Sreekumar Parameswaran G, Nam Mi-Hyun, Hong Elise, Kannan Ram, Nagaraj Ram H
Doheny Eye Institute, Pasadena, California, United States.
Sue Anschutz-Rodgers Eye Center and Department of Ophthalmology, School of Medicine, University of Colorado, Aurora, Colorado, United States.
Invest Ophthalmol Vis Sci. 2025 Jun 2;66(6):30. doi: 10.1167/iovs.66.6.30.
Subretinal fibrosis, a complication of neovascular age-related macular degeneration (nAMD), involves the epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells as a contributing mechanism. The receptor for advanced glycation end products (RAGE) is a multiligand receptor implicated in fibrotic diseases, but its role in subretinal fibrosis has not been studied. This study investigated the role of RAGE in subretinal fibrosis.
Subretinal fibrosis was induced in male RAGE-/- and wild-type (WT) mice via laser photocoagulation, and fibrosis lesion volume was assessed on day 35 using optical coherence tomography and immunostaining. In vitro, EMT was induced in primary human RPE cells with transforming growth factor-beta 2 (TGF-β2). The role of RAGE in EMT was studied in cells pretreated with RAGE antagonists (FPS-ZM1 or azeliragon), followed by cotreatment with TGF-β2 for 48 hours. Signaling studies were conducted by pretreatment with FPS-ZM1 for 2 hours, cotreatment with TGF-β2 for 60 minutes, and subsequent immunoblot analysis.
In RAGE-/- mice, subretinal fibrosis after laser-induced choroidal neovascularization was significantly reduced, with a smaller fibrosis volume, less inflammation, decreased activation of pSmad2, and reduced deposition of fibrotic markers (αSMA, collagen I) compared to WT mice. In vitro treatment with TGF-β2 in human RPE cells increased mitochondrial reactive oxygen species and upregulated EMT markers (αSMA, collagen I, and fibronectin), which were inhibited by cotreatment with FPS-ZM1 or azeliragon. FPS-ZM1 blocked TGF-β2-induced Smad2-dependent signaling and EMT without affecting the extracellular signal-regulated kinase (ERK) pathway.
Our findings indicate that RAGE plays a role in RPE cell EMT in subretinal fibrosis and that RAGE antagonists attenuate this process, making RAGE a promising therapeutic target for subretinal fibrosis in nAMD.
视网膜下纤维化是新生血管性年龄相关性黄斑变性（nAMD）的一种并发症，其涉及视网膜色素上皮（RPE）细胞的上皮-间质转化（EMT），是一种促成机制。晚期糖基化终产物受体（RAGE）是一种多配体受体，与纤维化疾病有关，但其在视网膜下纤维化中的作用尚未得到研究。本研究调查了RAGE在视网膜下纤维化中的作用。
通过激光光凝在雄性RAGE基因敲除（RAGE-/-）小鼠和野生型（WT）小鼠中诱导视网膜下纤维化，并在第35天使用光学相干断层扫描和免疫染色评估纤维化病变体积。在体外，用转化生长因子-β2（TGF-β2）诱导原代人RPE细胞发生EMT。在用RAGE拮抗剂（FPS-ZM1或阿兹利酮）预处理的细胞中研究RAGE在EMT中的作用，随后与TGF-β2共同处理48小时。通过用FPS-ZM1预处理2小时、与TGF-β2共同处理60分钟以及随后的免疫印迹分析进行信号传导研究。
在RAGE-/-小鼠中，激光诱导脉络膜新生血管形成后的视网膜下纤维化明显减轻，与WT小鼠相比，纤维化体积更小、炎症更少、pSmad2激活减少以及纤维化标志物（αSMA、胶原蛋白I）沉积减少。在人RPE细胞中用TGF-β2进行体外处理会增加线粒体活性氧并上调EMT标志物（αSMA、胶原蛋白I和纤连蛋白），而与FPS-ZM1或阿兹利酮共同处理可抑制这些标志物。FPS-ZM1阻断了TGF-β2诱导的Smad2依赖性信号传导和EMT，而不影响细胞外信号调节激酶（ERK）途径。
我们的研究结果表明，RAGE在视网膜下纤维化的RPE细胞EMT中起作用，并且RAGE拮抗剂可减弱这一过程，使RAGE成为nAMD视网膜下纤维化的一个有前景的治疗靶点。
