DOI: 10.5061/dryad.hx3ffbgmv
title: Data from: Inactivation of adenosine receptor 2A suppresses endothelial-to-mesenchymal transition and inhibits subretinal fibrosis in mice

description: 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 angiogenesis. Mechanistically, endothelial-to-mesenchymal
 transition (EndMT) played a major role in the development of subretinal
 fibrosis in these models. Deficiency of Adora2a in choroidal endothelial
 cells (CECs) suppressed induction of EndMT and resulted in decreased
 subretinal fibrosis. The metabolomic profile of cultured CECs showed
 decreased succinate under ADORA2A deficiency, a product associated with
 succinate dehydrogenase (SDH) of the tricarboxylic acid pathway,
 suggesting a role for the ADORA2A-SDH-succinate axis in subretinal
 fibrosis. Pharmacologic inhibition of ADORA2A with KW6002 recapitulated
 the subretinal fibrotic phenotypes previously observed in mice with
 genetic Adora2a deficiency. Therefore, this study indicated that ADORA2A
 inhibition may be an approach to prevent or treat subretinal fibrosis
 associated with nAMD.
description: <strong>Cells</strong>: 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.
 <strong>Metabolomics assay</strong>: HCECs were
 cultured in Human Choroidal Endothelial Cells Complete Growth medium and
 transfected with si<em>CTRL</em> 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. <strong>Extraction</strong>:
 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 protein
 quantitation, the cell pellets were dissolved in an 8 M urea solution. The
 supernatant obtained from the metabolite extraction was desiccated into a
 pellet using a SpeedVac from Eppendorf (Hamburg, Germany), employing a
 heat-free technique. Before analysis, the dried pellets were re-suspended
 in 20 μL of HPLC grade water [1].
 <strong>Ref:</strong>
 <strong>[1]</strong> Ma Q, Yang Q, Xu J, Zhang X, Kim
 D, Liu Z, Da Q, Mao X, Zhou Y, Cai Y, Pareek V, Kim HW, Wu G, Dong Z, Song
 WL, Gan L, Zhang C, Hong M, Benkovic SJ, Weintraub NL, Fulton D Jr, Asara
 JM, Ben-Sahra I, Huo Y. ATIC-Associated De Novo Purine Synthesis Is
 Critically Involved in Proliferative Arterial Disease. Circulation. 2022
 Nov 8;146(19):1444-1460. doi:10.1161/CIRCULATIONAHA.121.058901. Epub 2022
 Sep 8. PMID:36073366. <strong>Mass
 Spectrometry</strong>: A volume of 5-7 μL of the resulting
 resuspension was injected and subjected to analysis using a cutting-edge
 hybrid 6500 QTRAP triple quadrupole mass spectrometer from AB/SCIEX (MA,
 USA), which was coupled to a Prominence UFLC HPLC system from Shimadzu
 (Kyoto, Japan). The analysis was carried out through selected reaction
 monitoring (SRM), targeting a comprehensive set of 298 endogenous
 water-soluble metabolites, enabling a thorough examination of the
 steady-state characteristics of the samples. Some metabolites were
 targeted in both positive and negative ion mode for a total of 309 SRM
 transitions using 8 positive/negative ion polarity switching. ESI voltage
 was +4950 V in positive ion mode and -4500 V in negative ion mode. The
 dwell time was 3 ms per SRM transition and the total cycle time was 1.55
 s. Approximately 9-12 data points were acquired per detected
 metabolite. <strong>Data
 transformation</strong>: MultiQuant v3.0 software (AB/SCIEX) was
 used for data integration and quantitation.
 <strong>Metabolite identification</strong>: Peak
 areas from the total ion current for each metabolite SRM transition were
 integrated using MultiQuant v3.0 software (AB/SCIEX) and normalized with
 protein concentration.
description: # 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. Based on the changes in succinate abundance, we examined the expression of oxoglutarate dehydrogenase (OGDH) and succinate dehydrogenase (SDH) in TGFβ2-treated cells. TGFβ2 suppressed the expression of succinate dehydrogenase B (SDHB) but did not affect OGDH expression, thus inducing succinate accumulation. *ADORA2A* KD rescued the decreased SDHB expression, thereby reducing succinate production. Overexpression of *ADORA2A* in hCECs downregulated SDHB expression and did not change OGDH expression, resulting in increased succinate. To further examine the association of SDHB and succinate with EndMT, we treated hCECs with exogenous succinate and detected EndMT-relevant molecules. Exogenous succinate increased mesenchymal cell markers ACTA2 and SM22α, increased production of FN, and decreased endothelial cell marker CDH5. These results indicated that ADORA2A regulated SDHB, leading to subsequent changes in succinate production that contributed to EndMT induction in choroidal endothelial cells. ## Description of the data and file structure 1. **Raw data files: 032222QQM1-032222QQM20 (Files can be opened with OpenChrom)** 032222QQM1-M3: hCECs transfected with si*CTRL* for 24h, followed by vehicle treatment for another 24h. 032222QQM4-M6: hCECs transfected with si*ADORA2A* for 24h, followed by vehicle treatment for another 24h. 032222QQM7-M9: hCECs transfected with si*CTRL* for 24h, followed by 10ng/mL TGFb2 treatment for another 24h. 032222QQM10-M12: hCECs transfected with si*ADORA2A* for 24h, followed by 10ng/mL TGFb2 treatment for another 24h. 032222QQM13-M16: hCECs transfected with Ad-*CTRL* for 48h. 032222QQM17-M20: hCECs transfected with Ad-*ADORA2A* for 48h. 2. **032322QqmaMets1_20:** this file contains Peak areas from the total ion current for each metabolite. **And this file is easily accessible for user to analyze.** 3. **Polar Metabolite Targeted List KEGG IDs:** contains Metabolite name, formula, and PubChem Identifier (KEGG/HMDB). But the metabolites "O8P-O1P (octulose-8-phosphate)" and "octulose-1,8-bisphosphate (OBP)" are labeled as N/A (not applicable) because they currently do not have available codes or identifiers in public databases. 4. **m_z values metabolomics:** m_z values for each metabolite. If you attempt to import this file into R or any other program, please note that the file metabolomics m/z values contains two sheets. And they do not have units associated with the values in the third file. 5. **Sample Treatment and protein file:** Cell treatment and protein content (ug) for each sample. Peak areas from the total ion current for each metabolite SRM transition were integrated using MultiQuant v3.0 software (AB/SCIEX) and normalized with protein concentration. If you use this dataset, can open **032322QqmaMets1_20** with EXCEL. The unit of metabolite production is peak area. If you wish to compare metabolite levels across different groups, it is essential to normalize the peak area to protein concentration. Protein concentration can vary among samples or experimental groups due to factors such as cell density, growth phase, or tissue composition. Normalizing to protein helps mitigate these variations, ensuring a more accurate comparison of metabolite levels and allowing for a meaningful interpretation of the results.