Elucidating the role of EPOR signaling in an experimental model of neovascular age-related macular degeneration

Abstract

Purpose : Erythropoietin (EPO) signaling through its receptor (EPOR) is believed to exacerbate neovascular age-related macular degeneration (AMD) pathology, but it is unknown if EPOR signaling has a direct effect on either endothelial cells (ECs) or macrophages (MΦs) recruited to the choroid that release angiogenic factors. We addressed the hypothesis that EPOR signaling increases choroidal neovascularization (CNV) by direct effects on endothelial cells (ECs). We used the murine laser-induced CNV model comparing tamoxifen-inducible EC- or MΦ-specific EPOR knockout mice.

Methods : Offspring from Cdh5-CreERT2^+/+ or Cx3cr1-CreERT2^+/+ crossed with Rosa26-tdTomato^flox/flox mice were bred with EpoR^flox/flox mice to generate Cdh5-CreERT2^+/-;Rosa26-tdTomato^flox/flox;EpoR^flox/flox (EPOR^iΔEC) or Cx3cr1-CreERT2^+/-;Rosa26-tdTomato^flox/flox;EpoR^flox/flox (EPOR^iΔMΦ), respectively, and appropriate Cre-negative littermate controls (EPOR^fl). 4-week old EPOR^iΔEC, EPOR^iΔMΦ, and EPOR^fl received intraperitoneal tamoxifen treatment every other day for one week. Two weeks later, laser injury was performed with the Phoenix Micron IV laser module. One week after laser, choroids were dissected and stained with isolectin-B4 to label CNV. Z-stacks of CNV lesions were captured using a confocal microscope, and CNV volumes were calculated using IMARIS software and verified by a masked reviewer. Data were analyzed using a multilevel linear regression model with laser spots nested within the same eye and normalized to the EPOR^fl mice.

Results : EPOR^iΔEC mice had 20% smaller lesions than EPOR^fl mice (p=0.37), whereas EPOR^iΔMΦ mice had similar sized lesions as EPOR^fl mice (p=0.76). Stratification based on sex revealed a 47% reduction in average CNV volume in male EPOR^iΔEC compared to male EPOR^fl mice (p=0.03), but not in female EPOR^iΔEC compared to female EPOR^fl mice (p=0.62). Male EPOR^iΔMΦ compared to male EPOR^fl mice (p=0.35) or female EPOR^iΔMΦ compared to female EPOR^fl mice (p=0.18) did not have significant changes in CNV volume.

Conclusions : EPOR^iΔEC with reduced EPOR signaling in ECs developed smaller lesions in males, whereas EPOR^iΔMΦ with reduced EPOR signaling in MΦs had no difference. These results support the hypothesis that EPOR signaling in ECs is important in CNV. Further study is required to determine mechanisms involved in choroidal ECs and address potential tamoxifen effect on laser-induced CNV in female mice.

This is a 2021 ARVO Annual Meeting abstract.