Expression of Reverse Cholesterol Transport Proteins SR-BI, SR-BII and ABCA1 in the Human Retinal Pigment Epithelium

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