title: Multiple Growth Factors, But Not VEGF, Stimulate Glycosaminoglycan Hyperelongation in Retinal Choroidal Endothelial Cells.
abstract: 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.
authors: School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC 3083 Australia;
authors: Othman
authors: O
authors: Al Gwairi
authors: School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC 3083 Australia;; Department of Immunology, Monash University, Melbourne 3004 VIC, Australia.
authors: Narin
authors: N
authors: Osman
authors: School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC 3083 Australia;
authors: Robel
authors: R
authors: Getachew
authors: Faculty of Health Sciences, University of Macau, Taipa, Macau, China;; State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510006, China.
authors: Wenhua
authors: W
authors: Zheng
authors: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510006, China.
authors: X-L
authors: XL
authors: Liang
authors: School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC 3083 Australia;
authors: Danielle
authors: D
authors: Kamato
authors: School of Pharmacy. The University of Queensland, Wooloongabba, QLD 4102, Australia.
authors: Lyna
authors: L
authors: Thach
authors: School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC 3083 Australia;; School of Pharmacy. The University of Queensland, Wooloongabba, QLD 4102, Australia.
authors: Peter J
authors: PJ
authors: Little
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism*; D000882:Haplorhini; D010750:Phosphoproteins / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000166:cytology* / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: D006025:Glycosaminoglycans; D010750:Phosphoproteins; D011509:Proteoglycans; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D013917:Thrombin
keywords: Age related macular degeneration; Bruch's membrane; VEGF; growth factors; proteoglycans; xyloside.