Improving combination therapies: targeting A2B-adenosine receptor to modulate metabolic tumor microenvironment and immunosuppression.

BACKGROUND: We investigated the role of A2B-adenosine receptor in regulating immunosuppressive metabolic stress in the tumor microenvironment. Novel A2B-adenosine receptor antagonist PBF-1129 was tested for antitumor activity in mice and evaluated for safety and immunologic efficacy in a phase I clinical trial of patients with non-small cell lung cancer. METHODS: The antitumor efficacy of A2B-adenosine receptor antagonists and their impact on the metabolic and immune tumor microenvironment were evaluated in lung, melanoma, colon, breast, and epidermal growth factor receptor-inducible transgenic cancer models. Employing electron paramagnetic resonance, we assessed changes in tumor microenvironment metabolic parameters, including pO2, pH, and inorganic phosphate, during tumor growth and evaluated the immunologic effects of PBF-1129, including its pharmacokinetics, safety, and toxicity, in patients with non-small cell lung cancer. RESULTS: Levels of metabolic stress correlated with tumor growth, metastasis, and immunosuppression. Tumor interstitial inorganic phosphate emerged as a correlative and cumulative measure of tumor microenvironment stress and immunosuppression. A2B-adenosine receptor inhibition alleviated metabolic stress, downregulated expression of adenosine-generating ectonucleotidases, increased expression of adenosine deaminase, decreased tumor growth and metastasis, increased interferon γ production, and enhanced the efficacy of antitumor therapies following combination regimens in animal models (anti-programmed cell death 1 protein vs anti-programmed cell death 1 protein plus PBF-1129 treatment hazard ratio = 11.74 [95% confidence interval = 3.35 to 41.13], n = 10, P < .001, 2-sided F test). In patients with non-small cell lung cancer, PBF-1129 was well tolerated, with no dose-limiting toxicities; demonstrated pharmacologic efficacy; modulated the adenosine generation system; and improved antitumor immunity. CONCLUSIONS: Data identify A2B-adenosine receptor as a valuable therapeutic target to modify metabolic and immune tumor microenvironment to reduce immunosuppression, enhance the efficacy of immunotherapies, and support clinical application of PBF-1129 in combination therapies.

Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) impacts TGF-ß1 responses: insights into cardiac fibrosis and function following myocardial infarction.

Extracellular purine nucleotides and nucleosides released from activated or injured cells influence multiple aspects of cardiac physiology and pathophysiology. Ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1; CD39) hydrolyzes released nucleotides and thereby regulates the magnitude and duration of purinergic signaling. However, the impact of CD39 activity on post-myocardial infarction (MI) remodeling is incompletely understood. We measured the levels and activity of ectonucleotidases in human left ventricular samples from control and ischemic cardiomyopathy (ICM) hearts and examined the impact of ablation of Cd39 expression on post-myocardial infarction remodeling in mice. We found that human CD39 levels and activity are significantly decreased in ICM hearts (n = 5) compared with control hearts (n = 5). In mice null for Cd39, cardiac function and remodeling are significantly compromised in Cd39-/- mice following myocardial infarction. Fibrotic markers including plasminogen activator inhibitor-1 (PAI-1) expression, fibrin deposition, α-smooth muscle actin (αSMA), and collagen expression are increased in Cd39-/- hearts. Importantly, we found that transforming growth factor ß1 (TGF-ß1) stimulates ATP release and induces Cd39 expression and activity on cardiac fibroblasts, constituting an autocrine regulatory pathway not previously appreciated. Absence of CD39 activity on cardiac fibroblasts exacerbates TGF-ß1 profibrotic responses. Treatment with exogenous ectonucleotidase rescues this profibrotic response in Cd39-/- fibroblasts. Together, these data demonstrate that CD39 has important interactions with TGF-ß1-stimulated autocrine purinergic signaling in cardiac fibroblasts and dictates outcomes of cardiac remodeling following myocardial infarction. Our results reveal that ENTPD1 (CD39) regulates TGF-ß1-mediated fibroblast activation and limits adverse cardiac remodeling following myocardial infarction.NEW & NOTEWORTHY We show that CD39 is a critical modulator of TGF-ß1-mediated fibroblast activation and cardiac remodeling following myocardial infarction via modulation of nucleotide signaling. TGF-ß1-induced CD39 expression generates a negative feedback loop that attenuates cardiac fibroblast activation. In the absence of CD39 activity, collagen deposition is increased, elastin expression is decreased, and diastolic dysfunction is worsened. Treatment with ecto-apyrase attenuates the TGF-ß1-induced profibrotic cardiac fibroblast phenotype, revealing a novel approach to combat post-myocardial infarction cardiac fibrosis.

