Adenosine A2A Receptor Activation Research Articles

Adenosine A2A receptor activation regulates the M1 macrophages activation to initiate innate and adaptive immunity in psoriasis

Psoriasis is a common inflammatory systemic disease characterized by pro-inflammatory macrophages activation (M1 macrophage) infiltrated in the dermal layer. How M1 macrophage contributes to psoriasis remains unknown. In this study, we found that adenosine A2A receptor (A2AR) agonist CGS 21680 HCl alleviated the imiquimod (IMQ) and mouse IL-23 Protein (rmIL-23)-induced psoriasis inflammation through reducing infiltration of M1. Conversely, Adora2a deletion in mice exacerbated psoriasis-like phenotype. Mechanistically, A2AR activation inhibited M1 macrophage activation via the NF-κB-KRT16 pathway to reduce the secretion of CXCL10/11 and inhibit Th1/17 differentiation. Notably, the KRT16 expression was first found in M1 macrophage in our study, not only in keratinocytes (KCs). CXCL10/11 are first identified as primarily derived from macrophages and dendritic cells (DCs) rather than KCs in psoriasis using single cell RNA sequencing (scRNA-Seq). In total, the study emphasizes the importance of M1 as an innate immune cell in pathogenesis of psoriasis.

An agonist of the adenosine A2A receptor, CGS21680, promotes corneal epithelial wound healing via the YAP signalling pathway.

The adenosine A2A receptor (A2AR) is involved in various physiological and pathological processes in the eye; however, the role of the A2AR signalling in corneal epithelial wound healing is not known. Here, the expression, therapeutic effects and signalling mechanism of A2AR in corneal epithelial wound healing were investigated using the A2AR agonist CGS21680. A2AR localization and expression during wound healing in the murine cornea were determined by immunofluorescence staining, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting. The effect of CGS21680 on corneal epithelial wound healing in the lesioned corneal and cultured human corneal epithelial cells (hCECs) by modulating cellular proliferation and migration was critically evaluated. The role of Hippo-YAP signalling in mediating the CGS21680 effect on wound healing by pharmacological inhibition of YAP signalling was explored. A2AR expression was up-regulated after corneal epithelial injury. Topical administration of CGS21680 dose-dependently promoted corneal epithelial wound healing in the injured corneal epithelium by promoting cellular proliferation. Furthermore, CGS21680 accelerated the cellular proliferation and migration of hCECs in vitro. A2AR activation promoted early up-regulation and later down-regulation of YAP signalling molecules, and pharmacological inhibition of YAP signalling reverted CGS21680-mediated wound healing effect in vivo and in vitro. A2AR activation promotes wound healing by enhancing cellular proliferation and migration through the YAP signalling pathway. A2ARs play an important role in the maintenance of corneal epithelium integrity and may represent a novel therapeutic target for facilitating corneal epithelial wound healing.

Terpenes from Cannabis sativa induce antinociception in a mouse model of chronic neuropathic pain via activation of adenosine A2A receptors.

Terpenes are small hydrocarbon compounds that impart aroma and taste to many plants, including Cannabis sativa. A number of studies have shown that terpenes can produce pain relief in various pain states in both humans and animals. However, these studies were methodologically limited and few established mechanisms of action. In our previous work, we showed that the terpenes geraniol, linalool, β-pinene, α-humulene, and β-caryophyllene produced cannabimimetic behavioral effects via multiple receptor targets. We thus expanded this work to explore the potential antinociception and mechanism of these Cannabis terpenes in a mouse model of chronic pain. We first tested for antinociception by injecting terpenes (200 mg/kg, IP) into male and female CD-1 mice with mouse models of chemotherapy-induced peripheral neuropathy (CIPN) or lipopolysaccharide-induced inflammatory pain, finding that the terpenes produced roughly equal antinociception to 10 mg/kg morphine or 3.2 mg/kg WIN55,212. We further found that none of the terpenes produced reward as measured by conditioned place preference, while low doses of terpene (100 mg/kg) combined with morphine (3.2 mg/kg) produced enhanced antinociception vs either alone. We then used the adenosine A2A receptor (A2AR) selective antagonist istradefylline (3.2 mg/kg, IP) and spinal cord-specific CRISPR knockdown of the A2AR to identify this receptor as the mechanism for terpene antinociception in CIPN. In vitro cAMP and binding studies and in silico modeling studies further suggested that the terpenes act as A2AR agonists. Together these studies identify Cannabis terpenes as potential therapeutics for chronic neuropathic pain and identify a receptor mechanism for this activity.

