cadmium (Cd), released from cigarette smoke and metal industrial activities, is known to accumulate in human body organs including retina and is particularly higher in retinal tissues of age-related macular degeneration (AMD) eyes compared to non-AMD eyes. We have determined the cytotoxic effects of Cd on human retinal pigment epithelial (RPE) cells. Upon Cd treatment, there was a dose- and time-dependent decline in ARPE-19 cell viability as well as early apoptotic changes such as altered mitochondrial membrane potential (MMP) and Cytochrome C release in cytosol. Depletion of GSH by buthionine-[S,R]-sulfoximine (BSO) resulted in increased Cd toxicity in ARPE-19 cells. Cadmium also caused reactive oxygen species (ROS) generation and activation of mitogen-activated protein kinases (MAPKs) pathway including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (Erk1/2), and p38 in ARPE-19 cells. Antioxidants such as N-acetylcysteine (NAC) significantly reduced Cd-induced toxicity. These results indicate that elevated ROS-induced activation of the MAPK signaling pathway could be associated with Cd-induced RPE cell apoptosis, one of the major contributing factors in AMD. The toxic effects of Cd on ARPE-19 cells indicate that environmental heavy metals such as Cd could be important potential factors in RPE cells death associated retinal diseases particularly related to smoking.