Adenosine/TGFß axis in regulation of mammary fibroblast functions.

Cancer associated fibroblasts (CAF) play a key role in cancer progression and metastasis. Diminished TGFß response on CAF correlates with poor outcome and recurrence in cancer patients. Mechanisms behind lost TGFß signaling on CAF are poorly understood, but, utilizing MMTV-PyMT mouse model, we have previously demonstrated that in tumor microenvironment myeloid cells, producing adenosine, contribute to downregulated TGFß signaling on CAFs. In the current work, we performed serial in vitro studies to investigate the role of adenosine/TGFß axis in mouse mammary fibroblast functions, i.e., proliferation, protein expression, migration, and contractility. We found that adenosine analog NECA diminished TGFß-induced CCL5 and MMP9 expression. Additionally, we discovered that NECA completely inhibited effect of TGFß to upregulate αSMA, key protein of cytoskeletal rearrangements, necessary for migration and contractility of fibroblasts. Our results show that TGFß increases contractility of mouse mammary fibroblasts and human fibroblast cell lines, and NECA attenuates theses effects. Using pharmacological approach and genetically modified animals, we determined that NECA effects on TGFß pathway occur via A2A/A2B adenosine receptor-AC-PKA dependent manner. Using isolated CD11b+ cells from tumor tissue of CD73-KO and CD39-KO animals in co-culture experiments with ATP and AMP, we confirmed that myeloid cells can affect functions of mammary fibroblasts through adenosine signaling. Our data suggest a novel mechanism of interaction between adenosine and TGFß signaling pathways that can impact phenotype of fibroblasts in a tumor microenvironment.

Myeloid Cell-Derived TGFß Signaling Regulates ECM Deposition in Mammary Carcinoma via Adenosine-Dependent Mechanisms.

TGFß plays a crucial role in the tumor microenvironment by regulating cell-cell and cell-stroma interactions. We previously demonstrated that TGFß signaling on myeloid cells regulates expression of CD73, a key enzyme for production of adenosine, a protumorigenic metabolite implicated in regulation of tumor cell behaviors, immune response, and angiogenesis. Here, using an MMTV-PyMT mouse mammary tumor model, we discovered that deletion of TGFß signaling on myeloid cells (PyMT/TGFßRIILysM) affects extracellular matrix (ECM) formation in tumor tissue, specifically increasing collagen and decreasing fibronectin deposition. These changes were associated with mitigated tumor growth and reduced metastases. Reduced TGFß signaling on fibroblasts was associated with their proximity to CD73+ myeloid cells in tumor tissue. Consistent with these findings, adenosine significantly downregulated TGFß signaling on fibroblasts, an effect regulated by A2A and A2B adenosine receptors. METABRIC dataset analysis revealed that patients with triple-negative breast cancer and basal type harbored a similar signature of adenosine and ECM profiles; high expression of A2B adenosine receptors correlated with decreased expression of Col1 and was associated with poor outcome. Taken together, our studies reveal a new role for TGFß signaling on myeloid cells in tumorigenesis. This discovered cross-talk between TGFß/CD73 on myeloid cells and TGFß signaling on fibroblasts can contribute to ECM remodeling and protumorigenic actions of cancer-associated fibroblasts. SIGNIFICANCE: TGFß signaling on fibroblasts is decreased in breast cancer, correlates with poor prognosis, and appears to be driven by adenosine that accelerates tumor progression and metastasis via ECM remodeling.

ERBB signaling attenuates proinflammatory activation of nonclassical monocytes.

