Dataset Information

Utilizing Recombinant Spider Silk Proteins To Develop a Synthetic Bruch's Membrane for Modeling the Retinal Pigment Epithelium.

ABSTRACT: Spider silks are intriguing biomaterials that have a high potential as innovative biomedical processes and devices. The intent of this study was to evaluate the capacity of recombinant spider silk proteins (rSSps) as a synthetic Bruch's membrane. Nonporous silk membranes were prepared with comparable thicknesses (<10 μm) to that of native Bruch's membrane. Biomechanical characterization was performed prior to seeding cells. The ability of RPE cells (ARPE-19) to attach and grow on the membranes was then evaluated with bright-field and electron microscopy, intracellular DNA quantification, and immunocytochemical staining (ZO-1 and F-actin). Controls were cultured on permeable Transwell support membranes and characterized with the same methods. A size-dependent permeability assay, using FITC-dextran, was used to determine cell-membrane barrier function. Compared to Transwell controls, RPE cells cultured on rSSps membranes developed more native-like "cobblestone" morphologies, exhibited higher intracellular DNA content, and expressed key organizational proteins more consistently. Comparisons of the membranes to native structures revealed that the silk membranes exhibited equivalent thicknesses, biomechanical properties, and barrier functions. These findings support the use of recombinant spider silk proteins to model Bruch's membrane and develop more biomimetic retinal models.

SUBMITTER: Harris TI

PROVIDER: S-EPMC8078582 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Utilizing Recombinant Spider Silk Proteins To Develop a Synthetic Bruch's Membrane for Modeling the Retinal Pigment Epithelium.

Harris Thomas I TI Paterson Chase A CA Farjood Farhad F Wadsworth Ian D ID Caldwell Lori L Lewis Randolph V RV Jones Justin A JA Vargis Elizabeth E

ACS biomaterials science & engineering 20190716 8

Spider silks are intriguing biomaterials that have a high potential as innovative biomedical processes and devices. The intent of this study was to evaluate the capacity of recombinant spider silk proteins (rSSps) as a synthetic Bruch's membrane. Nonporous silk membranes were prepared with comparable thicknesses (<10 μm) to that of native Bruch's membrane. Biomechanical characterization was performed prior to seeding cells. The ability of RPE cells (ARPE-19) to attach and grow on the membranes w ...[more]

PMID: 33448804

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Project description:Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

Project description:PurposeTo describe and quantify Bruch's membrane (BM) and retinal pigment epithelium (RPE) separation using spectral-domain (SD) optical coherence tomography (OCT) in patients affected by inherited macular degenerations associated with BM thickening.MethodsPatients with molecularly confirmed Sorsby fundus dystrophy (SFD), dominant drusen (DD), and late-onset retinal degeneration (L-ORD) were included in this retrospective study. Each disease was classed as early stage if subjects were asymptomatic, intermediate stage if they had nyctalopia alone, and late stage if they described loss of central vision. The main outcome was measurement of BM-RPE separation on SD-OCT. The BM-RPE separation measurements were compared against those in normal age-matched controls.ResultsSeventeen patients with SFD, 22 with DD, and eight with L-ORD were included. BM-RPE separation on SD-OCT demonstrated a high test-retest and interobserver reproducibility (intraclass correlation coefficients >0.9). BM-RPE separation was not identified in normal subjects. In SFD, there was greater BM-RPE separation in late-stage disease compared with intermediate-stage patients both at subfoveal (P < 0.05) and juxtafoveal (P < 0.01) locations. In DD, there was increased BM-RPE separation in late-stage disease compared with early stage at subfoveal (P < 0.001) and juxtafoveal (P < 0.05) topographies. There was no significant difference in BM-RPE separation between disease stages in L-ORD.ConclusionsBM-RPE separation is a novel, quantifiable phenotype in the three monogenic macular dystrophies studied, and may be an optical correlate of the histopathological thickening in BM that is known to occur. BM-RPE separation, as measured by OCT, varies with stage of disease in SFD and DD, but not in L-ORD.Translational relevanceSFD, DD, and L-ORD are associated with BM thickening. In this group of patients, OCT assessment of macular structure identifies a separation of the usually single, hyperreflective line thought to represent BM and the overlying RPE. This separation is a novel and quantifiable feature of disease staging in SFD and DD.

Dataset Information

Utilizing Recombinant Spider Silk Proteins To Develop a Synthetic Bruch's Membrane for Modeling the Retinal Pigment Epithelium.

Publications

Utilizing Recombinant Spider Silk Proteins To Develop a Synthetic Bruch's Membrane for Modeling the Retinal Pigment Epithelium.

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