Glaucoma, long considered an ocular-limited, age-dependent and hypoxia-driven neurodegeneration, is here reframed as a systemic erythroid-inosine axis failure that originates in the bone marrow yet culminates in retinal ganglion cell (RGC) death. By mining UK Biobank datasets (n = 127,028) and validating our findings in an independent clinical cohort (n = 178), we reveal that glaucoma is preceded by dyserythropoiesis and a compensatory, AMPK-driven metabolic rewiring of mature erythrocytes that hypercatabolizes inosine to enhance oxygen unloading. This adaptation collapses when accelerated erythrocyte inosine metabolism drains systemic pools, starving high-energy demand hematopoietic progenitors, driving retinal microenvironment hypoxia and accelerating RGC loss. Genetic ablation of murine erythroid equilibrative nucleoside transporter 1 (ENT1) recapitulates the hallmark features of patients with glaucoma, including impaired erythropoiesis, reduced oxygen delivery, retinal hypoxia and RGC apoptosis in both age and intraocular pressure-induced glaucoma models. Conversely, inosine repletion reconstitutes erythroid output, restores oxygen delivery from mature erythrocytes and halts neurodegeneration in inducible glaucoma models. A ten-metabolite erythrocyte signature centered on inosine metabolism offers diagnostic potential. Altogether, our work redefines glaucoma as the first treatable systemic erythroid-driven hypoxic syndrome, positioning inosine as a pleiotropic metabolic rescue factor for neurodegeneration and a powerful biomarker for intercepting hypoxia-driven pathologies across organs.

Heart failure with preserved ejection fraction (HFpEF) accounts for nearly half of all heart failure cases and lacks effective therapies. Key features of HFpEF include endothelial dysfunction, fibrosis, and oxidative stress. Adenosine signaling, regulated by enzymes and receptors, is critical for vascular homeostasis and inflammation, but its role in HFpEF remains poorly understood. Adenosine receptors are abundantly expressed in the heart and kidney, modulating vascular, fibrotic, and tubular processes. Dysregulation of adenosine pathways in either organ may drive hypertension, microvascular dysfunction, and maladaptive cardio-renal crosstalk, highlighting the need to investigate adenosine signaling as a combined multi-organ target. HFpEF was induced in Dahl salt-sensitive rats by high-salt diet. Cardiac structure, function, fibrosis, oxidative stress, cytokines, renal adenosine receptors and cardiac adenosine pathway components were assessed using echocardiography, histology, proteome profiling and Western blotting. Human cardiac microvascular endothelial cells were treated with endothelin-1 in the presence of selective A2A or A2B agonists, or adenosine deaminase (ADA) inhibition, and profibrotic/oxidative stress genes were analyzed by qPCR. Hypertensive rats exhibited diastolic dysfunction with preserved systolic function, cardiac and renal fibrosis, oxidative/nitrative stress, and elevated pro-inflammatory cytokines. Cardiac expression of CD39, CD73, and ADA enzymes was significantly reduced, indicating impaired adenosine metabolism, while transporters ENT2 and CNT2 were also downregulated, reflecting impairment of both equilibrative and concentrative adenosine transport. Adenosine receptor profiles were altered: A1 expression increased, A2A decreased, and A2B and A3 selectively upregulated in hypertensive, but not HFpEF, animals. In the kidney, A1 and A2A receptor expression showed region-specific, time-dependent changes. In human endothelial cells, A2A activation or ADA inhibition suppressed endothelin-1-induced COL1, COL3, and TGF-β1 expression, whereas A2B had no effect. Both A2A and A2B restored MnSOD expression, while NOX4 was selectively increased by A2B. Only A2A activation induced CAT expression, highlighting its stronger antioxidant role. Impaired adenosine metabolism and transport, along with altered receptor signaling contribute to HFpEF progression. Selective A2A activation attenuates endothelial pro-fibrotic profiles and restores antioxidant defenses, supporting its therapeutic potential. Renal receptor changes reinforce maladaptive cardio-renal crosstalk, emphasizing the importance of multi-organ adenosine modulation in HFpEF and encouraging further translational studies.

