Am J Pathol. 2002 Jun;160(6):2231-44.
Synergistic up-regulation of vascular endothelial growth factor expression in murine macrophages by adenosine A(2A) receptor agonists and endotoxin.
Leibovich SJ, Chen JF, Pinhal-Enfield G, Belem PC, Elson G, Rosania A, Ramanathan M, Montesinos C, Jacobson M, Schwarzschild MA, Fink JS, Cronstein B. Department of Cell Biology and Molecular Medicine, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA. leibovic@umdnj.edu
Under normoxic conditions, macrophages from C57BL mice produce low levels of vascular endothelial growth factor (VEGF). Hypoxia stimulates VEGF expression by approximately 500%; interferon-gamma (IFN-gamma) with endotoxin [lipopolysaccharide (LPS)] also stimulates VEGF expression by approximately 50 to 150% in an inducible nitric oxide synthase (iNOS)-dependent manner. Treatment of normoxic macrophages with 5′-N-ethyl-carboxamido-adenosine (NECA), a nonselective adenosine A(2) receptor agonist, or with 2-[p-(2-carboxyethyl)-phenylethyl amino]-5′-N-ethyl-carboxamido-adenosine (CGS21680), a specific adenosine A(2A) receptor agonist, modestly increases VEGF expression, whereas 2-chloro-N(6)-cyclopentyl adenosine (CCPA), an adenosine A(1) agonist, does not. Treatment with LPS (0 to 1000 ng/ml), or with IFN-gamma (0 to 300 U/ml), does not affect VEGF expression. In the presence of LPS (EC(50) < 10 ng/ml), but not of IFN-gamma, both NECA and CGS21680 synergistically up-regulate VEGF expression by as much as 10-fold. This VEGF is biologically active in vivo in the rat corneal bioassay of angiogenesis. Inhibitors of iNOS do not affect this synergistic induction of VEGF, and macrophages from iNOS-/- mice produce similar levels of VEGF as wild-type mice, indicating that NO does not play a role in this induction. Under hypoxic conditions, VEGF expression is slightly increased by adenosine receptor agonists but adenosine A(2) or A(1) receptor antagonists 3,7-dimethyl-1-propargyl xanthine (DMPX), ZM241385, and 8-cyclopentyl-1,3-dipropylxanthine (DCPCX) do not modulate VEGF expression. VEGF expression is also not reduced in hypoxic macrophages from A(3)-/- and A(2A)-/- mice. Thus, VEGF expression by hypoxic macrophages does not seem to depend on endogenously released or exogenous adenosine. VEGF expression is strongly up-regulated by LPS/NECA in macrophages from A(3)-/- but not A(2A)-/- mice, confirming the role of adenosine A(2A) receptors in this pathway. LPS with NECA strongly up-regulates VEGF expression by macrophages from C(3)H/HeN mice (with intact Tlr4 receptors), but not by macrophages from C(3)H/HeJ mice (with mutated, functionally inactive Tlr4 receptors), implicating signaling through the Tlr4 pathway in this synergistic up-regulation. Finally, Western blot analysis of adenosine A(2A) receptor expression indicated that the synergistic interaction of LPS with A(2A) receptor agonists does not involve up-regulation of A(2A) receptors by LPS. These results indicate that in murine macrophages there is a novel pathway regulating VEGF production, that involves the synergistic interaction of adenosine A(2A) receptor agonists through A(2A) receptors with LPS through the Tlr4 pathway, resulting in the strong up-regulation of VEGF expression by macrophages in a hypoxia- and NO-independent manner.
PMID: 12057925 [PubMed – indexed for MEDLINE]

Am J Pathol. 2003 Aug;163(2):711-21.
An angiogenic switch in macrophages involving synergy between Toll-like receptors 2, 4, 7, and 9 and adenosine A(2A) receptors.
Pinhal-Enfield G, Ramanathan M, Hasko G, Vogel SN, Salzman AL, Boons GJ, Leibovich SJ.
