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Mutant Fibulin-3 Causes Proteoglycan Accumulation and Impaired Diffusion Across Bruch's Membrane

PURPOSE. The mutation R345W in EFEMP1 (fibulin-3) causes macular degeneration. This study sought to determine whether proteoglycan content and diffusion across Bruch's membrane are altered in Efemp1(ki/ki) mice carrying this mutation or in Efemp1(-/-) mice. METHODS. Proteoglycans in mouse Bruch's membranes were stained with Cupromeronic Blue (CB). Heparan sulfated proteoglycan (HSPG) and chondroitin/dermatan sulfate proteoglycan (C/DSPG) distributions were visualized following treatments with chondroitinase ABC (C-ABC) or nitrous acid. Total sulfated glycosaminoglycans (sGAGs) in Bruch's membrane/choroid (BrM/Ch) were measured with dimethylmethylene blue (DMMB). Matrix metalloprotease (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-3 were examined by immunofluorescence and quantified using Image J. Molecules with different Stokes radius (R-s) were allowed simultaneously to diffuse through mouse BrM/Ch mounted in a modified Ussing chamber. Samples were quantified using gel exclusion chromatography. RESULTS. HSPGs and C/DSPGs were markedly increased in Efemp1(ki/ki) Bruch's membrane, and MMP-2 and MMP-9 were decreased, but TIMP-3 was increased. Diffusion across Efemp1(ki/ki) Bruch's membrane was impaired. In contrast, the proteoglycan amount in Efemp1(-/-) Bruch's membrane was not significantly different, but the size of proteoglycans was much larger. MMP-2, MMP-3, and TIMP-3 levels were similar to that of Efemp1(+/+) mice, but they were localized diffusely in retinal pigment epithelium (RPE) cells instead of Bruch's membrane. Diffusion across Efemp1(-/-) Bruch's membrane was enhanced. CONCLUSIONS. Mutant fibulin-3 causes proteoglycan accumulation, reduction of MMP-2 and MMP-9, but increase of TIMP-3, and impairs diffusion across Bruch's membrane. Fibulin-3 ablation results in altered sizes of proteoglycans, altered distributions of MMP-2, MMP-9, and TIMP-3, and enhances diffusion across Bruch's membrane.

Identiferoai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/624956
Date20 June 2017
CreatorsZayas-Santiago, Astrid, Cross, Samuel D., Stanton, James B., Marmorstein, Alan D., Marmorstein, Lihua Y.
ContributorsUniv Arizona, Dept Ophthalmol & Vis Sci, Department of Pathology & Laboratory Medicine, Universidad Central del Caribe, Bayamón, Puerto Rico, Department of Ophthalmology, Mayo Clinic, Rochester, Minnesota, United States, Department of Ophthalmology & Vision Science, University of Arizona, Tucson, Arizona, United States, Department of Ophthalmology, Mayo Clinic, Rochester, Minnesota, United States, Department of Ophthalmology, Mayo Clinic, Rochester, Minnesota, United States
PublisherASSOC RESEARCH VISION OPHTHALMOLOGY INC
Source SetsUniversity of Arizona
LanguageEnglish
Detected LanguageEnglish
TypeArticle
RightsCopyright 2017 © The Authors. This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Relationhttp://iovs.arvojournals.org/article.aspx?doi=10.1167/iovs.17-21720

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