May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Expression of Reverse Cholesterol Transport Proteins SR-BI, SR-BII and ABCA1 in the Human Retinal Pigment Epithelium
Author Affiliations & Notes
  • J.M. Stewart
    Dept Ophthalmology, Univ Calif-San Francisco, San Francisco, CA, United States
  • K.R. Bailey
    Dept Ophthalmology, Univ Calif-San Francisco, San Francisco, CA, United States
  • K.G. Duncan
    Dept Ophthalmology, Univ Calif-San Francisco, San Francisco, CA, United States
  • J.P. Kane
    Cardiovasc Research Inst, Univ Calif-San Francisco, San Francisco, CA, United States
  • D.M. Schwartz
    Dept Ophthalmology, Veterans Affairs Medical Center, San Francisco, CA, United States
  • Footnotes
    Commercial Relationships  J.M. Stewart, None; K.R. Bailey, None; K.G. Duncan, None; J.P. Kane, None; D.M. Schwartz, None.
  • Footnotes
    Support  That Man May See, Inc., Research Grant and a Veterans Administration Merit Review Grant
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 1711. doi:
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      J.M. Stewart, K.R. Bailey, K.G. Duncan, J.P. Kane, D.M. Schwartz; Expression of Reverse Cholesterol Transport Proteins SR-BI, SR-BII and ABCA1 in the Human Retinal Pigment Epithelium . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1711.

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

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Abstract

Abstract: : Purpose: To determine whether human RPE cells in culture express the following reverse cholesterol transport proteins: scavenger receptor BI (SR-BI), scavenger receptor BII (SR-BII), and ATP-binding cassette protein A1 (ABCA1); to confirm this expression in fixed human tissue sections. Methods: Primary cultures of human RPE cells were grown for at least one week at confluence prior to RNA isolation or immunofluorescent staining. RNA expression was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) of total cellular RNA using primer sets specific to human SR-BI, SR-BII or ABCA1. Immunofluorescent staining was performed using a biotinylated secondary antibody and fluoresceinated avidin. Either affinity- purified antibodies specific to SR-BI and SR-BII peptides, or commercially available antiserum to ABCA1 were used as primary antibodies, with the appropriate controls. SR-BI, SR-BII and ABCA1 proteins were visualized in RPE cells by standard and confocal immunofluorescent microscopy. Proteins were visualized in paraffin embedded fixed human tissue by standard immunofluorescent microscopy. Results: Primary cultures of human RPE cells expressed mRNAs for SR-BI, SR-BII and ABCA1. Immunofluorescent microscopy confirmed that the protein for these mRNAs was also expressed in these cells. Confocal microscopy revealed a basolateral expression pattern. Staining of fixed human tissue sections confirmed that these proteins are expressed in vivo. Conclusions: Human RPE cells express mRNA and protein for the reverse cholesterol transport proteins SR-BI, SR-BII, and ABCA1. The proteins were expressed on basolateral cell membranes in vitro, consistent with their proposed function of transporting cholesterol and phospholipids of photoreceptor origin out of the RPE and into Bruch’s membrane.

Keywords: age-related macular degeneration • retinal pigment epithelium • immunohistochemistry 
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