Adenosine receptor 2A regulates subretinal fibrosis through SDH-Succinate signaling pathway

Abstract

Purpose : Many patients with neovascular age-related macular degeneration (nAMD) experience irreversible visual impairment even with anti-VEGF therapy, and subretinal fibrosis stand out as a critical factor contributing to this phenomenon. Here, we examined whether adenosine receptor 2A (ADORA2A), a gene previously implicated in cardiovascular diseases, participates in the development of subretinal fibrosis.

Methods : We histologically examined ADORA2A expression on sections of eyes from patients with wet AMD, and laser injury-induced mouse CNV and very low–density lipoprotein receptor-deficient (Vldlr^-/-) mouse models of subretinal fibrosis. We employed Adora2a global (Adora2a^iKO) and endothelial cell Adora2a (Adora2a^ΔiVEC) deficient mice, and Adora2a antagonist KW6002 to examine the role of Adora2a in the formation of subretinal fibrosis in vivo. We characterized subretinal fibrosis by qPCR, Western blot, and immunostaining of collagen I and ACTA2. Moreover, we cultured human choroidal endothelial cells (hCECs) and treated them with transforming growth factor beta-2 (TGFβ-2) to induce Endothelial-to-Mesenchymal Transition (EndMT) in vitro. With loss- and gain-of ADORA2A function approaches, we examined whether and how ADORA2A regulates EndMT with qPCR and Western blot. Results were statistically analyzed with two-tailed Student's t-tests or One-way ANOVA, with significance set at P < 0.05 to identify differences.

Results : ADORA2A expression was increased in human AMD lesions and two mouse models of subretinal fibrosis. Fibrosis development was suppressed in Adora2a^iKO and Adora2a^ΔiVEC mice, as well as in mouse subretinal fibrosis models treated with KW6002. TGFβ2 suppressed the expression of succinate dehydrogenase B (SDHB), thus inducing succinate accumulation. ADORA2A knockdown rescued the decreased SDHB expression, thereby reducing succinate production. Gain-of-function with overexpression of ADORA2A in hCECs downregulated SDHB expression and unchanged OGDH expression, enhancing succinate levels.

Conclusions : ADORA2A influences the SDHB signaling pathway, enhancing succinate production, inducing EndMT, and ultimately promoting the formation of subretinal fibrotic lesions. These findings strongly suggest the involvement of the ADORA2A-SDH-succinate axis in the development of subretinal fibrosis and define ADORA2A inhibition as a novel approach in subretinal fibrosis to nAMD.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.