Integrated multiomics revealed adenosine signaling predict immunotherapy response and regulate tumor ecosystem of melanoma.

Extracellular adenosine is extensively involved in regulating the tumor microenvironment. Given the disappointing results of adenosine-targeted therapy trials, personalized treatment might be necessary, tailored to the microenvironment status of individual patients. Here, we introduce the adenosine signaling score (ADO-score) model using non-negative matrix fraction identified patient subtypes using publicly available melanoma dataset, which aimed to profile adenosine signaling-related genes and construct a model to predict prognosis. We analyzed 580 malignant melanoma samples and demonstrated its robust value for prognosis. Further investigation in immune checkpoint inhibitor dataset suggests its potential as a stratified factor of immune checkpoint inhibitor efficacy. We validated the power of the ADO-score at the protein level immunofluorescence in a melanoma cohort from Xiangya Hospital. More importantly, single-cell and spatial transcriptomic data highlighted the cell-specific expression patterns of adenosine signaling-related genes and the existence of adenosine signaling-mediated crosstalk between tumor cells and immune cells in melanoma. Our study reveals a robust connection between adenosine signaling and clinical benefits in melanoma patients and proposes a universally applicable adenosine signaling model, the ADO-score, in gene expression profiles and histological sections. This model enables us to more precisely and conveniently select patients who are likely to benefit from immunotherapy.

Integrating bulk-RNA sequencing and single-cell sequencing analyses to characterize adenosine-enriched tumor microenvironment landscape and develop an adenosine-related prognostic signature predicting immunotherapy in lung adenocarcinoma.

The adenosine-signaling axis has been recognized as an important immunomodulatory pathway in tumor immunity. However, the biological role of the adenosine-signaling axis in the remodeling of the tumor microenvironment (TME) in lung adenocarcinoma (LUAD) remains unclear. Here, we quantified adenosine signaling (ado_sig) in LUAD samples using the GSVA method and assessed the prognostic value of adenosine in LUAD. Afterward, we explored the heterogeneity of the tumor-immune microenvironment at different adenosine levels. In addition, we analyzed the potential biological pathways engaged by adenosine. Next, we established single-cell transcriptional profiles of LUAD and analyzed cellular composition and cell-cell communication analysis under different adenosine microenvironments. Moreover, we established adenosine-related prognostic signatures (ARS) based on comprehensive bioinformatics analysis and evaluated the efficacy of ARS in predicting immunotherapy. The results demonstrated that adenosine signaling adversely impacted the survival of immune-enriched LUAD. The high-adenosine microenvironment exhibited elevated pro-tumor-immune infiltration, including M2 macrophages and displayed notably increased epithelial-mesenchymal transition (EMT) transformation. Furthermore, adenosine signaling displayed significant associations with the expression patterns and prognostic value of immunomodulators within the TME. Single-cell sequencing data revealed increased fibroblast occupancy and a prominent activation of the SPP1 signaling pathway in the high adenosine-signaling microenvironment. The ARS exhibited promising effectiveness in prognostication and predicting immunotherapy response in LUAD. In summary, overexpression of adenosine can cause a worsened prognosis in the LUAD with abundant immune infiltration. Moreover, increased adenosine levels are associated with pro-tumor-immune infiltration, active EMT transformation, pro-tumor angiogenesis, and other factors promoting cancer progression, which collectively contribute to the formation of an immunosuppressive microenvironment. Importantly, the ARS developed in this study demonstrate high efficacy in evaluating the response to immunotherapy.

Pilot study to evaluate the safety and effectiveness of etidronate treatment for arterial calcification due to deficiency of CD73 (ACDC).

