title: Synergistic up-regulation of vascular endothelial growth factor expression in murine macrophages by adenosine A(2A) receptor agonists and endotoxin.
abstract: 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.
authors.affiliation: 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
authors.forename: Samuel Joseph
authors.initials: SJ
authors.lastname: Leibovich
authors.forename: Jiang-Fan
authors.initials: JF
authors.lastname: Chen
authors.forename: Grace
authors.initials: G
authors.lastname: Pinhal-Enfield
authors.forename: Paula C
authors.initials: PC
authors.lastname: Belem
authors.forename: Genie
authors.initials: G
authors.lastname: Elson
authors.forename: Anthony
authors.initials: A
authors.lastname: Rosania
authors.forename: Madhuri
authors.initials: M
authors.lastname: Ramanathan
authors.forename: Carmen
authors.initials: C
authors.lastname: Montesinos
authors.forename: Marlene
authors.initials: M
authors.lastname: Jacobson
authors.forename: Michael A
authors.initials: MA
authors.lastname: Schwarzschild
authors.forename: J Stephen
authors.initials: JS
authors.lastname: Fink
authors.forename: Bruce
authors.initials: B
authors.lastname: Cronstein
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D029721:Drosophila Proteins; D016228:Endothelial Growth Factors / Q000096:biosynthesis*; D005260:Female; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008222:Lymphokines / Q000096:biosynthesis*; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000502:physiology*; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000502:physiology; D052247:Nitric Oxide Synthase Type II; D010627:Phenethylamines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D058906:Purinergic P1 Receptor Agonists; D012333:RNA, Messenger / Q000378:metabolism; D043705:Receptor, Adenosine A2A; D011956:Receptors, Cell Surface / Q000502:physiology*; D018047:Receptors, Purinergic P1 / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013805:Theobromine / Q000031:analogs & derivatives* / Q000494:pharmacology; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014227:Triazines / Q000494:pharmacology; D014230:Triazoles / Q000494:pharmacology; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D014970:Xanthines / Q000494:pharmacology
chemical_list: D029721:Drosophila Proteins; D016228:Endothelial Growth Factors; D008070:Lipopolysaccharides; D008222:Lymphokines; D008562:Membrane Glycoproteins; D010627:Phenethylamines; D047428:Protein Kinase Inhibitors; D058906:Purinergic P1 Receptor Agonists; D012333:RNA, Messenger; D043705:Receptor, Adenosine A2A; D011956:Receptors, Cell Surface; D018047:Receptors, Purinergic P1; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014227:Triazines; D014230:Triazoles; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D014970:Xanthines; C097270:ZM 241385; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D009569:Nitric Oxide; D019830:Adenosine-5'-(N-ethylcarboxamide); C058394:2-chloro-N(6)cyclopentyladenosine; C057837:3,7-dimethyl-1-propargylxanthine; D007371:Interferon-gamma; C051360:1,3-dipropyl-8-cyclopentylxanthine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D000241:Adenosine; D013805:Theobromine