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

Abstract

AbstractPurposeAngiogenesis 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.MethodsA 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 anin vitro3-D endothelial tubule formation assay, containing GFP-expressing human retinal endothelial cells, pericytes, and pro-angiogenic growth factors.ResultsIL-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.ConclusionOur 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.