BACKGROUND: Arterial calcification due to deficiency of CD73 (ACDC; OMIM 211800) is a rare genetic disease resulting in calcium deposits in arteries and small joints causing claudication, resting pain, severe joint pain, and deformities. Currently, there are no standard treatments for ACDC. Our previous work identified etidronate as a potential targeted ACDC treatment, using in vitro and in vivo disease models with patient-derived cells. In this study, we test the safety and effectiveness of etidronate in attenuating the progression of lower-extremity arterial calcification and vascular blood flow based on the computed tomography (CT) calcium score and ankle-brachial index (ABI). METHODS: Seven adult patients with a confirmed genetic diagnosis of ACDC were enrolled in an open-label, nonrandomized, single-arm pilot study for etidronate treatment. They took etidronate daily for 14 days every 3 months and were examined at the NIH Clinical Center bi-annually for 3 years. They received a baseline evaluation as well as yearly follow up after treatment. Study visits included imaging studies, exercise tolerance tests with ABIs, clinical blood and urine testing, and full dental exams. RESULTS: Etidronate treatment appeared to have slowed the progression of further vascular calcification in lower extremities as measured by CT but did not have an effect in reversing vascular and/or periarticular joint calcifications in our small ACDC cohort. CONCLUSIONS: Etidronate was found to be safe and well tolerated by our patients and, despite the small sample size, appeared to show an effect in slowing the progression of calcification in our ACDC patient cohort.(ClinicalTrials.gov Identifier NCT01585402).

Reciprocal upregulation of hypoxia-inducible factor-1α and persistently enhanced placental adenosine signaling contribute to the pathogenesis of preeclampsia.

Recent evidence indicates that elevated placental adenosine signaling contributes to preeclampsia (PE). However, the molecular basis for the chronically enhanced placental adenosine signaling in PE remains unclear. Here, we report that hypoxia-inducible factor-1α (HIF-1α) is crucial for the enhancement of placental adenosine signaling. Utilizing a pharmacologic approach to reduce placental adenosine levels, we found that enhanced adenosine underlies increased placental HIF-1α in an angiotensin receptor type 1 receptor agonistic autoantibody (AT1 -AA)-induced mouse model of PE. Knockdown of placental HIF-1α in vivo suppressed the accumulation of adenosine and increased ecto-5'-nucleotidase (CD73) and adenosine A2B receptor (ADORA2B) in the placentas of PE mouse models induced by AT1 -AA or LIGHT, a TNF superfamily cytokine (TNFSF14). Human in vitro studies using placental villous explants demonstrated that increased HIF-1α resulting from ADORA2B activation facilitates the induction of CD73, ADORA2B, and FLT-1 expression. Overall, we demonstrated that (a) elevated placental HIF-1α by AT1 -AA or LIGHT upregulates CD73 and ADORA2B expression and (b) enhanced adenosine signaling through upregulated ADORA2B induces placental HIF-1α expression, which creates a positive feedback loop that promotes FLT-1 expression leading to disease development. Our results suggest that adenosine-based therapy targeting the malicious cycle of placental adenosine signaling may elicit therapeutic effects on PE.

Neuronal modulation of hepatic lipid accumulation induced by bingelike drinking.

Excessive alcohol consumption, including binge drinking, is a common cause of fatty liver disease. Binge drinking rapidly induces hepatic steatosis, an early step in the pathogenesis of chronic liver injury. Despite its prevalence, the process by which excessive alcohol consumption promotes hepatic lipid accumulation remains unclear. Alcohol exerts potent effects on the brain, including hypothalamic neurons crucial for metabolic regulation. However, whether or not the brain plays a role in alcohol-induced hepatic steatosis is unknown. In the brain, alcohol increases extracellular levels of adenosine, a potent neuromodulator, and previous work implicates adenosine signaling as being important for the development of alcoholic fatty liver disease. Acute alcohol exposure also increases both the activity of agouti-related protein (AgRP)-expressing neurons and AgRP immunoreactivity. Here, we show that adenosine receptor A2B signaling in the brain modulates the extent of alcohol-induced fatty liver in mice and that both the AgRP neuropeptide and the sympathetic nervous system are indispensable for hepatic steatosis induced by bingelike alcohol consumption. Together, these results indicate that the brain plays an integral role in alcohol-induced hepatic lipid accumulation and that central adenosine signaling, hypothalamic AgRP, and the sympathetic nervous system are crucial mediators of this process.

Impaired dopamine- and adenosine-mediated signaling and plasticity in a novel rodent model for DYT25 dystonia.

