Gene expression as assessed by qRT-PCR.

<p><b>A</b>, NR. <b>B</b>, RPE. Key proteins involved in homeostatic regulation, synthesis, uptake and efflux of cholesterol were evaluated. Their gene expression was measured and normalized based on the expression of ACTB in the same sample. For each gene, the mean of the gene expression in 6 donors was then calculated and assigned a value of “1” on the Y-axis. The gene expression in the individual sample was then compared to this mean value giving a number of relative gene expression on Y-axis. Data are presented in the form of Whisker-box plots in which the box area encompasses middle 50% of expression level values, the dotted line represents the sample median and the whiskers represent upper 25% (top whisker) and lower 25% (bottom whisker) of expression level values.</p

Human Eye.

<p><b>A,</b> cross-section of a human eye. The neurosensory retina (central nervous system) and choroid (vascular bed for the photoreceptors and RPE) are part of the inner lining. The macula (a 6 mm diameter area responsible for central vision) and fovea (a depression in the macula) are bracketed. Schematic available at <a href="http://www.nei.nih.gov/health/eyediagram/index.asp" target="_blank">http://www.nei.nih.gov/health/eyediagram/index.asp</a>. <b>B,</b> human retina and choroid <i>in vivo</i>. Spectral domain optical coherence tomography with enhanced depth imaging. Scan of macula, courtesy of R.F. Spaide, MD. <b>C,</b> chorioretinal cells and layers. Cells: RPE, retinal pigment epithelium (nurse cells to the photoreceptors); C, cone photoreceptor; R, rod photoreceptor; H, horizontal cell (interneuron); B, bipolar cell (interneuron); M, Müller cell (radial glial cell); Am, amacrine cell (interneuron); DA, displaced amacrine cell (interneuron); G, ganglion cell (output neuron). Müller cells (M) extend almost the width of the retina; their apical processes form the ELM, and their foot processes partially form the ILM. Layers: ChC, choriocapillaris (capillary bed for RPE and photoreceptors); BrM, Bruch's membrane (vessel wall and RPE substratum); ELM, external limiting membrane (junctional complexes); ONL, outer nuclear layer; OPL, outer plexiform layer (synapses); INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, nerve fiber layer (ganglion cell axons); ILM, inner limiting membrane. Non-photoreceptor layers of the retina are supplied by the retinal circulation (not shown). Graphics by D. Fisher; inspired by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037926#pone-0037926-g004" target="_blank">Figure 4</a>–2 of Ryan SJ, editor. Retina: Mosby; 2006.</p

Histochemical detection of UC and EC with filipin.

<p>The three sections in each panel are the phase contrast image (left) and images with (middle) or without (right) the channel for propidium iodide (in red) to show nuclei. <b>A</b>, control for staining of UC (no treatment with filipin). <b>B</b>, staining of UC with filipin (in cyan). <b>C</b>, control for staining of EC (extracted with ethanol but not treated with cholesterol esterase or filipin). <b>D</b>, control for completeness of UC removal (extracted with ethanol and treated with filipin but not cholesterol esterase). <b>E</b>, staining of EC (extracted with ethanol and sequentially treated with cholesterol esterase and filipin). Exposure time in panels A and B was 15 msec and that in panels C–E was 400 msec. Faint fluorescence in panel C with no filipin treatment is due to increased exposure time as compared to panel A leading to detection of autofluorescence from the RPE. Fluorescence is not increased in panel D indicating complete removal of UC, yet is more pronounced in panel E indicating that EC is mainly present in BrM.</p

Gene expression of scavenger receptors as assessed by qRT-PCR.

<p><b>A,</b> NR. <b>B</b>, RPE. Donors, gene normalization and data presentation are the same as in Fig. 4.</p

Cholesterol homeostasis.

<p>Simplified representation of the coordinate regulation of the pathways of cholesterol (CHO) input and output indicating proteins investigated in the present work. HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase, the rate limiting enzyme in cholesterol biosynthesis; LDLR and CD36, receptors recognizing low density lipoproteins (LDL); LXR, the liver X receptor, transcription factor suppressing the expression of LDLR and activating the expression of SREBP1 and ABCA1; SREBP, SCAP and Insig, proteins activating the expression of HMGCR and LDLR; CYP27A1, CYP46A1 and CYP11A1, cytochromes P450 that metabolize cholesterol to 5-cholestenoic acid (27COOH), 27-hydroxycholesterol (27OH), 24-hydroxycholesterol (24OH) and 22R-hydroxycholesterol (22ROH), respectively; ABCA1, cholesterol efflux transporter; SR-BI and SR-BII, scavenger receptor SR-BI and its splice variant SR-BII recognizing HDL. Arrows and blunt ends indicate positive and negative regulators, respectively. The dumbbell-shaped object in the middle of the figure shows the SREBP pathways when cholesterol levels are high (bottom compartment) and low (top compartment). SREBPs are synthesized on the endoplasmic reticulum (ER) and form a complex with the escort protein SCAP. When sterol levels are low (top compartment), SCAP transports SREBPs to the Golgi, where the active form of SREBP is generated and initiates the transcription of target genes in the nucleus. When sterol concentrations are high (bottom compartment), cholesterol binds to SCAP triggering its interaction with the ER-resident protein Insig, whereas oxysterols bind to Insig eliciting its complex formation with SCAP. As a result, the SREBP/SCAP/Insig complex is retained in the ER. The cartoon showing the regulation of cholesterol biosynthesis is reproduced/adapted with permission from Meer, G. and Kroon, A. (2011) J. Cell Sci., 124, 5–8 (<a href="http://jcs.biologists.org/content/124/1/5.long" target="_blank">http://jcs.biologists.org/content/124/1/5.long</a>).</p

Gene expression of cholesterol-catabolizing P450s as assessed by qRT-PCR.

<p><b>A,</b> NR. <b>B</b>, RPE. <b>C</b>, brain. In <b>A</b> and <b>B,</b> data presentation is the same as in Fig. 4; in <b>C,</b> each bar represents the mean ± SD of the independent measurements in 6 donors of the retina and 4 donors of the brain. The latter are not the same as donors of the retina. Information on brain donors could be found in ref. 56. In all panels, gene normalization is as in Fig. 4.</p