Normal glucose dmem

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).

The HLEC line (FHL124) was kindly provided by Dr. J.R. Reddan (Oakland University). The cell line was maintained in HG Dulbecco's modified Eagle’s medium (DMEM; 25.0 mM glucose; Gibco, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Beyotime Biotechnology, China) in a humidified atmosphere at 37°C with 5% CO₂. The cell culture medium was changed every 2 days and passaged at 80% to 90% confluence. The medium was replaced by normal-glucose DMEM (5.5 mM glucose; Gibco) with 2% FBS before HG stimulation. In the normal glucose (NG) group, HLEC medium contained 5.5 mM glucose, while in the HG groups, additional glucose (Sigma-Aldrich, St. Louis, MO, USA; Merck KGaA) was added to bring the final concentration of glucose to 25 to 150 mM. The same gradient concentrations of mannitol (Aladdin, China) were added into the media as osmotic control groups.
MCC950 (MCE, USA), an NLRP3 inflammasome inhibitor, was dissolved in normal-glucose DMEM at a concentration of 1 mM as a storage solution. SRT1720 (Topscience, China), a selective SIRT1 activator, stock solution was prepared in DMSO and at a concentration of 10 mM. The final working concentrations of MCC950 and SRT1720 were 1 µM and 0.5 µM, respectively. The above two compounds were added 1 hour before HG stress.