Dystonia is a neurological movement disorder characterized by sustained or intermittent involuntary muscle contractions. Loss-of-function mutations in the GNAL gene have been identified to be the cause of "isolated" dystonia DYT25. The GNAL gene encodes for the guanine nucleotide-binding protein G(olf) subunit alpha (Gαolf), which is mainly expressed in the olfactory bulb and the striatum and functions as a modulator during neurotransmission coupling with D1R and A2AR. Previously, heterozygous Gαolf -deficient mice (Gnal+/-) have been generated and showed a mild phenotype at basal condition. In contrast, homozygous deletion of Gnal in mice (Gnal-/-) resulted in a significantly reduced survival rate. In this study, using the CRISPR-Cas9 system we generated and characterized heterozygous Gnal knockout rats (Gnal+/-) with a 13 base pair deletion in the first exon of the rat Gnal splicing variant 2, a major isoform in both human and rat striatum. Gnal+/- rats showed early-onset phenotypes associated with impaired dopamine transmission, including reduction in locomotor activity, deficits in rotarod performance and an abnormal motor skill learning ability. At cellular and molecular level, we found down-regulated Arc expression, increased cell surface distribution of AMPA receptors, and the loss of D2R-dependent corticostriatal long-term depression (LTD) in Gnal+/- rats. Based on the evidence that D2R activity is normally inhibited by adenosine A2ARs, co-localized on the same population of striatal neurons, we show that blockade of A2ARs restores physiological LTD. This animal model may be a valuable tool for investigating Gαolf function and finding a suitable treatment for dystonia associated with deficient dopamine transmission.

Multiprotein Complex With TRPC (Transient Receptor Potential-Canonical) Channel, PDE1C (Phosphodiesterase 1C), and A2R (Adenosine A2 Receptor) Plays a Critical Role in Regulating Cardiomyocyte cAMP and Survival.

BACKGROUND: cAMP plays a critical role in regulating cardiomyocyte survival. Various cAMP signaling pathways behave distinctly or in opposition. We have previously reported that activation of cAMP hydrolysis by cyclic nucleotide phosphodiesterase 1C (PDE1C) promotes cardiomyocytes death/apoptosis, yet the underlying molecular mechanism remains unknown. In this study, we aimed to identify the specific cAMP signaling pathway modulated by PDE1C and determine the mechanism by which Ca2+/calmodulin-stimulated PDE1C is activated. METHODS: To study cardiomyocyte death/apoptosis, we used both isolated mouse adult cardiomyocytes in vitro and doxorubicin-induced cardiotoxicity in vivo. We used a variety of pharmacological activators and inhibitors as well as genetically engineered molecular tools to manipulate the expression and activity of proteins of interest. RESULTS: We found that the protective effect of PDE1C inhibition/deficiency on Ang II or doxorubicin-induced cardiomyocyte death/apoptosis is dependent on cAMP-generating adenosine A2 receptors (A2Rs), suggesting that PDE1C's cAMP-hydrolyzing activity selectively modulates A2R-cAMP signaling in cardiomyocytes. In addition, we found that the effects of PDE1C activation on Ang II-mediated cAMP reduction and cardiomyocyte death are dependent on transient receptor potential-canonical (TRPC) channels, in particular TRPC3. We also observed synergistic protective effects on cardiomyocyte survival from the combination of A2R stimulation together with PDE1 or TRPC inhibition. Coimmunostaining and coimmunoprecipitation studies showed that PDE1C is localized in proximity with A2R and TRPC3 in the plasma membrane and perhaps T tubules. It is important to note that we found that doxorubicin-induced cardiac toxicity and dysfunction in mice are attenuated by the PDE1 inhibitor IC86340 or in PDE1C knockout mice, and this protective effect is significantly diminished by A2R antagonism. CONCLUSIONS: We have characterized a novel multiprotein complex comprised of A2R, PDE1C, and TRPC3, in which PDE1C is activated by TRPC3-derived Ca2+, thereby antagonizing A2R-cAMP signaling and promoting cardiomyocyte death/apoptosis. Targeting these molecules individually or in combination may represent a compelling therapeutic strategy for potentiating cardiomyocyte survival.

Safety, tolerability, and pharmacology of AB928, a novel dual adenosine receptor antagonist, in a randomized, phase 1 study in healthy volunteers.

