Sphingosine-1-phosphate (S1P) is a lysophospholipid mediator, promoting angiogenesis and inflammation via interactions with its receptors (S1P1-5), but the receptors and signaling pathways responsible for the progression of choroidal neovascularization (CNV) remain unknown. We investigated the roles of S1P/S1P receptors in RPE cells. ARPE-19 cells were treated with S1P dissolved in carrier proteins of albumin or apolipoprotein M (ApoM). The mRNA expression levels of interleukin-8 (IL-8), C-C motif chemokine ligand 2 (CCL2), and vascular endothelial growth factor (VEGF) were evaluated using quantitative real-time polymerase chain reaction. The protein level of hypoxia-inducible factor (HIF)-1α was assessed via enzyme-linked immunosorbent assay. HIF transcriptional activity was evaluated with a dual-reporter luciferase assay. Cellular barrier integrity was evaluated using transepithelial electrical resistance and the FITC-dextran permeability assay. The suppressive effect of an S1P antagonist on CNV progression was investigated with a laser-induced CNV model in mice. The increase in expression of IL-8, CCL2, and VEGF due to albumin-bound S1P was significantly mitigated by an S1P2 antagonist. The expression of HIF-1α significantly decreased with inhibition of S1P2 and S1P3. In addition, albumin-bound S1P disrupted the barrier integrity of retinal pigment epithelial cells via S1P2, whereas integrity was strengthened by ApoM-bound S1P. CNV lesions were significantly reduced in the mouse model with intravitreal injection of S1P2 antagonist. This study demonstrated that S1P significantly promotes angiogenesis, inflammation, and barrier integrity, which was attenuated by inhibition of S1P2 or S1P3, suggesting that regulation of S1P2 and S1P3 is a novel therapeutic target for CNV.

中文翻译：

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1-磷酸鞘氨醇受体对视网膜色素上皮细胞屏障功能和血管生成作用的作用。

鞘氨醇-1-磷酸酯（S1P）是一种溶血磷脂介质，通过与其受体（S1P1-5）的相互作用促进血管生成和炎症，但是负责脉络膜新血管形成（CNV）进程的受体和信号传导途径仍然未知。我们调查了RPE细胞中S1P / S1P受体的作用。用溶解在白蛋白或载脂蛋白M（ApoM）的载体蛋白中的S1P处理ARPE-19细胞。使用定量实时聚合酶链反应评估白细胞介素8（IL-8），CC基序趋化因子配体2（CCL2）和血管内皮生长因子（VEGF）的mRNA表达水平。通过酶联免疫吸附试验评估缺氧诱导因子（HIF）-1α的蛋白水平。HIF转录活性用双重报告荧光素酶测定法进行评估。使用跨上皮电阻和FITC-葡聚糖渗透性测定法评估细胞屏障的完整性。用小鼠激光诱导的CNV模型研究了S1P拮抗剂对CNV进展的抑制作用。由于白蛋白结合的S1P，IL-8，CCL2和VEGF的表达增加被S1P2拮抗剂明显缓解。HIF-1α的表达随S1P2和S1P3的抑制而显着降低。此外，结合白蛋白的S1P通过S1P2破坏了视网膜色素上皮细胞的屏障完整性，而结合了ApoM的S1P增强了完整性。玻璃体内注射S1P2拮抗剂可在小鼠模型中显着减少CNV病变。这项研究表明，S1P可显着促进血管生成，炎症和屏障完整性，