Exogenous H₂S reverses high glucose-induced endothelial progenitor cells dysfunction via regulating autophagy

This study aims to determine the effect of exogenous hydrogen sulfide (H₂S) under high glucose (HG)-induced injury in endothelial progenitor cells (EPCs), and to explore the possible underlying mechanisms. Mononuclear cells were isolated from the peripheral blood of healthy volunteers by density-gradient centrifugation and identified as late EPCs by immunofluorescence and flow cytometry. EPCs were treated with high concentrations of glucose, H₂S, Baf-A1, 3-MA or rapamycin. Cell proliferation, cell migration and tube formation were measured using cell counting kit-8, Transwell migration and tube formation assays, respectively. Cellular autophagy flux was detected by RFP-GFP-LC3, and Western blotting was used to examine the protein expression levels of LC3B, P62, and phosphorylated endothelial nitric oxide synthase (eNOS) at Thr495 (p-eNOS^Thr495). Reactive oxygen species (ROS) levels were measured using a DHE probe. H₂S and rapamycin significantly reversed the inhibitory effects of HG on the proliferation, migration, and tube formation of EPCs. Moreover, H₂S and rapamycin led to an increase in the number of autophagosomes accompanied by a failure in lysosomal turnover of LC3-II or p62 and p-eNOS^Thr495 expression and ROS production under the HG condition. However, Baf-A1 and 3-MA reversed the effects of H₂S on cell behavior. Collectively, exogenous H₂S ameliorated HG-induced EPC dysfunction by promoting autophagic flux and decreasing ROS production by phosphorylating eNOS^Thr495.