Pathophysiological significance of adrenomedullin-RAMP2 system in age-related macular degeneration

Abstract

Purpose : Age-related macular degeneration (AMD) is a leading cause of vision loss worldwide. Anti-VEGF injections have been established as the standard therapy for AMD. However, fibrotic changes which ensue over time have been implicated in the failure to more effectively preserve visual function. Adrenomedullin (AM) is a peptide having various physiological activities such as vasoprotective and anti-inflammatory actions. AM’s receptor CLR binds with 1 of the 3 subtypes of receptor activity-modifying protein (RAMP). Using knockout mice of AM and RAMPs, we showed that AM-RAMP2 system is essential for vascular development and homeostasis. AM and RAMP2 are highly expressed in the eye, and we reported AM-RAMP2 system is also involved in oxygen-induced retinopathy. In this study, we examined pathophysiological significance of AM-RAMP2 system in AMD by using RAMP2 knockout mice (RAMP2KO) and laser-induced choroidal neovascularization (LI-CNV) model. In addition, by using human retinal pigment epithelial cells (ARPE19), we investigated the effect of AM on epithelial-mesenchymal transition (EMT), which is suggested to contribute to retinal fibrosis.

Methods : LI-CNV model was prepared using 9-12 week-old male wild-type mice (WT) and RAMP2KO. Seven days after laser-irradiation, mice were perfused with fluorescein-labeled dextran, retinal flat-mounts were prepared and CNV and αSMA-immunostaining positive area were measured. With or without 24h AM-pretreatment, EMT of ARPE19 was induced by TGF-β2 (5 ng/ml) with TNF-α(10 ng/ml). After 48h, phalloidin-staining and ZO-1-immunostaining were performed to visualize actin fibers and tight junctions, respectively.

Results : CNV area / laser scar area ratio was significantly higher in RAMP2KO (27.8 ± 3.1%) than WT (16.9 ± 2.5%) (p = 0.01).αSMA-positive area was significantly higher in RAMP2KO (26461 ± 540μm²) than WT (9677 ± 1495μm²) (p = 0.01). In ARPE19, intracellular actin fiber density was 13.6 ± 1.5% in control but significantly increased to 36.9 ± 1.7% by TGF-β2 and TNF-α (p <0.01), which was suppressed to 22.0 ± 2.2% by AM-administration (p<0.01). ZO-1-positive area was 5.5 ± 0.32% in control but significantly decreased to 2.0 ± 0.15% by TGF-β2 and TNF-α (p <0.01), which was upregulated to 3.5 ± 0.20% by AM administration (p<0.01).

Conclusions : AM-RAMP2 system is expected as a therapeutic target of AMD by suppressing CNV formation, retinal fibrosis and EMT of RPE.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.