Saturday, May 11, 2024
5/11/2024
PROJECT SUMMARY Subretinal fibrosis, an end-stage fibrous scar of neovascular age-related macular degeneration (nAMD), compromises highly organized anatomical layers and tightly coordinated cellular interactions, inevitably leading to irreversible visual impairment. The current treatment for subretinal fibrosis is limited and therefore, new therapeutic strategies for the inhibition of subretinal fibrosis are imperative. Multiple cell types, including endothelial cells (ECs), retinal pigment epithelium (RPE) cells, macrophages and glial cells, contribute to subretinal fibrosis by either differentiating into mesenchymal-like cells and further differentiating into a-smooth muscle actin-positive myofibroblasts and/or producing profibrotic and proinflammatory factors. However, the underlying mechanisms for these cellular and molecular activities remain poorly defined. Adenosine receptor 2A (Adora2a) has been implicated in various vascular diseases and inflammation. Our preliminary data here show that (i) the level of Adora2a expression was increased in subretinal lesions of laser-induced CNV in mice; (ii) the size of subretinal fibrosis was markedly decreased in lesions of laser–induced CNV in Adora2a-deficient mice; (iii) endothelial-to-mesenchymal transition (EndMT) occurred to choroidal ECs (CECs) and EndMT participated in the formation of subretinal fibrosis in laser-induced mouse CNV; (iv) Tgfb2-induced EndMT was decreased for Adora2a-deficient CECs; (v) macrophage-to-myofibroblast transition (MMT) in laser-induced subretinal fibrotic lesions was markedly reduced in Adora2a-deficient mice; (vi) Adora2a-deficient bone marrow derived macrophages (BMDMs) had a compromised production of profibrotic factors after stimulation with Tgfb2; and (vii) the levels of hypoxia-inducible factor (Hif) 1a or 2a dynamically correlated with those of Adora2a in the above pathological alterations. Thus, we hypothesize that Adora2a- mediated Hif signaling in CECs and infiltrated macrophages enhance fibrotic effects leading to increased formation of fibrotic lesions in CNV. To test our hypothesis, we have generated mice with inducible global Adora2a deficiency in Vldlr-/- mice, endothelial lineage tracing mice, inducible endothelial Adora2a deficiency in C57BL/6j mice, and myeloid Adora2a deficiency in C57BL/6j mice. We established an ex vivo approach to culture mouse CECs and in vitro approaches to generate BMDMs. We will investigate the effect of Adora2a inactivation in CECs and myeloid cells in subretinal fibrosis using specific genetic and pharmacological tools and assess subretinal fibrosis using an integrated approach of in vivo, ex vivo, and in vitro models. Our study will define the role of Adora2a in the development of subretinal fibrosis and provide the basis for using ADORA2A inhibition as a novel approach in the prevention and treatment of blinding retinal disease.
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