Department of Cell Biology and Molecular Medicine, New Jersey Medical School, UMDNJ, Newark, New Jersey 07013, USA.
Adenosine A(2A) receptor (A(2A)R) agonists synergize with Escherichia coli (E. coli) LPS [toll-like receptor (TLR)4 agonist] to up-regulate vascular endothelial growth factor (VEGF) expression in murine macrophages. Here, we demonstrate that TLR2, TLR7, and TLR9, but not TLR3 and TLR5 agonists, also synergize with A(2A)R agonists and adenosine to up-regulate VEGF, while simultaneously strongly down-regulating TNFalpha expression. In the absence of adenosine or A(2A)R agonists, Porphyromonas gingivalis (P. gingivalis) LPS and PAM(3)CAG (TLR2 agonists), resiquimod (R848) (TLR7 agonist), and non-methylated CpG DNA (TLR9 agonist) strongly up-regulate TNFalpha expression, with no effect on VEGF. In the presence of adenosine or A(2A)R agonists, but not A(1)R agonists, TLR2, 4, 7, and 9 agonists strongly up-regulate VEGF expression, while simultaneously down-regulating TNFalpha. C57BL/10ScN (TLR4 deletion mutant) macrophages produce TNFalpha in response to TLR2, 3, 7, and 9 agonists, but not the TLR4 agonist E. coli LPS. With adenosine or A(2A)R agonists, TLR2, 7, and 9, but not TLR4 agonists, also synergistically up-regulate VEGF, while down-regulating TNFalpha expression. Polyinosinic-polycytidilic acid (poly(I:C)) (TLR3 agonist) stimulates TNFalpha expression in macrophages from both C57BL/10ScSn and C57BL/10ScN mice, but has little effect on VEGF expression in the presence of adenosine or A(2A)R agonists. R-flagellins from Serratia marcescens (S. marcescens) and Salmonella muenchen (S. muenchen) do not stimulate TNFalpha expression in either C57BL/10ScSn or C57BL10/ScN mice, and have no effect on VEGF production in the presence of adenosine or A(2A)R agonists. While adenosine and A(2A)R agonists strongly down-regulate TNFalpha protein expression induced by TLR2, 3, 4, 7, and 9 agonists, TNFalpha mRNA and NF-kappaB activation are not reduced. We propose a novel signaling pathway in murine macrophages involving synergy between TLRs 2, 4, 7, and 9 and A(2A)Rs, that up-regulates VEGF and down-regulates TNFalpha expression, thus acting as an angiogenic switch. This angiogenic switch may play an important role in ischemia when TLR agonists are present, providing an interface between innate immunity and wound healing.
PMID: 12875990 [PubMed – indexed for MEDLINE]

J Leukoc Biol. 2009 Sep;86(3):681-9. Epub 2009 May 28.
Differential regulation of HIF-1alpha isoforms in murine macrophages by TLR4 and adenosine A(2A) receptor agonists.
Ramanathan M, Luo W, Csóka B, Haskó G, Lukashev D, Sitkovsky MV, Leibovich SJ.
Department of Cell Biology and Molecular Medicine and The Cardiovascular Research Institute, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA.
