Al Gwairi Othman, Osman Narin, Getachew Robel, Zheng Wenhua, Liang X-L, Kamato Danielle, Thach Lyna, Little Peter J
School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC 3083 Australia;
School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC 3083 Australia;; Department of Immunology, Monash University, Melbourne 3004 VIC, Australia.
Int J Biol Sci. 2016 Jul 18;12(9):1041-51. doi: 10.7150/ijbs.16134. eCollection 2016.
A major feature of early age-related macular degeneration (AMD) is the thickening of Bruch's membrane in the retina and an alteration in its composition with increased lipid deposition. In certain pathological conditions proteoglycans are responsible for lipid retention in tissues. Growth factors are known to increase the length of glycosaminoglycan chains and this can lead to a large increase in the interaction between proteoglycans and lipids. Using choroidal endothelial cells, we investigated the effects of a number of AMD relevant growth factors TGFβ, thrombin, PDGF, IGF and VEGF on proteoglycan synthesis. Cells were characterized as of endothelial origin using the specific cell markers endothelial nitric oxide synthesis and von Willebrand factor and imaged using confocal microscopy. Cells were treated with growth factors in the presence and absence of the appropriate inhibitors and were radiolabeled with [35S]-SO4. Proteoglycans were isolated by ion exchange chromatography and sized using SDS-PAGE. Radiosulfate incorporation was determined by the cetylpyridinium chloride (CPC) precipitation technique. To measure cellular glycosaminoglycan synthesizing capacity we added xyloside and assessed the xyloside-GAGs by SDS-PAGE. TGFβ, thrombin, PDGF & IGF dose-dependently stimulated radiosulfate incorporation and GAG elongation as well as xyloside-GAG synthesis, however VEGF treatment did not stimulate any changes in proteoglycan synthesis. VEGF did not increase pAKT but caused a large increase in pERK relative to the response to PDGF. Thus, AMD relevant agonists cause glycosaminoglycan hyperelongation of proteoglycans synthesised and secreted by retinal choroidal endothelial cells. The absence of a response to VEGF is intriguing and identifies proteoglycans as a novel potential target in AMD. Future studies will examine the relevance of these changes to enhanced lipid binding and the development of AMD.
早期年龄相关性黄斑变性（AMD）的一个主要特征是视网膜中布鲁赫膜增厚，其成分发生改变，脂质沉积增加。在某些病理条件下，蛋白聚糖负责组织中的脂质潴留。已知生长因子会增加糖胺聚糖链的长度，这会导致蛋白聚糖与脂质之间的相互作用大幅增加。我们使用脉络膜内皮细胞，研究了多种与AMD相关的生长因子TGFβ、凝血酶、血小板衍生生长因子（PDGF）、胰岛素样生长因子（IGF）和血管内皮生长因子（VEGF）对蛋白聚糖合成的影响。使用特异性细胞标志物内皮型一氧化氮合成酶和血管性血友病因子将细胞鉴定为内皮来源，并使用共聚焦显微镜成像。在有和没有适当抑制剂的情况下，用生长因子处理细胞，并用[35S]-SO4进行放射性标记。通过离子交换色谱法分离蛋白聚糖，并使用SDS-PAGE进行大小分析。通过十六烷基吡啶氯化物（CPC）沉淀技术测定放射性硫酸盐掺入量。为了测量细胞糖胺聚糖的合成能力，我们添加了木糖苷，并通过SDS-PAGE评估木糖苷-糖胺聚糖。TGFβ、凝血酶、PDGF和IGF剂量依赖性地刺激放射性硫酸盐掺入、糖胺聚糖延长以及木糖苷-糖胺聚糖合成，然而VEGF处理并未刺激蛋白聚糖合成发生任何变化。相对于对PDGF的反应，VEGF没有增加pAKT，但导致pERK大幅增加。因此，与AMD相关的激动剂会导致视网膜脉络膜内皮细胞合成和分泌的蛋白聚糖的糖胺聚糖过度延长。对VEGF无反应这一点很有趣，并将蛋白聚糖确定为AMD中的一个新的潜在靶点。未来的研究将探讨这些变化与增强脂质结合及AMD发展的相关性。
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