Deficiency of S100A8/A9 attenuates pulmonary microvascular leakage in septic mice.

BACKGROUND: We have reported a positive correlation between S100 calcium-binding protein (S100) A8/S100A9 and sepsis-induced lung damage before. However, limited knowledge exists concerning the biological role of S100A8/A9 in pulmonary vascular endothelial barrier dysfunction, as well as the diagnostic value of S100A8/A9 in sepsis. METHODS: Sepsis was induced in C57BL/6J mice and S100A9-knockout (KO) mice through the cecal ligation and puncture (CLP). Pulmonary vascular leakage was determined by measuring extravasated Evans blue (EB). Reverse transcription polymerase chain reaction and the histological score were used to evaluate inflammation and lung injury, respectively. Recombinant S100A8/A9 (rhS100A8/A9) was used to identify the effects of S100A8/A9 on endothelial barrier dysfunction in human umbilical vein endothelial cells (HUVECs). Additionally, the diagnostic value of S100A8/A9 in sepsis was assessed using receiver operating characteristic. RESULTS: S100A8/A9 expression was up-regulated in the lungs of CLP-operated mice. S100A9 KO significantly reversed CLP-induced hypothermia and hypotension, resulting in an improved survival rate. S100A9 KO also decreased the inflammatory response, EB leakage, and histological scores in the lungs of CLP-operated mice. Occludin and VE-cadherin expressions were decreased in the lungs of CLP-operated mice; However, S100A9 KO attenuated this decrease. Moreover, CLP-induced signal transducer and activator of transcription 3 (STAT3) and p38/extracellular signal-regulated kinase (ERK) signalling activation and apoptosis were mitigated by S100A9 KO in lungs. In addition, rhS100A8/A9 administration significantly decreased occludin and VE-cadherin expressions, increased the phosphorylated (p)-ERK/ERK, p-p38/p38, and B-cell leukaemia/lymphoma 2 protein (Bcl-2)-associated X protein/Bcl-2 ratios in HUVECs. CONCLUSION: The present study demonstrated S100A8/A9 aggravated sepsis-induced pulmonary inflammation, vascular permeability, and lung injury. This was achieved, at least partially, by activating the P38/STAT3/ERK signalling pathways. Moreover, S100A8/A9 showed the potential as a biomarker for sepsis diagnosis.

Dihydroxyeicosatrienoic Acid, a Metabolite of Epoxyeicosatrienoic Acids Upregulates Endothelial Nitric Oxide Synthase Expression Through Transcription: Mechanism of Vascular Endothelial Function Protection.

The present study aimed to investigate the impacts and underlying mechanisms of 14,15-DHETs on eNOS and vascular endothelial functions. Bovine aortic endothelial cells (BAECs) were treated with various concentrations of 14, 15-DHET. The expressions of eNOS protein and mRNA were observed at different time points. The eNOS expression and phosphorylation were subsequently detected administered with 8,9-DHET, 11,12-DHET, and 14,15-DHET, respectively. Meanwhile, 14,15-DHET action on tube formation was observed in human umbilical vein endothelial cells (HUVECs). Finally, the aorta of male C57BL/6 mice was injected with 14,15-DHET via the tail vein. The impacts of 14,15-DHET (0.4 mg/kg body weight) on the expressions of eNOS protein and mRNA and endothelium-dependent vasodilation (EDV) were detected following 24 h. The expression of eNOS was greatly improved with the 14,15-DHET treatment compared with the BAECs, and eNOS phosphorylation sites at Ser1179, Ser635, and Thr497 were elevated. However, no statistically significant difference was revealed on total eNOS among the 8,9-DHET, 11,12-DHET, and 14,15-DHET treatment groups. Based on the upregulation of eNOS protein, eNOS mRNA levels were increased in BAECs and thoracic aorta of the male C57BL/6 mice treated with 14,15-DHET, suggesting that transcriptional activation was achieved in vascular eNOS. Moreover, 14,15-DHET enhanced tube formation abilities in HUVECs and acetylcholine(ACh)-induced EDV. These findings indicated that 14,15-DHET could improve the vascular endothelial diastolic functions of male C57BL/6 mice, and enhance the ability of tube formation, which might be related to the increase of eNOS expression.

Contribution of the NLRP3/IL-1ß axis to impaired vasodilation in sepsis through facilitation of eNOS proteolysis and the protective role of melatonin.

Endothelial dysfunction is a typical characteristic of sepsis. Endothelial nitric oxide synthase (eNOS) is important for maintaining endothelial function. Our previous study reported that the NLRP3 inflammasome promoted endothelial dysfunction by enhancing inflammation. However, the effects of NLRP3 on eNOS require further investigation. Therefore, the present study aimed to investigate the role of NLRP3 on eNOS expression levels in cecal ligation and puncture-induced impaired endothelium-dependent vascular relaxation and to determine the protective effects of melatonin. eNOS expression levels were discovered to be downregulated in the mesenteric arteries of sepsis model mice. Inhibiting NLRP3 with 10 mg/ kg MCC950 or inhibiting IL-1ß with 100 mg diacerein rescued the eNOS expression and improved endothelium-dependent vascular relaxation. In vitro, IL-1ß stimulation downregulated eNOS expression levels in human aortic endothelial cells (HAECs) in a concentration- and time-dependent manner, while pretreatment with 1 µM of the proteasome inhibitor MG132 reversed this effect. In addition, treatment with 10 mg/kg MG132 also prevented the proteolysis of eNOS and improved endothelium-dependent vascular relaxation in vivo. Notably, treatment with 30 mg/kg melatonin downregulated NLRP3 expression levels and decreased IL-1ß secretion, subsequently increasing the expression of eNOS and improving endothelium-dependent vascular relaxation. In conclusion, the findings of the present study indicated that the NLRP3/IL-1ß axis may impair vasodilation by promoting the proteolysis of eNOS and melatonin may protect against sepsis-induced endothelial relaxation dysfunction by inhibiting the NLRP3/IL-1ß axis, suggesting its pharmacological potential in sepsis.

