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Sirt1 Protects Endothelial Cells against LPS-Induced Barrier Dysfunction.

ABSTRACT: Sepsis is a threatening health problem and characterized by microvascular dysfunction. In this study, we verified that LPS caused the downregulation of Sirt1 and the hyperpermeability of endothelial cells. Inhibition of Sirt1 with ex527 or Sirt1 siRNA displayed a higher permeability, while activation of Sirt1 with SRT1720 reversed the LPS-induced hyperpermeability, formation of fiber stress, and disruption of VE-cadherin distribution. In pulmonary microvascular vein endothelial cells isolated from wild-type mice, Sirt1 was attenuated upon LPS, while Sirt1 was preserved in a receptor of advanced glycation end product-knockout mice. The RAGE antibody could also diminish the downregulation and ubiquitination of Sirt1 in LPS-exposed human umbilical vein endothelial cells. An LPS-induced decrease in Sirt1 activity was attenuated by the RAGE antibody and TLR4 inhibitor. In vivo study also demonstrated the attenuating role of Sirt1 and RAGE knockout in LPS-induced increases in dextran leakage of mesenteric venules. Furthermore, activation of Sirt1 prevented LPS-induced decreases in the activity and expression of superoxide dismutase 2, as well as the increases in NADPH oxidase 4 and reactive oxygen species, while inhibition of Sirt1 aggravated the SOD2 decline. It also demonstrated that Sirt1-deacetylated p53 is required for p53 inactivation, which reversed the downregulation of ?-catenin caused by LPS.

SUBMITTER: Zhang W

PROVIDER: S-EPMC5676476 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Sirt1 Protects Endothelial Cells against LPS-Induced Barrier Dysfunction.

Zhang Weijin W Zhang Yaoyuan Y Guo Xiaohua X Zeng Zhenhua Z Wu Jie J Liu Yanan Y He Jing J Wang Ruiting R Huang Qiaobing Q Chen Zhongqing Z

Oxidative medicine and cellular longevity 20171025

Sepsis is a threatening health problem and characterized by microvascular dysfunction. In this study, we verified that LPS caused the downregulation of Sirt1 and the hyperpermeability of endothelial cells. Inhibition of Sirt1 with ex527 or Sirt1 siRNA displayed a higher permeability, while activation of Sirt1 with SRT1720 reversed the LPS-induced hyperpermeability, formation of fiber stress, and disruption of VE-cadherin distribution. In pulmonary microvascular vein endothelial cells isolated fr ...[more]

PMID: 29209448

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Project description:BackgroundCell-based therapeutic strategies have been proposed as an alternative for brain repair after stroke, but their clinical application has been hampered by potential adverse effects in the long term. The present study was designed to test the effect of the secretome of endothelial progenitor cells (EPCs) from stroke patients (scCM) on in vitro human models of angiogenesis and vascular barrier.MethodsTwo different scCM batches were analysed by mass spectrometry and a proteome profiler. Human primary CD34+-derived endothelial cells (CD34+-ECs) were used for designing angiogenesis studies (proliferation, migration, and tubulogenesis) or in vitro models of EC monolayer (confluent monolayer ECs-CMECs) and blood-brain barrier (BBB; brain-like ECs-BLECs). Cells were treated with scCM (5 μg/mL) or protein-free endothelial basal medium (scEBM-control). CMECs or BLECs were exposed (6 h) to oxygen-glucose deprivation (OGD) conditions (1% oxygen and glucose-free medium) or normoxia (control-5% oxygen, 1 g/L of glucose) and treated with scCM or scEBM during reoxygenation (24 h).ResultsThe analysis of different scCM batches showed a good reproducibility in terms of protein yield and composition. scCM increased CD34+-EC proliferation, tubulogenesis, and migration compared to the control (scEBM). The proteomic analysis of scCM revealed the presence of growth factors and molecules modulating cell metabolism and inflammatory pathways. Further, scCM decreased the permeability of CMECs and upregulated the expression of the junctional proteins such as occludin, VE-cadherin, and ZO-1. Such effects were possibly mediated through the activation of the interferon pathway and a moderate downregulation of Wnt signalling. Furthermore, OGD increased the permeability of both CMECs and BLECs, while scCM prevented the OGD-induced vascular leakage in both models. These effects were possibly mediated through the upregulation of junctional proteins and the regulation of MAPK/VEGFR2 activity.ConclusionOur results suggest that scCM promotes angiogenesis and the maturation of newly formed vessels while restoring the BBB function in ischemic conditions. In conclusion, our results highlight the possibility of using EPC-secretome as a therapeutic alternative to promote brain angiogenesis and protect from ischemia-induced vascular leakage.