Adenosine suppresses antitumor immune responses via A2a and A2b receptors expressed on intratumoral immune cells. This effect is mediated by increased cyclic adenosine 5'-monophosphate (AMP) levels and phosphorylation of cyclic AMP response element binding protein (CREB). We conducted a phase 1, placebo-controlled, single-ascending-dose (SAD) and multiple-ascending-dose (MAD) study to assess the safety, tolerability, pharmacokinetics (PK), including food effect (FE), and pharmacodynamics (PD) of oral AB928, a novel dual A2aR/A2bR antagonist, in healthy volunteers. AB928 doses between 10 and 200 mg once daily and 100 mg twice daily were evaluated. The study enrolled 85 subjects (randomized 3:1, AB928:placebo), 40 each in the SAD and MAD cohorts, and 5 in the FE cohort. AB928 was well tolerated up to the highest dose tested and did not affect any physiologic parameters potentially sensitive to adenosine inhibition. No safety concern was identified. The PK profile of AB928 was linear and dose-proportional, and a clear PK/PD correlation was demonstrated. Significant inhibition of adenosine receptor-mediated phosphorylated CREB was observed at peak plasma concentrations in all dose cohorts and at trough plasma concentrations in the higher-dose cohorts. AB928 plasma levels ≥1 µM were associated with ≥90% adenosine receptor inhibition. In the postprandial state, the rate of AB928 absorption decreased but the extent of absorption was unchanged. Together, these data support further clinical development of oral AB928 in cancer patients.

Adenosine: An endogenous mediator in the pathogenesis of gynecological cancer.

Extracellular concentration of adenosine increases in the hypoxic tumor microenvironment. Adenosine signaling regulates apoptosis, angiogenesis, metastasis, and immune suppression in cancer cells. Adenosine-induced cell responses depend upon different subtypes of adenosine receptors activation and type of cancer. Suppression of adenosine signaling via inhibition of adenosine receptors or adenosine generating enzymes including CD39 and CD73 on ovarian or cervical cancer cells is a potentially novel therapeutic approach for gynecological cancer patients. This review summarizes the role of adenosine in the pathogenesis of gynecological cancer for a better understanding and hence a better management of this disease.

Role of adenosine signaling in coordinating cardiomyocyte function and coronary vascular growth in chronic fetal anemia.

Fetal anemia causes rapid and profound changes in cardiac structure and function, stimulating proliferation of the cardiac myocytes, expansion of the coronary vascular tree, and impairing early contraction and relaxation. Although hypoxia-inducible factor-1α is sure to play a role, adenosine, a metabolic byproduct that increases coronary flow and growth, is implicated as a major stimulus for these adaptations. We hypothesized that genes involved in myocardial adenosine signaling would be upregulated in chronically anemic fetuses and that calcium-handling genes would be downregulated. After sterile surgical instrumentation under anesthesia, gestationally timed fetal sheep were made anemic by isovolumetric hemorrhage for 1 wk (16% vs. 35% hematocrit). At 87% of gestation, necropsy was performed to collect heart tissue for PCR and immunohistochemical analysis. Anemia increased mRNA expression levels of adenosine receptors ADORA 1, ADORA2A, and ADORA2B in the left and right ventricles (adenosine receptor ADORA3 was unchanged). In both ventricles, anemia also increased expression of ectonucleoside triphosphate diphosphohydrolase 1 and ecto-5'-nucleotidase. The genes for both equilibrative nucleoside transporters 1 and 2 were expressed more abundantly in the anemic right ventricle but were not different in the left ventricle. Neither adenosine deaminase nor adenosine kinase cardiac levels were significantly changed by chronic fetal anemia. Chronic fetal anemia did not significantly change cardiac mRNA expression levels of the voltage-dependent L-type calcium channel, ryanodine receptor 1, sodium-calcium exchanger, sarcoplasmic/endoplasmic reticulum calcium transporting ATPase 2, phospholamban, or cardiac calsequestrin. These data support local metabolic integration of vascular and myocyte function through adenosine signaling in the anemic fetal heart.

Nanomaterial exposure and sterile inflammatory reactions.