Immune activation in chronic systolic heart failure (HF) correlates with disease severity and prognosis. Recombinant neuregulin-1 (rNRG-1) is being developed as a possible therapy for HF, based on the activation of ERBB receptors in cardiac cells. Work in animal models of HF led us to hypothesize that there may be direct effects of NRG-1 on immune system activation and inflammation. We investigated the expression of ERBB receptors and the effect of rNRG-1 isoform glial growth factor 2 (GGF2) in subpopulations of peripheral blood mononuclear cells (PB MNCs) in subjects with HF. We found that human monocytes express both ERBB2 and ERBB3 receptors, with high interindividual variability among subjects. Monocyte surface ERBB3 and TNF-α mRNA expression were inversely correlated in subjects with HF but not in human subjects without HF. GGF2 activation of ERBB signaling ex vivo inhibited LPS-induced TNF-α production, specifically in the CD14lowCD16+ population of monocytes in a phosphoinositide 3-kinase-dependent manner. GGF2 suppression of TNF-α correlated directly with the expression of ERBB3. In vivo, a single dose of intravenous GGF2 reduced TNF-α expression in PB MNCs of HF subjects participating in a phase I safety study of GGF2. These results support a role for ERBB3 signaling in the regulation of TNF-α production from CD14lowCD16+ monocytes and a need for further investigation into the clinical significance of NRG-1/ERBB signaling as a modulator of immune system function.NEW & NOTEWORTHY This study identified a novel role of neuregulin-1 (NRG-1)/ERBB signaling in the control of proinflammatory activation of monocytes. These results further improve our fundamental understanding of cardioprotective effects of NRG-1 in patients with heart failure.

Increased Number of Circulating CD8/CD26 T Cells in the Blood of Duchenne Muscular Dystrophy Patients Is Associated with Augmented Binding of Adenosine Deaminase and Higher Muscular Strength Scores.

Duchenne muscular dystrophy (DMD) is an X-linked disorder that leads to cardiac and skeletal myopathy. The complex immune activation in boys with DMD is incompletely understood. To better understand the contribution of the immune system into the progression of DMD, we performed a systematic characterization of immune cell subpopulations obtained from peripheral blood of DMD subjects and control donors. We found that the number of CD8 cells expressing CD26 (also known as adenosine deaminase complexing protein 2) was increased in DMD subjects compared to control. No differences, however, were found in the levels of circulating factors associated with pro-inflammatory activation of CD8/CD26 cells, such as tumor necrosis factor-α (TNFα), granzyme B, and interferon-γ (IFNγ). The number of CD8/CD26 cells correlated directly with quantitative muscle testing (QMT) in DMD subjects. Since CD26 mediates binding of adenosine deaminase (ADA) to the T cell surface, we tested ADA-binding capacity of CD8/CD26 cells and the activity of bound ADA. We found that mononuclear cells (MNC) obtained from DMD subjects with an increased number of CD8/CD26 T cells had a greater capacity to bind ADA. In addition, these MNC demonstrated increased hydrolytic deamination of adenosine to inosine. Altogether, our data demonstrated that (1) an increased number of circulating CD8/CD26 T cells is associated with preservation of muscle strength in DMD subjects, and (2) CD8/CD26 T cells from DMD subjects mediated degradation of adenosine by adenosine deaminase. These results support a role for T cells in slowing the decline in skeletal muscle function, and a need for further investigation into contribution of CD8/CD26 T cells in the regulation of chronic inflammation associated with DMD.

Extracellular nucleotide regulation and signaling in cardiac fibrosis.

Following myocardial infarction, purinergic nucleotides and nucleosides are released via non-specific and specific mechanisms in response to cellular activation, stress, or injury. These extracellular nucleotides are potent mediators of physiologic and pathologic responses, contributing to the inflammatory and fibrotic milieu within the injured myocardium. Via autocrine or paracrine signaling, cell-specific effects occur through differentially expressed purinergic receptors of the P2X, P2Y, and P1 families. Nucleotide activation of the ionotropic (ligand-gated) purine receptors (P2X) and several of the metabotropic (G-protein-coupled) purine receptors (P2Y) or adenosine activation of the P1 receptors can have profound effects on inflammatory cell function, fibroblast function, and cardiomyocyte function. Extracellular nucleotidases that hydrolyze released nucleotides regulate the magnitude and duration of purinergic signaling. While there are numerous studies on the role of the purinergic signaling pathway in cardiovascular disease, the extent to which the purinergic signaling pathway modulates cardiac fibrosis is incompletely understood. Here we provide an overview of the current understanding of how the purinergic signaling pathway modulates cardiac fibroblast function and myocardial fibrosis.

