RAGE ligand upregulation of VEGF secretion in ARPE-19 cells
RAGE ligand upregulation of VEGF secretion in ARPE-19 cells
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DOI：
10.1167/iovs.06-0738
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发表时间：
2007-03-01
影响因子：
4.4
通讯作者：
Barile, Gaetano R.
中科院分区：
文献类型：
--
作者：
Ma, Wanchao;Lee, Song Eun;Barile, Gaetano R.
目的. VEGF在刺激新生血管性年龄相关性黄斑变性（AMD）中的重要性是公认的，但诱导VEGF局部上调的起始因素仍不清楚。本研究旨在验证以下假设：AGES（晚期糖基化终产物受体[AGES]）通过其配体（包括AGES、淀粉样蛋白-P肽（A β）和S100 B/钙颗粒蛋白，其中一些是玻璃疣和布鲁赫膜沉积物的已知组分）激活调节视网膜色素上皮（RPE）细胞分泌VEGF。ARPE-19细胞用于所有实验。用编码人RAGE的信号转导突变体的构建体转染细胞以评估细胞内信号传导的RAGE依赖性。通过ELISA和定量实时PCR评估VEGF分泌和基因表达。采用SDS-PAGE和分子排阻色谱分析了S100B在变性和非变性条件下巯基氧化后的结构变化。NF-κ B活化通过电泳迁移率变动分析（EMSA）评估。使用小白菊内酯评估NF-κ B抑制的影响。ARPE-19细胞在正常细胞培养条件下基本分泌VEGF。固定化配体的EGF的分泌增加RAGE依赖性的方式。相反，可溶性AGE-BSA、新鲜A β和S100 B在增加VEGF分泌方面效果较差。对A β的研究表明，A β的寡聚体和表面固定形式，而不是A β的可溶性单体形式，是VEGF分泌的有效上调剂。氧化S100 B的巯基导致形成的寡聚体，显示出不同的生物活性与简单的二聚体形式相比。RAGE介导的ARPE-19细胞VEGF分泌的上调在很大程度上依赖于NF-κ B，如小白菊内酯研究所示。VEGF的固定化或寡聚化配体诱导R-PE细胞增加VEGF分泌。NF-κ B B在RAGE依赖性RPE分泌VEGF中起重要作用。在AMD。在RPE细胞中，VEGF轴的激活可能导致VEGF的上调，从而潜在地诱发或传播新生血管性黄斑疾病。
PURPOSE. The importance of VEGF in stimulating neovascular age-related macular degeneration (AMD) is well-recognized, but the initiating factors that induce local upregulation of VEGF remain unclear. The current study was conducted to test the hypothesis that activation of RAGE (receptor for advanced glycation end products [AGES]) by its ligands, including AGES, amyloid-P peptide (A beta), and S100B/calgranulins, some of which are known components of drusen and Bruch's membrane deposits, modulate secretion of VEGF by retinal pigment epithelial (RPE) cells.METHODS. ARPE-19 cells were used for all experiments. The cells were transfected with constructs encoding a signal transduction mutant of human RAGE to assess the RAGE-dependence of intracellular signaling. VEGF secretion and gene expression were assessed by ELISA and quantitative real-time PCR. SDS-PAGE and size exclusion chromatography were performed to analyze the structural changes of S100B after oxidation of its thiol groups under denaturing and nondenaturing conditions, respectively. NF-kappa B activation was assessed via electrophoretic mobility shift assay (EMSA). The impact of the NF-kappa B inhibition was assessed by using parthenolide.RESULTS. ARPE-19 cells basally secreted VEGF under normal cell culture conditions. Immobilized ligands of RAGE increased EGF secretion in a RAGE-dependent manner. In contrast, soluble AGE-BSA, fresh A beta, and S100B were less effective in increasing VEGF secretion. Studies with A beta demonstrated that oligomeric and surface-immobilized forms of A beta, but not soluble monomeric forms of A beta, were effective upregulators of VEGF secretion Via RAGE. Oxidation of S100B's thiol groups resulted in the formation of oligomers that displayed distinct RAGE biological activity compared with the simple dimeric form. RAGE-mediated upregulation of VEGF secretion by ARPE-19 cells was largely dependent on NF-kappa B, as indicated by studies with parthenolide.CONCLUSIONS. Immobilized or oligomerized ligands for RAGE induce R-PE cells to increase VEGF secretion. NF-kappa B plays a central role in RAGE-dependent RPE secretion of VEGF. In AMD. activation of the RAGE axis in RPE cells may contribute to upregulation of VEGF, potentially inciting or propagating neovascular macular disease.