LPS-treated Human Umbilical Vein Endothelial Cells Research Articles

Anti-Inflammatory Activities of (+)-Afzelechin against Lipopolysaccharide-Induced Inflammation.

In this study, we investigated the potential protective effects of (+)-afzelechin (AZC), a natural compound that is derived from Bergenia ligulata, on lipopolysaccharide (LPS)-induced inflammatory responses. AZC is known to have antioxidant, anticancer, antimicrobial, and cardiovascular protective properties. However, knowledge regarding the therapeutic potential of AZC against LPS-induced inflammatory responses is limited. Thus, we investigated the protective attributes of AZC against inflammatory damage caused by LPS exposure. We examined the effects of AZC on heme oxygenase (HO)-1, cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) in LPS-activated human umbilical vein endothelial cells (HUVECs). In addition, the effects of AZC on the expression of iNOS, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β were analyzed in the lung tissues of LPS-injected mice. Data revealed that AZC promoted the production of HO-1, inhibited the interaction between luciferase and nuclear factor (NF)-κB, and reduced the levels of COX-2/PGE2 and iNOS/NO, thereby leading to a decrease in the signal transducer and activator of transcription (STAT)-1 phosphorylation. Moreover, AZC facilitated the nuclear translocation of Nrf2, increased the binding activity between Nrf2 and the antioxidant response elements (AREs), and lowered the expression of IL-1β in the LPS-treated HUVECs. In the animal model, AZC significantly reduced the expression of iNOS in the lung tissue structure and the TNF-α level in the bronchoalveolar lavage fluid. These findings demonstrate that AZC possesses anti-inflammatory properties that regulate iNOS through the inhibition of both NF-κB expression and p-STAT-1. Consequently, AZC has potential as a future candidate for the development of new clinical substances for the treatment of pathological inflammation.

Ethanolic Extract of Garcinia kola Stem Bark Inhibits LDL-Uptake and LPS-Induced Anxa-1 and ICAM-1 Expression in Human Umbilical Vein Endothelial Cells (HUVECS)

The endothelial cells’ dysfunction linked to the development of atherosclerosis plays an important role in the regulation of inflammatory responses. Previous studies demonstrated the antiatherogenic effects of Garcinia kola seed extracts based on their lipid lowering effects. Our recent studies showed the in vitro antioxidant and anti-inflammatory activities of the ethanolic extract of Garcinia kola stem barks (EE). For more insight on the antiatherogenic effect of EE, we investigated its activity on some key points of the atherosclerosis process. The cytotoxicity of EE as well as its effects on LDL-uptake, LPS-induced InterCellular Adhesion Molecule (ICAM-1) and Annexin-1 (Anxa-1) expression and LPS-induced DNA damage were evaluated in Human Umbilical Vein Endothelial Cells (HUVECs). EE significantly (p<0.0001) increased the cell viability in both naive and LPS-treated HUVECs. At the concentrations of 50 and 100 µg/ml, EE significantly reduced LDL-uptake by endothelial cells stimulated with LPS. The immunohistochemistry results showed a significant (p<0.01) decrease in the ICAM-1 expression at the EE concentration of 250 µg/ml. EE also showed a significant (p<0.0001) concentration-dependent reduction of Annexin-1 expression in LPS-treated HUVECs. Besides, EE exhibited significant inhibition on LPS-induced genotoxicity marked by a decrease in tail DNA expression (p<0.0001) and tail movement expression (p<0.001) for the concentrations of 50 and 100 µg/ml. These findings showed that EE may mitigate the atherogenic process by reducing LDL-uptake and the expression of adhesion molecules. Thus, the EE of Garcinia kola turns out to be a potential candidate for the treatment of atherosclerosis with a lower risk of toxicity.
 Keywords: Garcinia kola, LDL-uptake, LPS-induced inflammation, comet assay.

Protective Effects of Cirsilineol against Lipopolysaccharide-Induced Inflammation; Insights into HO-1, COX-2, and iNOS Modulation.