Astrocytes in preoptic area regulate acute nociception-induced hypothermia through adenosine receptors.

The preoptic area (POA) of the hypothalamus, crucial in thermoregulation, has long been implicated in the pain process. However, whether nociceptive stimulation affects body temperature and its mechanism remains poorly studied. We used capsaicin, formalin, and surgery to induce acute nociceptive stimulation and monitored rectal temperature. Optical fiber recording, chemical genetics, confocal imaging, and pharmacology assays were employed to confirm the role and interaction of POA astrocytes and extracellular adenosine. Immunofluorescence was utilized for further validation. Acute nociception could activate POA astrocytes and induce a decrease in body temperature. Manipulation of astrocytes allowed bidirectional control of body temperature. Furthermore, acute nociception and astrocyte activation led to increased extracellular adenosine concentration within the POA. Activation of adenosine A1 or A2A receptors contributed to decreased body temperature, while inhibition of these receptors mitigated the thermo-lowering effect of astrocytes. Our results elucidate the interplay between acute nociception and thermoregulation, specifically highlighting POA astrocyte activation. This enriches our understanding of physiological responses to painful stimuli and contributes to the analysis of the anatomical basis involved in the process.

Glia-derived adenosine in the ventral hippocampus drives pain-related anxiodepression in a mouse model resembling trigeminal neuralgia

Glial activation and dysregulation of adenosine triphosphate (ATP)/adenosine are involved in the neuropathology of several neuropsychiatric illnesses. The ventral hippocampus (vHPC) has attracted considerable attention in relation to its role in emotional regulation. However, it is not yet clear how vHPC glia and their derived adenosine regulate the anxiodepressive-like consequences of chronic pain. Here, we report that chronic cheek pain elevates vHPC extracellular ATP/adenosine in a mouse model resembling trigeminal neuralgia (rTN), which mediates pain-related anxiodepression, through a mechanism that involves synergistic effects of astrocytes and microglia. We found that rTN resulted in robust activation of astrocytes and microglia in the CA1 area of the vHPC (vCA1). Genetic or pharmacological inhibition of astrocytes and connexin 43, a hemichannel mainly distributed in astrocytes, completely attenuated rTN-induced extracellular ATP/adenosine elevation and anxiodepressive-like behaviors. Moreover, inhibiting microglia and CD39, an enzyme primarily expressed in microglia that degrades ATP into adenosine, significantly suppressed the increase in extracellular adenosine and anxiodepressive-like behaviors. Blockade of the adenosine A2A receptor (A2AR) alleviated rTN-induced anxiodepressive-like behaviors. Furthermore, interleukin (IL)-17A, a pro-inflammatory cytokine probably released by activated microglia, markedly increased intracellular calcium in vCA1 astrocytes and triggered ATP/adenosine release. The astrocytic metabolic inhibitor fluorocitrate and the CD39 inhibitor ARL 67156, attenuated IL-17A-induced increases in extracellular ATP and adenosine, respectively. In addition, astrocytes, microglia, CD39, and A2AR inhibitors all reversed rTN-induced hyperexcitability of pyramidal neurons in the vCA1. Taken together, these findings suggest that activation of astrocytes and microglia in the vCA1 increases extracellular adenosine, which leads to pain-related anxiodepression via A2AR activation. Approaches targeting astrocytes, microglia, and adenosine signaling may serve as novel therapies for pain-related anxiety and depression.

