Nuclear Receptor Atlases of Choroidal Tissues Reveal Candidate Receptors Associated with Age-Related Macular Degeneration

Abstract

The choroid is a vulnerable tissue site in the eye, impacted in several blinding diseases including age related macular degeneration (AMD), which is the leading cause of central vision loss in the aging population. Choroidal thinning and choriocapillary dropout are features of the early form of AMD, and endothelial dysfunction and vascular changes are primary characteristics of the neovascular clinical sub-type of AMD. Given the importance, the choroidal endothelium and outer vasculature play in supporting visual function, a better understanding of baseline choroidal signaling pathways engaged in tissue and cellular homeostasis is needed. Nuclear receptors are a large family of transcription factors responsible for maintaining various cellular processes during development, aging and disease. Herein we developed a comprehensive nuclear receptor atlas of human choroidal endothelial cells and freshly isolated choroidal tissue by examining the expression levels of all members of this transcription family using quantitative real time PCR. Given the close relationship between the choroid and retinal pigment epithelium (RPE), this data was cross-referenced with the expression profile of nuclear receptors in human RPE cells, to discover potential overlap versus cell-specific nuclear receptor expression. Finally, to identify candidate receptors that may participate in the pathobiology of AMD, we cataloged nuclear receptor expression in a murine model of wet AMD, from which we discovered a subset of nuclear receptors differentially regulated following neovascularization. Overall, these databases serve as useful resources establishing the influence of nuclear receptor signaling pathways on the outer vascular tissue of the eye, while providing a list of receptors, for more focused investigations in the future, to determine their suitability as potential therapeutic targets for diseases, in which the choroid is affected.

Keywords: age-related macular degeneration; choroidal endothelial cells; choroidal injury; nuclear receptor atlas.

Figure 1

Characterization of human primary choroidal endothelial cells (1°CEC). (A) 1°CEC stain 83% positive for CD31 as demonstrated by flow cell cytometry analysis. (B) 1°CEC grown to confluence maintain a “cobblestone-like” appearance as observed with light microscopy. (C) 1°CEC stain positive for pan-endothelial surface marker CD31 (green) and Von Willebrand Factor (vWF, red). (D) Tube formation assay of 1°CECs. FSC-A = forward scatter area; aquablue = live-dead stain for live cell gating. Magnification bar = 50 μm.

Figure 2

Nuclear receptor expression profiles by qPCR analysis of primary human choroidal endothelial cells (1°CEC) and freshly isolated human choroid (fCh). Expression profiles of nuclear receptors and related transcription factors are categorized into steroid hormone nuclear receptors (A), non-steroid hormone receptors (B), adopted orphan nuclear receptors (C), AhR and ARNT (D), which are not classical nuclear receptors but important transcription factors involved in toxin clearance and orphan nuclear receptors (E). Data for nuclear receptors are presented as mean arbitrary expression ratios ± SEM for 1°CEC and fCh. The levels of nuclear receptor expression in 1°CEC (F) and fCh (G) are displayed in pie charts on the left, and the nuclear receptor common names are listed according to their detectable levels on the right. Normalized nuclear receptor mRNA expression levels were defined as absent if the Ct value was ≥35, low if the level was less than 0.75, medium if the level was between 0.75 and 1.0, and high if the level was greater than 1. NR = nuclear receptor.

Figure 3

Nuclear receptor expression in human choroidal endothelial cells compared to the choroid and biological pathway analyses. (A) Venn diagram illustrates overlapping expression of nuclear receptors in primary choroidal endothelial cells (1°CEC) and freshly isolated choroidal endothelial cells (fCh). Diagram was made using Venny 2.1.0. (B) Gene Ontology analysis of 32 common nuclear receptors in the 1°CEC and fCh were significantly enriched in a number of pathways. Stacked bar graph shows % of genes in the pathway (red), −log10 p value of each category in green and number of genes next to each bar graph. Analysis was performed using the DAVID functional annotational tool with the Reactome database.

Figure 4

Commonly expressed nuclear receptors in the human choroidal tissue models versus RPE models. Commonly expressed nuclear receptors are shown in a four-set Venn diagram, with an overlap of freshly isolated choroid tissue (fCh), cultured primary choroidal endothelial cells (1°CEC), freshly isolated retinal pigment epithelial cells (fRPE) and cultured primary RPE is presented here. The Venn diagram was drawn using VENNY 2.1.0. Common nuclear receptors expressed in all four categories are listed in the table with color coded legend. * 26 common nuclear receptors found in all model systems are shown in the adjoining table.

Figure 5

Laser induced CNV mouse disease model paradigm. (A) Schematic of the experimental plan. On day 0 lesions in mice eyes are induced with laser and imaged at day 3 with optical coherence tomography (OCT), fundus and fluorescein angiography. On day 4, eye tissues are collected for flatmount staining with isolectin GS-IB4 and propidium iodide and choroid is isolated for RNA extraction. In vivo evaluation of mice 3 days post lasering occurred in 15–17 month mice. Representative images are shown here. (B,G) Fundus images of the posterior eye showing the extent of the CNV lesions. (C,H) OCT images displaying cross-sections of the lesions (corresponding to the green line in ‘(B)’ or ‘(G)’). (D,I) Regions of leakage (dotted line circles) from the CNV lesions are visible in fluorescein angiography images. (E,J). A montage of the entire choroidal flat mount was created by overlapping and combing 4–5 images captured at lower magnification (4×). Choroidal flat mounts from control and CNV lasered mice stained with propidium iodide and (F,K) isolectin GS-IB4Representative image is shown to demonstrate CNV lesions (dotted line circles demarcate lesions). CNV: Choroidal neovascularization; ON: Optic nerve head; RNFL/GCL: retinal nerve fiber layer/ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; IS/OS: inner segment/outer segment; RPE: retinal pigment epithelium; Chor: choroid.

Figure 6

Heatmap of nuclear receptor expression in the choroid of aged laser induced CNV mice. Clustergram shows expression intensity results of cluster analysis of 84 genes from the PCR array in the control and laser induced CNV mice cohorts (n = 12 arrays per group). Each colored band represents expression of a single gene from a sample, with higher expression in shown in red and lower expression shown in green.

Figure 7

Nuclear receptor expression in the choroid of age laser induced CNV mice. (A) Scatter plot showing gene expression changes of nuclear receptors and co-regulators in lasered mice as compared to control group. The scatter plot compares the normalized expression of every gene on the PCR array between the two selected groups by plotting them against one another to visualize large gene expression changes. The center diagonal line indicates unchanged gene expression, while genes with red data points above in the upper left and yellow data points in lower right corners are up-regulated or down-regulated, respectively by more than 2-fold regulation threshold in the y-axis CNV Group relative to the x-axis Control Group. Grey data points represent within 2-fold cutoff threshold indicated by red lines. (B) Fold regulation of genes in CNV laser versus control mice using ΔΔCT method. Fold regulation of genes are categorized into different nuclear receptor classes like steroid hormone receptor, non-steroid hormone receptor, orphan receptors and adopted orphan receptors. NR = nuclear receptor.