Abstract
Purpose :
Mechanisms underlying abnormal choriocapillaris vascular changes, seen in wet age-related macular degeneration, are unknown. Nuclear receptors (NR) are a family of transcription factors responsible for various cellular homeostatic mechanisms and vascular changes during development/disease. We created a NR atlas of human choroidal endothelial cells (CEC), freshly isolated choroid (FI), and choroid from a wet AMD mouse model, to identify candidate receptors that may play a role in vascular stability and/or disease.
Methods :
Samples: (a) CECs isolated from human donor eyes (n=6 males and 2 females, ages 47-90, normal ophthalmic history), through positive enrichment by VE-cadherin cell sorting; b) FI human choroid (n=3 males and 3 females, ages 61-90) and (c) choroid from aged laser induced CNV and control C57BL/6J mice (n=2-3 females and 11 males/cohort). CECs were plated on gelatin/fibronectin til confluence, dissociated, and sorted with a CD31 antibody to ensure a pure cell population. RNA was isolated (n=3 passages up to passage 5), integrity evaluated with an Agilent Bioanalyzer, and cDNA synthesized. Expression of all 48 NRs plus the aryl-hydrocarbon receptor (AhR) and AhR translocator (ARNT), in human CECs and FI, were determined by relative absolute qRT-PCR (arbitrary expression ratio=arbitrary mass of housekeeping gene/NR±SEM) and by PCR array for mouse samples.
Results :
The highest expressed NRs came from the thyroid hormone receptor-like subfamily [LXRβ (CEC: 1.22±0.02; FI: 1.04±0.02), PPARβ/δ1 (CEC: 1.01±0.02; FI: 1.02±0.05), PPARβ/δ2 (FI: 1.09±0.08), RARα (CEC: 1.03±0.04; FI: 1.15±0.05), RARβ (CEC: 1.47±0.05; FI: 1.80±0.12), RARγ (CEC: 1.34±0.07; FI: 1.15±0.05), Rev-ErbAβ (CEC: 1.14±0.03; FI: 1.04±0.07), RORα (FI: 1.15±0.04), RORβ (FI: 1.15±0.06), and RORγ (FI: 1.15±0.06)]. Highly expressed RXR-like NRs included RXRγ (FI: 1.44±0.04), COUP-TFII (CEC: 1.02±0.02), V-erbA-related (CEC: 1.17±0.03), and TR4 (CEC: 1.01±0.01; FI: 1.10±0.05). AhR (CEC: 1.41±0.04; FI: 1.33±0.12) and ARNT (FI: 1.00±0.02) were also highly expressed. Remaining NRs were categorized into intermediate (CEC, n=12 NRs; FI, n=10 NRs), low (CEC, n=13 NRs; FI, n=17 NRs), and absent (CEC, n=15 NRs; FI, n=12 NRs) expression bins.
Conclusions :
With this atlas we identified candidate receptors (e.g. LXR, AHR, RARα, RARβ, RARγ, COUP-TFII, REV-ERBAα, REV-ERBAβ, PPARβ/δ1, PPARβ/δ2, RORα, and RXRγ) as potential regulators in vasculature homeostasis/disease.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.