Abstract
Purpose :
A number of patients with neovascular AMD (nAMD) do not respond well to standard anti-vascular endothelial growth factor (VEGF) therapy. One of underlying mechanisms is development of fibrosis in nAMD lesion. Thus, therapeutic strategies for the inhibition of subretinal fibrosis are imperative. Our past work shows that knockout/blockade of adenosine receptor 2A (ADORA2A for human/Adora2a for rodents) exhibits anti-inflammatory and anti-angiogenic effects. In this study, we examined whether and how knockout/blockade of ADORA2A/Adora2a suppresses the development of subretinal fibrosis.
Methods :
Laser injury-induced mouse choroidal neovascularization (CNV) displays subretinal fibrosis lesions. We performed this model in mice deficient in Adora2a globally or in choroidal endothelial cells (CECs) selectively. We collected RPE/choroid complex of mice at day 7, 21- and 35-days post laser injury, and characterized subretinal fibrosis by immunostaining RPE/choroid complex with antibodies against isolectin B4, collagen I and Acta2. In vitro, we initiated EndMT by treating cultured human CECs with TGFβ-2. With loss- and gain-of function approaches, we examined underlying mechanism with quantitative PCR and Western blotting. Two-tailed Student's t-test or One-way ANOVA was used for statistical analysis.
Results :
Adora2a/ADORA2A expression was significantly increased in mouse subretinal fibrotic lesions and TGFβ-2-treated CECs. Compared with littermate control mice, mice with deficiency of global and EC-specific Adora2a exhibited 50-60% reduction in the size of CNV and subretinal fibrosis at day 7, 21 and 35 post laser injury. Mechanistically, ADORA2A knockdown in CECs suppressed TGFβ-2-induced EndMT and HIF signaling, and ADORA2A overexpression in CECs increased the levels of HIF and SNAIL1. ADORA2A antagonist KW6002 inhibited TGFB2-induced induction of EndMT, and inhibited subretinal fibrosis in mice of the laser injury-induced CNV model.
Conclusions :
Our results demonstrate that ADORA2A-mediated activation of HIF signaling in CECs promotes their transition to mesenchymal cells and then myofibroblasts, and increases production of profibrotic and proinflammatory factors, resulting in formation of subretinal fibrotic lesions. These findings provide the basis for using ADORA2A inhibition as a novel approach in the prevention and treatment of blinding retinal disease.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.