Adenosine A(2A)R and TLR agonists synergize to induce an “angiogenic switch” in macrophages, down-regulating TNF-alpha and up-regulating VEGF expression. This switch involves transcriptional regulation of VEGF by HIF-1, transcriptional induction of HIF-1alpha by LPS (TLR4 agonist), and A(2A)R-dependent post-transcriptional regulation of HIF-1alpha stability. Murine HIF-1alpha is expressed as two mRNA isoforms: HIF-1alphaI.1 and -I.2, which contain alternative first exons and promoters. HIF-1alphaI.2 is expressed ubiquitously, and HIF-1alphaI.1 is tissue-specific. We investigated the regulation of these isoforms in macrophages by TLR4 and A(2A)R agonists. HIF-1alphaI.1 is induced strongly compared with HIF-1alphaI.2 upon costimulation with LPS and A(2A)R agonists (NECA or CGS21680). In unstimulated cells, the I.1 isoform constituted approximately 4% of HIF-1alpha transcripts; in LPS and NECA- or CGS21680-treated macrophages, this level was approximately 15%, indicating a substantial contribution of HIF-1alphaI.1 to total HIF-1alpha expression. The promoters of both isoforms were induced by LPS but not enhanced further by NECA, suggesting A(2A)R-mediated post-transcriptional regulation. LPS/NECA-induced expression of HIF-1alphaI.1 was down-regulated by Bay 11-7085 (NF-kappaB inhibitor) and ZM241385 (A(2A)R antagonist). Although VEGF and IL-10 expression by HIF-1alphaI.1-/- macrophages was equivalent to that of wild-type macrophages, TNF-alpha, MIP-1alpha, IL-6, IL-12p40, and IL-1beta expression was significantly greater, suggesting a role for HIF-1alphaI.1 in modulating expression of these cytokines. A(2A)R expression in unstimulated macrophages was low but was induced rapidly by LPS in a NF-kappaB-dependent manner. LPS-induced expression of A(2A)Rs and HIF-1alpha and A(2A)R-dependent HIF-1alpha mRNA and protein stabilization provide mechanisms for the synergistic effects of LPS and A(2A)R agonists on macrophage VEGF expression.
PMID: 19477908 [PubMed – indexed for MEDLINE]

Mol Biol Cell. 2007 Jan;18(1):14-23. Epub 2006 Oct 25.
Synergistic up-regulation of vascular endothelial growth factor (VEGF) expression in macrophages by adenosine A2A receptor agonists and endotoxin involves transcriptional regulation via the hypoxia response element in the VEGF promoter.
Ramanathan M, Pinhal-Enfield G, Hao I, Leibovich SJ
Department of Cell Biology and Molecular Medicine, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA.
Macrophages are an important source of vascular endothelial growth factor (VEGF). Adenosine A2A receptor (A2AR) agonists with Toll-like receptor (TLR) 2, 4, 7, and 9 agonists synergistically induce macrophage VEGF expression. We show here using VEGF promoter-luciferase reporter constructs that the TLR4 agonist Escherichia coli lipopolysaccharide (LPS) and the A2AR agonists NECA and CGS21680 synergistically augment VEGF transcription in macrophages and that the HRE in the VEGF promoter is essential for this transcription. We examined whether LPS and/or NECA induce HIF-1alpha expression. HIF-1alpha mRNA levels were increased in LPS-treated macrophages in an NF-kappaB-dependent manner; NECA strongly increased these levels in an A2AR-dependent manner. LPS induced luciferase expression from a HIF-1alpha promoter-luciferase construct in an A2AR-independent manner. Further stimulation with NECA did not increase HIF-1alpha promoter activity, indicating that the A2AR-dependent increase in HIF-1alpha mRNA is post-transcriptional. LPS/NECA treatment also increased HIF-1alpha protein and DNA binding levels. Deletion of putative NF-kappaB-binding sites from the VEGF promoter did not affect LPS/NECA-induced VEGF promoter activity, suggesting that NF-kappaB is not directly involved in VEGF transcription. Taken together, these data indicate that LPS/NECA-induced VEGF expression involves transcriptional regulation of the VEGF promoter by HIF-1alpha through the HRE. HIF-1alpha is transcriptionally induced by LPS and post-transcriptionally up-regulated in an A2AR-dependent manner.
PMID: 17065555 [PubMed – indexed for MEDLINE]

J Pharmacol Exp Ther. 2006 Jan;316(1):71-8. Epub 2005 Sep 27.
Activation of the adenosine A3 receptor in RAW 264.7 cells inhibits lipopolysaccharide-stimulated tumor necrosis factor-alpha release by reducing calcium-dependent activation of nuclear factor-kappaB and extracellular signal-regulated kinase 1/2.
Martin L, Pingle SC, Hallam DM, Rybak LP, Ramkumar V.
