Bone Marrow-derived Macrophage (BMDM) Extracellular Vesicles (EVs) From Diabetes Impair Angiogenesis In Ischemic Hind Limb Of Diabetic Mice

Critical limb ischemia (CLI), which is increased in diabetes, is a life-threatening condition that leads to high mortality and disability. The underlying mechanisms linking diabetes and severity of CLI remain unclear. Induction of hind limb ischemia (HLI) in diabetic Akita (C57BL/6-Ins2 Akita /J) vs. non-diabetic (NDM, C57BL/6J) mice was performed by ligation of the left femoral artery. Blood perfusion in the ischemic hind limb, measured by Laser Doppler Imaging, revealed reduced blood flow recovery in diabetic vs. NDM mice. Macrophage extracellular vesicles (EVs) contribute to cell-to-cell communications and diabetic complications. BMDMs from male NDM and Akita mice were isolated, cultured in Exo-free complete DMEM media for 48 hours and EVs isolated by ultracentrifugation followed by size, zeta potential and concentration measurements by ZetaView®-nanoparticle tracking analysis. EVs size of Akita BMDMs was similar to that of NDM BMDM EVs (diameter, 20-300nm). The zeta potential for the Akita EVs was -18.62±0.18 mV vs -8.38±0.14 mV in NDM EVs. Western blot confirmed positive exosomal markers (CD81, Flotillin) in NDM and Akita EVs; the negative control marker (calnexin) was not identified. To identify the proteome cargo, we employed LC-MS/MS. Comparing Akita vs. NDM BMDM EVs, a total of 3277 proteins was identified and quantified. 821 proteins were significantly differentially expressed in Akita vs. NDM EVs. These significantly altered proteins (Upregulated and downregulated) are involved in: metabolic functions, immune system processes, developmental and biological processes. Enrichment analysis (GO, KEGG and Reactome) highlighted pathways predominantly related to oxidative stress, insulin signaling, actin cytoskeleton, CLEAR (Coordinated Lysosomal Expression And Regulation), lipid metabolism, phagosome maturation, RNA binding protein and DNA replication pathways. In vitro, endothelial tube formation was impaired in human iliac artery endothelial cells treated with diabetic vs. NDM BMDM EVs. Targeting macrophage EVs composition might identify therapeutic approaches for the prevention and treatment of CLI in diabetes.