"Sterile inflammation" is a type of pathogen-free inflammation which may be induced by various physical, chemical, or metabolic insults, including environmental particles e.g. silica dioxide crystals and asbestos. The possible role of nanomaterials as danger signals able to trigger sterile inflammatory responses is not yet fully understood. Therefore, the aim of this review was to investigate conditions of sterile inflammation induced by nanomaterial exposure with a specific focus on possible modes of action. Metal-, metal-oxide- and carbon-based nanomaterials may trigger a sterile inflammatory cascade by means of different damage-associated molecular patterns, including chromatin associated protein high-mobility group box-1 secretion, ATP, ADP and adenosine purinergic signaling, interleukin-1α alarmin, and NLPR-3 inflammasome activation. However, such preliminary results do not enable us to draw definite conclusions regarding the sterile inflammatory potential of nanomaterials and possible underlying molecular mechanisms. Therefore, further research is required to understand the complex "interplay" between the multitude of nanomaterial intrinsic or acquired physico-chemical properties and the extremely changeable immunological competence of organisms. The identification of possible nanomaterial modes of action in sterile inflammation could provide early indicators of the biological reactivity of nano-sized chemicals and could direct "safe by design" efforts for improving occupational safety and that of the general population. Moreover, the possible pathological consequences of nanomaterial-induced sterile inflammatory reactions need to be clarified in order to define suitable preventive measures to protect the health of chronically exposed subjects.

A hypoxic episode during cardiogenesis downregulates the adenosinergic system and alters the myocardial anoxic tolerance.

To what extent hypoxia alters the adenosine (ADO) system and impacts on cardiac function during embryogenesis is not known. Ectonucleoside triphosphate diphosphohydrolase (CD39), ecto-5'-nucleotidase (CD73), adenosine kinase (AdK), adenosine deaminase (ADA), equilibrative (ENT1,3,4), and concentrative (CNT3) transporters and ADO receptors A1, A2A, A2B, and A3 constitute the adenosinergic system. During the first 4 days of development chick embryos were exposed in ovo to normoxia followed or not followed by 6 h hypoxia. ADO and glycogen content and mRNA expression of the genes were determined in the atria, ventricle, and outflow tract of the normoxic (N) and hypoxic (H) hearts. Electrocardiogram and ventricular shortening of the N and H hearts were recorded ex vivo throughout anoxia/reoxygenation ± ADO. Under basal conditions, CD39, CD73, ADK, ADA, ENT1,3,4, CNT3, and ADO receptors were differentially expressed in the atria, ventricle, and outflow tract. In H hearts ADO level doubled, glycogen decreased, and mRNA expression of all the investigated genes was downregulated by hypoxia, except for A2A and A3 receptors. The most rapid and marked downregulation was found for ADA in atria. H hearts were arrhythmic and more vulnerable to anoxia-reoxygenation than N hearts. Despite downregulation of the genes, exposure of isolated hearts to ADO 1) preserved glycogen through activation of A1 receptor and Akt-GSK3ß-GS pathway, 2) prolonged activity and improved conduction under anoxia, and 3) restored QT interval in H hearts. Thus hypoxia-induced downregulation of the adenosinergic system can be regarded as a coping response, limiting the detrimental accumulation of ADO without interfering with ADO signaling.

CD38 symmetric dimethyl site R58 promotes malignant tumor cell immune escape by regulating the cAMP-GSK3ß-PD-L1 axis.