Single-molecule visualization of human A2A adenosine receptor activation by a G protein and constitutively activating mutations

Mutations that constitutively activate G protein-coupled receptors (GPCRs), known as constitutively activating mutations (CAMs), modify cell signaling and interfere with drugs, resulting in diseases with limited treatment options. We utilize fluorescence imaging at the single-molecule level to visualize the dynamic process of CAM-mediated activation of the human A2A adenosine receptor (A2AAR) in real time. We observe an active-state population for all CAMs without agonist stimulation. Importantly, activating mutations significantly increase the population of an intermediate state crucial for receptor activation, notably distinct from the addition of a partner G protein. Activation kinetics show that while CAMs increase the frequency of transitions to the intermediate state, mutations altering sodium sensitivity increase transitions away from it. These findings indicate changes in GPCR function caused by mutations may be predicted based on whether they favor or disfavor formation of an intermediate state, providing a framework for designing receptors with altered functions or therapies that target intermediate states.

Adenosinergic System and BDNF Signaling Changes as a Cross-Sectional Feature of RTT: Characterization of Mecp2 Heterozygous Mouse Females.

Rett Syndrome is an X-linked neurodevelopmental disorder (RTT; OMIM#312750) associated to MECP2 mutations. MeCP2 dysfunction is seen as one cause for the deficiencies found in brain-derived neurotrophic factor (BDNF) signaling, since BDNF is one of the genes under MeCP2 jurisdiction. BDNF signaling is also dependent on the proper function of the adenosinergic system. Indeed, both BDNF signaling and the adenosinergic system are altered in Mecp2-null mice (Mecp2-/y), a representative model of severe manifestation of RTT. Considering that symptoms severity largely differs among RTT patients, we set out to investigate the BDNF and ADO signaling modifications in Mecp2 heterozygous female mice (Mecp2+/-) presenting a less severe phenotype. Symptomatic Mecp2+/- mice have lower BDNF levels in the cortex and hippocampus. This is accompanied by a loss of BDNF-induced facilitation of hippocampal long-term potentiation (LTP), which could be restored upon selective activation of adenosine A2A receptors (A2AR). While no differences were observed in the amount of adenosine in the cortex and hippocampus of Mecp2+/- mice compared with healthy littermates, the density of the A1R and A2AR subtype receptors was, respectively, upregulated and downregulated in the hippocampus. Data suggest that significant changes in BDNF and adenosine signaling pathways are present in an RTT model with a milder disease phenotype: Mecp2+/- female animals. These features strengthen the theory that boosting adenosinergic activity may be a valid therapeutic strategy for RTT patients, regardless of their genetic penetrance.

Adenosine A2A receptors in the rostral ventrolateral medulla participate in blood pressure decrease with electroacupuncture in hypertensive rats.

Acupuncture is increasingly used to manage high blood pressure (BP) as a complementary therapy. However, the mechanisms underlying its hypotensive effects remain unclear. Our previous studies have shown that electroacupuncture (EA) at the ST36-37 acupoints, overlying the deep peroneal nerve, attenuates pressor responses through adenosine A2A receptors (A2AR) in the rostral ventrolateral medulla (rVLM). However, it is uncertain whether rVLM A2AR contributes to EA's BP-lowering effect in sustained hypertension. We hypothesized that a course of EA treatment lowers BP, in part, through the activation of adenosine A2AR in the rVLM in hypertensive rats. To mimic essential hypertension in the clinic, we performed EA in conscious Dahl salt-sensitive hypertensive rats (DSHRs). EA (0.1-0.4 mA, 2 Hz) was applied at ST36-37 for 30 min twice weekly for four weeks, while sham-EA was conducted in a similar manner but without electrical input. In hypertensive rats, BP was reduced by EA (n = 14) but neither by sham-EA (n = 14) nor in the absence of needling (n = 8). Following four weeks of eight treatments and then under anesthesia, EA's modulatory effect on elevated BP was reversed by unilateral rVLM microinjection of SCH 58261 (1 mM in 50 nl; an A2AR antagonist; n = 7; P < 0.05) but not the vehicle (n = 5) in EA-treated DSHRs. Activation of rVLM A2AR in DSHRs treated with sham-EA by an A2AR agonist, CGS-21680 (0.4 mM in 50 nl; n = 8), decreased BP. Unilateral administration of SCH 58261 or CGS-21680 into the rVLM did not alter basal BP in Dahl salt-sensitive rats fed a regular diet with normal BP. The A2AR level in the rVLM after EA was increased compared to the sham-EA and untreated DSHRs (n = 5 in each group; all P < 0.05). These data suggest that a 4-week twice weekly EA treatment reduced BP in salt-sensitive hypertensive rats likely through adenosine-mediated A2AR in the rVLM.