Dept. of Pharmacology, Southern Illinois University School of Medicine, P.O. Box 19230, Springfield, IL 62794-1222, USA.
Bacterial lipopolysaccharide (LPS) activates the immune system and promotes inflammation via Toll-like receptor (TLR) 4, which regulates the synthesis and release of tumor necrosis factor (TNF)-alpha and other inflammatory cytokines. Previous studies have shown that the nucleoside adenosine suppresses LPS-stimulated TNF-alpha release in human UB939 macrophages by activating an adenosine A(3) receptor (A(3)AR) subtype on these cells. In this study, we examined the mechanism(s) underlying A(3)AR-dependent inhibition of TNF-alpha release in a mouse (RAW 264.7) cell line. Treatment of RAW 264.7 cells with LPS (3 mug/ml) increased TNF-alpha release, which was reduced in a dose-dependent manner by adenosine analogs N(6)-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) and R-phenylisopropyladenosine and reversed by selective A(3)AR blockade. The increase in TNF-alpha release was preceded by an increase in intracellular Ca(2+) levels. Inhibition of intracellular Ca(2+) release by IB-MECA, a selective agonist of the A(3)AR, or with BAPTA-AM, an intracellular Ca(2+) chelator, reduced LPS-stimulated TNF-alpha release. Activation of the A(3)AR or inhibition of intracellular Ca(2+) release also reduced LPS-stimulated nuclear factor-kappaB (NF-kappaB) activation and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Similar inhibition by A(3)AR was observed for LPS-stimulated inducible nitric-oxide synthase. These data support the contention that inhibition of LPS-stimulated release of inflammatory molecules, such as TNF-alpha and NO via the A(3)AR, involves suppression of intracellular Ca(2+)signaling, leading to suppression of NF-kappaB and
ERK1/2 pathways.
PMID: 16188954 [PubMed – indexed for MEDLINE]

Circ Res. 2007 Nov 26;101(11):1130-8. Epub 2007 Sep 27.
A1 adenosine receptor activation promotes angiogenesis and release of VEGF from monocytes.
Clark AN, Youkey R, Liu X, Jia L, Blatt R, Day YJ, Sullivan GW, Linden J, Tucker AL.
Source
Department of Internal Medicine, Cardiovascular Division, University of Virginia Health System, Charlottesville, VA 22908, USA.
Abstract
Adenosine is a proangiogenic purine nucleoside released from ischemic and hypoxic tissues. Of the 4 adenosine receptor (AR) subtypes (A1, A2A, A2B, and A3), the A2 and A3 have been previously linked to the modulation of angiogenesis. We used the chicken chorioallantoic membrane (CAM) model to determine whether A1 AR activation affects angiogenesis. We cloned and pharmacologically characterized chicken AR subtypes to evaluate the selectivity of various agonists and antagonists. Application of the A1 AR-selective agonist N6-cyclopentyladenosine (CPA; 100 nmol/L) to the CAM resulted in a 40% increase in blood vessel number (P<0.01), which was blocked by the A1 AR-selective antagonist C8-(N-methylisopropyl)-amino-N6-(5′-endohydroxy)-endonorbornan-2-yl-9-methyladenine (WRC-0571; 1 micromol/L). Selective A2A AR agonists did not stimulate angiogenesis in the CAM. In an ex vivo rat aortic ring model of angiogenesis that includes cocultured endothelial cells, fibroblasts, and smooth muscle cells, 50 nmol/L CPA did not directly stimulate capillary formation; however, medium from human mononuclear cells pretreated with CPA, but not vehicle, increased capillary formation by 48% (P<0.05). This effect was blocked by WRC-0571 (1.5 micromol/L) or anti-VEGF antibody (1 microg/mL). CPA (5 nmol/L) stimulated a 1.7-fold increase in VEGF release from the mononuclear cells. This is the first study to show that A1 AR activation induces angiogenesis. Stimulation of A2 ARs on endothelial cells results in proliferation and tube formation, and A2 and A3 ARs on inflammatory cells modulate release of angiogenic factors. We conclude that adenosine promotes a coordinated angiogenic response through its interactions with multiple receptors on multiple cell types.