Temporary, Systemic Inhibition of the WNT/ß-Catenin Pathway promotes Regenerative Cardiac Repair following Myocardial Infarct.

AIMS: The WNT/ß-catenin pathway is temporarily activated in the heart following myocardial infarction (MI). Despite data from genetic models indicating both positive and negative roles for the WNT pathway depending on the model used, the effect of therapeutic inhibition of WNT pathway on post-injury outcome and the cellular mediators involved are not completely understood. Using a newly available, small molecule, GNF-6231, which averts WNT pathway activation by blocking secretion of all WNT ligands, we sought to investigate whether therapeutic inhibition of the WNT pathway temporarily after infarct can mitigate post injury cardiac dysfunction and fibrosis and the cellular mechanisms responsible for the effects. METHODS AND RESULTS: Pharmacologic inhibition of the WNT pathway by post-MI intravenous injection of GNF-6231 in C57Bl/6 mice significantly reduced the decline in cardiac function (Fractional Shortening at day 30: 38.71 ± 4.13% in GNF-6231 treated vs. 34.89 ± 4.86% in vehicle-treated), prevented adverse cardiac remodeling, and reduced infarct size (9.07 ± 3.98% vs. 17.18 ± 4.97%). WNT inhibition augmented proliferation of interstitial cells, particularly in the distal myocardium, inhibited apoptosis of cardiomyocytes, and reduced myofibroblast proliferation in the peri-infarct region. In vitro studies showed that WNT inhibition increased proliferation of Sca1+ cardiac progenitors, improved survival of cardiomyocytes, and inhibited collagen I synthesis by cardiac myofibroblasts. CONCLUSION: Systemic, temporary pharmacologic inhibition of the WNT pathway using an orally bioavailable drug immediately following MI resulted in improved function, reduced adverse remodeling and reduced infarct size in mice. Therapeutic WNT inhibition affected multiple aspects of infarct repair: it promoted proliferation of cardiac progenitors and other interstitial cells, inhibited myofibroblast proliferation, improved cardiomyocyte survival, and reduced collagen I gene expression by myofibroblasts. Our data point to a promising role for WNT inhibitory therapeutics as a new class of drugs to drive post-MI repair and prevent heart failure.

Wnt10b Gain-of-Function Improves Cardiac Repair by Arteriole Formation and Attenuation of Fibrosis.

RATIONALE: Myocardial infarction causes irreversible tissue damage, leading to heart failure. We recently discovered that canonical Wnt signaling and the Wnt10b ligand are strongly induced in mouse hearts after infarction. Wnt10b regulates cell fate in various organs, but its role in the heart is unknown. OBJECTIVE: To investigate the effect of Wnt10b gain-of-function on cardiac repair mechanisms and to assess its potential to improve ventricular function after injury. METHODS AND RESULTS: Histological and molecular analyses showed that Wnt10b is expressed in cardiomyocytes and localized in the intercalated discs of mouse and human hearts. After coronary artery ligation or cryoinjury in mice, Wnt10b is strongly and transiently induced in peri-infarct cardiomyocytes during granulation tissue formation. To determine the effect of Wnt10b on neovascularization and fibrosis, we generated a mouse line to increase endogenous Wnt10b levels in cardiomyocytes. We found that gain of Wnt10b function orchestrated a recovery phenotype characterized by robust neovascularization of the injury zone, less myofibroblasts, reduced scar size, and improved ventricular function compared with wild-type mice. Wnt10b stimulated expression of vascular endothelial growth factor receptor 2 in endothelial cells and angiopoietin-1 in vascular smooth muscle cells through nuclear factor-κB activation. These effects coordinated endothelial growth and smooth muscle cell recruitment, promoting robust formation of large, coronary-like blood vessels. CONCLUSION: Wnt10b gain-of-function coordinates arterial formation and attenuates fibrosis in cardiac tissue after injury. Because generation of mature blood vessels is necessary for efficient perfusion, our findings could lead to novel strategies to optimize the inherent repair capacity of the heart and prevent the onset of heart failure.

Expression of adenosine receptors in monocytes from patients with bronchial asthma.