Adenosine A2A receptors control generalization of contextual fear in rats

Fear learning is essential to survival, but traumatic events may lead to abnormal fear consolidation and overgeneralization, triggering fear responses in safe environments, as occurs in post-traumatic stress disorder (PTSD). Adenosine A2A receptors (A2AR) control emotional memory and fear conditioning, but it is not known if they affect the consolidation and generalization of fear, which was now investigated. We now report that A2AR blockade through systemic administration of the A2AR antagonist SCH58261 immediately after contextual fear conditioning (within the consolidation window), accelerated fear generalization. Conversely, A2AR activation with CGS21680 decreased fear generalization. Ex vivo electrophysiological recordings of field excitatory post-synaptic potentials (fEPSPs) in CA3-CA1 synapses and of population spikes in the lateral amygdala (LA), showed that the effect of SCH58261 is associated with a reversion of fear conditioning-induced decrease of long-term potentiation (LTP) in the dorsal hippocampus (DH) and with increased amplitude of LA LTP in conditioned animals. These data suggest that A2AR are engaged during contextual fear consolidation, controlling long-term potentiation mechanisms in both DH and LA during fear consolidation, impacting on fear generalization; this supports targeting A2AR during fear consolidation to control aberrant fear processing in PTSD and other fear-related disorders.

Electroacupuncture stimulating Zusanli (ST36), Sanyinjiao (SP6) in mice with collagen-induced arthritis leads to adenosine A2A receptor-mediated alteration of p38α mitogen-activated protein kinase signaling and inhibition of osteoclastogenesis.

To observe the effect of electroacupuncture (EA) stimulating Zusanli (ST36), Sanyinjiao (SP6) on inhibition of osteoclastogenesis and the role of the adenosine A2A receptor (A2AR) and the p38α Mitogen-Activated Protein Kinase (MAPK) signaling pathway in mediating this effect. Mice with collagen induced arthritis (CIA) received different treatments. Immunohistochemistry and western blotting were used to determine the levels of multiple signaling molecules in these joints [receptor activator of nuclear transcription factor-κB (NF-κB) ligand (RANKL), receptor activator of NF-κB (RANK), tumor necrosis factor receptor associated factor 6 (TRAF6), p38α, NF-κB, and nuclear factor of activated T cells C1 (NFATc1)]. Osteoclasts were identified using tartrate-resistant acid phosphatase (TRAP) staining. The immunohistochemistry results indicated upregulation of p38α, NF-κB, and NFATc1 in the CIA-control and CIA-EA-SCH58261 groups, but reduced levels in the CIA-EA group. Western blotting indicated upregulation of RANKL, RANK, TRAF6, p38α, NF-κB, and NFATc1 in the CIA-control and CIA-EA-SCH58261 groups, but reduced expression in the CIA-EA group. Osteoclasts were more abundant in the CIA-control and CIA-EA-SCH58261 groups than in the CIA-EA group. EA treatment enhanced the A2AR activity and inhibited osteoclast formation by inhibition of RANKL, RANK, TRAF6, p38α, NF-κB, and NFATc1. SCH58261 reversed the effect of EA. These results suggest that EA regulated p38α-MAPK signaling by increasing A2AR activity, which inhibited osteoclastogenesis.

