Synergistic up-regulation of vascular endothelial growth factor expression in murine macrophages by adenosine A(2A) receptor agonists and endotoxin.
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.
Leibovich
Samuel Joseph
SJ
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
Chen
Jiang-Fan
JF
Pinhal-Enfield
Grace
G
Belem
Paula C
PC
Elson
Genie
G
Rosania
Anthony
A
Ramanathan
Madhuri
M
Montesinos
Carmen
C
Jacobson
Marlene
M
Schwarzschild
Michael A
MA
Fink
J Stephen
JS
Cronstein
Bruce
B
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
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