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Una L Kelly, Michael Landowski, Nikolai Skiba, Jian Liu, Catherine Bowes Rickman; Lipoprotein synthesis and deposition in the eye is regulated by the complement system and glycosaminoglycans (GAGs).. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5249.
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© ARVO (1962-2015); The Authors (2016-present)
We have shown that aged mice, heterozygous for complement factor H (Cfh+/-) and fed a high fat, high cholesterol (HFC) diet develop significantly more sub-RPE deposits than age-matched C57Bl/6J mice. We now show these mice produce higher plasma concentrations of VLDL than control mice and demonstrate that this is likely due to an increase in complement activation. We hypothesize that lipoprotein changes may be responsible for the increase in sub-RPE accumulation of lipid/protein aggregates and molecules that compete with these larger lipoproteins for binding to the glycosaminoglycans (GAGs) in the extracellular matrix (ECM) may be useful for reducing these deposits.
C57Bl/6J mice were fed a HFC diet with or without cobra venom factor (CVF) to activate complement. Blood was taken 1 and 3 days after treatment. Plasma lipoproteins were separated by FPLC and fractions assayed for cholesterol. A binding assay that coats 96-well plates with primary porcine RPE-derived ECM was developed. The heparan sulfate (HS) present in this ECM was compared to the HS isolated from human Bruch’s membrane using disaccharide analysis with a LTQ Orbitrap-MS. Human Bruch’s membrane/choroid tissue was incubated with various GAGs or buffer controls and the lipoproteins remaining on the tissue were analyzed using FPLC fractionation and cholesterol and apolipoprotein measurements.
Activating the complement system in C57Bl/6J mice with CVF resulted in cleavage of C3 and an increase in VLDL in plasma. ECM derived from primary porcine RPE was found to have a similar HS fingerprint to that found in both porcine and human Bruch’s membrane making it a valuable binding matrix to model molecular binding to Bruch’s membrane. VLDL, LDL and CFH can bind to this RPE-derived ECM, the technique is sensitive enough to detect differences in oxidized LDL binding versus native LDL and subtle CFH variant differences in binding were observed. As shown previously CFH does compete with lipoproteins for binding sites in the ECM as do short HS oligosaccharides. These GAGs can also remove lipoproteins from human donor Bruch’s membrane/choroid.
Short HS oligosaccharides may be a potential new class of therapeutic molecule that could disrupt lipoprotein/protein aggregates at an early stage in age-related macular degeneration.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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