Adenosine reuptake inhibition reduces diabetes-induced glomerular hyperfiltration via the adenosine A2a receptor.

Diabetes-induced glomerular hyperfiltration is an early alteration in kidney function in diabetes. Previous studies have shown that reduced adenosine A2a receptor signaling contributes to diabetes-induced glomerular hyperfiltration. The present study investigated the effects of enhanced interstitial adenosine concentration by inhibition of cellular adenosine reuptake, thereby promoting endogenous adenosine signaling. Insulinopenic diabetes was induced by streptozotocin in adult male Sprague-Dawley rats. Two weeks after diabetes induction, kidney function in terms of glomerular filtration rate, and total, cortical, and medullary renal blood flows were evaluated under thiobutabarbital anesthesia during baseline and after renal artery infusion of two doses of the adenosine reuptake inhibitor dilazep. Dilazep did not affect mean arterial pressure indicating that the effects of the interventions were intrarenal. Diabetics had increased glomerular filtration rate compared with controls and dilazep dose-dependently decreased glomerular filtration rate in diabetics, whereas it had no significant effect in controls. Dilazep increased cortical renal blood flows in controls, whereas medullary blood flow was not significantly changed. Dilazep did not affect total renal blood flow in any of the groups but decreased cortical blood flow in diabetics, resulting in decreased filtration fraction by dilazep in diabetics. Pretreatment with the adenosine A2a antagonist ZM241385 prevented intrarenal dilazep-mediated effects on glomerular filtration rate and filtration fraction in diabetics. In conclusion, enhancing intrarenal adenosine signaling by dilazep normalizes diabetes-induced glomerular hyperfiltration at least in part by activation of adenosine A2a receptors.

Adenosine A2A Receptor Regulates microRNA-181b Expression in Aorta: Therapeutic Implications for Large-Artery Stiffness.

Background The identification of large-artery stiffness as a major, independent risk factor for cardiovascular disease-associated morbidity and death has focused attention on identifying therapeutic strategies to combat this disorder. Genetic manipulations that delete or inactivate the translin/trax microRNA-degrading enzyme confer protection against aortic stiffness induced by chronic ingestion of high-salt water (4%NaCl in drinking water for 3 weeks) or associated with aging. Therefore, there is heightened interest in identifying interventions capable of inhibiting translin/trax RNase activity, as these may have therapeutic efficacy in large-artery stiffness. Methods and Results Activation of neuronal adenosine A2A receptors (A2ARs) triggers dissociation of trax from its C-terminus. As A2ARs are expressed by vascular smooth muscle cells (VSMCs), we investigated whether stimulation of A2AR on vascular smooth muscle cells promotes the association of translin with trax and, thereby increases translin/trax complex activity. We found that treatment of A7r5 cells with the A2AR agonist CGS21680 leads to increased association of trax with translin. Furthermore, this treatment decreases levels of pre-microRNA-181b, a target of translin/trax, and those of its downstream product, mature microRNA-181b. To check whether A2AR activation might contribute to high-salt water-induced aortic stiffening, we assessed the impact of daily treatment with the selective A2AR antagonist SCH58261 in this paradigm. We found that this treatment blocked aortic stiffening induced by high-salt water. Further, we confirmed that the age-associated decline in aortic pre-microRNA-181b/microRNA-181b levels observed in mice also occurs in humans. Conclusions These findings suggest that further studies are warranted to evaluate whether blockade of A2ARs may have therapeutic potential in treating large-artery stiffness.

A2AR-mediated lymphangiogenesis via VEGFR2 signaling prevents salt-sensitive hypertension.

