Isolation and Culture of Endothelial Progenitor Cells

The isolation, ex vivo expansion and culture of EPCs was performed as previously described (17 (link)). In brief, under anesthesia, peripheral blood samples were collected from the mice and mononuclear cells were isolated and purified from the blood. Freshly isolated mononuclear cells from peripheral blood were characterized using flow cytometry, as previously described (18 (link)). EPCs were identified by labeling with Dil-acetylated low density lipoprotein (Dil-acLDL; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and fluorescein isothiocyanate-labeled Ulex europaeus agglutinin (Sigma-Aldrich, St. Louis, MO, USA). In order to mimic the in vivo environment of EPCs, EPCs from diabetic mice were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM; 30 mM D-glucose; cat no. 11965-126; Thermo Fisher Scientific, Inc). EPCs from control mice were cultured in normal glucose DMEM (5 mM D-glucose; cat no. 10567-014; Thermo Fisher Scientific, Inc).