Adenosine is generated from adenosine triphosphate, which is released by stressed and damaged cells. Adenosine levels are significantly increased in patients with bronchial asthma (BA) and mediate mast cell degranulation and bronchoconstriction. Over the last decade, increasing evidence has shown that adenosine can modulate the innate immune response during monocytes differentiation towards mature myeloid cells. These adenosine-differentiated myeloid cells, characterized by co-expression of monocytes/macrophages and dendritic cell markers such as CD14 and CD209, produce high levels of pro-inflammatory cytokines, thus contributing to the pathogenesis of BA and chronic obstructive pulmonary disease. We found that expression of ADORA2A and ADORA2B are increased in monocytes obtained from patients with BA, and are associated with the generation of CD14(pos)CD209(pos) pro-inflammatory cells. A positive correlation between expression of ADORA2B and IL-6 was identified in human monocytes and may explain the increased expression of IL-6 mRNA in asthmatics. Taken together, our results suggest that monocyte-specific expression of A2 adenosine receptors plays an important role in pro-inflammatory activation of human monocytes, thus contributing to the progression of asthma.

Anti-remodeling and anti-fibrotic effects of the neuregulin-1ß glial growth factor 2 in a large animal model of heart failure.

BACKGROUND: Neuregulin-1ß (NRG-1ß) is a growth factor critical for cardiac development and repair with therapeutic potential for heart failure. We previously showed that the glial growth factor 2 (GGF2) isoform of NRG-1ß improves cardiac function in rodents after myocardial infarction (MI), but its efficacy in a large animal model of cardiac injury has not been examined. We therefore sought to examine the effects of GGF2 on ventricular remodeling, cardiac function, and global transcription in post-MI swine, as well as potential mechanisms for anti-remodeling effects. METHODS AND RESULTS: MI was induced in anesthetized swine (n=23) by intracoronary balloon occlusion. At 1 week post-MI, survivors (n=13) received GGF2 treatment (intravenous, biweekly for 4 weeks; n=8) or were untreated (n=5). At 5 weeks post-MI, fractional shortening was higher (32.8% versus 25.3%, P=0.019), and left ventricular (LV) end-diastolic dimension lower (4.5 versus 5.3 cm, P=0.003) in GGF2-treated animals. Treatment altered expression of 528 genes, as measured by microarrays, including collagens, basal lamina components, and matricellular proteins. GGF2-treated pigs exhibited improvements in LV cardiomyocyte mitochondria and intercalated disk structures and showed less fibrosis, altered matrix structure, and fewer myofibroblasts (myoFbs), based on trichrome staining, electron microscopy, and immunostaining. In vitro experiments with isolated murine and rat cardiac fibroblasts demonstrate that NRG-1ß reduces myoFbs, and suppresses TGFß-induced phospho-SMAD3 as well as αSMA expression. CONCLUSIONS: These results suggest that GGF2/NRG-1ß prevents adverse remodeling after injury in part via anti-fibrotic effects in the heart.

Role of TGF-ß signaling in generation of CD39+CD73+ myeloid cells in tumors.

There is growing evidence that generation of adenosine from ATP, which is mediated by the CD39/CD73 enzyme pair, predetermines immunosuppressive and proangiogenic properties of myeloid cells. We have previously shown that the deletion of the TGF-ß type II receptor gene (Tgfbr2) expression in myeloid cells is associated with decreased tumor growth, suggesting protumorigenic effect of TGF-ß signaling. In this study, we tested the hypothesis that TGF-ß drives differentiation of myeloid-derived suppressor cells into protumorigenic terminally differentiated myeloid mononuclear cells (TDMMCs) characterized by high levels of cell-surface CD39/CD73 expression. We found that TDMMCs represent a major cell subpopulation expressing high levels of both CD39 and CD73 in the tumor microenvironment. In tumors isolated from mice with spontaneous tumor formation of mammary gland and conditional deletion of the type II TGF-ß receptor in mammary epithelium, an increased level of TGF-ß protein was associated with further increase in number of CD39(+)CD73(+) TDMMCs compared with MMTV-PyMT/TGFßRII(WT) control tumors with intact TGF-ß signaling. Using genetic and pharmacological approaches, we demonstrated that the TGF-ß signaling mediates maturation of myeloid-derived suppressor cells into TDMMCs with high levels of cell surface CD39/CD73 expression and adenosine-generating capacity. Disruption of TGF-ß signaling in myeloid cells resulted in decreased accumulation of TDMMCs, expressing CD39 and CD73, and was accompanied by increased infiltration of T lymphocytes, reduced density of blood vessels, and diminished progression of both Lewis lung carcinoma and spontaneous mammary carcinomas. We propose that TGF-ß signaling can directly induce the generation of CD39(+)CD73(+) TDMMCs, thus contributing to the immunosuppressive, proangiogenic, and tumor-promoting effects of this pleiotropic effector in the tumor microenvironment.

