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

ABSTRACT: ABSTRACT 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-eNOSThr495). 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-eNOSThr495 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 eNOSThr495.

SUBMITTER: Ma W

PROVIDER: S-EPMC8805971 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:As discovered in our previous study, autologous endothelial progenitor cells (EPCs) protect against acute focal ischemia rat via the promotion of angiogenesis. However, it is unknown whether the EPCs that reached the deficient region were transplanted ones or the products of other auto-conversion cells they had promoted. This study aimed to gather direct evidence for determining if exogenous transplanted EPCs directly participate in angiogenesis in ischemic areas and attempted to clarify the related mechanism. Methods:First, EPCs were extracted in vitro from male rats, which were characterized by uptake of fluorescently labeled acetylated low-density lipoprotein (ac-LDL) intake and Ulex europaeus agglutinin (UEA-1) and subsequently introduced to middle cerebral artery occlusion (MCAO) female rats for 7 days after ischemia surgery. The EPC-treated animals received approximately 1×106 cells, while the control animals received phosphate buffered saline (PBS). The animals behavior function recovery were by a rotarod (TOR) test, while infarct volume was assessed by brain magnetic resonance imaging (MRI). CD31 antibody was used to determine the presence of EPCs in the ischemic zone, and sex-determining region Y (SRY) gene in-situ hybridization (ISH) traced the EPC process. In addition, immunohistochemistry and Western blot were used to assess B-cell lymphoma 2 (Bcl-2) expression in the ischemic brain. Results:Behavior tests and MRI of all ischemic stroke groups on postoperative day 14 indicated that EPCs were more effective in behavior function recovery and reducing infarct volume and gliosis status than the control group. Cluster of differentiation (CD31) immunofluorescent staining and SRY gene ISH demonstrated that EPCs yielded a better outcome in both angiogenesis and exogenous cell homing status. We also observed increased Bcl-2 distribution and higher plasma Bcl-2 levels in the EPC-treated group compared to the control group. Conclusions:Our results provide direct evidence that exogenous EPCs can participate in angiogenesis to improve neurological outcome and revascularization directly after stroke, with Bcl-2 playing an important role in this process.

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

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