Adenosine receptors and angiogenesis.Auchampach JA. Circ Res. 2007 Nov 26; 101(11):1075-7.
PMID:17901362[PubMed – indexed for MEDLINE]

Mol Interv. 2003 Oct;3(7):370-4.
Adenosine receptors and mammalian toll-like receptors: synergism in macrophages.
Olah ME, Caldwell CC.
Source
Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Cincinnati, OH 45267, USA. Mark.Olah@UC.edu
Abstract
Adenosine is known to participate in tissue protection subsequent to ischemic events. New evidence points to a role for adenosine in promoting neovascularization through a mechanism that requires interaction with the Toll-like receptor (TLR) signaling pathway. In macrophages, the adenosine receptor subtype 2A (A(2A)R) synergizes with some but not all of the Toll-like receptors, leading to increased expression of vascular endothelial growth factor (VEGF). Simultaneously, the expression of tumor necrosis factor-alpha (TNFalpha) is decreased; this phenomenon depends on the presence of AR agonists; however, the activation of transcription factor nuclear factor-kappaB (NF-kappaB) is not attenuated in the presence of A(2A)R agonists. It appears that the addition of adenosine or other A(2A)R agonists can mediate the “angiogenic switch,” in macrophages, from TNFalpha protein expression to expression of components necessary for angiogenesis. Although these observations might have important implications for wound healing, it will be important to discern whether this interaction between ARs and TLRs is necessary for angiogenesis associated with tumor growth.
PMID:14993458[PubMed – indexed for MEDLINE]

J Pharmacol Exp Ther. 2006 Apr;317(1):172-80. Epub 2005 Dec 9.
Adenosine inhibits tumor necrosis factor-alpha release from mouse peritoneal macrophages via A2A and A2B but not the A3 adenosine receptor.
Kreckler LM, Wan TC, Ge ZD, Auchampach JA.
Source
Department of Pharmacology, Medical College of Wisconsin, Milwaukee, 53226, USA.
Abstract
Adenosine is elaborated in injured tissues where it suppresses inflammatory responses of essentially all immune cells, including production of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). Most of the anti-inflammatory actions of adenosine have been attributed to signaling through the A(2A) adenosine receptor (A(2A)AR). Previously, however, it has been shown that the A(3)AR agonist N(6)-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (IB-MECA) potently inhibited TNF-alpha release from macrophages obtained from A(2A)AR “knockout” (A(2A)KO) mice, suggesting that the A(3)AR may also regulate cytokine expression. Here, we confirmed that the A(2A)AR is the primary AR subtype that suppresses TNF-alpha release from thioglycollate-elicited mouse peritoneal macrophages induced by both Toll-like receptor-dependent (TLR) and TLR-independent stimuli, but we determined that the A(2B)AR rather than the A(3)AR mediates the non-A(2A)AR actions of adenosine since 1) the ability of IB-MECA to inhibit TNF-alpha release was not altered in macrophages isolated from A(3)KO mice, and 2) the A(2B)AR antagonist 1,3-dipropyl-8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]xanthine (MRS 1754) blocked the ability of the nonselective AR agonist adenosine-5′-N-ethylcarboxamide (NECA) to inhibit TNF-alpha release from macrophages isolated from A(2A)KO mice. Although A(2B)ARs seem capable of inhibiting TNF-alpha release, the A(2A)AR plays a dominant suppressive role since MRS 1754 did not block the ability of NECA to inhibit TNF-alpha release from macrophages isolated from wild-type (WT) mice. Furthermore, the potency and efficacy of adenosine to inhibit TNF-alpha release from WT macrophages were not influenced by blocking A(2B)ARs with MRS 1754. The data indicate that adenosine suppresses TNF-alpha release from macrophages primarily via A(2A)ARs, although the A(2B)AR seems to play an underlying inhibitory role that may contribute to the anti-inflammatory actions of adenosine under select circumstances.
