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L. Wang, M. E. Clark, D. K. Crossman, K. Kojima, J. D. Messinger, J. A. Mobley, C. A. Curcio; Abundant Lipid and Protein Components of Drusen. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6168.
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© ARVO (1962-2015); The Authors (2016-present)
Drusen are extracellular lesions characteristic of aging and age-related maculopathy (ARM) known to contain ~150 components, both lipids and proteins. We determined the relative proportions of these components in extra-macular drusen isolated from aged human retina.
Drusen capped with retinal pigment epithelium (RPE) and RPE were isolated manually from 36 donor eyes obtained <6 hr after death and either frozen (prep1), preserved in paraformaldehyde (prep2), or paraformaldehyde and glutaraldehyde (prep3). Total protein was determined by bicinchoninic acid (prep1). Component proteins (prep2) were extracted using novel methods, separated, subjected to tryptic digestion and LC-MS(MS)2 analysis using an ion trap mass spectrometer, and identified with reference to SEQUEST, X!TANDEM, and MASCOT databases. Major lipid classes were separated from extracts (prep3) using thin layer chromatography and solvent systems for neutral or polar lipids and quantified by densitometry. Protein and lipid quantities were converted to ng/druse. Transmission electron microscopy (prep3) and indirect immunofluorescence (prep2) were used to localize lipid-containing components and specific proteins, respectively.
Major druse components are esterified cholesterol (EC), phosphatidylcholine, and total protein (37.47±13.70, 36.89±12.86, and 42.96±11.48 ng/druse, respectively). Lipid-containing particles were electron dense and dispersed (median 78 nm), occupying 37-44% of druse volume. Of 20 major proteins, 19 had been previously identified through proteomics or immunohistochemistry in drusen (e.g., vitronectin, TIMP-3, complement components 5, 8 and 9, apoE) or RPE (e.g., retinol dehydrogenase, scavenger receptor BII). All proteins were found in drusen and RPE, with higher ion intensities in drusen. C8 was localized to drusen.
These data validate new extraction and de-cross-linking techniques suitable for preserved tissues, reveal druse proteins that are abundant, survive fixation, and ionize well, and suggest that a catalogue of major druse proteins is attainable. Knowledge of druse components will inform the creation of model lesions and the therapeutic goal of mitigating or eliminating them. This effort will require accommodating the substantial volume of neutral lipids, particularly EC, which likely derive from large lipoprotein particles secreted by RPE into Bruch’s membrane (Wang IOVS 50:870, 2009).
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