Monomethylfumarate Induces γ-Globin Expression and Fetal Hemoglobin Production in Cultured Human Retinal Pigment Epithelial (RPE) and Erythroid Cells, and in Intact Retina
Monomethylfumarate Induces γ-Globin Expression and Fetal Hemoglobin Production in Cultured Human Retinal Pigment Epithelial (RPE) and Erythroid Cells, and in Intact Retina
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DOI：
10.1167/iovs.14-14179
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发表时间：
2014-08-01
影响因子：
4.4
通讯作者：
Martin, Pamela M.
中科院分区：
文献类型：
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作者：
Promsote, Wanwisa;Makala, Levi;Martin, Pamela M.
目的.镰状视网膜病变（SR）是镰状细胞病（SCD）视力丧失的主要原因。没有预防SR的策略，治疗方法也非常有限。本研究评价了（1）视网膜色素上皮（RPE）细胞作为血红蛋白生产者和胎儿血红蛋白（HbF）诱导的新细胞靶点，以及（2）富马酸单甲酯（MMF）作为HbF诱导治疗和SCD视网膜中氧化应激和炎症的消除剂。在人RPE细胞系ARPE-19和从Townes人源化SCD小鼠分离的原代RPE细胞中通过RT-定量（q）PCR评估人珠蛋白基因表达。在用0 - 1000 μ M富马酸二甲酯、MMF或羟基脲（HU;阳性对照）处理的KU 812稳定双荧光素酶报告基因表达细胞中，通过双荧光素酶测定监测γ-珠蛋白启动子活性。使用逆转录酶-qPCR、荧光激活细胞分选（FACS）、免疫荧光和蛋白质印迹技术来评价原代人红系祖细胞ARPE-19和经类似处理的正常血红蛋白产生（HbAA）和纯合β突变（HbSS）RPE中以及注射MMF（1000 μ M）的HbAA和HbSS视网膜中的γ-珠蛋白表达和HbF产生。还分析了二氢乙锭标记和核因子（红细胞衍生2）样2（Nrf 2）、IL-1 β和VEGF表达。视网膜色素上皮细胞表达珠蛋白基因并合成成人和胎儿血红蛋白。在培养的RPE和红系细胞以及HbSS小鼠视网膜中，MMF刺激γ-珠蛋白表达和HbF产生，并降低氧化应激和炎症。RPE产生血红蛋白表明这种细胞类型可能参与SR的病因学。富马酸单甲酯影响与SCD中视网膜健康改善一致的多个参数，因此可能在SR治疗中具有治疗潜力。
PURPOSE. Sickle retinopathy (SR) is a major cause of vision loss in sickle cell disease (SCD). There are no strategies to prevent SR and treatments are extremely limited. The present study evaluated (1) the retinal pigment epithelial (RPE) cell as a hemoglobin producer and novel cellular target for fetal hemoglobin (HbF) induction, and (2) monomethylfumarate (MMF) as an HbF-inducing therapy and abrogator of oxidative stress and inflammation in SCD retina.METHODS. Human globin gene expression was evaluated by RT-quantitative (q)PCR in the human RPE cell line ARPE-19 and in primary RPE cells isolated from Townes humanized SCD mice. gamma-Globin promoter activity was monitored in KU812 stable dual luciferase reporter expressing cells treated with 0 to 1000 mu M dimethylfumarate, MMF, or hydroxyurea (HU; positive control) by dual luciferase assay. Reverse transcriptase-qPCR, fluorescence-activated cell sorting (FACS), immunofluorescence, and Western blot techniques were used to evaluate gamma-globin expression and HbF production in primary human erythroid progenitors, ARPE-19, and normal hemoglobin producing (HbAA) and homozygous beta(s) mutation (HbSS) RPE that were treated similarly, and in MMF-injected (1000 mu M) HbAA and HbSS retinas. Dihydroethidium labeling and nuclear factor (erythroid-derived 2)-like 2 (Nrf2), IL-1 beta, and VEGF expression were also analyzed.RESULTS. Retinal pigment epithelial cells express globin genes and synthesize adult and fetal hemoglobin MMF stimulated gamma-globin expression and HbF production in cultured RPE and erythroid cells, and in HbSS mouse retina where it also reduced oxidative stress and inflammation.CONCLUSIONS. The production of hemoglobin by RPE suggests the potential involvement of this cell type in the etiology of SR. Monomethylfumarate influences multiple parameters consistent with improved retinal health in SCD and may therefore be of therapeutic potential in SR treatment.