May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Basal Reverse Cholesterol Transport of Retinal Pigment Epithelium Cell Digested Photoreceptor Outer Segment Lipids
Author Affiliations & Notes
  • K.R. Bailey
    Ophthalmology,
    University of California, San Francisco, CA
  • B.Y. Ishida
    Cardiovascular Research Institute,
    University of California, San Francisco, CA
  • K.G. Duncan
    Ophthalmology,
    University of California, San Francisco, CA
  • J.P. Kane
    Cardiovascular Research Institute,
    University of California, San Francisco, CA
  • D.M. Schwartz
    Ophthalmology,
    University of California, San Francisco, CA
    Surgery, Veterans Affairs Medical Center, San Francisco, CA
  • Footnotes
    Commercial Relationships  K.R. Bailey, None; B.Y. Ishida, None; K.G. Duncan, None; J.P. Kane, None; D.M. Schwartz, None.
  • Footnotes
    Support  Veterans Affairs Medical Center Merit Review Grant (DMS)
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 1799. doi:
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      K.R. Bailey, B.Y. Ishida, K.G. Duncan, J.P. Kane, D.M. Schwartz; Basal Reverse Cholesterol Transport of Retinal Pigment Epithelium Cell Digested Photoreceptor Outer Segment Lipids . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1799.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: To characterize factors involved in regulation of basal reverse cholesterol and phospholipid transport of digested photoreceptor outer segment (POS) lipids in human retinal pigment epithelium (RPE) cells in culture. Methods: Human RPE cells were grown at confluence on laminin coated plates for at least one week at confluence prior to treatment. Regulation of apolipoprotein E (apoE) and ABCA1 expression was assessed after incubation of RPE cells in medium containing 10–7 M cis–retinoic acid (RA) and 2.5x10–6 M 22(R) hydroxycholesterol for 12 to 36 hours. RNA was detected by reverse transcriptase–polymerase chain reaction (RT–PCR) using primer sets specific to human apo E, ABCA1, and scavenger receptors BI and BII (SR–BI, and SR–BII). Secreted apo E was detected by western blotting of cell culture medium. ABCA1, SR–BI and SR–BII were detected by western blotting or immune precipitation of cell extracts. Lipidation state of apoE was determined by density centrifugation followed by western blotting, and in some cases electron microscopy. To assess POS lipid transport, RPE cells were grown on Transwell (Costar) plates and incubated with medium containing [14C]–POS in the apical chambers and medium containing different size fractions of human high density lipoprotein (HDL) in the basal chambers. After 24 hrs lipoproteins were purified from basal medium by centrifugation. [14C]–Lipids in HDL were quantified by scintillation counting. Results: RT–PCR and western blotting revealed that RPE cells express several proteins known to be involved in reverse cholesterol and phospholipid transport including apoE, SR–BI, SR–BII , and ABCA1. Expression of apoE and ABCA1 was up–regulated (∼1.5 to 2–fold) by ligands for retinoid X receptor (RXR) and liver X receptor (LXR). RPE cells secrete partially lipidated apoE, consistent with phospholipid disc form. The presence of HDL in the basal culture medium stimulated basal transport of [14C]–labeled POS lipids. Large HDL species were good acceptors of basally transported [14C]–labeled POS lipids; small (pre–beta) HDL was a poor acceptor of [14C]–labeled POS lipids. Conclusions: The results imply that reverse cholesterol and phospholipid transport is an important mechanism for trafficking digested POS lipids by the RPE. The finding that HDL stimulates basal lipid transport and that large HDL species are better lipid acceptors than the small HDL species such as pre–beta HDL may be relevant to the development of dry AMD.

Keywords: age–related macular degeneration • retinal pigment epithelium • lipids 
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