Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin International Joint Research and Development Center of Ophthalmology and Vision Science, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin Medical University Eye Institute, 251 Fukang Road, Nankai, Tianjin, 300384, People's Republic of China.
Department of Ophthalmology and Optometry, Eye and ENT Hospital, Fudan University, Shanghai, China.
J Mol Med (Berl). 2021 Feb;99(2):225-240. doi: 10.1007/s00109-020-01995-8. Epub 2020 Nov 14.
Diabetic retinopathy (DR), currently considered as a neurovascular disease, has become the major cause of blindness. More and more scholars believe that DR is no longer just a kind of microvascular disease, but accompanied by retinal neurodegenerative changes. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs is a classic treatment for DR; however, anti-VEGF drugs can exacerbate fibrosis and eventually lead to retinal detachment. The aim of this study was to explore the pathogenesis of DR and identified new treatments that can provide dual-target intervention for angiogenesis and fibrosis.
We explored changes in gene expression in high glucose-induced vascular endothelial cells using RNA sequencing (RNA-seq) technology. We identified bone morphogenetic protein 4 (BMP4) and SMAD family member 9 (SMAD9) among 449 differentially expressed genes from RNA-seq data and confirmed the expression of these two genes in the blood of diabetes patients by RT-PCR and in streptozotocin-induced rat retinas by RT-PCR, immunofluorescence, and western blot. Moreover, considering that DR is a multifactorial and multicellular disease, we used hydrogen peroxide (HO), advanced glycation end products (AGEs), CoCl, 4-hydroxynonenal (4-HNE), and hypoxia to induce three human retinal cell types (Müller, retinal pigment epithelium, and human retinal capillary endothelial cells) to simulate the pathogenesis of DR, and MTT experiment, scratch experiment, Transwell experiment, and lumen formation experiment were used to test whether the model was successfully established. Then, we verified the overexpression of these two genes in the cell models by RT-PCR, immunofluorescence, and western blot. We further tested the effects of BMP4 on retinal cells. We use BMP4 to stimulate retinal cells and observe the effect of BMP4 on retinal cells by MTT experiment, scratch experiment, and RT-PCR.
The results demonstrated that BMP4 and SMAD9 were highly expressed in both in vivo and in vitro models, while BMP4 could significantly upregulate the expression of SMAD9 and promote the expression of VEGF and fibrosis factors.
This study is the first to analyze the mechanism by which high glucose levels affect retinal vascular endothelial cells through RNA transcriptome sequencing and indicates that BMP4 may be a potential target for the dual-target treatment (anti-VEGF and anti-fibrosis) of DR.
• High-glucose effect on vascular endothelial cell was analyzed by RNA-seq. • KEGG analysis revealed enrichment of TGF-beta signaling pathway. • SMAD9 and BMP4 expression was upregulated in all samples. • Dual-target therapy of PDR by antagonizing BMP4.
糖尿病视网膜病变（DR）目前被认为是一种神经血管疾病，已成为主要的致盲原因。越来越多的学者认为，DR 不再仅仅是一种微血管疾病，而是伴有视网膜神经退行性改变。玻璃体内注射抗血管内皮生长因子（VEGF）药物是治疗 DR 的经典方法；然而，抗 VEGF 药物会加重纤维化，最终导致视网膜脱离。本研究旨在探讨 DR 的发病机制，并寻找新的治疗方法，为血管生成和纤维化提供双重靶向干预。
我们使用 RNA 测序（RNA-seq）技术探索高糖诱导的血管内皮细胞中基因表达的变化。我们从 RNA-seq 数据中确定了 449 个差异表达基因中的骨形态发生蛋白 4（BMP4）和 SMAD 家族成员 9（SMAD9），并通过 RT-PCR 证实了这两个基因在糖尿病患者血液中的表达，通过 RT-PCR、免疫荧光和 Western blot 证实了这两个基因在链脲佐菌素诱导的大鼠视网膜中的表达。此外，考虑到 DR 是一种多因素、多细胞疾病，我们使用过氧化氢（HO）、晚期糖基化终产物（AGEs）、CoCl、4-羟基壬烯醛（4-HNE）和缺氧来诱导三种人视网膜细胞类型（Müller 细胞、视网膜色素上皮细胞和人视网膜毛细血管内皮细胞），以模拟 DR 的发病机制，并通过 MTT 实验、划痕实验、Transwell 实验和管腔形成实验来测试模型是否成功建立。然后，我们通过 RT-PCR、免疫荧光和 Western blot 验证了这两个基因在细胞模型中的过表达。我们进一步测试了 BMP4 对视网膜细胞的作用。我们用 BMP4 刺激视网膜细胞，通过 MTT 实验、划痕实验和 RT-PCR 观察 BMP4 对视网膜细胞的影响。
结果表明，BMP4 和 SMAD9 在体内和体外模型中均高表达，而 BMP4 可显著上调 SMAD9 的表达，并促进 VEGF 和纤维化因子的表达。
本研究首次通过 RNA 转录组测序分析了高糖水平对视网膜血管内皮细胞的影响，并表明 BMP4 可能是 DR 双重靶向治疗（抗 VEGF 和抗纤维化）的潜在靶点。
通过 RNA-seq 分析高葡萄糖对血管内皮细胞的影响。
KEGG 分析显示 TGF-β信号通路富集。
所有样本中 SMAD9 和 BMP4 的表达均上调。
通过拮抗 BMP4 进行 PDR 的双重靶向治疗。
