Synergistic Up-Regulation of Vascular Endothelial Growth Factor Expression in Murine Macrophages by Adenosine A2A 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.

Signaling pathways involving adenosine A2A and A2B receptors in wound healing and fibrosis

“…Adenosine, acting at its receptors, promotes granulation tissue formation both directly and indirectly [14][15][16]. It has been shown that A 1 R and A 2A R hasten the resolution of acute inflammation by a variety of mechanisms [17][18][19].…”

“…Stimulation of A 2A R and A 2B R directly stimulates endothelial cell proliferation and A 2A R stimulation leads to tube formation in vitro. Moreover, A 2B R occupancy upregulates VEGF, bFGF, and Il-8 in human endothelial cells via G q and possibly G 12/13 and activation of phospholipase C. A 2A R also upregulates VEGF in macrophages in a PKA-independent mechanism [14,15]. Additionally, A 2A receptor stimulation suppresses thrombospondin-1 expression in microvascular endothelial cells, and thrombospondin-1 suppresses angiogenesis in vitro in microvascular endothelial cells [20] although others have reported that A 2A and A 2B receptor stimulation promotes angiogenesis through increased macrophage expression of thrombospondin-1 [21].…”

An Angiogenic Switch in Macrophages Involving Synergy between Toll-Like Receptors 2, 4, 7, and 9 and Adenosine A2A Receptors

“…This synergistic up-regulation is independent of hypoxia, NO, and protein kinase-A and is stronger than that induced by hypoxia alone. 3 Mammalian toll-like receptors (TLRs) are members of a family of proteins that resemble the Drosophila toll protein. 4,5 Toll plays a role in dorsal-ventral patterning in the developing fly embryo, and also plays a key role in regulating the innate immune response of adult flies to fungi.…”

“…For the synthesis of PAM 3 Cys and PAM 3 CAG, reagents were purchased from Alfa Aesar (Ward Hill, MA) or Aldrich (Milwaukee, WI). N-Fmoc-protected alanine and the preloaded Wang resin were from NovaBiochem (Darmstad, Germany).…”

Adenosine as an endogenous immunoregulator in cancer pathogenesis: where to go?

“…Adenosine increases the VEGF release from macrophages, thus facilitating the process of angiogenesis [103]. Adenosine and LPS via A 2A receptors and toll-like receptor 4 (TLR4) synergistically upregulate the production of VEGF by macrophages in hypoxia-and NOindependent manner [104].…”