Impaired adenosine-mediated angiogenesis in preeclampsia: potential implications for fetal programming.

Preeclampsia is a pregnancy-specific syndrome, defined by such clinical hallmarks as the onset of maternal hypertension and proteinuria after 20 weeks of gestation. The syndrome is also characterized by impaired blood flow through the utero-placental circulation and relative placental ischemia, which in turn, may generate feto-placental endothelial dysfunction. Endothelial dysfunction in offspring born from preeclamptic pregnancies has been associated with an increased risk of cardiovascular disease, including hypertension, later in life. Interestingly, diminished endothelial function, manifested by low angiogenic capacity, leads to hypertension in animal studies. Recently, we have shown that the adenosine receptor A2A/nitric oxide/vascular endothelial growth factor axis is reduced in human umbilical vein endothelial cells derived from preeclamptic pregnancies, an effect correlated with gestational age at onset of preeclampsia. We and others suggested that impaired vascular function might be associated with high cardiovascular risk in offspring exposed to pregnancy diseases. However, we are not aware of any studies that examine impaired adenosine-mediated angiogenesis as a possible link to hypertension in offspring born from preeclamptic pregnancies. In this review, we present evidence supporting the hypothesis that reduced adenosine-mediated angiogenesis during preeclamptic pregnancies might be associated with development of hypertension in the offspring.

Role of adenosine A2B receptor signaling in contribution of cardiac mesenchymal stem-like cells to myocardial scar formation.

Adenosine levels increase in ischemic hearts and contribute to the modulation of that pathological environment. We previously showed that A2B adenosine receptors on mouse cardiac Sca1(+)CD31(-) mesenchymal stromal cells upregulate secretion of paracrine factors that may contribute to the improvement in cardiac recovery seen when these cells are transplanted in infarcted hearts. In this study, we tested the hypothesis that A2B receptor signaling regulates the transition of Sca1(+)CD31(-) cells, which occurs after myocardial injury, into a myofibroblast phenotype that promotes myocardial repair and remodeling. In vitro, TGFß1 induced the expression of the myofibroblast marker α-smooth muscle actin (αSMA) and increased collagen I generation in Sca1(+)CD31(-) cells. Stimulation of A2B receptors attenuated TGFß1-induced collagen I secretion but had no effect on αSMA expression. In vivo, myocardial infarction resulted in a rapid increase in the numbers of αSMA-positive cardiac stromal cells by day 5 followed by a gradual decline. Genetic deletion of A2B receptors had no effect on the initial accumulation of αSMA-expressing stromal cells but hastened their subsequent decline; the numbers of αSMA-positive cells including Sca1(+)CD31(-) cells remained significantly higher in wild type compared with A2B knockout hearts. Thus, our study revealed a significant contribution of cardiac Sca1(+)CD31(-) cells to the accumulation of αSMA-expressing cells after infarction and implicated A2B receptor signaling in regulation of myocardial repair and remodeling by delaying deactivation of these cells. It is plausible that this phenomenon may contribute to the beneficial effects of transplantation of these cells to the injured heart.

Role of JunB in adenosine A2B receptor-mediated vascular endothelial growth factor production.

