Synergistic Up-Regulation of Vascular Endothelial Growth Factor (VEGF) Expression in Macrophages by Adenosine A sub(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 sub(2A) receptor (A sub(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 sub(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 sub(2A)R-dependent manner. LPS induced luciferase expression from a HIF-1 alpha promoter-luciferase construct in an A sub(2A)R-independent manner. Further stimulation with NECA did not increase HIF- 1 alpha promoter activity, indicating that the A sub(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 sub(2A)R-dependent manner.