VEGF Production in Macrophages Is Enhanced by Anti-Inflammatory Stimuli

Abstract

Purpose: : Following stimulation, macrophages can produce vascular endothelial growth factor (VEGF) which may play a role in pathological neovascularization. After they exit from the bone marrow, macrophages undergo a period of maturation that is directed by the local microenvironment. For example interferon gamma (IFN-γ) induces a M1 phenotype and IL-4 and IL-13 induce a M2 subtype. These populations are characterized by the productions of inducible nitric oxide synthase (iNOS) and arginase I respectively. In this study we have examined the relation between VEGF production and macrophage stimulation and investigated how macrophage phenotype influences to VEGF production.

Methods: : Macrophages were derived from the bone marrow (BMDMs) or peritoneal cavity of C57BL/6 mice. BMDMs were generated by culture in the presence of M-CSF for 8 days in Teflon bags. These cells are naïve until they are transferred to tissue culture plastic. Cells were stimulated by prostaglandin E₂ (PGE₂), IFN- γ, lipopolysaccharide (LPS), and CGS21680 (CGS), a selective A₂A receptor agonist. After 48 hrs supernatant was collected and we measured nitrite production by using the Griess reaction and IL-6, IL-10 and VEGF levels by ELISA. Levels of VEGF mRNA in stimulated macrophages were analyzed by Q-PCR and arginase I expression was determined by FACS.

Results: : Nitrite and IL-6 levels were up-regulated by LPS but decreased by addition of PGE₂, which therefore exhibited anti-inflammatory properties in this situation. IL-10 production was stimulated by LPS and markedly up-regulated in the present of PGE₂. Moreover, arginase was induced by PGE₂ and CGS. This suggests that PGE₂ and CGS are able to deliver anti-inflammatory signals to macrophages. Furthermore, PGE2 and CGS in combination with LPS triggered increased levels of VEGF production. In contrast, PGE₂ also increased VEGF mRNA levels compared with activation with LPS/IFN-γ stimulation Taken together, this suggests that VEGF producing macrophages may be of an anti-inflammatory M2-like phenotype.

Conclusions: : We conclude that macrophages that mature in an anti-inflammatory microenvironment may be a more effective source of VEGF than those that develop in a proinflammatory environment. This may partly explain the low level of neovascularization in uveitis. Further work will determine whether VEGF can itself modulate macrophage activation and phenotype.

Keywords: vascular endothelial growth factor • neovascularization • nitric oxide