In this study, the potential protective effects of cirsilineol (CSL), a natural compound found in Artemisia vestita, were examined on lipopolysaccharide (LPS)-induced inflammatory responses. CSL was found to have antioxidant, anticancer, and antibacterial properties, and was lethal to many cancer cells. We assessed the effects of CSL on heme oxygenase (HO)-1, cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) in LPS-activated human umbilical vein endothelial cells (HUVECs). We also examined the effects of CSL on the expression of iNOS, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β in the pulmonary histological status of LPS-injected mice. The results showed that CSL increased HO-1 production, inhibited luciferase-NF-κB interaction, and reduced COX-2/PGE2 and iNOS/NO levels, leading to a decrease in signal transducer and activator of transcription (STAT)-1 phosphorylation. CSL also enhanced the nuclear translocation of Nrf2, elevated the binding activity between Nrf2 and antioxidant response elements (AREs), and reduced IL-1β expression in LPS-treated HUVECs. We found that CSL's suppression of iNOS/NO synthesis was restored by inhibiting HO-1 through RNAi. In the animal model, CSL significantly decreased iNOS expression in the pulmonary biostructure, and TNF-α level in the bronchoalveolar lavage fluid. These findings indicate that CSL has anti-inflammatory properties by controlling iNOS through inhibition of both NF-κB expression and p-STAT-1. Therefore, CSL may have potential as a candidate for developing new clinical substances to treat pathological inflammation.

Jujuboside B Inhibited High Mobility Group Box Protein 1-Mediated Severe Inflammatory Responses in Human Endothelial Cells and Mice.

High mobility group box protein 1 (HMGB1) is a biomolecule that acts as an alerting signal of late sepsis by accelerating the production of proinflammatory cytokines, and eventually leads to various inflammation-related symptoms. When released into plasma at high concentration, it disrupts precise diagnosis and prognosis and worsens the survival of patients with systemic inflammatory conditions. Jujuboside B (JB) is a natural compound pressed from the seed of Zizyphi Spinosi Semen, which is known for its medical efficacies in treating various conditions such as hyperlipidemia, hypoxia, and platelet aggregation. Nevertheless, the medicinal activity of JB on HMGB1-involved inflammatory response in vascular cells in the human body is still ambiguous. Therefore, we hypothesized that JB could regulate the lipopolysaccharide (LPS)-induced dynamics of HMGB1 and its mediated cascade in inflammatory responses in human umbilical vein endothelial cells (HUVECs). In this experiment, JB and HMGB1 were administered in that order. In vitro and in vivo permeability, and cell viability, adhesion, and excavation of leukocytes, development of cell adhesion molecules, and lastly production of proinflammatory substances were investigated on human endothelial cells and mouse disease models to investigate the efficacy of JB in inflammatory condition. JB substantially blocked the translocation of HMGB1 from HUVECs and controlled HMGB1-induced adhesion and extravasation of the neutrophils through LPS-treated HUVECs. Moreover, JB decreased the formation of HMGB1 receptors and continually prevented HMGB1-induced proinflammatory mechanisms by blocking transcription of nuclear factor-κB and synthesis of tumor necrosis factor-α. In conclusion, JB demonstrated preventive effects against inflammatory pathologies and showed the potential to be a candidate substance for various inflammatory diseases by regulating HMGB1-mediated cellular signaling.

Anti-Inflammatory Effect of Sparstolonin B through Inhibiting Expression of NF-κB and STAT-1.

Sparstolonin B (SsnB), which is found in Sparganium stoloniferum, prevents the synthesis of inflammatory mediators and is related to functional pathways of survival. In this study, we assessed the possible protective functions of SsnB on lipopolysaccharide (LPS)-induced inflammatory responses. We determined the functions of SsnB on controlling heme oxygenase (HO)-1, cyclooxygenase (COX-)2, and inducible nitric oxide synthase (iNOS) in LPS-activated human umbilical vein endothelial cells (HUVECs). Furthermore, the distinct function of SsnB on the expression of iNOS and well-known pro-inflammatory mediators, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, were assessed in the pulmonary histological status of LPS-injected mice. SsnB upregulated the HO-1 production, inhibited luciferase-NF-κB interaction, and lowered COX-2/PGE2 and iNOS/NO, which lead to the reduction of STAT-1 phosphorylation. Moreover, SsnB enhanced the nuclear translocation of Nrf2, elevated the binding activity between Nrf2 and antioxidant response elements (AREs), and weakened IL-1β expression on LPS-treated HUVECs. SsnB-suppressed iNOS/NO synthesis was restored by the process of the RNAi inhibition of HO-1. In experiment with an LPS-injected animal model, SsnB remarkably decreased the iNOS expression in the pulmonary biostructure and TNF-α level in the bronchoalveolar lavage fluid (BALF). Therefore, these results demonstrate that SsnB is responsible for inflammation ameliorative activity by controlling iNOS through inhibition of both NF-κB expression and p-STAT-1. Therefore, SsnB could be a candidate for promoting novel clinical substances to remedy pathologic inflammation.

