Modulating Macrophage Phenotype for the Promotion of Angiogenesis in an Ischemic Environment
Authors:   , Risser Gregory Eric , Spiller Kara L 
Published: February 2023

Angiogenesis, the growth of blood vessels, is an extremely important process that occurs during wound healing. Blood vessels are necessary to transport cells and nutrients into a wound while also moving waste products out. Upon injury, blood vessels grow into the wound, and macrophages are believed to be one of the key cell types that aids in this ingrowth. Macrophages can play a role in the complex angiogenesis process because they exist on a spectrum of phenotypes. As pro-inflammatory M1 macrophages, they are thought to promote the formation of brand-new vasculature through various protein secretions and other mechanisms. As the wound healing progresses, there is an increased numbers of M2 pro-resolution macrophages in the site of injury. This M2 phenotype is believed to support the newly formed vasculature and promote the maturation of the blood vessels. The goals of this work were to: (1) make a biomaterial that could promote the M2 phenotype and then deliver the biomaterial to promote angiogenesis at the site of injury, and (2) sequentially deliver an M1 promoting protein to generate a more inflammatory wound site and then deliver the M2-promoting biomaterial to modulate macrophage phenotype to enhance angiogenesis. Poly(lactic-co-glycolic acid) microparticles (MPs) were fabricated with the M2 promoting protein interleukin-4 (IL-4) inside and given to macrophages. The microparticles promoted increased expression of M2 markers in vitro, although this effect was blunted in the presence of pro-inflammatory stimuli. When co-delivered with fluorescently labeled M2 macrophages 3 days after injury in a murine model of hindlimb ischemia, IL-4-releasing microparticles increased the numbers of larger blood vessels, but not smaller blood vessels, pointing to a potential stabilizing role. These effects are attributed to polarization of host macrophages, as opposed to administered macrophages, because host macrophages increased expression of the M2 marker Arg1 and decreased expression of the M1 marker CD86, while very few administered macrophages could be detected at 3 days after administration. Multidimensional analysis of flow cytometry data showed that macrophages existed as multiple complex phenotypes expressing traditional M1 and M2 markers, but that IL4 treatment primarily increased the numbers of cells expressing high levels of the M2 marker Arg1. Once the M2 promoting biomaterial was proven to work, the M1 promoting protein interferon gamma (IFN[gamma]) was tested next for phenotype modulating behavior. The IFN[gamma] was injected into mice shortly after injury, wherein the host macrophages had a subtle response towards more inflammatory marker expression. Then, a head-to-head comparison was made between sequential delivery of IFN[gamma] followed by IL-4 MPs versus just a delivery of IL-4 MPs alone was performed. Sequentially delivering IFN[gamma], followed by MPs containing IL-4 performed no differently than just IL-4 MPs alone at modulating macrophage phenotype or angiogenesis. The IL-4 microparticle system was then evaluated further in depth by assessing a range of IL-4 doses in vivo, macrophage phenotype, and blood vessel development. A minimum dose of IL-4 MPs was established to generate a phenotypic change in macrophage profile for up to 7 days. Upon further investigation of the phenotype caused by the treatments using clustering algorithms, we found that macrophages expressing M1/M2 markers emerged as a primary group of interest. The effects of the IL-4 MPs and macrophages on angiogenesis was more complicated. Host macrophages polarized by IL-4 MPs alone to an M2-like phenotype had no effect on angiogenesis. However, when macrophages were included in the treatment, an increase in blood vessel diameter was observed. Interestingly, when blood vessel volume was measured via microCT, macrophages or IL-4 MPs alone caused an increase. Additionally, macrophages were observed interacting with blood vessels and quantification revealed that blood vessel size was positively correlated with number of macrophages in close proximity. Altogether, these findings indicate that delivery of IL-4 and macrophages can be beneficial for angiogenesis but achieving the proper control of phenotype is vital. Future studies should investigate the use of this immunomodulation strategy in immunocompromised wounds, where the immune system needs guidance.