RPE cells internalize low-density lipoprotein (LDL) and oxidized LDL (oxLDL) in large quantities in vitro and in vivo.
OBJECTIVE
To determine whether plasma low-density lipoprotein (LDL) could be internalized by the RPE and which receptors may be involved. A secondary objective was to determine whether ARPE19 cells could be used as a model to investigate cholesterol processing in the RPE.


METHODS
Commercially available human LDL was labeled with rhodamine or AlexaFluor 568. Immunofluorescence was performed using commercially available antibodies to LDL-R, CD36, and LOX-1. Cells and tissues were imaged using epifluorescence and confocal fluorescence microscopy. Immunoblot analysis and RT-PCR were performed using published techniques.


RESULTS
Intravenously injected rhodamine-labeled LDL (rhoLDL) was detected in the rat RPE by fluorescence confocal microscopy 24 hours after injection. The rhoLDL was present in some areas and absent in others. Cultured ARPE19 cells were also found to internalize LDL and oxidized LDL (oxLDL) readily. Using AlexaFluor 568-labeled LDL we determined that the average cultured RPE cell could internalize approximately 12 to 16 pg of LDL and oxLDL in 24 hours. Immunoblots readily detected the presence of CD36 and LDL-R in the cultured RPE cells but not LOX-1, whereas RT-PCR detected mRNA for all three receptors. Dual-labeling experiments using AlexaFluor 568-labeled LDL and AlexaFluor 488 for the immunolocalization of the receptors showed colocalization of LDL-R with the internalized LDL and CD36 with oxLDL particles.


CONCLUSIONS
Plasma LDL readily enters the RPE through the choriocapillaris but is not found homogeneously throughout the retina. This may suggest some form of regulation to the permeability of the fenestrated choroidal endothelial cell junctions. ARPE19 cells are a good model for studying the internalization mechanisms of LDL and oxLDL in vitro. LDL may be used as a vector to carry hydrophobic molecules into the RPE.
Laboratory of Retinal Cell and Molecular Biology, Section on Mechanisms of Retinal Diseases, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Nataliya
N
Gordiyenko
Maria
M
Campos
Jung Wha
JW
Lee
Robert N
RN
Fariss
Jorge
J
Sztein
Ignacio R
IR
Rodriguez
D000818:Animals; D015153:Blotting, Western; D018955:CD36 Antigens / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010084:Oxidation-Reduction; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018955:CD36 Antigens; D005456:Fluorescent Dyes; D008077:Lipoproteins, LDL; D012333:RNA, Messenger; D011973:Receptors, LDL; C099252:oxidized low density lipoprotein