Hydrogen-rich medium ameliorates lipopolysaccharides-induced mitochondrial fission and dysfunction in human umbilical vein endothelial cells (HUVECs) via up-regulating HO-1 expression

BackgroundSepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. It has been showed that the change of mitochondrial dynamics has been proved to be one of the main causes of death in patients with severe sepsis. And hydrogen has been proved to exert its protective effects against sepsis via heme oxygenase-1 (HO-1). This study was designed to demonstrate that whether the benefit effects of hydrogen can maintain the dynamic process of mitochondrial fusion/fission to mitigate human umbilical vein endothelial cells (HUVECs) injury exposed to endotoxin through HO-1. MethodsHUVECs cells cultured with medium which contained Lipopolysaccharides (LPS), Saline, hydrogen, Mdivi-1 (a dynamin-related protein 1 [Drp1] inhibitor) or zinc protoporphyrin IX (Znpp) (a HO-1 inhibitor) were also used in the research. Cell death and apoptosis were assessed using FITC annexin V and PI. Mitochondria were stained with Mitotracker orange and observed by confocal microscope. Oxygen consumption rate was assessed by seahorse xf24 extracellular analyzer. Mitochondrial membrane potential monitored by JC-1 dye. The expressions of Drp1 and HO-1 were tested by Western blot. The co-localization of Drp1 and mitochondria was determined by immunofluorescence. ResultsLPS caused a decrease in ATP content, mitochondrial membrane potential, and maximal respiration rate. At the same time, increased expression of Drp1 were observed in LPS-stimulated HUVECs, concomitantly with excessive mitochondrial fission. We found that hydrogen-rich medium can increase ATP content, mitochondrial membrane potential and maximal respiration rate, and decrease the expression of Drp1 in LPS-treated HUVECs. Meanwhile, hydrogen can ameliorate excessive mitochondrial fission caused by LPS. Furthermore, hydrogen-rich medium had a similar effect to Mdivi-1, a mitochondrial fission blocker. Both of them rescued the up-regulation of Drp1 and mitochondrial fission induced by LPS, then normalized mitochondrial shape after LPS stimulation. But after Znpp pretreatment, HO-1 expression was inhibited and the protective effects of hydrogen were abrogated. ConclusionsHydrogen-rich medium can alleviate the LPS-induced mitochondrial fusion/fission and dysfunction in HUVECs via HO-1 up-regulation.

The Pyruvate Dehydrogenase Complex Mitigates LPS-Induced Endothelial Barrier Dysfunction by Metabolic Regulation.

Sepsis is a fatal health issue induced by an aberrant host response to infection, and it correlates with organ damage and a high mortality rate. Endothelial barrier dysfunction and subsequent capillary leakage play major roles in sepsis-induced multiorgan dysfunction. Anaerobic glycolysis is the primary metabolic mode in sepsis and the pyruvate dehydrogenase complex (PDHC) serves as a critical hub in energy regulation. Therefore, it is important to understand the role of PDHC in metabolic regulation during the development of sepsis-induced endothelial barrier dysfunction.In present study, human umbilical vein endothelial cells (HUVECs) and C57 BL/6 mice were treated with lipopolysaccharide (LPS) as models of endotoxemia. LPS increased basal glycolysis, compensatory glycolysis, and lactate secretion, indicating increased glycolysis level in endothelial cells (ECs). Activation of PDHC with dichloroacetate (DCA) reversed LPS-induced glycolysis, allowing PDHC to remain in the active dephosphorylated state, thereby preventing lactic acid production and HUVECs monolayers barrier dysfunction, as assessed by transendothelial electrical resistance and Fluorescein Isothiocyanate-labeled dextran. The in vivo study also showed that the lactate level and vascular permeability were increased in LPS-treated mice, but pretreatment with DCA attenuated these increases. The LPS-treated HUVEC model showed that DCA reversed LPS-induced phosphorylation of pyruvate dehydrogenase E1α Ser293 and Ser300 to restore PDHC activity. Immunoprecipitation results showed that LPS treatment increased the acetylation level of PDH E1α in HUVECs.Our study suggested that activation of PDHC may represent a therapeutic target for treatment of LPS-induced endothelial barrier dysfunction.

Human Amnion Epithelial Cells and Their Derived Exosomes Alleviate Sepsis-Associated Acute Kidney Injury via Mitigating Endothelial Dysfunction.