Metabolic reprogramming allows cancer cells to survive and proliferate under nutrient-deprived conditions. Uridine, a central molecule in pyrimidine metabolism, supports both nucleotide biosynthesis and redox homeostasis. However, high-sensitivity imaging tools for equilibrative nucleoside transporter 1 (ENT1)-mediated uridine transport are lacking, limiting applications in precise diagnosis and intraoperative guidance. This study aimed to develop and validate a novel dual-modality imaging platform targeting ENT1-mediated uridine transport for tumor imaging and surgical navigation. We synthesized [68Ga]Ga-DOTA-FZUD and ICG-FZUD probes for PET and NIR-II fluorescence imaging, respectively. Small-animal PET/CT and NIR-II fluorescence imaging were performed, and biodistribution were analyzed. Ex vivo NIR-II fluorescence imaging using ICG-FZUD was performed on surgical specimens from three gastric cancer patients to confirm tumor targeting. [68Ga]Ga-DOTA-FZUD exhibited excellent radiochemical purity. In pancreatic cancer models with relatively higher ENT1 expression (AsPC-1, Panc-1) compared with lower ENT1 expression models (MiaPaCa-2, BxPC-3), [68Ga]Ga-DOTA-FZUD demonstrated markedly greater tumor uptake. Similar uptake was also observed in gastric, breast, and glioblastoma models, with tumor-to-muscle ratios consistently exceeding 3.5. ICG-FZUD enabled high-contrast NIR-II imaging and clearly delineated tumor margins. Notably, ICG-FZUD penetrated the blood-brain barrier and visualized orthotopic glioblastoma. Ex vivo imaging of human gastric cancer tissues confirmed selective tumor uptake, consistent with histopathological findings. This ENT1-targeted uridine transport PET/NIRF dual-modality imaging platform complements conventional glucose-based imaging and provides real-time intraoperative navigation. It holds significant promise for early cancer diagnosis and precision surgery with strong translational potential.

ABSTRACT Introduction Restless Legs Syndrome (RLS) is a sensorimotor disorder linked to brain iron deficiency and dopaminergic dysfunction. Increasing evidence suggests that alterations in adenosine signaling may represent the missing link connecting dopaminergic and glutamatergic abnormalities. The ‘adenosine hypothesis’ proposes that brain iron deficiency induces a hypoadenosinergic state, primarily through adenosine A1 receptor downregulation, resulting in cortical hyperexcitability, hyperarousal, and periodic limb movements. Areas covered This critical perspective examines experimental, neurophysiological, and clinicaldata supporting the adenosine hypothesis of RLS. Findings from animal models demonstrate altered A1/A2A receptor balance in cortico-striatal terminals, promoting glutamatergic hyperactivity. In particular, neurophysiological studies further support the adenosine hypothesis, highlighting a pattern of cortical hyperexcitability and impaired inhibitory control in RLS. In this context, preliminary clinical trials with dipyridamole, an equilibrative nucleoside transporter inhibitor that enhances extracellular adenosine, showed symptomatic improvement, supporting adenosinergic enhancement as a therapeutic approach. Expert opinion The adenosine hypothesis provides an integrative framework uniting dopaminergic, glutamatergic, and iron-related mechanisms in RLS. Targeting adenosine transmission could complement existing therapies and mitigate augmentation. Future research should prioritize receptor-selective ligands, multimodal biomarkers, and controlled trials to translate this hypothesis into mechanism-based neurotherapeutic strategies.

Nonarteritic anterior ischemic optic neuropathy (NAION) is a leading cause of sudden, painless vision loss in the elderly, yet no proven intervention exists. Ischemic preconditioning (IPC) is a promising neuroprotective strategy, but defining an effective clinical protocol remains a major challenge in fulfilling its translational potential. We recently discovered that 40 Hz flicker induces extracellular adenosine, a key neurochemical underpinning of IPC, in the visual pathway, suggesting a previously unexplored non-invasive IPC approach. Here, we demonstrated that 3-day 40 Hz flicker preconditioning significantly protected against NAION by reducing retinal ganglion cell loss, preserving ganglion cell layer structure, improving visual function, and attenuating microglial activation. Protection was strongest when ischemia occurred 12 hours after preconditioning, remained moderate at 24 hours, and persisted for at least 4 weeks. This effect was specific to preconditioning and flicker frequency-dependent (effective at 40 Hz, but not at 20 Hz or 80 Hz). Furthermore, neuroprotection by 40 Hz flicker was abolished by treatment with the equilibrative nucleoside transporter inhibitor dipyridamole and the A1 receptor antagonist DPCPX. These findings establish 40 Hz flicker as a non-invasive, adenosine-mediated IPC strategy, suggesting a potentially safe and translational approach for protecting against NAION and other ocular ischemic disorders.

