Adenosine receptor 2A in subretinal fibrosis

项目总结 视网膜下纤维化，新生血管性老年性黄斑变性(NAMD)的终末期纤维瘢痕， 妥协了高度组织的解剖层和紧密协调的细胞相互作用，不可避免地导致 到不可逆转的视力损害。目前对视网膜下纤维化的治疗是有限的，因此，新的 抑制视网膜下纤维化的治疗策略势在必行。 多种细胞类型，包括内皮细胞(ECs)、视网膜色素上皮(RPE)细胞、巨噬细胞 和神经胶质细胞，通过分化为间充质样细胞和进一步 分化为α-平滑肌肌动蛋白阳性的肌成纤维细胞和/或产生促纤维化和 促炎因子。然而，这些细胞和分子活动的潜在机制仍然存在 定义不明确。腺苷受体2a(Adora2a)与多种血管疾病和 发炎。我们的初步数据显示：(1)Adora2a在视网膜下的表达水平增加 激光诱导的小鼠CNV的损伤；(Ii)视网膜下纤维化的大小在激光诱导的CNV损伤中明显减小 激光诱导Adora2a基因缺陷小鼠的CNV；(Iii)血管内皮细胞向间充质细胞的转变 脉络膜内皮细胞和EndMT参与激光诱导的小鼠视网膜下纤维化的形成 在缺乏Adora2a的CECs中，Tgfb2诱导的EndMT减少；(V)巨噬细胞转化为肌成纤维细胞 在Adora2a缺乏的小鼠中，激光诱导的视网膜下纤维化损伤的过渡(MMT)显著减少；(Vi) 缺乏Adora2a的骨髓源性巨噬细胞(BMDM)纤维蛋白原的产生受到影响 Tgfb2刺激后的因子；以及(Vii)动态缺氧诱导因子(Hif)1a或2a的水平 上述病理改变与Adora2a的表达呈正相关。因此，我们假设阿多拉2a- CEC和巨噬细胞中介导的HIF信号增强纤维化效应，导致 新生血管内纤维化病变的形成。为了验证我们的假设，我们产生了可诱导的全局 Vldlr-/-小鼠的Adora2a缺陷，内皮细胞谱系追踪小鼠，诱导性内皮Adora2a缺陷 C57BL/6J小鼠和C57BL/6J小鼠的髓系Adora2a缺乏症。我们建立了一种体外培养方法 小鼠内皮细胞和体外生成BMDM的方法。我们将调查Adora2a失活的影响 在视网膜下纤维化的CECs和髓样细胞中使用特定的遗传学和药理学工具并评估 采用体内、体外和体外模型相结合的方法研究视网膜下纤维化。我们的研究将定义 Adora2a在视网膜下纤维化发生发展中的作用，为使用ADORA2a抑制剂作为 预防和治疗致盲视网膜疾病的新方法。

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 α-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.