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.

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 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].

Ref:

[1] 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.

Mass Spectrometry: 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.

Data transformation: MultiQuant v3.0 software (AB/SCIEX) was used for data integration and quantitation.

Metabolite identification: 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.