Circular RNAs (circRNAs) are associated with various biological features of cancer, including chemosensitivity and the structural characteristics of circRNAs indicate their potential as liquid biomarkers. Gemcitabine is a cornerstone treatment for pancreatic cancer (PC). A deeper understanding of gemcitabine sensitivity and the exploration of clinically valuable liquid biomarkers that are predictive of gemcitabine sensitivity may contribute to the development of improved-tailored treatment strategies for PC. The aim of the present study was to identify a candidate circRNA associated with gemcitabine sensitivity, investigate its biological functions and evaluate its potential as a liquid biomarker in predicting gemcitabine sensitivity. circRNA sequencing analysis was conducted to identify candidate circRNAs and the function of a candidate circRNA in modulating gemcitabine sensitivity was investigated in vitro. Further, the potential of this circRNA in predicting gemcitabine sensitivity in patients with PC who received gemcitabine-based neoadjuvant chemotherapy was evaluated using pre-treatment serum samples. circ72309 was identified as the candidate circRNA and its overexpression in gemcitabine-resistant PC cell lines increased gemcitabine-induced apoptosis and markedly increased gemcitabine sensitivity in vitro. Furthermore, circ72309 decreased cytidine deaminase by increasing reactive oxygen species activity and increasing human equilibrative nucleoside transporter 1 expression via regulation of target miRNAs. Patients with high serum circ72309 had markedly improved progression-free survival (PFS) and high serum circ72309 was an independent prognostic predictor of a favorable PFS in patients with PC. circ72309 affected multiple steps in the gemcitabine metabolic pathway and its overexpression resulted in markedly increased gemcitabine sensitivity. Therefore, circ72309 expression in the pre-treatment serum samples may serve as a predictor of gemcitabine sensitivity in patients with PC.

Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with epileptic seizures caused by genetic mutations in either TSC1 or TSC2 gene. Multiple genetic, epigenetic, and environmental factors can affect the phenotypical outcomes of TSC individuals. Accumulating evidence has shown that the seizures occurred in early life may contribute to the epileptogenesis and aggravate the neurological setting and neuropsychiatric symptoms of TSC. Therefore, treatments targeting seizures and/or epileptogenesis have always been the main focus on TSC therapies. Current anti-epileptic drugs and mTOR inhibitors show some efficacy, yet up to one-third of TSC-epileptic individuals are classified as refractory epilepsy. Vigabatrin, which has been used as the first-line therapy for infantile spasms in TSC, has demonstrated to delay the onset and lower the overall incidence of seizures in infants with TSC when it was used as a preventive treatment. Recently, because of its efficacy, cannabidiol, which targets adenosine signaling pathway, has been approved by the U.S. FDA for the treatment of TSC-associated epilepsy, suggesting an anti-epilepsy strategy other than mTOR inhibition is also plausible for TSC. To this end, we sought for a preventative treatment of an adenosine pathway-targeted therapeutic strategy. In this study, we pretreated Tsc2+/– mice with J4, an equilibrative nucleoside transporter 1 inhibitor, before the initiation of kindling epileptogenesis driven by the repetitive PTZ induction paradigm. We found that J4 reduced the seizure behavior severity in Tsc2+/– mice, as well as decreased mossy fiber sprouting resulted from the aberrant neurogenesis upon PTZ injurious insults. We also found that J4 increased the expression of GluR2, inhibited the astrogliosis and microgliosis, and eventually prevented the neuronal cell loss due to the excitotoxicity. The present study provides a new alternative therapeutic concept for pretreating TSC-related epilepsy before the epileptogenesis process.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-025-01518-3.