In recent years, immunotherapy has emerged as an effective approach for treating tumors, with programmed cell death ligand 1 (PD-L1)/programmed cell death protein-1 (PD-1) immune checkpoint blockade (ICB) being a promising strategy. However, suboptimal therapeutic efficacy limits its clinical benefit. Understanding the regulation mechanism of PD-L1 expression is crucial for improving anti-PD-L1/PD-1 therapy and developing more effective tumor immunotherapy. Previous studies have revealed that resistance to PD-L1/PD-1 blockade therapy arises from the upregulation of CD38 on tumor cells induced by ATRA and IFN-ß, which mediates the inhibition of CD8+ T cell function through adenosine receptor signaling, thereby promoting immune evasion.Yet, the precise role of CD38 in regulating PD-L1 on malignant tumor cells and its impact on CD8+ T cells through PD-L1 remain unclear. Here, we demonstrate that CD38 is highly expressed in malignant tumors (lung cancer, nasopharyngeal carcinoma, cervical cancer) and upregulates PD-L1 protein expression, impairing CD8+ T cell function. Mechanistically, CD38 phosphorylates GSK3ß via the adenosine-activated cAMP-PKA signaling pathway, leading to GSK3ß inactivation and enhanced PD-L1 stability and expression, facilitating tumor immune escape. Furthermore, we identify PRMT5 as a novel CD38-interacting molecule that symmetrically dimethylates CD38 arginine position 58, augmenting PD-L1 stability and expression through the ADO-cAMP-GSK3ß signaling axis. This inhibits CD8+ T cell-mediated tumor cell killing, enabling tumor cells to evade immune surveillance. Our findings suggest that targeting the CD38 R58 site offers a new avenue for enhancing anti-PD-L1/PD-1 therapy efficacy in tumor treatment.

Impaired olfactory performance and anxiety-like behavior in a rat model of multiple sclerosis are associated with enhanced adenosine signaling in the olfactory bulb via A1R, A2BR, and A3R.

The present study shows that animals with experimental autoimmune encephalomyelitis (EAE) exhibit olfactory dysfunction and impaired general cognitive abilities, as well as anxiety-like behavior. Olfactory dysfunction occurs on average at 2 dpi, well before the onset of the first motor signs of EAE (8-10 dpi). After the initial olfactory dysfunction, the EAE animals show a fluctuation in olfactory performance that resembles the relapsing-remitting course of human MS. The study also shows severe neuroinflammation in the olfactory bulb (OB), with numerous infiltrated CD4+ T cells and peripheral macrophages in the superficial OB layers, marked microgliosis, and massive induction of TNF-α, IL-1ß, and IL-6. Reduced tyrosine hydroxylase activity in the glomerular layer, pronounced granule cell atrophy, and reduced numbers of type B neuroblasts in the rostral migratory stream also indicate altered plasticity of the neuronal network in the OB. Considering the exceptionally high purinome expression in the OB, the possible involvement of purinergic signaling was also investigated. The study shows that macrophages infiltrating the OB overexpress A3R, while highly reactive microglia overexpress the adenosine-producing enzyme eN/CD73 as well as A2BR, A3R, and P2X4R. Given the simultaneous induction of complement component C3, the results suggest that the microglial cells develop a functional phenotype of phagocytizing microglia. The study also demonstrates transcriptional and translational upregulation of A1R in mitral and tufted cells, which likely influence resting network activity in OB and likely contribute to olfactory dysfunction in EAE. Overall, our study shows that olfactory dysfunction and altered social and cognitive behavior in EAE are associated with increased adenosine signaling via A1R, A2BR, and A3R.

Metabolic and neurobehavioral disturbances induced by purine recycling deficiency in Drosophila.

Adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) are two structurally related enzymes involved in purine recycling in humans. Inherited mutations that suppress HGPRT activity are associated with Lesch-Nyhan disease (LND), a rare X-linked metabolic and neurological disorder in children, characterized by hyperuricemia, dystonia, and compulsive self-injury. To date, no treatment is available for these neurological defects and no animal model recapitulates all symptoms of LND patients. Here, we studied LND-related mechanisms in the fruit fly. By combining enzymatic assays and phylogenetic analysis, we confirm that no HGPRT activity is expressed in Drosophila melanogaster, making the APRT homolog (Aprt) the only purine-recycling enzyme in this organism. Whereas APRT deficiency does not trigger neurological defects in humans, we observed that Drosophila Aprt mutants show both metabolic and neurobehavioral disturbances, including increased uric acid levels, locomotor impairments, sleep alterations, seizure-like behavior, reduced lifespan, and reduction of adenosine signaling and content. Locomotor defects could be rescued by Aprt re-expression in neurons and reproduced by knocking down Aprt selectively in the protocerebral anterior medial (PAM) dopaminergic neurons, the mushroom bodies, or glia subsets. Ingestion of allopurinol rescued uric acid levels in Aprt-deficient mutants but not neurological defects, as is the case in LND patients, while feeding adenosine or N6-methyladenosine (m6A) during development fully rescued the epileptic behavior. Intriguingly, pan-neuronal expression of an LND-associated mutant form of human HGPRT (I42T), but not the wild-type enzyme, resulted in early locomotor defects and seizure in flies, similar to Aprt deficiency. Overall, our results suggest that Drosophila could be used in different ways to better understand LND and seek a cure for this dramatic disease.