Excess dietary sodium intake and retention lead to hypertension. Impaired dermal lymphangiogenesis and lymphatic dysfunction-mediated sodium and fluid imbalance are pathological mechanisms. The adenosine A2A receptor (A2AR) is expressed in lymphatic endothelial cells (LECs), while the roles and mechanisms of LEC-A2AR in skin lymphangiogenesis during salt-induced hypertension are not clear. The expression of LEC-A2AR correlated with lymphatic vessel density in both high-salt diet (HSD)-induced hypertensive mice and hypertensive patients. Lymphatic endothelial cell-specific A2AR knockout mice fed HSD exhibited 17 ± 2% increase in blood pressure and 17 ± 3% increase in Na+ content associated with decreased lymphatic density (-19 ± 2%) compared with HSD-WT mice. A2AR activation by agonist CGS21680 increased lymphatic capillary density and decreased blood pressure in HSD-WT mice. Furthermore, this A2AR agonist activated MSK1 directly to promote VEGFR2 activation and endocytosis independently of VEGF as assessed by phosphoprotein profiling and immunoprecipitation assays in LECs. VEGFR2 kinase activity inhibitor fruquintinib or VEGFR2 knockout in LECs but not VEGF-neutralizing antibody bevacizumab suppressed A2AR activation-mediated decrease in blood pressure. Immunostaining revealed phosphorylated VEGFR2 and MSK1 expression in the LECs were positively correlated with skin lymphatic vessel density and A2AR level in hypertensive patients. The study highlights a novel A2AR-mediated VEGF-independent activation of VEGFR2 signaling in dermal lymphangiogenesis and sodium balance, which might be a potential therapeutic target in salt-sensitive hypertension.

Adenosine A2A Receptor Activation Regulates Niemann-Pick C1 Expression and Localization in Macrophages.

Adenosine plays an important role in modulating immune cell function, particularly T cells and myeloid cells, such as macrophages and dendritic cells. Cell surface adenosine A2A receptors (A2AR) regulate the production of pro-inflammatory cytokines and chemokines, as well as the proliferation, differentiation, and migration of immune cells. In the present study, we expanded the A2AR interactome and provided evidence for the interaction between the receptor and the Niemann-Pick type C intracellular cholesterol transporter 1 (NPC1) protein. The NPC1 protein was identified to interact with the C-terminal tail of A2AR in RAW 264.7 and IPMФ cells by two independent and parallel proteomic approaches. The interaction between the NPC1 protein and the full-length A2AR was further validated in HEK-293 cells that permanently express the receptor and RAW264.7 cells that endogenously express A2AR. A2AR activation reduces the expression of NPC1 mRNA and protein density in LPS-activated mouse IPMФ cells. Additionally, stimulation of A2AR negatively regulates the cell surface expression of NPC1 in LPS-stimulated macrophages. Furthermore, stimulation of A2AR also altered the density of lysosome-associated membrane protein 2 (LAMP2) and early endosome antigen 1 (EEA1), two endosomal markers associated with the NPC1 protein. Collectively, these results suggested a putative A2AR-mediated regulation of NPC1 protein function in macrophages, potentially relevant for the Niemann-Pick type C disease when mutations in NPC1 protein result in the accumulation of cholesterol and other lipids in lysosomes.

Dual mechanisms of cholesterol-GPCR interactions that depend on membrane phospholipid composition

Cholesterol is a critical component of mammalian cell membranes and an allosteric modulator of G protein-coupled receptors (GPCRs), but divergent views exist on the mechanisms by which cholesterol influences receptor functions. Leveraging the benefits of lipid nanodiscs, i.e., quantitative control of lipid composition, we observe distinct impacts of cholesterol in the presence and absence of anionic phospholipids on the function-related conformational dynamics of the human A2A adenosine receptor (A2AAR). Direct receptor-cholesterol interactions drive activation of agonist-bound A2AAR in membranes containing zwitterionic phospholipids. Intriguingly, the presence of anionic lipids attenuates cholesterol's impact through direct interactions with the receptor, highlighting a more complex role for cholesterol that depends on membrane phospholipid composition. Targeted amino acid replacements at two frequently predicted cholesterol interaction sites showed distinct impacts of cholesterol at different receptor locations, demonstrating the ability to delineate different roles of cholesterol in modulating receptor signaling and maintaining receptor structural integrity.