It remains unclear how excess adipose tissue in obesity leads to inflammation, insulin resistance, and other comorbidities. Extracellular nucleosides can induce inflammation through the activation of immune cell toll-like and purinergic receptors. The present study quantified nucleoside release from adipocytes and adipose tissue. Cultured mouse adipocytes released many nucleosides used in RNA/DNA. Adipose tissue from obese mice released more nucleosides than that from control nonobese mice ex vivo and had higher interstitial fluid concentrations in vivo. Consistent with the mouse study, human adipose tissue also showed significant release of adenosine/deoxyadenosine, guanosine/deoxyguanosine, and uridine ex vivo. Adipocytes release nucleosides in part through the equilibrative nucleoside transporter 1, though other pathways also appear to contribute to extracellular nucleoside concentrations. Extracellular nucleosides induce adipose tissue expression of inflammatory cytokines Tnfα, Il6, and Il1β. These data uncover a previously unknown phenomenon of adipocyte release of nucleosides, which contribute to adipose tissue inflammation in obesity.NEW & NOTEWORTHY Adipose tissue inflammation contributes to the morbidity and mortality of obesity. Adipocytes are known to release uridine and adenosine, but information on other nucleosides is lacking. As nucleosides can induce inflammation, we characterized nucleoside release from mouse and human adipose tissue. Adipose tissue released adenosine/deoxyadenosine, guanosine/deoxyguanosine, and uridine ex vivo. Nucleoside secretion was associated with adipose tissue expression of inflammatory cytokines. This represents a new mechanism by which obese adipose tissue may develop inflammation.

Myopathy encompasses a group of diseases characterized by abnormalities in both muscle function and structure. However, the underlying regulatory mechanisms of newly formed myofiber development remain poorly defined. No promising therapeutic approach has been developed, but numerous medication options are available to alleviate symptoms. Our previous studies demonstrated that adenosine kinase (ADK) is critical in regulating adenosine metabolism, pathological angiogenesis, pathological vascular remodeling, and vascular inflammatory diseases. Adenosine dynamically distributes between extracellular and intracellular, and adenosine concentration regulates ADK expression. However, the mechanism by which adenosine triggers an ADK-dependent intracellular signaling pathway to regulate skeletal muscle regeneration is not well defined. This study aimed to evaluate whether the adenosine-induced intracellular signaling pathway is involved in regulating myopathy, and how it regulates the development of newly formed myofibers. In this study, an intramuscular injection of cardiotoxin was used to induce a skeletal muscle injury model; satellite cells and C2C12 cells were employed. Whether adenosine regulates satellite cell activity, new myofiber formation and differentiation, as well as fusion of myofibers, were determined by H&E staining, BrdU incorporation assay, and spheroid sprouting assay. Interaction between ADK and PFKFB3 was evaluated by IF staining, PPI network analysis, molecular docking simulation, and CO-immunoprecipitation assay. The results demonstrated that adenosine dynamically distributes between extracellular and intracellular through concentrative nucleoside transports or equilibrative nucleoside transporters, and it rapidly induces an ADK-dependent intracellular signaling pathway, which interacts with PFKFB3-mediated glycolytic metabolism to promote satellite cell activity, new myofiber formation, differentiation, and fusion, and eventually enhances skeletal muscle regeneration after injury stress. The remarkable endogenous regeneration capacity of skeletal muscle, which is regulated by adenosine-triggered intracellular signaling, presents a promising therapeutic strategy for treating muscle trauma and muscular dystrophies.

ENT1 inhibition enhances weight control and metabolic health in diet-induced obesity.

ENT3: A lysosomal urate transporter regulating urate disposition and macrophage inflammation.

5-O-Methylvisammioside, a novel inhibitor of CNT2, improves hyperuricemia by inhibiting intestinal purine nucleoside absorption with a safe profile.

Genetic diversity of Plasmodium falciparum equilibrative nucleoside transporters PfENT1 and PfENT4: Implications for purine-based antimalarial drug development.

