Vascular derived endothelin receptor A controls endothelin-induced retinal ganglion cell death

Abstract

Endothelin (EDN, also known as ET) signaling has been suggested to be an important mediator of retinal ganglion cell (RGC) death in glaucoma. Antagonism of EDN receptors (EDNRA and EDNRB, also known as ET-A and ET-B) prevented RGC death in mouse models of chronic ocular hypertension, and intravitreal injection of EDN ligand was sufficient to drive RGC death. However, it remains unclear which cell types EDN ligands directly affect to elicit RGC death. Multiple cell types in the retina and optic nerve express EDNRA and EDNRB and thus could respond to EDN ligands in the context of glaucoma. Here, we systematically deleted Edn receptors from specific cell types to identify the critical EDN receptor mediating RGC death in vivo. Deletion of both Ednra and Ednrb from retinal neurons (including RGCs) and macroglia did not prevent RGC loss after exposure to EDN1 ligands, suggesting EDN1 ligands cause RGC death via an indirect mechanism involving a secondary cell type. Deletion of Ednra from the full body, and then specifically from vascular mural cells, prevented EDN1-induced vasoconstriction and RGC death. Together, these data suggest EDN ligands cause RGC death via a mechanism initiated by vascular mural cells. It is possible RGC death is a consequence of vascular mural cell-induced vasoconstriction and its pathological sequelae. These results highlight the potential importance of neurovascular dysfunction in glaucoma.

Fig. 1. EDN1 did not cause RGC death directly or via macroglia-expressed receptors.

A Fluorescein angiography of retinal vasculature in naive and EDN1-injected eyes from WT, Six3-cre⁺Ednra^fl/flEdnrb^+/+, Six3-cre⁺Ednra^+/+Ednrb^fl/fl, and Six3-cre⁺Ednra^fl/flEdnrb^fl/fl animals. Deletion of either or both Edn receptors with Six3-cre did not prevent EDN1-induced vasoconstriction (n ≥ 3). B Retinal flat mounts and quantification of cleaved caspase 3+ (cCASP3+, red) RBPMS+ (green) cells from WT, Six3-cre⁺Ednra^+/+Ednrb^fl/fl, Six3-cre⁺Ednra^fl/flEdnrb^+/+, Six3-cre⁺Ednra^fl/flEdnrb^fl/fl 5 days post-EDN1 or PBS (vehicle control) injection. Each genotype group had significant increases in cCASP3+ RGCs compared to PBS controls. No significant difference in cCASP3+ RGCs was observed between genotype groups after EDN1. cCASP3+ RGCs/mm² ± SEM: PBS: 0.8 ± 0.3, WT: 32.0 ± 7.2, Six3-cre⁺Ednra^fl/flEdnrb^+/+: 23.7 ± 8.6, Six3-cre⁺Ednra^+/+Ednrb^fl/fl: 32.3 ± 9.5, Six3-cre⁺Ednra^fl/flEdnrb^fl/fl: 19.8 ± 6.0 (n ≥ 7 per genotype, *P < 0.05, Kruskal–Wallis test). Scale bars, 50 μm.

Fig. 2. Cag-creER^T2 robustly recombined floxed alleles in retinal and optic nerve DAPI+ cells.

A Cag-creER^T2+Tdtomato⁺ retinal and optic nerve head sections depicting cell-type-specific expression of Tdtomato. Cag-creER^T2 robustly recombined floxed alleles in DAPI+ cells, including RBPMS+ RGCs, SOX2+ Müller glia, GFAP+ retinal astrocytes, CD31+ vascular cells, and SOX2+ GFAP+ optic nerve head (ONH) astrocytes (n = 3). Scale bars, 50 μm. B Fluorescein angiography of retinal vasculature in naive and EDN1-injected eyes from WT, Cag-creER^T2+Ednrb^fl/fl, and Cag-creER^T2+Ednra^fl/fl animals. Full body deletion of Ednra ablated EDN1-induced vasoconstriction (n ≥ 5).

Fig. 3. EDN1 ligand acted through EDNRA to drive RGC death.

A Retinal flat mounts from WT, Cag-creER^T2+Ednrb^fl/fl, and Cag-creER^T2+Ednra^fl/fl mice immunoassayed for cCASP3 and RBPMS 5 days post-EDN1. Cag-creER^T2+Ednrb^fl/fl retinas had similar numbers of cCASP3+ RGCs compared to WT controls, while Cag-creER^T2+Ednra^fl/fl mice had significantly reduced cCASP3+ RGCs compared to both WT and Cag-creER^T2+Ednrb^fl/fl retinas. cCASP3+ RGCs/mm² ± SEM: WT: 24.2 ± 8.9 Cag-creER^T2+Ednrb^fl/fl: 18.0 ± 7.4, Cag-creER^T2+Ednra^fl/fl: 0.7 ± 0.1 (n ≥ 6, *P < 0.01, Kruskal-Wallis test). Scale bar, 50 μm. B Flat mounted WT and Cag-creER^T2+Ednra^fl/fl retinas immunoassayed for RBPMS 28 days post-EDN1 injection. Full body deletion of Ednra prevented EDN1-induced RGC loss. %RBPMS+ cell survival±SEM for WT and Cag-creER^T2+Ednra^fl/fl respectively: PBS: 100.0 ± 3.4, 100.0 ± 2.7; EDN1: 82.0 ± 2.7, 97.0 ± 3.8 (n ≥ 6, *P < 0.05, two-way ANOVA, Holm–Sidak post hoc). Scale bars, 50 μm.

Fig. 4. Myh11-creER^T2 recombined floxed alleles in vascular mural cells.

A Myh11-creER^T2+Tdtomato⁺ fluorescein angiography overlayed with TdTomato fluorescence demonstrating TdTomato localization to retinal arteries (n = 4). B Myh11-creER^T2Tdtomato⁺ retinal flat mounts counterstained with CD31 to visualize retinal vasculature. Tdtomato was robustly and specifically expressed by arterial cells and capillaries, but not by RBPMS+ RGCs or any other observable cell type. C Tdtomato+ cells surrounding retinal capillaries were apparent in superficial, intermediate, and deep layers of the retina (n = 3). Scale bars, 50 μm. D Fluorescein angiography of retinal vasculature in naive and EDN1-injected eyes from WT and Myh11-creER^T2+Ednra^fl/fl animals. Mural cell-specific deletion of Ednra ablated EDN1-induced vasoconstriction (n ≥ 5).

Fig. 5. EDNRA expressed by vascular mural cells elicited RGC death in response to EDN1.

A Flat mounted retinas immunoassayed for cCASP3 and RBPMS 5 days post-EDN1 injection. Ednra deletion from vascular mural cells significantly reduced numbers of cCASP3+ RGCs after EDN1 injury. cCASP3+ RGCs/mm²: WT: 23.6 ± 7.6, Myh11-creER^T2+Ednra^fl/fl: 6.0 ± 4.4 (n ≥ 9, *P = 0.009, Mann–Whitney test). B Flat mounted WT and Myh11-creER^T2+Ednra^fl/fl retinas immunoassayed for RBPMS 28 days post-EDN1 injection. Mural cell deletion of Ednra prevented EDN-induced RGC loss. %RBPMS+ cell survival ± SEM for WT and Myh11-creER^T2+Ednra^fl/fl respectively: PBS: 100.0 ± 2.3, 100.0 ± 1.4; EDN1: 85.0 ± 4.3, 101.4 ± 1.3 (n ≥ 8, *P ≤ 0.001, two-way ANOVA, Holm–Sidak post hoc). Scale bars, 50 μm.