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I.R. Rodriguez, N. Gordiyenko, M. Campos, J.W. Lee, R.N. Farris, J. Sztein, S. Alam; RPE cell internalize LDL and oxLDL–cholesterol in large quantities in vitro and in vivo. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1842.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine if plasma LDL could be internalized by the RPE and which receptors may be involved. A secondary objective is to determine if 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 Alexa 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. Immunoblots and RT–PCR were performed using published techniques. Results: Intravenously injected rhodamine–labeled LDL was detected in the rat RPE by fluorescent confocal microscopy 24 hrs after injection. The rho–LDL was present in some areas and absent in others. Bright deposits were also observed in Bruch's membrane. Cultured ARPE19 cells were also found to readily internalize LDL and oxLDL. Using Alexa 568–labeled LDL we determined that the average cultured RPE cell could internalize approximately 6–8 pg of LDL and oxLDL in 24 hrs. Immunoblots detected the presence of CD36 and LDL–R in the culture RPE cells but not LOX–1 while RT–PCR detected mRNA for all three receptors. Dual labeling experiments using Alexa 568 labeled LDL and Alexa 488 for the immunolocalization of the receptors showed co–localization LDL–R with the internalized LDL and CD36 with oxLDL particles. Some of the LDL and oxLDL failed to co–localize with either receptor indicating that other receptors are also involved in the internalization. Conclusions: Plasma LDL readily enters the RPE in vivo via the choriocapillaris but is not found homogeneously throughout the retina. This may suggest some form of signaling mechanism initiated by the RPE to regulate choroidal endothelial cell junctions. ARPE19 cells are a good model for studying the internalization mechanisms of LDL and oxLDL in vitro. Multiple receptors seem to be involved in the LDL and oxLDL internalization. LDL may be used a vector to carry hydrophobic molecules into the RPE.
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