BackgroundSepsis is characterized by organ dysfunction resulting from a patient’s dysregulated response to infection. Sepsis-associated acute kidney injury (S-AKI) is the most frequent complication contributing to the morbidity and mortality of sepsis. The prevention and treatment of S-AKI remains a significant challenge worldwide. In the recent years, human amnion epithelial cells (hAECs) have drawn much attention in regenerative medicine, yet the therapeutic efficiency of hAECs in S-AKI has not been evaluated.MethodsSeptic mice were induced by cecal ligation and puncture (CLP) operation. hAECs and their derived exosomes (EXOs) were injected into the mice via tail vein right after CLP surgery. The 7-day survival rate was observed. Serum creatinine level was measured and H&E staining of tissue sections were performed 16 h after CLP. Transmission electron microscopy was used to examine the renal endothelial integrity in CLP mice. Human umbilical vein endothelial cells (HUVECs) were treated with lipopolysaccharide (LPS) and EXOs. Zonula occludens-1 (ZO-1) localization was observed by immunofluorescence staining. Expression of phosphor-p65 (p-p65), p65, vascular cell adhesion molecule-1 (VCAM-1), and ZO-1 in the kidney were determined by Western blot.ResultshAECs decreased the mortality of CLP mice, ameliorated septic injury in the kidney, and improved kidney function. More precisely, hAECs suppressed systemic inflammation and maintained the renal endothelial integrity in septic animals. EXOs from hAECs exhibited similar renal protective effects as their parental cells. EXOs maintained endothelial cell adhesion junction in vitro and inhibited endothelial cell hyperactivation in vivo. Mechanistically, EXOs suppressed proinflammatory nuclear factor kappa B (NF-κB) pathway activation in LPS-treated HUVECs and in CLP mice kidneys.ConclusionOur results indicate that hAECs and their derived EXOs may ameliorate S-AKI via the prevention of endothelial dysfunction in the early stage of sepsis in mice. Stem cell or exosome-based therapy targeting endothelial disorders may be a promising alternative for treatment of S-AKI.

Meteorin-like protein (METRNL)/IL-41 improves LPS-induced inflammatory responses via AMPK or PPARδ–mediated signaling pathways

PurposeMeteorin-like protein (METRNL) (also known as IL-41), recently identified as a myokine, is released in response to muscle contraction. It improves the skeletal muscle insulin sensitivity through exerting a beneficial anti-inflammatory effect. However, no independent studies have been published to verify the effects of METRNL on human umbilical vein endothelial cells (HUVECs) and THP-1 human monocytes. Materials and methodsThe levels of NFκB and IκB phosphorylation as well as the expression of adhesion molecules were assessed by Western blotting analysis. Cell adhesion assay demonstrated the interactions between HUVEC and THP-1 ​cells. We used enzyme-linked immunosorbent assay (ELISA) to measure the levels of TNFα and MCP-1 in culture medium. ResultsTreatment with METRNL suppressed the secretion of TNFα and MCP-1 as well as NFκB and IκB phosphorylation and inflammatory markers in lipopolysaccharide (LPS)-treated HUVECs and THP-1 ​cells. Furthermore, treatment with METRNL ameliorated LPS-induced attachment of THP-1 monocytes to HUVECs via inhibition of adhesion molecule expression and apoptosis. Treatment of HUVEC and THP-1 ​cells with METRNL enhanced AMPK phosphorylation and PPARδ expression in a dose-dependent manner. Small interference (si) RNA-mediated suppression of AMPK or PPARδ restored all these changes. ConclusionsIt has therefore been shown that METRNL ameliorates inflammatory responses through AMPK and PPARδ-dependent pathways in LPS-treated HUVEC. In sum, the current study may suggest the suppressive potential of METRNL against endothelial inflammation.

TUG1 Represses Apoptosis, Autophagy, and Inflammatory Response by Regulating miR-27a-3p/SLIT2 in Lipopolysaccharide-Treated Vascular Endothelial Cells

