The Effect Of Exosomes Derived From Macrophages Upon Cobalt Ions On Endothelial Cell Functions

Although hard tissue implants have been used clinically,the ability of osseointegration is still insufficient.Angiogenesis around the implant is a prerequisite for osteogenesis and osseointegration.Therefore,it’s widely concerned to promote osseointegration by improving the angiogenesis ability of endothelial cells around the implant.As an important immune cell of the organism,macrophages have been confirmed to play an immunomodulatory function in angiogenesis.The hypoxic microenvironment can stabilize the hypoxia-inducible factor-1α（HIF-1α）of macrophages and endothelial cells to promote the expression and secretion of vascular endothelial growth factor（VEGF）and induce angiogenesis.However,it is still unclear whether exosomes,a new carrier for cell-cell communication,are involved in this process and its mechanism.Although some studies have shown that exosomes derived from macrophages are involved in angiogenesis,it’s unknown whether changes in the cellular microenvironment cause changes in exosomes secreted by macrophages.In this study,different concentrations of cobalt ions(Co²⁺)（0-200μM）were used to mimic hypoxic microenvironment under the condition of lipopolysaccharide（LPS）stimulation.Firstly,the effects of Co²⁺on macrophage viability,proliferation,morphology,skeleton,adhesion and phenotype gene expression were studied.On this basis,the exosomes derived from macrophages stimulated by different Co²⁺concentrations were extract by ultracentrifugation,and the exosomes were identified by scanning electron microscopy（SEM）,particle size analysis and western blot.The effects of exosomes on endothelial cell viability,proliferation,morphology,skeleton,adhesion,migration,angiogenesis in vitro and vivo,nitric oxide（NO）synthesis,VEGF secretion,angiogenesis-related genes and integrinβ1 trafficking were evaluated.The specific research results are as follows:（1）Co/LPS has no significant effect on macrophage viability,but the increase of Co²⁺concentration can promote macrophage proliferation.Co/LPS can stimulate the formation of macrophage pseudopodium,thereby promoting the adhesion of macrophages to the substrate.With the increase of Co²⁺concentration,the expression of M1 phenotype genes（CD86 and CD11）and M2 phenotype genes（CD206,interleukin-10（IL-10）and arginine（ARG））of macrophages are down regulated,but the expression of M1 phenotype genes（inducible nitric oxide synthase（i NOS））is up-regulated.Co/LPS has no significant effect on the total protein content in the supernatant of macrophages.（2）By analyzing exosome morphology and particle size,macrophage-derived exosomes are mainly distributed in the range of 30～150 nm.The results of western blot show that macrophage-derived exosomes highly expressed TSG101 and CD81,and low expressed GAPDH.These results indicate that the exosomes were successfully extracted.BCA is used to measure the secretion of macrophage-derived exosomes in each group,and the results show that different Co²⁺concentrations have no significant effect on the secretion of macrophage-derived exosomes.（3）Macrophage-derived exosomes can be ingested by endothelial cells,the exosomes have no effect on endothelial cell viability and adhesion,but they can inhibit cell proliferation.The exosomes derived from macrophages upon Co/LPS can upregulate the expression of endothelial nitric oxide synthase（e NOS）,VEGFA and angiogenin（ANG）genes in endothelial cells to facilitate NO synthesis and VEGF secretion,and further promote the formation endothelial cell pseudopodia and spreading,thereby promoting endothelial cell angiogenesis in vitro and vivo.In addition,Exo-Co200 can inhibit the trafficking and degradation of endothelial cell surface integrinβ1 to target lysosomes,thus promoting endothelial cell migration and angiogenesis.