PMID:16339914[PubMed – indexed for MEDLINE] PMCID:PMC1409807

Biochem Biophys Res Commun. 2010 Feb 12;392(3):351-6
Adenosine up-regulates vascular endothelial growth factor in human macrophages.
Ernens I, Léonard F, Vausort M, Rolland-Turner M, Devaux Y, Wagner DR.
Source
Laboratory of Cardiovascular Research, Centre de Recherche Public-Santé, Luxembourg.
Abstract
It is known from animal models that the cardioprotective nucleoside adenosine stimulates angiogenesis mainly through up-regulation of vascular endothelial growth factor (VEGF). Since macrophages infiltrate the heart after infarction and because adenosine receptors behave differently across species, we evaluated the effect of adenosine on VEGF in human macrophages. Adenosine dose-dependently up-regulated VEGF expression and secretion by macrophages from healthy volunteers. VEGF production was also increased by blockade of extracellular adenosine uptake with dipyridamole. This effect was exacerbated by the toll-like receptor-4 ligands heparan sulfate, hyaluronic acid and lipopolysaccharide, and was associated with an increase of hypoxia inducible factor-1alpha expression, the main transcriptional inducer of VEGF in hypoxic conditions. The agonist of the adenosine A2A receptor CGS21680 reproduced the increase of VEGF and the antagonist SCH58261 blunted it. In conclusion, these results provide evidence that activation of adenosine A2A receptor stimulates VEGF production in human macrophages. This study suggests that adenosine is a unique pro-angiogenic molecule that may be used to stimulate cardiac repair.
PMID:20067761[PubMed – indexed for MEDLINE]

Am J Physiol Regul Integr Comp Physiol. 2005 Aug;289(2):R283-R296.
Growth regulation of the vascular system: an emerging role for adenosine.
Adair TH.
Source
Dept. of Physiology and Biophysics, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS 39216-4505, USA. tadair@physiology.umsmed.edu
Abstract
The importance of metabolic factors in the regulation of angiogenesis is well understood. An increase in metabolic activity leads to a decrease in tissue oxygenation causing tissues to become hypoxic. The hypoxia initiates a variety of signals that stimulate angiogenesis, and the increase in vascularity that follows promotes oxygen delivery to the tissues. When the tissues receive adequate amounts of oxygen, the intermediate effectors return to normal levels, and angiogenesis ceases. An emerging concept is that adenosine released from hypoxic tissues has an important role in driving the angiogenesis. The following feedback control hypothesis is proposed: AMP is dephosphorylated by ecto-5′-nucleotidase, producing adenosine under hypoxic conditions in the extracellular space adjacent to a parenchymal cell (e.g., cardiomyocyte, skeletal muscle fiber, hepatocyte, etc.). Extracellular adenosine activates A(2) receptors, which stimulates the release of vascular endothelial growth factor (VEGF) from the parenchymal cell. VEGF binds to its receptor (VEGF receptor 2) on endothelial cells, stimulating their proliferation and migration. Adenosine can also stimulate endothelial cell proliferation independently of VEGF, which probably involves modulation of other proangiogenic and antiangiogenic growth factors and perhaps an intracellular mechanism. In addition, hemodynamic factors associated with adenosine-induced vasodilation may have a role in the development and remodeling of the vasculature. Once a new capillary network has been established, and the diffusion/perfusion capabilities of the vasculature are sufficient to supply the parenchymal cells with adequate amounts of oxygen, adenosine and VEGF as well as other proangiogenic and antiangiogenic growth factors return to near-normal levels, thus closing the negative feedback loop. The available data indicate that adenosine might be an essential mediator for up to 50-70% of the hypoxia-induced angiogenesis in some situations; however, additional studies in intact animals will be required to fully understand the quantitative importance of adenosine.
PMID:16014444[PubMed – indexed for MEDLINE]