Inactivation of adenosine receptor 2A suppresses endothelial-to-mesenchymal transition and inhibits subretinal fibrosis in mice

The introduction of anti-vascular endothelial growth factor therapy has had a substantial impact on the treatment of choroidal neovascularization (CNV) in patients with neovascular age-related macular degeneration (nAMD), the leading cause of vision loss in older adults. Nonetheless, despite treatment, many patients with nAMD still develop severe and irreversible visual impairment due to the development of subretinal fibrosis. Therefore, therapeutic strategies that prevent or inhibit subretinal fibrosis are needed. We recently reported the anti-inflammatory and anti-angiogenic effects of inhibiting adenosine receptor 2A (ADORA2A), a gene previously implicated in cardiovascular diseases. Here we used laser injury-induced CNV and very low-density lipoprotein receptor-deficient mice as models of subretinal fibrosis and found that subretinal fibrosis was reduced in these mice in the absence of Adora2a globally or endothelial-specifically. This decreased fibrosis was independent of angiogene..., Cells: Primary human choroidal endothelial cell (HCEC) was purchased from Celprogen Inc. company (36052-03, Celprogen Inc.) and cultured with Human Choroidal Endothelial Cells Complete Growth (M36052-03S, Celprogen Inc.) in a humidified incubator with 5% CO2 at 37°C. HCECs were treated with human TGFβ2 (100-35B, PeproTech) to induce EndMT.
Metabolomics assay: HCECs were cultured in Human Choroidal Endothelial Cells Complete Growth medium and transfected with siCTRL or siRNA ADORA2A for 24 h, followed by vehicle or 10 ng/mL TGF2 treatment for another 24 h, refreshing cell medium and treatments at 2 h before sample collection. Then the metabolites were extracted and stored as described in Extraction.
Extraction: Metabolites were extracted using 1 mL of ice-cold 80% methanol on dry ice. Subsequently, the samples were centrifuged at 14,000 rpm for 5 min. To ensure thorough extraction, the cell pellets were subjected to an additional extraction with 0.5 mL of 80% methanol. For accurate prote..., , # Inactivation of adenosine receptor 2A suppresses endothelial-to-mesenchymal transition and inhibits subretinal fibrosis in mice

[https://doi.org/10.5061/dryad.hx3ffbgmv](https://doi.org/10.5061/dryad.hx3ffbgmv)

To explore the mechanism whereby ADORA2A regulates endothelial to mesenchymal cell transition (EndMT), we examined the metabolomics profiles of human primary choroidal endothelial cells (hCECs) transfected with si*ADORA2A* and treated with transforming growth factor β2 (TGFβ2, 10 ng/mL) for 48 h by analyzing the cell extracts with liquid chromatography-tandem mass spectrometry (LC-MS/MS).

**Results:** TGFβ2 treatment changed the amounts of many metabolites in pathways of glycolysis, pentose phosphate pathway (PPP), and hexosamine synthesis but not nucleic acid metabolic pathway. *ADORA2A* knockdown (KD) did not affect these metabolic pathways in hCECs. However, elevated succinate was observed in TGFβ2-treated hCECs, and this increased succinate was reduced by *ADORA2A* KD. B...