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Kimberly A. Toops, Jin Xu, Aparna Lakkaraju; Interactions Between Apolipoprotein E Isoforms, Hepatic Lipase, and Lipoprotein Lipase In The Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6499.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate how Apolipoprotein E isoforms modulate the activities of hepatic lipase (HL, LIPC gene) and lipoprotein lipase (LpL) in retinal pigment epithelial (RPE) cells. Abnormal cholesterol homeostasis contributes to age-related macular degeneration (AMD): Bruch’s membrane drusen is cholesterol-rich and we have shown that the lipofuscin fluorophore A2E causes RPE cholesterol storage. ApoE, HL and LpL regulate serum HDL levels and are associated with AMD. ApoE is a cholesterol transporter that occurs in three isoforms in humans: ApoE2, E3 and E4. ApoE2 increases AMD risk and ApoE4 is protective. HL and LpL facilitate lipoprotein uptake into cells and hydrolyze HDL triglycerides. However, the relationship between serum HDL and AMD risk is unclear. Because ApoE isoforms affect the ligand-binding and catalytic activities of HL and LpL in circulation, our study focuses on understanding the functional interplay between these proteins in the RPE.
Expression of ApoE, HL and LpL was measured by immunoblot. To visualize intracellular trafficking, primary pig RPE cells were transfected with GFP-tagged ApoE2, E3, and E4 and mCherry-tagged HL or LpL. Live imaging was performed using spinning disk confocal microscopy. Cholesterol levels were measured biochemically and by microscopy using filipin staining for free cholesterol and bodipy 493/503 for lipid droplets. HL and LpL activities were measured using fluorogenic substrates.
Outer segment phagocytosis, lipofuscin accumulation, treatment with statins and LXR/PPARg agonists influence protein expression, cholesterol levels and lipid droplet stores in RPE cells. In RPE cells expressing ApoE3, statin treatment significantly reduced cholesterol levels. HL and LpL expressed by the RPE are catalytically active and sensitive to inhibition by tetrahydrolipstatin. Analysis of live imaging data is ongoing.
Understanding how ApoE isoforms interact with HL and LpL is important to gain further insight into how cholesterol and lipoproteins are handled by the RPE as this has direct bearing on the delivery of dietary carotenoids to the retina. Our data could also help explain some of the variations seen in AMD risk, progression and response to drugs like statins in patients with different ApoE alleles.
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