Sun Yiwen, Song Ruixia, Ai Yanliang, Zhu Jianjun, He Jun, Dang Minyan, Li Hui
Department of Ophthalmology, Chengwu County People's Hospital, No. 66, Bole Street, Heze, Shandong 274200, China.
Department of Biomedical Sciences, City University of Hong Kong, Kowloon Tong, Hong Kong.
Saudi J Biol Sci. 2020 Oct;27(10):2770-2777. doi: 10.1016/j.sjbs.2020.06.037. Epub 2020 Jun 27.
Neovascular age-related macular degeneration (nvAMD) is one of the main pathological features of wet AMD. Apolipoprotein E2 is involved in the formation of nvAMD but the molecular mechanism has not been reported.
The APOE alleles in AMD patients were detected by genotyping. Mouse models were divided into 4 groups according to transfection different gene segments and laser-induced treatment. APOE2, VEGF, PDGF-BB, b-FGF and inflammatory cytokines (including p-NF-κB, TNF-α, IL-1β and IL-6) were tested by ELISA in mice retinal lysate. The formation of nvAMD in the indicated treatment groups at 3rd, 7th and 14th day after laser-induced damage were detected by FFA. Besides, qRT-PCR was used to determine the mRNA levels of p38, JNK and ERK in ARPE-19 cells. Finally, the inflammatory cytokines and MAPK proteins (including P38, p-P38, JNK, p-JNK, ERK and p-ERK) were detected by western blot.
The statistics of APOE alleles showed that APOE2 allele carriers were more likely to nvAMD. VEGF, PDGF-BB, b-FGF and related inflammatory cytokines were up-regulated significantly after treatment with APOE2, which were reduced after silencing the MAPK family genes, however. Further, the expression levels of neovascular growth factors and inflammatory cytokines were highly consistent between mouse models and ARPE-19 cells. Besides, the phosphorylation levels of p38, JNK and ERK were affected by APOE2.
nvAMD was affected directly by the overexpression of VEGF, PDGF-BB and b-FGF, which were regulated by APOE2 through activating MAPKs pathway.
新生血管性年龄相关性黄斑变性（nvAMD）是湿性年龄相关性黄斑变性（wet AMD）的主要病理特征之一。载脂蛋白E2（APOE2）参与nvAMD的形成，但其分子机制尚未见报道。
通过基因分型检测AMD患者的APOE等位基因。根据转染不同基因片段和激光诱导处理将小鼠模型分为4组。采用酶联免疫吸附测定（ELISA）法检测小鼠视网膜裂解液中APOE2、血管内皮生长因子（VEGF）、血小板衍生生长因子-BB（PDGF-BB）、碱性成纤维细胞生长因子（b-FGF）及炎性细胞因子（包括磷酸化核因子κB（p-NF-κB）、肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和白细胞介素-6（IL-6））。通过荧光素眼底血管造影（FFA）检测激光诱导损伤后第3、7和14天各处理组nvAMD的形成情况。此外，采用实时定量聚合酶链反应（qRT-PCR）法测定视网膜色素上皮细胞（ARPE-19细胞）中p38、应激活化蛋白激酶（JNK）和细胞外信号调节激酶（ERK）的信使核糖核酸（mRNA）水平。最后，通过蛋白质免疫印迹法检测炎性细胞因子和丝裂原活化蛋白激酶（MAPK）蛋白（包括P38、磷酸化P38（p-P38）、JNK、磷酸化JNK（p-JNK）、ERK和磷酸化ERK（p-ERK））。
APOE等位基因统计显示，APOE2等位基因携带者更易患nvAMD。用APOE2处理后，VEGF、PDGF-BB、b-FGF及相关炎性细胞因子显著上调，但沉默MAPK家族基因后则降低。此外，小鼠模型与ARPE-19细胞中新生血管生长因子和炎性细胞因子的表达水平高度一致。此外，p38、JNK和ERK的磷酸化水平受APOE2影响。
nvAMD直接受VEGF、PDGF-BB和b-FGF过表达的影响，而APOE2通过激活MAPKs通路对其进行调控。