Proteolysis-targeting chimeras (PROTACs) are gathering considerable interest due to their ability to address previously undruggable targets. We were keen to understand the potential for these very large molecules to interact with transporters that may influence absorption, distribution, metabolism, and excretion or toxicity properties and to what extent this may be predictable using machine learning models. Consequently, we tested a set of PROTACs against several human drug transporters, namely the equilibrative nucleoside (ENT) family transporters ENT1 and ENT2, which have been directly implicated in the uptake of anticancer or antiviral drugs into target cells. We describe the dramatic inhibition observed for ENT1 and ENT2 but not for the unrelated transporter organic anion transporter 4. In addition, we report dose-response relationships for ENT1 to show some PROTACs are nanomolar inhibitors. We also explored the chemistry space of small molecules tested against ENT1 and ENT2 and compared them with PROTACs to illustrate that they are found on the periphery and close to other larger small molecules. While PROTACs are thought of as a dissimilar class to small molecules, it may be possible to bring them closer to those Food and Drug Administration-approved orally available large molecules, and in turn, increase their oral bioavailability. The outcomes of these combined in vitro and computational assessments could influence PROTAC development, be useful for their repurposing as ENT1 inhibitors for several disease indications beyond their primary one, and be used for transporter machine learning model generation and evaluation. SIGNIFICANCE STATEMENT: Proteolysis-targeting chimeras are an increasingly popular class of molecules for which we do not have a complete picture of their absorption, distribution, metabolism, and excretion or toxicity properties. For example, their interactions with uptake and efflux transporters are unknown. Here, we provide evidence that many proteolysis-targeting chimeras act as inhibitors of equilibrative nucleoside transporters 1 and 2. We hope to stimulate further study of their potential for inhibition of other transporters.

Adenosine deprivation modulates purine metabolism and enhances Trichomonas vaginalis cytotoxicity.

Endophytic colonization of Arabidopsis (Arabidopsis thaliana) by the beneficial root endophyte Serendipita indica is characterized by an initial biotrophic phase, followed by a confined host cell death phase that facilitates fungal accommodation. However, the host molecular pathways that restrict S. indica proliferation and regulate symbiosis-associated cell death remain largely unknown. Our study demonstrates that autophagy, a key cellular degradation pathway that maintains homeostasis, is locally activated during colonization and is required to limit fungal proliferation and immunometabolic stress. Autophagy-deficient mutants exhibit elevated basal root cell death, increased colonization, and hypersensitivity to the fungal-derived purine metabolite 2'-deoxyadenosine (dAdo), an immunometabolic signal that modulates host cell viability and reprograms immune and metabolic responses via ENT3 (equilibrative nucleoside transporter 3)-mediated uptake. In ent3 and atg5 ent3 mutants, suppression of dAdo import reduces S. indica-induced cell death, confirming the central role of ENT3-mediated uptake. Despite increased colonization and stress sensitivity, autophagy-deficient plants retain S. indica-mediated root growth promotion, indicating that mutualistic benefits can occur independently of immunometabolic stress resilience. Based on these findings, we propose that autophagy-mediated pro-survival responses are essential for maintaining symbiotic homeostasis by integrating immunometabolic signals and preserving host cell viability.

Equilibrative nucleoside transporters (ENTs) 1 and 2 are considered critical to the cellular uptake of purine and pyrimidine analogs used to treat cancer and viral infections. However, a detailed understanding of the discrete and overlapping roles of these ENT subtypes in drug activity remains limited. A significant barrier to progress has been the absence of model systems that enable functional characterization of individual nucleoside transporters in the context of their native environment. To address this, we developed and characterized a panel of CRISPR/cas9-engineered human embryonic kidney 293 cell lines with selective deletion of ENT subtypes: ENT1 knockout, ENT2 knockout, and dual knockout. These models were used to dissect subtype-specific roles of ENT1 and ENT2 in nucleoside/nucleobase analog uptake and cytotoxicity. Our data show that ENT1 and ENT2 in their endogenous environment have a similar affinity for a range of both endogenous and chemotherapeutic nucleoside and nucleobase analogs. Deletion of ENT1 generally enhanced the sensitivity of cells to these drugs, particularly the nucleobase analogs, likely due to reduced nucleoside salvage by the cells via ENT1. Deletion of ENT2, on the other hand, dramatically reduced the ability of a number of the tested drugs to impact cell viability, by mechanisms beyond those related to reduced cellular uptake of the drugs. This study highlights distinctive roles of ENT1 and ENT2 in the actions of nucleoside/nucleobase analog drugs.Significance StatementA panel of genetically modified human embryonic kidney 293 cells has been created as a model to screen novel nucleoside transporter inhibitors and substrates. Using these cell lines, it was revealed that ENT2 may play a more functionally significant role in nucleoside analog chemotherapeutic drug activity than previously appreciated.

