Hydrogen Sulfide Protects Against High Glucose-Induced Human Umbilical Vein Endothelial Cell Injury Through Activating PI3K/Akt/eNOS Pathway.

PURPOSE: Dysfunction of endothelial cells plays a key role in the pathogenesis of diabetic atherosclerosis. High glucose (HG) has been found as a key factor in the progression of diabetic complications, including atherosclerosis. PI3K/Akt/eNOS signaling pathway has been shown to involve in HG-induced vascular injuries. Hydrogen sulfide (H2S) has been found to exhibit protective effects on HG-induced vascular injuries. Moreover, H2S activates PI3K/Akt/eNOS pathway in endothelial cells. Thus, the present study aimed to determine if H2S exerts protective effects against HG-induced injuries of human umbilical vein endothelial cells (HUVECs) via activating PI3K/Akt/eNOS signaling. MATERIALS AND METHODS: The endothelial protective effects of H2S were evaluated and compared to the controlled groups. Cell viability, cell migration and tube formation were determined by in vitro functional assays; protein levels were evaluated by Western blot assay and ELISA; cell apoptosis was determined by Hoechst 33258 nuclear staining; Reactive oxygen species (ROS) production was evaluated by the ROS detection kit. RESULTS: HG treatment significantly inhibited PI3K/Akt/eNOS signaling in HUVECs, which was partially reversed by the H2S treatment. HG treatment inhibited cell viability of HUVECs, which were markedly prevented by H2S or PI3K agonist Y-P 740. HG treatment also induced HUVEC cell apoptosis by increasing the protein levels of cleaved caspase 3, Bax and Bcl-2, which were significantly attenuated by H2S or 740 Y-P. ROS production and gp91phox protein level were increased by HG treatment in HUVECs and this effect can be blocked by the treatment with H2S or Y-P 740. Moreover, HG treatment increased the protein levels of pro-inflammatory cytokines, caspase-1 and phosphorylated JNK, which was significantly attenuated by H2S or Y-P 740. Importantly, the cytoprotective effect of H2S against HG-induced injury was inhibited by LY294002 (an inhibitor of PI3K/Akt/eNOS signaling pathway). CONCLUSION: The present study demonstrated that exogenous H2S protects endothelial cells against HG-induced injuries by activating PI3K/Akt/eNOS pathway. Based on the above findings, we proposed that reduced endogenous H2S levels and the subsequent PI3K/Akt/eNOS signaling impairment may be the important pathophysiological mechanism underlying hyperglycemia-induced vascular injuries.

Proteolytic degradation of nitric oxide synthase isoforms by calpain is modulated by the expression levels of HSP90.

Ca2+ loading of Jurkat and bovine aorta endothelium cells induces the degradation of the neuronal and endothelial nitric oxide synthases that are selectively expressed in these cell lines. For neuronal nitric oxide synthase, this process involves a conservative limited proteolysis without appreciable loss of catalytic activity. By contrast, endothelial nitic oxide synthase digestion proceeds through a parallel loss of protein and catalytic activity. The chaperone heat shock protein 90 (HSP90) is present in a large amount in Jurkat cells and at significantly lower levels in bovine aorta endothelium cells. The differing ratios of HSP90/nitric oxide synthase (NOS) occurring in the two cell types are responsible for the conservative or nonconservative digestion of NOS isozymes. Consistently, we demonstrate that, in the absence of Ca2+, HSP90 forms binary complexes with NOS isozymes or with calpain. When Ca2+ is present, a ternary complex containing the three proteins is produced. In this associated state, HSP90 and NOS forms are almost completely resistant to calpain digestion, probably due to a structural hindrance and a reduction in the catalytic efficiency of the protease. Thus, the recruitment of calpain in the HSP90-NOS complexes reduces the extent of the proteolysis of these two proteins. We have also observed that calpastatin competes with HSP90 for the binding of calpain in reconstructed systems. Digestion of the proteins present in the complexes can occur only when free active calpain is present in the system. This process can be visualized as a novel mechanism involving the association of NOS with HSP90 and the concomitant recruitment of active calpain in ternary complexes in which the proteolysis of both NOS isozymes and HSP90 is significantly reduced.

17Beta-oestradiol partially attenuates the inhibition of nitric oxide synthase-3 by advanced glycation end-products in human platelets.

1. Diabetes mellitus predisposes to and female sex protects against arterial thrombosis. The aim of the present study was to determine whether advanced glycation end-products (AGE), which accumulate in diabetes, impair platelet function through effects on platelet nitric oxide (NO) generation and whether this can be prevented by 17beta-oestradiol. 2. Aggregation responses of human platelet-rich plasma to ADP were determined in the absence or presence of 200 mg/L AGE-modified albumin (AGE-albumin), 10(-5) mol/L 17beta-oestradiol and 10(-5) mol/L ICI 182 780 (the pure oestrogen receptor antagonist). 3. Intraplatelet cGMP, an index of bioactive NO, was measured by radioimmunoassay and expression of nitric oxide synthase (NOS)-3, phosphoserine-1177-NOS-3 and O-glycosylated NOS-3 was quantified by western blotting in response to these same treatments. 4. Advanced glycation end-products-albumin increased platelet aggregatory responses to ADP. This increase was largely prevented by 17beta-oestradiol. Advanced glycation end-products-albumin decreased and 17beta-oestradiol increased intraplatelet NO-attributable cGMP and 17beta-oestradiol attenuated the AGE-albumin-induced decrease in NO-attributable cGMP. Despite no effect on NOS-3 expression, AGE-albumin decreased and 17beta-oestradiol increased phosphoserine-1177-NOS-3 and 17beta-oestradiol largely prevented the decrease in phosphoserine-1177-NOS-3 induced by AGE-albumin. Alone, AGE-albumin increased O-glycosylation of NOS-3 by N-acetylglucosamine, an effect largely inhibited by 17beta-oestradiol. 5. In conclusion, AGE-albumin inhibits platelet NO biosynthesis through effects on serine phosphorylation and O-glycosylation of platelet NOS-3 and this may explain, at least in part, the increase in platelet aggregability induced by AGE-albumin. These effects of AGE-albumin are largely prevented by 17beta-oestradiol. These actions may contribute to the effects of diabetes and sex on arterial thrombosis in vivo.