Melatonin Alleviates Cardiac Dysfunction Via Increasing Sirt1-Mediated Beclin-1 Deacetylation and Autophagy During Sepsis.

Cardiac dysfunction is a major cause leading to multiple organ failure in sepsis. Beclin-1-dependent autophagy has been evidenced to exert protective effects on hearts in sepsis. However, the mechanisms on how Beclin-1 and autophagy are regulated remains enigmatic. To explore the detailed mechanisms controlling Beclin-1-dependent autophagy in septic heart and whether melatonin could protect against sepsis via regulating cardiac autophagy, adult Sprague-Dawley (SD) rats were subjected to cecal ligation and puncture (CLP) to induce sepsis. Rats were intraperitoneally administrated with 30 mg/kg melatonin within 5-min post-CLP surgery. Our data showed that sepsis induced Becline-1 acetylation and inhibited autophagy in hearts, resulting in impaired cardiac function. However, melatonin treatment facilitated Beclin-1 deacetylation and increased autophagy in septic hearts, thus improved cardiac function. Moreover, melatonin increased the expression and activity of Sirtuin 1 (Sirt1), and inhibition of Sirt1 abolished the protective effects of melatonin on Beclin-1 deacetylation and cardiac function. In conclusion, increased Beclin-1 acetylation was involved in impaired autophagy in septic hearts, while melatonin contributed to Beclin-1 deacetylation via Sirt1, leading to improved autophagy and cardiac function in sepsis. Our study sheds light on the important role of Beclin-1 acetylation in regulating autophagy in sepsis and suggests that melatonin is a potential candidate drug for the treatment of sepsis.

Disrupted eNOS activity and expression account for vasodilator dysfunction in different stage of sepsis.

AIMS: Sepsis is a severe endothelial dysfunction syndrome. The role of endothelial nitric oxide synthase (eNOS) in endothelial dysfunction induced by sepsis is controversial. To explore the role of eNOS in vascular dysfunction. MAIN METHODS: The effect of sepsis on vasodilation and eNOS levels was examined in septic mouse arteries and in cell models. KEY FINDINGS: In early sepsis mouse arteries, endothelium-dependent relaxation decreased and phosphorylation of the inhibitory Thr495 site in endothelial nitric oxide synthase increased. Mechanically, the phosphorylation of endothelial nitric oxide synthase at Thr497 in bovine aortic endothelial cells occurred in a protein kinase C-α dependent manner. In late sepsis, both nitric oxide-dependent relaxation responses and endothelial nitric oxide synthase levels were decreased in septic mice arteries. Endothelial nitric oxide synthase levels expression levels decreased in tumor necrosis factor-α-treated human umbilical vein endothelial cells and this could be prevented by the ubiquitin proteasome inhibitor (MG-132). MG-132 could reverse the decrease in endothelial nitric oxide synthase expression and improve nitric oxide-dependent vasodilator dysfunction in septic mice arteries. SIGNIFICANCE: These data indicate that vasodilator dysfunction is induced by the increased phosphorylation of endothelial nitric oxide synthase in early sepsis and its degradation in late sepsis.

Regulation of O-GlcNAcylation on endothelial nitric oxide synthase by glucose deprivation and identification of its O-GlcNAcylation sites.

As an energy-sensitive post-translational modification, O-GlcNAcylation plays a major role in endothelial nitric oxide synthase (eNOS) activity regulation. However, effects of glucose deprivation on eNOS O-GlcNAcylation and the presence of novel O-GlcNAcylation sites of eNOS under glucose deprivation remain unknown. Hence, we aim to determine the effects of glucose deprivation on O-GlcNAcylation and novel O-GlcNAcylation sites of eNOS. Bovine aortic endothelial cells (BAECs) and Sprague-Dawley rats were induced by glucose deprivation and their eNOS O-GlcNAcylation was subjected to immunoblotting. eNOS and transfected eNOS were purified by pull-down assay and immunoprecipitation respectively. Novel O-GlcNAcylation sites of eNOS were predicted by HPLC-MS and MS/MS Ion and determined by immunoblotting. eNOS activity was detected by Elisa and isotope labeling method. In BAECs and rat thoracic aorta, low glucose-associated activation of eNOS was accompanied by elevated O-GlcNAcylation, which did not affect O-linked serine phosphorylation at 1179/1177 residues. Changes in this post-translational modification were associated with increased O-GlcNAc transferase (OGT) expression and were reversed by AMPK knockdown. Immunoblot analysis of cells expressing His-tagged wild-type human eNOS and human eNOS carrying a mutation at the Ser1177 phosphorylation site confirmed an increase in O-GlcNAcylation by glucose deprivation. A marked increase in O-GlcNAcylation indicated that eNOS contained novel O-GlcNAcylation sites that were activated by glucose deprivation. Immunoblot analysis of cells expressing His-tagged human eNOS carrying a mutation at Ser738 and Ser867 confirmed an increase in O-GlcNAcylation by glucose deprivation. Conversely, in His-tagged human eNOS carrying a mutation at Thr866, O-GlcNAcylation was unaffected by glucose deprivation. Differences in culture conditions were identified using two-way analysis of variance (ANOVA), one-way ANOVA, and unpaired Student's t-test. Glucose deprivation increases O-GlcNAcylation and activity of eNOS, potentially by the AMPK-OGT pathway, suggesting that Thr866 is a novel O-GlcNAcylation site involved in glucose-deprivation mediated eNOS activation.