Interstitial adenosine stimulates neovascularization in part through A2B adenosine receptor-dependent upregulation of vascular endothelial growth factor (VEGF). In the current study, we tested the hypothesis that A2B receptors upregulate JunB, which can contribute to stimulation of VEGF production. Using the human microvascular endothelial cell line, human mast cell line, mouse cardiac Sca1-positive stromal cells, and mouse Lewis lung carcinoma (LLC) cells, we found that adenosine receptor-dependent upregulation of VEGF production was associated with an increase in VEGF transcription, activator protein-1 (AP-1) activity, and JunB accumulation in all cells investigated. Furthermore, the expression of JunB, but not the expression of other genes encoding transcription factors from the Jun family, was specifically upregulated. In LLC cells expressing A2A and A2B receptor transcripts, only the nonselective adenosine agonist NECA (5'-N-ethylcarboxamidoadenosine), but not the selective A2A receptor agonist CGS21680 [2-p-(2-carboxyethyl) phenylethylamino-5'-N-ethylcarboxamidoadenosine], significantly increased JunB reporter activity and JunB nuclear accumulation, which were inhibited by the A2B receptor antagonist PSB603 [(8-[4-[4-((4-chlorophenzyl)piperazide-1-sulfonyl)phenyl]]-1-propylxanthine]. Using activators and inhibitors of intracellular signaling, we demonstrated that A2B receptor-dependent accumulation of JunB protein and VEGF secretion share common intracellular pathways. NECA enhanced JunB binding to the murine VEGF promoter, whereas mutation of the high-affinity AP-1 site (-1093 to -1086) resulted in a loss of NECA-dependent VEGF reporter activity. Finally, NECA-dependent VEGF secretion and reporter activity were inhibited by the expression of a dominant negative JunB or by JunB knockdown. Thus, our data suggest an important role of the A2B receptor-dependent upregulation of JunB in VEGF production and possibly other AP-1-regulated events.

Adenosine A2B receptors on cardiac stem cell antigen (Sca)-1-positive stromal cells play a protective role in myocardial infarction.

Transplantation of mesenchymal stem-like cells to the heart is known to improve cardiac recovery in animal models of myocardial infarction (MI). Because stimulation of A2B adenosine receptors on mouse cardiac stem cell antigen (Sca)-1(+)CD31(-) mesenchymal stem-like cells significantly up-regulates their secretion of pro-angiogenic factors, we hypothesized that ablation of the A2B receptor signaling in these cells would reduce their ability to improve vascularization of the infarct area seen after transplantation. Wild-type (WT) C57BL/6 mice underwent permanent left coronary artery ligation and received intramyocardial injections of Sca-1(+)CD31(-) cells generated from WT or A2B receptor knockout (A2BKO) mice or the same volume of cell-free saline. Only 12% to 16% of injected cells remained in the ventricles 1 week later; there was no significant difference between WT and A2BKO cell survival. Transplantation of WT, but not A2BKO, cells significantly reduced both post-MI decline in cardiac function and adverse remodeling compared with that seen in control hearts. Morphological analysis conducted 4 weeks after MI revealed significantly increased vascularization of the infarct areas and reduced myocardial scarring in animals treated with WT, but not with A2BKO, cells compared with control. Thus, our study demonstrated that the A2B receptor signaling linked to up-regulation of pro-angiogenic factors in cardiac Sca-1(+)CD31(-) stromal cells is essential for overall improvement of cardiac recovery seen after their transplantation to the injured heart.

Role of A2B adenosine receptors in regulation of paracrine functions of stem cell antigen 1-positive cardiac stromal cells.

The existence of multipotent cardiac stromal cells expressing stem cell antigen (Sca)-1 has been reported, and their proangiogenic properties have been demonstrated in myocardial infarction models. In this study, we tested the hypothesis that stimulation of adenosine receptors on cardiac Sca-1(+) cells up-regulates their secretion of proangiogenic factors. We found that Sca-1 is expressed in subsets of mouse cardiac stromal CD31(-) and endothelial CD31(+) cells. The population of Sca-1(+)CD31(+) endothelial cells was significantly reduced, whereas the population of Sca-1(+)CD31(-) stromal cells was increased 1 week after myocardial infarction, indicating their relative functional importance in this pathophysiological process. An increase in adenosine levels in adenosine deaminase-deficient mice in vivo significantly augmented vascular endothelial growth factor (VEGF) production in cardiac Sca-1(+)CD31(-) stromal cells but not in Sca-1(+)CD31(+) endothelial cells. We found that mouse cardiac Sca-1(+)CD31(-) stromal cells predominantly express mRNA encoding A(2B) adenosine receptors. Stimulation of adenosine receptors significantly increased interleukin (IL)-6, CXCL1 (a mouse ortholog of human IL-8), and VEGF release from these cells. Using conditionally immortalized Sca-1(+)CD31(-) stromal cells obtained from wild-type and A(2B) receptor knockout mouse hearts, we demonstrated that A(2B) receptors are essential for adenosine-dependent up-regulation of their paracrine functions. We found that the human heart also harbors a population of stromal cells similar to the mouse cardiac Sca-1(+)CD31(-) stromal cells that increase release of IL-6, IL-8, and VEGF in response to A(2B) receptor stimulation. Thus, our study identified A(2B) adenosine receptors on cardiac stromal cells as potential targets for up-regulation of proangiogenic factors in the ischemic heart.

