M2 Microglial Cells Promote Angiogenesis Via Secretion Of Exosomes

Ischemic stroke is a sudden cerebral blood circulation disorders, because of the high incidence of stroke, finding a good treatment is urgent. Angiogenesis is a key factor in the prognosis of stroke, and microglia in the central nervous system is extremely important innate immune cells by monitoring changes in the environment, maintain the homeostasis of the central nervous system. Ischemic stroke can cause rapid activation of microglia, activated microglia rapidly gather and proliferate in the damage zone. Microglia exhibit different phenotypes in different environments. Our previous results showed that M2 type microglia can improve stroke infarct size in mice, and participate in upregulation of angiogenesis-related molecules, so in this experiment,we use the 3D technology to study the angiogenesis by co-culture of M2 microglia and HUVEC endothelial cells, and analyze whether its possible mechanism is through the secretion of exosomes. This study was designed to further investigate the mechanisms of M2 type of microglia in promoting angiogenesis to improve stroke.Therefore, the study was performed from the following aspects:1. The polarization and characterization of microglia.We use LPS and IL-4 to polarize microglia BV2 to M1 and M2 type type respectively, and identify phenotypes by immunofluorescence, PCR, flow cytometry method to show the polarization is success, different phenotypes BV2 can be used for subsequent experiments.2. Detect the effect of microglia pro-angiogenicWe co-cultured the polarized BV2 with endothelial cells HUVEC in 3D matrigel, and observed under a microscope by a number of pipe structure to detect angiogenic ability of different BV2 phenotypes. The results showed that M2 type BV2 can better promte angiogenesis than BV2 unpolarized and M1 type BV2 relatively. Further we detected the expression of angiogenesis-related factors. The results showed that there was no difference in expression of the angiogenesis-related factors, such as VEGF, FGF, HGF etc.in the three kinds phenotypes of BV2. By reading a lot of literature,we found that tumor cells, mesenchymal stem cells, etc. can play a role in promoting angiogenesis by releasing vesicles called the exosomes, so we guess whether M2 type BV2 have an effect of pro-angiogenic by releasing these vesicles.3. The detection of microglia pro-angiogenic effect by secretion of exosomesExtract the secretion of exosomes from different phenotypes of BV2 supernatant, After identifying the different phenotypes BV2 derived exosomes by the electron microscopy and western method, we co-cultured exosomes with endothelial cells HUVEC in 3D matrigel respectively. Then we observed and counted the generated tube-like structure by microscope, and the results showed, M2 type BV2 derived exosomes has a better ability to generate pro-tube-like structure.4. The mechanism of exosomes’ s effect of pro-angiogenicSince the exosomes has an extremely complex contents, and in recent years many reported micro RNA played an important role in the pro-angiogenic, so we try to find what micro RNA expressed high and related with angiogenesis in M2 type BV2 derieved exosomes by microarray methods.From the studies of above, we made the following conclusions:1. BV2 microglia was successfully polarized to M1 and M2 type when treated with LPS, IL-4 respectively.2. M2 type BV2 have a stronger ability to promote angiogenesis than M0 type and M1 type BV2.3. M2 type BV2 mainly implement this pro-angiogenic effect through the release of exosomes vesicles.4. The micro RNA in exosomes is one of the mechanisms in the pro-angiogenic.M2 type BV2 derived exosomes displayed, mi RNA-26 a is the mainly micro RNA in pro-angiogenic effect.This study reveals that the M2 type microglia exerts angiogenic effect mainly by releasing the exosomes, and the micro RNA in it, which may as a key mechanism of improving stroke.This provides bases and new ideas for treatment of stroke.