BackgroundThe dysfunction of vascular endothelial cells is associated with sepsis development. Long noncoding RNAs take part in the regulation of vascular endothelial cell function. This study aimed to explore the role and mechanism of long noncoding RNA taurine-upregulated gene 1 (TUG1) in lipopolysaccharide (LPS)-induced endothelial cell injury. MethodsLPS-treated human umbilical vein endothelial cells (HUVECs) were used as a model of sepsis in vitro. Quantitative real-time polymerase chain reaction was performed to detect the expression of TUG1, microRNA-27a-3p (miR-27a-3p) and slit guidance ligand 2 (SLIT2) messenger RNA. Western blot was conducted to measure the protein levels of SLIT2 as well as those involved in apoptosis, autophagy, and inflammatory response. Flow cytometry was used to detect cell apoptotic rate. The targets of TUG1 and miR-27a-3p were predicted via starBase (http://starbase.sysu.edu.cn/index.php). Dual-luciferase reporter, RNA immunoprecipitation, and pull-down assays were carried out to validate the target correlation between miR-27a-3p and TUG1/SLIT2. ResultsTUG1 expression was decreased after the treatment of LPS in HUVECs. Overexpression of TUG1 decreased LPS-induced apoptosis, autophagy, and inflammatory response. TUG1 was a sponge of miR-27a-3p. Upregulation of miR-27a-3p reversed the suppressive effect of TUG1 overexpression on LPS-induced apoptosis, autophagy, and inflammatory response. SLIT2 was a target of miR-27a-3p. Knockdown of miR-27a-3p could inhibit LPS-induced injury by increasing SLIT2 in HUVECs. TUG1 could enhance SLIT2 expression by competitively sponging miR-27a-3p. ConclusionsTUG1 could repress cell apoptosis, autophagy, and inflammatory response in LPS-treated HUVECs by sponging miR-27a-3p to target SLIT2, providing a potential target for the treatment of sepsis.

Decidual mesenchymal stem/stromal cell-derived extracellular vesicles ameliorate endothelial cell proliferation, inflammation, and oxidative stress in a cell culture model of preeclampsia.

To determine whether DMSC-secreted, extracellular vesicles (DMSC_EV) restore endothelial cell function and reduce oxidative stress. DMSC were isolated from the placentae of uncomplicated term pregnancies and DMSC_EV prepared by ultracentrifugation. Human umbilical vein endothelial cells (HUVEC) were treated with bacterial lipopolysaccharide (LPS), or with serum from PE patients, to model the effects of PE. DMSC_EV were then added to treated HUVEC and their growth profiles, inflammatory state, and oxidative stress levels measured. DMSC_EV displayed characteristic features of extracellular vesicles. In both LPS- and PE serum-treatment models, addition of DMSC_EV significantly increased HUVEC cell attachment and proliferation, and significantly reduced production of pro-inflammatory cytokine IL-6. The addition of DMSC_EV to LPS-treated HUVEC had no significant effect on total antioxidant capacity, superoxide dismutase levels or on lipid peroxidation levels. In contrast, the addition of DMSC_EV to PE serum-treated HUVEC resulted in a significant reduction in levels of lipid peroxidation. Addition of DMSC_EV had beneficial effects in both LPS- and PE serum- treated HUVEC but the two treatment models to induce endothelial cell dysfunction showed differences. The LPS treatment of HUVEC model may not accurately model the endothelial cell dysfunction characteristic of PE. Human cell culture models of PE show that DMSC_EV improve endothelial cell dysfunction in PE, but testing in in vivo models of PE is required.

Secoisolariciresinol Diglucoside Exerts Anti-Inflammatory and Antiapoptotic Effects through Inhibiting the Akt/IκB/NF-κB Pathway on Human Umbilical Vein Endothelial Cells.

Inflammation is a key regulator in the progression of atherosclerosis (AS) which extremely affects people's health. Secoisolariciresinol diglucoside (SDG), a plant lignan, is relevant to angiogenesis and cardioprotection against ischemia-reperfusion injury and improves vascular disorders. However, the effect of SDG on cardiovascular disorder is not clear. In the present study, we aimed to investigate the effects of SDG on lipopolysaccharide- (LPS-) stimulated Human Umbilical Vein Endothelial Cells (HUVECs) and elucidate the underlying mechanism. The LPS-stimulated HUVEC cellular model was established. The cell viability, the cell tube formation activity, the nitric oxide (NO) release, the levels of inflammatory cytokine interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), the activation of nuclear factor kappa-B (NF-κB) pathway, and the expression of protein kinase B (Akt) were determined using Cell Counting Kit-8, cell tube-formation assay, western blotting, and enzyme-linked immunosorbent assay. Our results revealed that SDG reduces the angiogenic capacity of HUVECs and inhibited LPS-mediated HUVEC injury and apoptosis. In addition, SDG increased NO release and decreased the levels of IL-1β, IL-6, and TNF-α in LPS-treated HUVECs. Meanwhile, SDG inhibited the NF-κB pathway and downregulated Akt expression in LPS-induced HUVECs. Our results indicated that SDG relieves LPS-mediated HUVEC injury by inhibiting the NF-κB pathway which is partly dependent on the disruption of Akt activation. Therefore, SDG exerts its cytoprotective effects in the context of LPS-treated HUVECs via regulation of the Akt/IκB/NF-κB pathway and may be a potential treatment drug for cardiovascular disease.

