Differentially polarized macrophages show diverse proangiogenic characteristics under normo- and hyperglycemic conditions

Purpose: Angiogenesis is a vital process involved in the formation of new blood vessels from existing ones. Macrophages play a crucial role in initiating endothelial activation and inflammation, and are involved in the pathological angiogenesis. Traditionally, macrophages have been classified, with the pro-angiogenic activity attributed to the M2 phenotype. However, recent evidence challenges the notion that only M2 macrophages possess pro-angiogenic properties. This study aims to investigate the pro- and anti-angiogenic properties of human polarized macrophages in normo- and hyper-glycemic conditions, in order to gain a better insight into the angiogenic capacity of M1- and M2-like macrophages in diabetes. Methods: A comprehensive bioinformatic analysis of pro- and anti-angiogenic gene expression profiles related in M1- vs. M2- polarized macrophages was performed based on a large previously published dataset. The most contributing differentially expressed genes in angiogenesis were selected for further validation. Macrophages were generated and polarized by culturing CD14+ monocytes and their stimulation with any of IFN-γ, IL-4, or IL-6 cytokines. Polarized macrophages were immunophenotyped using flow cytometry, and their expression of the selected genes were measured using qPCR. Finally, the proangiogenic capacity of the cells was assessed in an in vitro 3-D endothelial tubule formation assay, containing GFP-expressing human retinal endothelial cells, pericytes, and pro-angiogenic growth factors. Results: IL-4 and IL-6 induce distinct M2-like phenotypes in macrophages with mixed pro- and anti-angiogenic gene expressions. Hyperglycemia has a mild negative effect on the expression of M2-associated markers, however it does not significantly affect the angiogenic properties of macrophages. Conclusion: Our data support the concept of a spectrum model for macrophage polarization, indicating that the angiogenic status of polarized macrophages is not limited to the M2-phenotype, but is rather mediated by microenvironmental cues, and can result in diverse phenotypic characteristics. The effect of hyperglycemia on the angiogenic capacity of macrophages requires more comprehensive investigation. ### Competing Interest Statement The authors have declared no competing interest.