Biological implications of extracellular adenosine in hepatic ischemia and reperfusion injury.

The purine nucleoside adenosine is clinically employed in the treatment of supraventricular tachycardia. In addition, it has direct coronary vasodilatory effects, and may influence platelet aggregation. Experimental observations mechanistically link extracellular adenosine to cellular adaptation to hypoxia. Adenosine generation has been implicated in several pathophysiologic processes including angiogenesis, tumor defenses and neurodegeneration. In solid organ transplantation, prolonged tissue ischemia and subsequent reperfusion injury may lead to profound graft dysfunction. Importantly, conditions of limited oxygen availability are associated with increased production of extracellular adenosine and subsequent tissue protection. Within the rapidly expanding field of adenosine biology, several enzymatic steps in adenosine production have been characterized and multiple receptor subtypes have been identified. In this review, we briefly examine the biologic steps involved in adenosine generation and chronicle the current state of adenosine signaling in hepatic ischemia and reperfusion injury.

Engineering of an Osteoinductive and Growth Factor-Free Injectable Bone-Like Microgel for Bone Regeneration.

Bone autografts remain the gold standard for bone grafting surgeries despite having increased donor site morbidity and limited availability. Bone morphogenetic protein-loaded grafts represent another successful commercial alternative. However, the therapeutic use of recombinant growth factors has been associated with significant adverse clinical outcomes. This highlights the need to develop biomaterials that closely approximate the structure and composition of bone autografts, which are inherently osteoinductive and biologically active with embedded living cells, without the need for added supplements. Here, injectable growth factor-free bone-like tissue constructs are developed, that closely approximate the cellular, structural, and chemical composition of bone autografts. It is demonstrated that these micro-constructs are inherently osteogenic, and demonstrate the ability to stimulate mineralized tissue formation and regenerate bone in critical-sized defects in-vivo. Furthermore, the mechanisms that allow human mesenchymal stem cells (hMSCs) to be highly osteogenic in these constructs, despite the lack of osteoinductive supplements, are assessed, whereby Yes activated protein (YAP) nuclear localization and adenosine signaling appear to regulate osteogenic cell differentiation. The findings represent a step toward a new class of minimally invasive, injectable, and inherently osteoinductive scaffolds, which are regenerative by virtue of their ability to mimic the tissue cellular and extracellular microenvironment, thus showing promise for clinical applications in regenerative engineering.

The expression of the adenosine pathway markers CD39 and CD73 in salivary gland carcinomas harbors the potential for novel immune checkpoint inhibition.

BACKGROUND: In salivary gland carcinomas (SGC), there is only a small fraction of entities that appears to profit from immune checkpoint inhibition (ICI). Recent findings connected the activation of adenosine-signaling with a tolerogenic microenvironment. Therefore, the inhibition of adenosine pathway markers (CD39 and/or CD73) can augment ICI and/or display a novel immunotherapeutic strategy beyond ICI. Here, we assessed the immuno-histochemical expression of CD39 and CD73 across a wide spectrum of SGCs. METHODS: In total, 114 patients with SGCs consecutively diagnosed between 2001 and 2021 were assessed for clinicopathological baseline characteristics and underwent confirmatory histopathological review. Immunohistochemical expression levels of CD39 and CD73 were assessed by applying the tumor proportion score (TPS) and the immune proportional score (IPS) comparable to PD-L1 expression analysis in routine clinical practice. Additionally, findings were correlated with PD-L1 expression levels. RESULTS: The median age was 60.6 and 51.8% patients were female. The cohort covered a spectrum of eight distinct entities. Advanced-stage disease (UICC/AJCC III/IVA-IVC) at initial diagnosis was present in the majority of patients (64/114). Immunohistochemical staining revealed positivity for CD39 and CD73 in 48.2% and 21.1% on tumor cells (TPS ≥ 1%) as well as 46.4% and 42.9% within the immune cell infiltrate (IPS ≥ 1%), respectively. Further comparative analyses revealed immune-cold entities such adenoid cystic carcinoma (AdCC), immune-hot tumors such as adenocarcinoma, not otherwise specified (AC (NOS)) and entities with intermediate immunologic features such as acinic cell carcinoma (ACC). CONCLUSION: Current results indicate entity-specific adenosine signaling signatures. These findings suggest that the adenosine pathway plays a decisive role in tumor immunity among the major spectrum of SGCs. Targeting the adenosine pathway might pose a promising therapeutic option for selected entities.