Maslinic Acid Ameliorates Inflammation via the Downregulation of NF-κB and STAT-1.

Maslinic acid (MA), a natural compound of the triterpenoid group derived from olive, prevents the generation of pro-inflammatory cytokines and oxidative stress. In human umbilical vein endothelial cells (HUVECs) treated with lipopolysaccharide (LPS), we characterized the effects of MA on the regulation of heme oxygenase (HO)-1, cyclooxygenase (COX-)2, and inducible nitric oxide synthase (iNOS). MA was tested in the lung tissues of LPS-treated mice, to determine its effect on levels of iNOS expression and representative inflammatory mediators such as interleukin (IL)-1α and tumor necrosis factor (TNF)-α. We show that MA induced the expression of HO-1, reduced LPS-induced NF-κB-luciferase activity, and inhibited iNOS/NO and COX-2/PGE2, resulting in the downregulation of STAT-1 phosphorylation. Furthermore, our data show that MA induced the nuclear translocation of Nrf2, increased the binding of Nrf2 to ARE, and decreased IL-1α production in LPS-treated HUVECs. The MA-induced reduction in iNOS/NO expression was reversed by RNAi suppression of HO-1. In mice treated with LPS, MA significantly downregulated levels of iNOS in lung tissue and TNF-α in the bronchoalveolar lavage fluid. Taken together, our findings indicate that MA exerts a critical anti-inflammatory effect by modulating iNOS via the downregulation of NF-κB and p-STAT-1. Thus, we propose that MA may be an ideal substance to treat inflammatory diseases.

Resveratrol Inhibits MMP3 and MMP9 Expression and Secretion by Suppressing TLR4/NF-κB/STAT3 Activation in Ox-LDL-Treated HUVECs.

Aim Resveratrol is a natural plant polyphenol. The present study investigated the effects of resveratrol on the Toll-like receptor 4- (TLR4-) mediated expression and secretion of matrix metalloproteinases (MMPs) in oxidized low-density lipoprotein- (ox-LDL-) treated human umbilical vein endothelial cells (HUVECs). Methods Protein expression was analyzed by immunoblotting. The secretion of MMPs was measured by an enzyme-linked immunosorbent assay. The animal experiments were performed with and without resveratrol treatment in high-fat chow-fed mice. Results Resveratrol inhibited the expression of TLR4, MMP3, and MMP9 in ox-LDL- and lipopolysaccharide- (LPS-) treated HUVECs. Resveratrol reduced the secretion of MMP3 and MMP9 that was induced by ox-LDL and LPS. The TLR4 inhibitor CLI-095 similarly suppressed the expression and secretion of MMP3 and MMP9 in ox-LDL- and LPS-treated HUVECs. Resveratrol attenuated the phosphorylation of the transcription factors nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) that was induced by ox-LDL and LPS. Resveratrol recovered Sirt1 expression. In the animal experiments, resveratrol decreased TLR4 expression in the aorta, MMP9 levels in plasma, and vascular structural changes in high-fat chow-fed mice, with no significant effect on plasma MMP3 levels. Conclusion Resveratrol inhibited the TLR4-mediated expression and secretion of MMP3 and MMP9 in ox-LDL-treated HUVECs. The mechanism of action of resveratrol may be associated with the suppression of NF-κB and STAT3 phosphorylation and restoration of Sirt1 expression. Resveratrol exerts protective effects against vascular structural changes in high-fat chow-fed mice.

In vitro effect of flaxseed oil and α-linolenic acid against the toxicity of lipopolysaccharide (LPS) to human umbilical vein endothelial cells.

Consumption of α-linolenic acid (ALA)-rich flaxseed oil is a possible way to supplement ω-3 polyunsaturated fatty acids, but the beneficial effects to cardiovascular systems are still controversial. In this study, human umbilical vein endothelial cells (HUVECs) treated with 100ng/mL lipopolysaccharide (LPS) were used as the in vitro model and the potential beneficial effects of well-characterized flaxseed oil and ALA were studied. Exposure of HUVECs to LPS for 24h significantly promoted inflammatory response as release of interleukin 6 (IL-6), soluble intercellular cell adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1) as well as adhesion of THP-1 monocytes, but did not induce cytotoxicity or oxidative stress. ALA, but not flaxseed oil, significantly reduced LPS-induced release of sICAM-1 and sVCAM-1, without effect on THP-1 adhesion. No radical scavenging activity was observed after flaxseed oil or ALA exposure. Rather, ALA at high concentrations promoted intracellular superoxide associated with damages to lysosomes, which was not observed in flaxseed oil exposed cells. These results indicated that ALA at high concentrations could inhibit inflammatory responses in LPS-treated HUVECs in vitro but might also promote a modest effect in cytotoxicity and oxidative stress.

