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Diabetic vascular complications are the leading causes of death and disability in patients with diabetes. Alpha-mangostin has been reported to have anti-diabetic capacity in recent years. Here, we investigated the protective function of alpha-mangostin on endothelium in vitro and in vivo experiments. We also observed that alpha-mangostin improved impaired endothelium-dependent vasodilation (EDV) of diabetic animals while it limited the aSMase/ceramide pathway and up-regulated eNOS/NO pathway in aortas from diabetic mice. Meanwhile, alpha-mangostin inhibited elevated aSMase/ceramide pathway and reversed impaired EDV induced by high glucose in isolated mouse aortas. In addition, alpha-mangostin increased phosphorylation of eNOS and NO production in high glucose-treated aortas. Alpha-mangostin normalized high glucose-induced activation of aSMase/ceramide pathway and improved eNOS/NO pathway in endothelial cells with high glucose. In conclusion, alpha-mangostin regulates eNOS/NO pathway and improves EDV in aortas of diabetic mice through inhibiting aSMase activity and endogenous ceramide accumulation., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

This study investigated the effects of high glucose levels on human vascular endothelial cells and the expression of apoptosis-associated signaling molecules. Cell proliferation of human umbilical vein endothelial cells (HUVECs) was analyzed by colorimetric assay and cell number counting. Apoptosis was measured by Annexin V/FITC staining and flow cytometry. Gene knockdown was established by transfection of synthesized small interfering RNA. Caspase-3 activation was inhibited by a caspase-3 inhibitor. Protein expression of signaling molecules was measured by Western blot. Glucose significantly decreased HUVEC viability, induced cell apoptosis, and elevated levels of intracellular reactive oxygen species in a concentration and time-dependent manner. Glucose significantly upregulated the Nox4 isoform of nicotinamide adenine dinucleotide phosphate oxidase and phosphatase and tensin homolog (PTEN) protein expression, increased PTEN phosphorylation, and activated caspase-3 in a concentration-dependent manner. Silencing Nox4 and PTEN gene expression and inhibiting caspase-3 activation significantly protected HUVECs from glucose-induced cell apoptosis. Silencing Nox4 significantly normalized the levels of reactive oxygen species in glucose-treated cells; 20 mM glucose obviously upregulated Nox4, PTEN, phosphor-PTEN, and Bax levels, but significantly reduced integrin-linked kinase (ILK) activity, Bcl-2 (B cell lymphoma 2) expression, and protein kinase B (Akt) phosphorylation at serine 473. High glucose levels can reduce cell viability and induce apoptosis in HUVECs through Nox4-produced reactive oxygen species. Elevated levels of reactive oxygen species decreased Bcl-2 expression and increased PTEN expression and phosphorylation, which lead to the subsequent inhibition of ILK-Akt signaling, elevation of Bax expression, and activation of caspase-3.

Aims: The present study was to investigate the effect of hydrogen peroxide (H2O2) on bone marrow stem cells and their endothelial differentiation and the underlying mechanisms in vitro., Main Methods: Rat bone marrow multipotent adult progenitor cells (MAPCs) were used as the source of bone marrow stem cells, and treated with H2O2 (with the final concentration from 0 to 50 μM) with or without N-acetylcysteine (NAC, 0.1 mM). Reactive oxygen species (ROS) was measured by electron paramagnetic resonance (EPR) and fluorescent microscope. Flow cytometry and immunoblotting were used to determine apoptosis and differentiation of MAPCs., Key Findings: H2O2 generated a significant amount of intracellular and extracellular ROS in the culture system, substantially inhibited the proliferation of MAPCs and Oct-4 expression, and induced their apoptosis in a dose-dependent manner. Exposure to H2O2 also significantly attenuated the endothelial differentiation of MAPCs with reduced expression of endothelial markers CD31 and FLK-1 as well as impaired in vitro vascular structure formation. Both intracellular and extracellular ROS production from H2O2 were blocked by NAC. NAC treatment effectively prevented H2O2-induced reduction of Oct-4 expression in the cells. However, NAC treatment only partially prevented H2O2-induced apoptosis, and inhibition of cell proliferation and endothelial differentiation of MAPCs., Significance: H2O2 exposure suppressed Oct-4 expression in MAPCs through ROS-dependent mechanism, while increasing the apoptosis of MAPCs and inhibiting their proliferation and endothelial differentiation with a mechanism partially due to ROS generation in vitro., (Copyright © 2014. Published by Elsevier Inc.)

