Abstract Body: Introduction:
Diabetes causes macro and microvascular complications, and the full range of mediating mechanisms remains unclear. Research suggests that small extracellular vesicles (EVs) (5- 200 nm) can transfer key cargo between cells and organs to play crucial roles in communication between cells and tissues and are involved in the development and progression of diabetes complications.
Methods:
Murine BMDMs were grown to isolate EVs and characterized. Integrated Proteomics and miRNAseq analyses of EVs from diabetic (Ins2^Akita) (DM) and non-diabetic (NDM) littermate mice were employed. Macrophage inflammation and cellular OXPHOS were measured by qRT-PCR and Seahorse analyzer. MicroRNA inhibitors (antagomirs) and Scramble controls were employed. Cell viability, migration and tube formation assays were employed. Hindlimb ischemia (HLI) was induced by left femoral artery ligation. Blood flow recovery and capillary density were evaluated by Laser Doppler imaging and immunofluorescent analyses.
Results:
By Zetaview, BMDMs-EV size was similar in DM and NDM mice (diameter, 20-200nm) with comparable cup-shaped morphology by TEM. Western blot confirmed known exosomal markers. Comparing DM vs. NDM BMDM EVs, a total of 3,277 proteins were identified by proteomics; 821 proteins were significantly differentially expressed. A total of 912 EV miRNAs, 39 were significantly up-regulated and 19 were down-regulated. MiRNA–proteomic interactome showed novel microRNA targets and network clusters. Mir-7217-5p was highly expressed in DM vs. NDM EVs and validated by qRT-PCR and multiple putative proteome targets of this miRNA were differentially expressed; a number of which are associated with endothelial function and angiogenesis. Impairment of endothelial tube formation was observed in human ECs treated with DM vs. NDM BMDM EVs, which was prevented by treatment with antagomirs of mir-7217-5p versus Scramble in BMDMs. EVs from miR-7217-5p inhibitor-treated BMDMs promoted proliferation and migration in mouse ECs. BMDMs treated with DM vs. NDM EVs displayed increased expression of Tnf, IL6 and reduced oxygen consumption rate. In vivo, the gastrocnemius and tibialis anterior muscles were injected with Mir-7217-5p antagomir DM BMDM sEVs or scramble DM BMDM sEVs on days 0,3,7,14 after HLI. Mir-7217-5p antagomir restored blood flow, increased capillary density and promoted angiogenesis.
Conclusion:
Targeting DM macrophage EVs miR-7217-5p may be a treatment strategy for PAD in diabetes.