Project description:Increased oxidative stress by hyperglycemia is a major cause of vascular complications in diabetes. Bird's nest, which is made from the saliva of swiftlets has both medicinal and nutritional values dated back to ancient China. However, its role in improving endothelial dysfunction due to diabetes is yet to be elucidated. The present study examined the protective effect and mechanism of action of the aqueous extract of hydrolyzed edible bird nest (HBN) on endothelium in models of diabetes, in vitro and in vivo. Male db/m+ and db/db mice were orally administered with or without HBN and glibenclamide for 28 days, followed by vascular reactivity studies in mouse aortas. Human umbilical vein endothelial cells (HUVECs) and isolated mouse aorta from C57BL/6J were treated with high glucose (HG), HBN, sialic acid (SA), glibenclamide, and apocynin, respectively. The effects of HBN on reactive oxygen species (ROS) production and nitric oxide (NO) bioavailability were assessed by Western blot, 2',7'-dichlorofluorescin-diacetate (DCF-DA), and 4-amino-5-methylamino-2',7' difluorofluorescein (DAF-FM DA) in HUVECs, isolated mouse aorta, and db/db diabetic mice. HBN significantly reversed the endothelial dysfunction in diabetic mice and isolated mouse aorta. HBN normalized ROS over-production of NOX2 and nitrotyrosine, reversed the reduction of anti-oxidant marker, SOD-1 as well as restored NO bioavailability in both HUVECs challenged with HG and in db/db diabetic mice. Similarly, HG-induced elevation of oxidative stress in HUVECs were reversed by SA, glibenclamide, and apocynin. This attests that HBN restores endothelial function and protects endothelial cells against oxidative damage induced by HG in HUVECs, isolated mouse aorta, and db/db diabetic mice via modulating ROS mechanism, which subsequently increases NO bioavailability. This result demonstrates the potential role of HBN in preserving endothelial function and management of micro- or macrovascular complications in diabetes.

Project description:ObjectivesAcute respiratory distress syndrome represents the devastating result of acute lung injury, with high mortality. Limited methods are available for rehabilitation of lungs affected by acute respiratory distress syndrome. Our laboratory has demonstrated rehabilitation of sepsis-injured lungs via normothermic ex vivo and in vivo perfusion with Steen solution (Steen). However, mechanisms responsible for the protective effects of Steen remain unclear. This study tests the hypothesis that Steen directly attenuates pulmonary endothelial barrier dysfunction and inflammation induced by lipopolysaccharide.MethodsPrimary pulmonary microvascular endothelial cells were exposed to lipopolysaccharide for 4 hours and then recovered for 8 hours in complete media (Media), Steen, or Steen followed by complete media (Steen/Media). Oxidative stress, chemokines, permeability, interendothelial junction proteins, and toll-like receptor 4-mediated pathways were assessed in pulmonary microvascular endothelial cells using standard methods.ResultsLipopolysaccharide treatment of pulmonary microvascular endothelial cells and recovery in Media significantly induced reactive oxygen species, lipid peroxidation, expression of chemokines (eg, chemokine [C-X-C motif] ligand 1 and C-C motif chemokine ligand 2) and cell adhesion molecules (P-selectin, E-selectin, and vascular cell adhesion molecule 1), permeability, neutrophil transmigration, p38 mitogen-activated protein kinase and nuclear factor kappa B signaling, and decreased expression of tight and adherens junction proteins (zonula occludens-1, zonula occludens-2, and vascular endothelial-cadherin). All of these inflammatory pathways were significantly attenuated after recovery of pulmonary microvascular endothelial cells in Steen or Steen/Media.ConclusionsSteen solution preserves pulmonary endothelial barrier function after lipopolysaccharide exposure by promoting an anti-inflammatory environment via attenuation of oxidative stress, toll-like receptor 4-mediated signaling, and conservation of interendothelial junctions. These protective mechanisms offer insight into the advancement of methods for in vivo lung perfusion with Steen for the treatment of severe acute respiratory distress syndrome.

Dataset Information

Sirt1 Protects Endothelial Cells against LPS-Induced Barrier Dysfunction.

Publications

Sirt1 Protects Endothelial Cells against LPS-Induced Barrier Dysfunction.

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