Lateral septum adenosine A2A receptors control stress-induced depressive-like behaviors via signaling to the hypothalamus and habenula

Major depressive disorder ranks as a major burden of disease worldwide, yet the current antidepressant medications are limited by frequent non-responsiveness and significant side effects. The lateral septum (LS) is thought to control of depression, however, the cellular and circuit substrates are largely unknown. Here, we identified a subpopulation of LS GABAergic adenosine A2A receptors (A2AR)-positive neurons mediating depressive symptoms via direct projects to the lateral habenula (LHb) and the dorsomedial hypothalamus (DMH). Activation of A2AR in the LS augmented the spiking frequency of A2AR-positive neurons leading to a decreased activation of surrounding neurons and the bi-directional manipulation of LS-A2AR activity demonstrated that LS-A2ARs are necessary and sufficient to trigger depressive phenotypes. Thus, the optogenetic modulation (stimulation or inhibition) of LS-A2AR-positive neuronal activity or LS-A2AR-positive neurons projection terminals to the LHb or DMH, phenocopied depressive behaviors. Moreover, A2AR are upregulated in the LS in two male mouse models of repeated stress-induced depression. This identification that aberrantly increased A2AR signaling in the LS is a critical upstream regulator of repeated stress-induced depressive-like behaviors provides a neurophysiological and circuit-based justification of the antidepressant potential of A2AR antagonists, prompting their clinical translation.

Berberine attenuates epithelial mesenchymal transition in bleomycin-induced pulmonary fibrosis in mice via activating A2aR and mitigating the SDF-1/CXCR4 signaling

AimsBerberine is endowed with anti-oxidant, anti-inflammatory and anti-fibrotic effects. This study explored the role of adenosine A2a receptor (A2aR) activation and SDF-1/CXCR4 signaling suppression in the protective effects of berberine in bleomycin-induced pulmonary fibrosis in mice. Main methodsPulmonary fibrosis was generated in mice by injecting bleomycin (40 U/kg, i.p.) on days 0, 3, 7, 10 and 14. Mice were treated with berberine (5 mg/kg, i.p.) from day 15 to day 28. Key findingsSevere lung fibrosis and increased collagen content were observed in the bleomycin-challenged mice. Pulmonary A2aR downregulation was documented in bleomycin-induced pulmonary fibrosis animals and was accompanied by enhanced expression of SDF-1/CXCR4. Moreover, TGF-β1elevation and pSmad2/3 overexpression were reported in parallel with enhanced epithelial mesenchymal transition (EMT) markers expression, vimentin and α-SMA. Besides, bleomycin significantly elevated the inflammatory and pro-fibrogenic mediator NF-κB p65, TNF-α and IL-6. Furthermore, bleomycin administration induced oxidative stress as depicted by decreased Nrf2, SOD, GSH and catalase levels. Interestingly, berberine administration markedly ameliorated the fibrotic changes in lungs by modulating the purinergic system through the inhibition of A2aR downregulation, mitigating EMT and effectively suppressing inflammation and oxidative stress. Strikingly, A2aR blockade by SCH 58261, impeded the pulmonary protective effect of berberine. SignificanceThese findings indicated that berberine could attenuate the pathological processes of bleomycin-induced pulmonary fibrosis at least partially via upregulating A2aR and mitigating the SDF-1/CXCR4 related pathway, suggesting A2aR as a potential therapeutic target for the management of pulmonary fibrosis.

Sub-millisecond conformational dynamics of the A2A adenosine receptor revealed by single-molecule FRET

The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Förster Resonance Energy Transfer (smFRET) is well suited to quantify dynamics for individual protein molecules; however, its application to GPCRs is challenging. Therefore, smFRET has been limited to studies of inter-receptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A2A adenosine receptor (A2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A2AAR, explaining the receptor’s constitutive activity. For the agonist-bound A2AAR, we detected faster (390 ± 80 µs) ligand efficacy-dependent dynamics. Our work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.

