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Synergistic Up-Regulation of Vascular Endothelial Growth Factor Expression in Murine Macrophages by Adenosine A2A Receptor Agonists and Endotoxin

https://doi.org/10.1016/S0002-9440(10)61170-4Get rights and content

Under normoxic conditions, macrophages from C57BL mice produce low levels of vascular endothelial growth factor (VEGF). Hypoxia stimulates VEGF expression by ∼500%; interferon-γ (IFN-γ) with endotoxin [lipopolysaccharide (LPS)] also stimulates VEGF expression by ∼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 A2 receptor agonist, or with 2-[p-(2-carboxyethyl)-phenylethyl amino]-5′-N-ethyl-carboxamido-adenosine (CGS21680), a specific adenosine A2A receptor agonist, modestly increases VEGF expression, whereas 2-chloro-N6-cyclopentyl adenosine (CCPA), an adenosine A1 agonist, does not. Treatment with LPS (0 to 1000 ng/ml), or with IFN-γ (0 to 300 U/ml), does not affect VEGF expression. In the presence of LPS (EC50 < 10 ng/ml), but not of IFN-γ, 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 A2 or A1 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 A3−/− and A2A−/− 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 A3−/− but not A2A−/− mice, confirming the role of adenosine A2A receptors in this pathway. LPS with NECA strongly up-regulates VEGF expression by macrophages from C3H/HeN mice (with intact Tlr4 receptors), but not by macrophages from C3H/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 A2A receptor expression indicated that the synergistic interaction of LPS with A2A receptor agonists does not involve up-regulation of A2A 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 A2A receptor agonists through A2A 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.

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Supported in part by the United States Public Health Service [grants RO1-GM57982 (to S. J. L.), GM56268 (to B. C.), and AR41911 (to B. C.)], a focused giving grant from Johnson and Johnson (to S. J. L.), and a grant from King Pharmaceuticals Inc. (to B. C.).

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