Aspirin ameliorates atherosclerotic immuno-inflammation through regulating the Treg/Th17 axis and CD39-CD73 adenosine signaling via remodeling the gut microbiota in ApoE-/- mice.

The gut microbiome has been implicated in the development of cardiovascular disease (CVD) and atherosclerosis (AS), a chronic inflammatory condition. Aspirin may improve the immuno-inflammatory status in AS by regulating microbiota dysbiosis. However, the potential role of aspirin in modulating gut microbiota and microbial-derived metabolites remains less explored. In this study, we investigated the effect of aspirin treatment on AS progression by modulating gut microbiota and microbial-derived metabolites in apolipoprotein E-deficient (ApoE-/-) mice. We analyzed the fecal bacterial microbiome and targeted metabolites, including short-chain fatty acids (SCFAs) and bile acids (BAs). The immuno-inflammatory status of AS was evaluated by analyzing regulatory T cells (Tregs), Th17 cells, and the CD39-CD73 adenosine signaling pathway involved in purinergic signaling. Our results indicated that aspirin altered gut microbiota, leading to an increase in the phylum Bacteroidetes and a decrease in the Firmicutes to Bacteriodetes (F/B) ratio. Aspirin treatment also increased levels of targeted SCFA metabolites, such as propionic acid, valeric acid, isovaleric acid, and isobutyric acid. Furthermore, aspirin impacted BAs by reducing the level of harmful deoxycholic acid (DCA) and increasing the levels of beneficial isoalloLCA and isoLCA. These changes were accompanied by a rebalancing of the ratio of Tregs to Th17 cells and an increase in the expression of ectonucleotidases CD39 and CD73, thereby ameliorating inflammation. These findings suggest that aspirin has an athero-protective effect with an improved immuno-inflammatory profile, partially attributed to its manipulation of the gut microbiota.

Pharmacological Characterization of P626, a Novel Dual Adenosine A2A/A2B Receptor Antagonist, on Synaptic Plasticity and during an Ischemic-like Insult in CA1 Rat Hippocampus.

In recent years, the use of multi-target compounds has become an increasingly pursued strategy to treat complex pathologies, including cerebral ischemia. Adenosine and its receptors (A1AR, A2AAR, A2BAR, A3AR) are known to play a crucial role in synaptic transmission either in normoxic or ischemic-like conditions. Previous data demonstrate that the selective antagonism of A2AAR or A2BAR delays anoxic depolarization (AD) appearance, an unequivocal sign of neuronal injury induced by a severe oxygen-glucose deprivation (OGD) insult in the hippocampus. Furthermore, the stimulation of A2AARs or A2BARs by respective selective agonists, CGS21680 and BAY60-6583, increases pre-synaptic neurotransmitter release, as shown by the decrease in paired-pulse facilitation (PPF) at Schaffer collateral-CA1 synapses. In the present research, we investigated the effect/s of the newly synthesized dual A2AAR/A2BAR antagonist, P626, in preventing A2AAR- and/or A2BAR-mediated effects by extracellular recordings of synaptic potentials in the CA1 rat hippocampal slices. We demonstrated that P626 prevented PPF reduction induced by CGS21680 or BAY60-6583 and delayed, in a concentration-dependent manner, AD appearance during a severe OGD. In conclusion, P626 may represent a putative neuroprotective compound for stroke treatment with the possible translational advantage of reducing side effects and bypassing differences in pharmacokinetics due to combined treatment.