Preservation of vascular endothelial glycocalyx and barrier by activation of adenosine A2A receptor (A2AR) improved renal dysfunction in cirrhotic rats

Cirrhosis-related hepatic and renal endothelial dysfunction is characterized by macrophage-endothelium adhesion-mediated inflammation, glycocalyx/barrier damage, and impaired vasodilation. Activation of adenosine A2A receptor (A2AR) protects cirrhotic rats from impairment of hepatic microcirculation post hepatectomy. This study evaluates the effects of A2AR activation on the cirrhosis-related hepatic and renal endothelial dysfunction in biliary cirrhotic rats receiving two weeks of A2AR agonist PSB0777 [bile duct ligated (BDL)+PSB0777] treatment.Endothelial dysfunction in cirrhotic liver, renal vessels, and kidney is characterized by downregulation of the A2AR expressions, decreased vascular endothelial vasodilatory (p-eNOS)/anti-inflammatory (IL-10/IL-10R)/barrier [VE-cadherin (CDH5) and β-catenin (CTNNB1)]/glycocalyx [syndecan-1 (SDC1) and hyaluronan synthase-2 (HAS2)] markers, and increased leukocyte-endothelium adhesion molecules (F4/80, CD68, ICAM-1, and VCAM-1). In BDL rats, PSB0777 treatment improves hepatic and renal endothelial dysfunction, ameliorates portal hypertension, and attenuates renal hypoperfusion by restoring of the vascular endothelial anti-inflammatory, barrier, glycocalyx markers and vasodilatory response as well as inhibiting the leukocyte-endothelium adhesion. In an in vitro study, conditioned medium (CM) of bone marrow-derived macrophage (BMDM) of BDL rats [BMDM-CM (BDL)] induced barrier/glycocalyx damage, which was reversed by the PSB0777 pre-treatment. The A2AR agonist is a potential agent that can simultaneously correct cirrhosis-related hepatic and renal endothelial dysfunction, portal hypertension, renal hypoperfusion, and renal dysfunction.

Regadenoson Reduces Soluble Receptor for Advanced Glycation End-Products in Lung Recipients

BackgroundThe selective adenosine A2A receptor (A2AR) agonist regadenoson reduces inflammation due to lung ischemia-reperfusion injury (IRI). The objective of this study was to investigate molecular and cellular mechanisms by which regadenoson reduces IRI in lung transplant recipients. MethodsFourteen human lung transplant recipients were infused for 12 hours with regadenoson and 7 more served as untreated controls. Plasma levels of high mobility group box 1 and its soluble receptor for advanced glycation end-products (sRAGE) were measured by Luminex. Matrix metalloproteinase (MMP) 2 and 9 were measured by gelatin zymography. Tissue inhibitor of metalloproteinase 1 was measured by mass spectroscopy. A2AR expression on leukocytes was analyzed by flow cytometry. MMP-9–mediated cleavage of RAGE was evaluated using cultured macrophages in vitro. ResultsRegadenoson treatment during lung transplantation significantly reduced levels of MMP-9 (P < .05), but not MMP-2, and elevated levels of tissue inhibitor of metalloproteinase 1 (P < .05), an endogenous selective inhibitor of MMP-9. Regadenoson infusion significantly reduced plasma levels of sRAGE (P < .05) during lung reperfusion compared with control subjects. A2AR expression was highest on invariant natural killer T cells and higher on monocytes than other circulating immune cells (P < .05). The shedding of RAGE from cultured monocytes/macrophages was increased by MMP-9 stimulation and reduced by an MMP inhibitor or by A2AR agonists, regadenoson or ATL146e. ConclusionsIn vivo and in vitro studies suggest that A2AR activation reduces sRAGE in part by inhibiting MMP-9 production by monocytes/macrophages. These results suggest a novel molecular mechanism by which A2AR agonists reduce primary graft dysfunction.