Secreted phosphoprotein 1 expression in retinal mononuclear phagocytes links murine to human choroidal neovascularization. Secreted phosphoprotein 1 expression in retinal mononuclear phagocytes links murine to human choroidal neovascularization

Neovascular age-related macular degeneration represents the most common cause of blindness in the western world. Alterations of the outer Blood-retina barrier integrity and a localized inflammatory microenvironment lead to sprouting of choroidal neovascularization in intimate contact with surrounding myeloid cells and ultimately lead to visual impairment. The discovery of novel targets interfering with angiogenesis and inflammation is vital for the future treatments in AMD patients. To identify novel potential targets in the local phagocytes of the retina, microglia, we performed a comprehensive RNA-seq analysis in the mouse model of laser-induced choroidal neovascularization (mCNV). Here, we identified the angiogenic factor Osteopontin (Opn), also known as "secreted phosphoprotein 1” (Spp1), to be one of the most highly expressed genes in retinal microglia in the course of CNV formation. We could confirm the presence of SPP1 at the lesion site in recruited retinal microglia of Cx3cr1CreER:Rosa26-Tomato reporter mice using immunohistochemistry and in whole retinal tissue lysates by ELISA compared to controls highlighting a massive local production of SPP1. Inhibition of SPP1 by intravitreal injection of anti-SPP1 antibody significantly increased the lesion size compared to IgG-treated control eyes. In line with the results in rodents, we found an increased SPP1 mRNA expression in surgically extracted human choroidal neovascular (hCNV) membranes by the quantitative RNA-seq approach of massive analysis of cDNA ends (MACE) and found numerous IBA1+SPP1+ myeloid cells in human CNV membranes. Taken together, these results highlight the importance of SPP1 in the formation of CNV and potentially offer new opportunities for therapeutic intervention by inhibiting the SPP1 pathway. Overall design: RNA sequencing of 6 laser samples (retinal microglia, mouse, laser CNV) and 6 unlasered controls.

新生血管性年龄相关性黄斑变性（neovascular age-related macular degeneration）是西方世界最常见的致盲病因。血视网膜屏障（Blood-retina barrier）外层完整性受损以及局部炎症微环境，可诱导脉络膜新生血管（choroidal neovascularization）出芽并与周围髓系细胞（myeloid cells）紧密结合，最终引发视力损害。筛选可干预血管生成与炎症的新型靶点，对于年龄相关性黄斑变性（age-related macular degeneration, AMD）患者的未来治疗至关重要。为了识别视网膜局部吞噬细胞——小胶质细胞（microglia）中的新型潜在治疗靶点，我们在激光诱导脉络膜新生血管（laser-induced choroidal neovascularization, mCNV）小鼠模型中开展了全面的RNA测序（RNA-seq）分析。本研究鉴定出血管生成因子骨桥蛋白（Osteopontin, Opn，又称「分泌型磷蛋白1」（Spp1））是脉络膜新生血管形成过程中小鼠视网膜小胶质细胞中表达水平最高的基因之一。我们借助免疫组织化学（immunohistochemistry）技术，在Cx3cr1CreER:Rosa26-Tomato报告小鼠的募集性视网膜小胶质细胞的病变部位验证了SPP1的表达；同时与对照组相比，全视网膜组织裂解物的酶联免疫吸附试验（ELISA）结果也证实了SPP1的大量局部产生。与经免疫球蛋白G（IgG）处理的对照眼相比，通过玻璃体内注射抗SPP1抗体抑制SPP1，可显著增大病变体积。与啮齿类动物（rodents）的研究结果一致，我们通过cDNA末端大规模分析（MACE）的定量RNA测序方法，在手术提取的人脉络膜新生血管膜（human choroidal neovascular membranes, hCNV）中检测到SPP1 mRNA表达上调，并在人CNV膜中发现大量IBA1+SPP1+髓系细胞。综上，本研究结果凸显了SPP1在脉络膜新生血管形成中的关键作用，为通过抑制SPP1通路开展治疗干预提供了全新的潜在机遇。实验总体设计：对6份激光处理样本（小鼠视网膜小胶质细胞、激光诱导CNV）与6份未激光处理的对照样本开展RNA测序。