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Samuel David Cross, Astrid Zayas, James Brett Stanton, Alan D Marmorstein, Lihua Marmortein; Diffusion across and proteoglycan content in Bruch’s membrane are altered in mice carrying an Efemp1 mutation.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2974.
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© ARVO (1962-2015); The Authors (2016-present)
A missense mutation R345W in EFEMP1 (fibulin-3) causes Malattia Leventinese/Doyne’s honeycomb retinal dystrophy (ML/DHRD), an autosomal dominant macular degenerative disease with strong similarities to age-related macular degeneration (AMD). Both ML/DHRD and AMD are characterized by sub-retinal pigment epithelium (RPE) deposits. Impairment of Bruch’s membrane’s diffusion properties is thought to contribute to the deposit formation. This study was to determine whether the diffusion across and proteoglycan content in Bruch’s membrane are altered using a mouse knock-in model carrying the R345W mutation in Efemp1.
Cupromeronic Blue coupled with chondroitinase ABC and nitrous acid treatment was used to determine the distribution patterns of proteoglycans in mouse Bruch’s membrane of wild type, knock-in carrying the R345W mutation, or knock-out lacking Efemp1. Cupromeronic Blue bound proteoglycans were visualized under electron microscopy as filaments with different sizes reflecting different types of proteoglycans. Isolated mouse Bruch’s membrane/chroid was mounted in a modified Ussing chamber with a small exposed surface area. A solution containing 4 different tracers was placed in one compartment of the Ussing chamber. Samples were collected from both chambers at various time points and the amount of each tracer determined using quantitative gel exclusion chromatography.
Two main types of filaments were found in mouse Bruch’s membrane: small rod filaments primarily in the basement membranes of the RPE and choroidal endothelium, and longer filaments in the collagenous/elastic layers. The longer filaments, which were eliminated by chondroitinase ABC, were chondroitin and dermatan sulfate proteoglycans. The small rod filaments, which were eliminated by nitrous acid treatment, were heparan sulfate proteoglycans. In Efemp1 knock-out mice, the quantity of longer filaments was similar to that of wild-type mice, but the small rod filaments decreased. In Efemp1 knock-in mice, there were higher amounts of both types of filaments. The diffusion of tracers was reduced across Bruch’s membrane of Efemp1 knock-in mice.
EFEMP1 affects the distribution of different proteoglycans. The R345W mutation causes an increase of both heparan and chondroitin/dermatan proteoglycans in Bruch’s membrane, and a decrease in permeability of Bruch’s membrane in mice.
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