Pivotal role of glutathione depletion in eNOS uncoupling of LPS-Treated HUVECs.

The present study investigated the relationship between uncoupling of endothelial nitric oxide synthase (eNOS) and vascular endothelial cell (VEC) oxidative stress (OS) during sepsis and the role of eNOS glutathionylation in eNOS uncoupling of septic VECs. Human umbilical vein endothelial cells (HUVECs) cultured in vitro (EA.hy269 cell line) were incubated with Dulbecco's modified Eagle's medium (DMEM) (normal control group), lipopolysaccharide (LPS) (sepsis group), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (glutathionylation group), and LPS+ dithiothreitol (DTT) (deglutathionylation sepsis group). As result, compared with the DMEM group, malondialdehyde (MDA) level and uncoupling eNOS activity significantly increased in the LPS and BCNU groups. However, in the LPS + DTT group, only the NO level increased. Compared with the LPS group, MDA level, NO concentration, and normal functional eNOS activity significantly decreased, and uncoupling eNOS activity significantly increased in the BCNU group. In the LPS + DTT group, MDA level and uncoupling eNOS activity significantly decreased, and NO concentration and normal functional eNOS activity significantly increased. During sepsis, the main mechanism for VEC OS was eNOS uncoupling mediated by eNOS glutathionylation.

Dynamic contributions of P- and E-selectins to β2-integrin-induced neutrophil transmigration.

Leukocyte transendothelial migration is a key step in their recruitment to sites of inflammation. However, synergic regulation of endothelium-expressed selectins on leukocyte transmigration remains unclear. In this study, an in vitro model was developed to investigate the dynamic contributions of P- and E-selectin to polymorphonuclear neutrophil (PMN) transmigration under static conditions. Human umbilical vein endothelial cells (HUVECs) were treated with LPS for 4 or 12 h to induce different expression of selectins and intercellular adhesion molecule (ICAM)-1. PMN transmigration was increased significantly by LPS stimulation, which was higher on 4-h than on 12-h LPS-treated HUVECs. Blocking and competitive tests indicated that P-selectin engages PSGL-1 to activate β2-integrin and initiate PMN transmigration within the first 15 min, whereas E-selectin engages CD44 to influence PMN transmigration after 15 min. P- and E-selectin-induced β2-integrin activation is likely conducted through the spleen tyrosine kinase signaling pathway. Complicated complementary and competitive mechanisms are involved in the interaction of P-/E-selectins and their ligands to promote PMN transmigration. These results provide direct evidence of the distinct and dynamic contribution of P- and E-selectins in mediating PMN transmigration and give new insight into PMN interaction with the vessel wall.-Gong, Y., Zhang, Y., Feng, S., Liu, X., Lü, S., Long, M. Dynamic contributions of P- and E-selectins to β2-integrin-induced neutrophil transmigration.

ET-1 Stimulates Superoxide Production by eNOS Following Exposure of Vascular Endothelial Cells to Endotoxin.