Objective: To investigate how ceramide mediates the effects of high-glucose-induced inhibition of the Akt/endothelial nitric oxide synthase (eNOS) signalling pathway in human vascular endothelial cells (HUVECs)., Materials and Methods: NO levels were determined by ELISA. Endogenous ceramide levels were determined using a liquid chromatography-mass spectrometry assay. Akt and eNOS protein expressions were determined by Western blotting., Results: High-glucose levels induce ceramide accumulation in a dose- and time-dependent manner (p<0.05). We also show that exposure of HUVECs to high-glucose conditions inhibits the insulin-mediated activation of Akt/eNOS signalling and the subsequent NO generation in a dose-dependent manner (p<0.05). Preventing de novo ceramide synthesis attenuated the antagonistic effects of high-glucose levels on the Akt/eNOS signalling pathway (p<0.05); conversely, inducing ceramide build-up augmented the inhibitory effects of high-glucose levels on the Akt/eNOS signalling pathway (p<0.05)., Conclusion: Ceramide is both necessary and sufficient for mediating the inhibition of the Akt/eNOS signalling pathway by high-glucose levels in endothelial cells.

Objective: To evaluate the effect of intermittent high glucose on the apoptosis of human umbilical vein endothelial cells (HUVECs) and its mechanism., Methods: Intermittent high glucose and constant high glucose were applied to HUVEC-12 for 7 days. Flow cytometer and fluorescent staining with Hoechst 33258 were used to detect apoptosis of HUVEC-12. The superoxide dismutase (SOD) activity and the content of malonaldehyde (MDA) in culture solution were detected with colorimetry, and the changes of p-JNK level were examined by Western blot., Results: The apoptosis rate was obviously higher in the intermittent high glucose group than that in the constant high glucose group (P < 0.05). The SOD activity was significantly lower in the intermittent high glucose group (P < 0.05), but MDA level was higher than those of constant high glucose(P < 0.05). SP600125, the inhibitor of JNK, decreased the apoptosis rate induced by intermittent high glucose (P < 0.05). Antioxidant (Vitamin C) inhibited the p-JNK, decreased the apoptosis rate (P < 0.05)., Conclusion: Intermittent high glucose is easier to worsen the proapoptotic effects on HUVECs than that of constant high glucose, which may account for the increased oxidative stress, and then activates JNK signal transduction pathway.

Objective: To compare the effect of intermittent and constant high glucose media on the synthesis of vascular relaxing factor nitric oxide (NO) in cultured human umbilical vein endothelial cells (HUVECs), and to investigate its mechanism., Methods: HUVECs were exposed to 5.5 mmol/L glucose (normal control), 20 mmol/L glucose (constant high glucose), and 5.5 mmol/L alternating with 20 mmol/L glucose (intermittent high glucose) for 7 days. The NO level was measured using Griess Reaction in cell culture supernatants; the expressions of PI3K, PKB and eNOS mRNA and protein were measured with RT-PCR or Western blot., Results: NO level in the intermittent high glucose group was significantly lower than that in the constant high glucose group (P<0.05). The expression of PI3K, PKB, and eNOS in the intermittent high glucose group was significantly lower than that in the constant high glucose group (P<0.05)., Conclusion: Intermittent high glucose could be more deleterious to endothelial cells than constant high glucose, and may lead to decrease synthesis of NO by inhibiting PI3K/PKB/eNOS pathway activation.