Lipoprotein–like Particles (LLP) in Human Bruch’s Membrane: Isolation and Initial Characterization

Abstract

Abstract: : Purpose: To isolate and characterize LLP from Bruch’s membrane (BrM)/ choroid (Ch) in human eyes. Methods: BrM/Ch was isolated from 20 eyes from 10 donors >60 yr old with grossly normal maculas. LLP were released by high–salt buffer, fractionated by density gradient ultracentrifugation, and characterized by determining 1) cholesterol, triglyceride, and phospholipid concentration (by enzymatic colorimetry, and for cholesterol, fluorimetry), 2) cholesteryl ester composition (by electrospray ionization mass spectrometry, ESI/MS), and 3) particle morphology (by negative stain electron microscopy). 4) Apolipoprotein (apo) gene expression was determined with RT–PCR, Western blot analysis, and immunofluorescence of retinal/choroidal cryosections. Results: A pooled fraction of LLP released from BrM/Ch (concentrated total LLP, d<1.24 g/mL fraction) was fractionated into 2 peaks. A large Peak 1 (with plasma LDL–HDL density range), containing predominantly phospholipid and unesterified cholesterol, was morphologically heterogeneous. A small Peak 2 (with plasma VLDL density range), enriched with esterified cholesterol, contained ∼100 nm diameter round electron–lucent particles. Both peaks contained apoB and apoA–I, RPE and retina contained apoA–I mRNA transcripts, and BrM and drusen contained apoA–I immunoreactivity. Both density peaks, native RPE, and BrM/Ch from fresh eyes were cholesteryl linoleate–enriched and contained a small amount of cholesteryl docosahexanoate. Conclusions: LLP isolated from BrM/Ch do not resemble plasma lipoproteins in either density profile, cholesterol distribution, or morphology. Peak 2 contains EC–rich particles resembling BrM particles in situ. As BrM/Ch (but not cornea and sclera) in preserved eyes is cholesteryl oleate–enriched (Curcio, ARVO2002; Haimovici IOVS2001), cholesteryl esters in BrM/Ch may respond to long–term storage differently than esters of plasma lipoprotein origin accumulated in other ocular tissues. Evidence for apoB and intraocular apoA–I expression supports an emerging hypothesis that the RPE assembles and secretes a large, possibly novel, lipoprotein particle.

Keywords: Bruch's membrane • lipids • drusen