It has been shown that microcirculation is hypersensitized to endothelin1 (ET-1) following endotoxin (lipopolysaccharide [LPS]) treatment leading to an increased vasopressor response. This may be related in part to decreased activation of endothelial nitric oxide synthase (eNOS) by ET-1. eNOS can also be uncoupled to produce superoxide (O2). This aberrant eNOS activity could further contribute to the hyperconstriction and injury caused by ET-1 following LPS. We therefore tested whether LPS affects ROS production by vascular endothelial cells and whether and how this effect is altered by ET-1. Human umbilical vein endothelial cells (HUVEC) or human microvascular endothelial cells (HMEC) were subjected to a 6-h treatment with LPS (250 ng/mL) or LPS and sepiapterin (100 μM) followed by a 30-min treatment with 100 μM L-Iminoethyl Ornithine (L-NIO) an irreversible eNOS inhibitor and 30-min treatment with ET-1 (10 nM). Conversion of [H]L-arginine to [H]L-citrulline was used to measure eNOS activity. Superoxide dismutase (SOD) inhibitable reduction of Cytochrome-C, dihydro carboxy fluorescein (DCF), and Mitosox was used to estimate ROS. LT-SDS PAGE was used to assess the degree of monomerization of the eNOS homodimer. Stimulation of HUVECs with ET-1 significantly increased NO synthesis by 1.4-fold (P < 0.05). ET-1 stimulation of LPS-treated HUVECs failed to increase NO production. Western blot for eNOS protein showed no change in eNOS protein levels. LPS alone resulted in an insignificant increase in ROS production as measured by cytochrome C that was increased 4.6-fold by ET-1 stimulation (P < 0.05). L-NIO significantly decreased ET-1-induced ROS production (P < 0.05). Sepiapterin significantly decreased ROS production in both; unstimulated and ET-1-stimulated LPS-treated groups, but did not restore NO production. DCF experiments confirmed intracellular ROS while Mitosox suggested a non-mitochondrial source. ET-1 treatment following a chronic LPS stress significantly monomerized the eNOS homodimer that was inhibited by sepiapterin loading. The two concomitant phenomena of decreased NO production and increased ROS formation seem to be multifactorial in nature with ROS production dependent upon pterin availability.

Berberine Protects Human Umbilical Vein Endothelial Cells against LPS-Induced Apoptosis by Blocking JNK-Mediated Signaling.

Endothelial dysfunction is a critical factor during the initiation of atherosclerosis. Berberine has a beneficial effect on endothelial function; however, the underlying mechanisms remain unclear. In this study, we investigated the effects of berberine on lipopolysaccharide- (LPS-) induced apoptosis in human umbilical vein endothelial cells (HUVECs) and the molecular mechanisms mediating the effect. The effects of berberine on LPS-induced cell apoptosis and viability were measured with 5-ethynyl-2′-deoxyuridine staining, flow cytometry, and Cell Counting Kit-8 assays. The expression and/or activation of proapoptotic and antiapoptotic proteins or signaling pathways, including caspase-3, poly(ADP-ribose) polymerase, myeloid cell leukemia-1 (MCL-1), p38 mitogen-activated protein kinase, C-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase, were determined with western blotting. The malondialdehyde levels, superoxide dismutase (SOD) activity, and production of proinflammatory cytokines were measured with enzyme-linked immunosorbent assays. The results demonstrated that berberine pretreatment protected HUVECs from LPS-induced apoptosis, attenuated LPS-induced injury, inhibited LPS-induced JNK phosphorylation, increased MCL-1 expression and SOD activity, and decreased proinflammatory cytokine production. The effects of berberine on LPS-treated HUVECs were prevented by SP600125, a JNK-specific inhibitor. Thus, berberine might be a potential candidate in the treatment of endothelial cell injury-related vascular diseases.

Early diagnosis of sepsis using serum hemoglobin subunit Beta.

The development of new sepsis-specific biomarkers is mandatory to improve the detection and monitoring of the disease. Hemoglobin is the main oxygen and carbon dioxide carrier in cells of the erythroid lineage and is responsible for oxygen delivery to the respiring tissues of the body. Hemoglobin subunit beta (HBβ) is a component of a larger protein called hemoglobin. The aim of this study was to evaluate blood levels of HBβ in septic patients. A prospective study of 82 patients with sepsis was conducted. Furthermore, C57BL/6 mice were subjected to cecal ligation and puncture (CLP) surgery. Alternatively, human umbilical vein endothelial cells (HUVECs) or C57BL/6 mice were exposed to lipopolysaccharide (LPS, 100 ng/ml to HUVECs or 10 mg/kg to mice). The data showed that LPS induced upregulation of the synthesis and secretion of HBβ in LPS-treated HUVECs and in LPS-injected and CLP mice. In patients admitted to the intensive care unit with sepsis, circulating levels of HBβ were significantly high (sepsis, 64.93-114.76 ng/ml, n = 30; severe sepsis, 157.37-268.69 ng/ml, n = 22; septic shock, 309.98-427.03 ng/ml, n = 30) when compared to the levels of control donors (9.76-12.28 ng/ml, n = 21). Patients with septic shock had higher HBβ levels when compared to patients with severe sepsis. Furthermore, the HBβ levels in septic patients were higher than those in healthy volunteers. These results suggest that in septic patients, HBβ blood level is related to the severity of sepsis and may represent a novel endothelial cell dysfunction marker. Moreover, HBβ can be used as a biomarker to determine the severity of sepsis.