Adenosinergic regulation of the expansion and immunosuppressive activity of CD11b+Gr1+ cells.

Extracellular adenosine and purine nucleotides are elevated in many pathological situations associated with the expansion of CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs). Therefore, we tested whether adenosinergic pathways play a role in MDSC expansion and functions. We found that A(2B) adenosine receptors on hematopoietic cells play an important role in accumulation of intratumoral CD11b(+)Gr1(high) cells in a mouse Lewis lung carcinoma model in vivo and demonstrated that these receptors promote preferential expansion of the granulocytic CD11b(+)Gr1(high) subset of MDSCs in vitro. Flow cytometry analysis of MDSCs generated from mouse hematopoietic progenitor cells revealed that the CD11b(+)Gr-1(high) subset had the highest levels of CD73 (ecto-5'-nucleotidase) expression (Δmean fluorescence intensity [MFI] of 118.5 ± 16.8), followed by CD11b(+)Gr-1(int) (ΔMFI of 57.9 ± 6.8) and CD11b(+)Gr-1(-/low) (ΔMFI of 12.4 ± 1.0) subsets. Even lower levels of CD73 expression were found on Lewis lung carcinoma tumor cells (ΔMFI of 3.2 ± 0.2). The high levels of CD73 expression in granulocytic CD11b(+)Gr-1(high) cells correlated with high levels of ecto-5'-nucleotidase enzymatic activity. We further demonstrated that the ability of granulocytic MDSCs to suppress CD3/CD28-induced T cell proliferation was significantly facilitated in the presence of the ecto-5'-nucleotidase substrate 5'-AMP. We propose that generation of adenosine by CD73 expressed at high levels on granulocytic MDSCs may promote their expansion and facilitate their immunosuppressive activity.

Role of adenosine A(2B) receptors in inflammation.

Recent progress in our understanding of the unique role of A(2B) receptors in the regulation of inflammation, immunity, and tissue repair was considerably facilitated with the introduction of new pharmacological and genetic tools. However, it also led to seemingly conflicting conclusions on the role of A(2B) adenosine receptors in inflammation with some publications indicating proinflammatory effects and others suggesting the opposite. This chapter reviews the functions of A(2B) receptors in various cell types related to inflammation and integrated effects of A(2B) receptor modulation in several animal models of inflammation. It is argued that translation of current findings into novel therapies would require a better understanding of A(2B) receptor functions in diverse types of inflammatory responses in various tissues and at different points of their progression.

Experimental myocardial infarction triggers canonical Wnt signaling and endothelial-to-mesenchymal transition.

Despite available therapies, myocardial infarction (MI) remains a leading cause of death worldwide. Better understanding of the molecular and cellular mechanisms that regulate cardiac repair should help to improve the clinical outcome of MI patients. Using the reporter mouse line TOPGAL, we show that canonical (ß-catenin-dependent) Wnt signaling is induced 4 days after experimental MI in subepicardial endothelial cells and perivascular smooth muscle actin (SMA)-positive (SMA(+)) cells. At 1 week after ischemic injury, a large number of canonical-Wnt-positive cells accumulated in the infarct area during granulation tissue formation. Coincidently with canonical Wnt activation, endothelial-to-mesenchymal transition (EndMT) was also triggered after MI. Using cell lineage tracing, we show that a significant portion of the canonical-Wnt-marked SMA(+) mesenchymal cells is derived from endothelial cells. Canonical Wnt signaling induces mesenchymal characteristics in cultured endothelial cells, suggesting a direct role in EndMT. In conclusion, our study demonstrates that canonical Wnt activation and EndMT are molecular and cellular responses to MI and that canonical Wnt signaling activity is a characteristic property of EndMT-derived mesenchymal cells that take part in cardiac tissue repair after MI. These findings could lead to new strategies to improve the course of cardiac repair by temporal and cell-type-specific manipulation of canonical Wnt signaling.