Synergistic up-regulation of vascular endothelial growth factor (VEGF) expression in macrophages by adenosine A(2A) receptor agonists and endotoxin involves transcriptional regulation via the hypoxia response element in the VEGF promoter

Macrophages are an important source of vascular endothelial growth factor (VEGF). Adenosine A(2A) receptor (A(2A)R) 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 A(2A)R 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-1 alpha expression. HIF-1 alpha mRNA levels were increased in LPS-treated macrophages in an NF-kappa B-dependent manner; NECA strongly increased these levels in an A(2A)R-dependent manner. LPS induced luciferase expression from a HIF-1 alpha promoter-luciferase construct in an A(2A)R-independent manner. Further stimulation with NECA did not increase HIF-1 alpha promoter activity, indicating that the A(2A)R-dependent increase in HIF-1 alpha mRNA is post-transcriptional. LPS/NECA treatment also increased HIF-1 alpha protein and DNA binding levels. Deletion of putative NF-kappa B-binding sites from the VEGF promoter did not affect LPS/NECA-induced VEGF promoter activity, suggesting that NF-kappa B 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-1 alpha through the HRE. HIF-1 alpha is transcriptionally induced by LPS and post-transcriptionally up-regulated in an A(2A)R-dependent manner.