Inflammatory signaling selectively disrupts GATA factor-regulated networks governing erythroid differentiation and hemoglobin synthesis

Direct-acting antiviral agents (DAAs) are safe and effective in eradicating hepatitis C virus (HCV) infection. Ribavirin is still used as an important part of modern HCV therapy. Main protein involved in ribavirin cellular uptake is encoded by solute carrier family 29, member1 (SLC29A1), which has a role in ribavirin uptake into erythrocytes. To evaluate the association of SLC29 Agene polymorphism (rs760370) with hematological changes and sustained virological response (SVR) in Egyptian chronic HCV patients treated with DAAs. Sixty chronic HCV infected patients receiving DAAs with ribavirin (group I; triple therapy) were classified as group Ia is associated with hematologic complication, group Ib without complications. Sixty chronic HCV infected patients receiving DAAs without ribavirin were included in (group II; dual therapy). Liver tests, complete blood counts, hemolytic markers (indirect bilirubin, reticulocytic count and LDH), HCV RNA level and SLC29A1 (rs760370) using allelic discrimination real time PCR were done for all subjects. Median age of the enrolled patients was 37 years old with female majority (69 patients). Patients carrying GG genotype had increased a significant risk of hemoglobin decline ≥ 2 g/dL as compared to AA genotype. Similar results were obtained in the dominant model which also had increased risk for Hb decline (OR = 4.26, 95% CI: 1.34-13.55, and p = 0.011), changes in reticulocyte count and platelet count (p = 0.022, and 0.020 respectively). Also, patients with dominant genetic models had an increase in SVR in patients receiving triple therapy. Regarding the change in hematological parameters revealed a highly significant decrease in delta Δ Hb, RBCs count and MCHC (p < 0.001) in group 1 compared to group 2 plus a statistically significant increase in delta Δ AST level, indirect bilirubin and LDH in group 1 compared to group 2 p-value (p = 0.019, p < 0.001, p < 0.001) respectively. SNP rs760370A > G at SLC29A1 gene influences the severity of ribavirin-induced anemia, possibly mirroring erythrocyte uptake of ribavirin and that increases SVR in chronic hepatitis C patients receiving DAAs and ribavirin.

Avid nutrient consumption is a metabolic hallmark of cancer and leads to regional depletion of key metabolites within the tumor microenvironment (TME). Cancer cells consequently employ diverse strategies to acquire the fuels needed for growth, including bulk uptake of the extracellular medium by macropinocytosis. Here, we show that breast and pancreatic cancer cells macropinocytically internalize extracellular DNA (exDNA), an abundant component of the TME, and deliver it to lysosomes for degradation. This provides a supply of nucleotides that sustains growth when de novo biosynthesis is impaired by glutamine restriction or pharmacological blockade. Mechanistically, this process is dependent on the non-redundant lysosomal equilibrative nucleoside transporter SLC29A3 (ENT3), which mediates the export of nucleosides from the lysosomal lumen into the cytosol. Accordingly, genetic ablation of SLC29A3 or pharmacological disruption of lysosomal function prevents exDNA scavenging and potently sensitizes breast tumors to antimetabolite chemotherapy in vivo . These findings reveal a previously unrecognized nutrient acquisition pathway through which cancer cells recycle exDNA into metabolic building blocks and highlight SLC29A3 as a mediator of metabolic flexibility and a potential target to improve chemotherapy response.