Cipepofol attenuates endothelial barrier dysfunction in acute lung injury through DUSP1-dependent suppression of MAPK signaling.

Differential Regulation of Pulmonary Endothelial Monolayer Integrity by Varying Degrees of Cyclic Stretch

Sepsis-induced acute respiratory distress syndrome (ARDS) is an independent risk factor for mortality in critically ill septic patients. However, effective therapeutic targets are still unavailable due to the lack of understanding of its unclear pathogenesis. With increasing understanding in the roles of circulating histones and endothelial dysfunction in sepsis, we aimed to investigate the mechanism of histone-induced endothelial dysfunction leading to sepsis-induced ARDS and to provide experimental support for histone-targeted treatment of sepsis-induced ARDS. First of all, in vitro experiments were conducted. Human umbilical vein endothelial cells (HUVEC) were stimulated with gradient concentrations of histones to explore for the optimal stimulation concentration in vitro. Then, HUVEC were exposed to histones at an optimal concentration with or without resatorvid (TAK-242), a selective inhibitor of Toll-like receptor 4 (TLR4), for 24 hours for modeling. The cells were divided into 4 groups: 1) the blank control group, 2) the blank control+TAK-242 intervention group, 3) the histone stimulation group, and 4) the histone+TAK-242 intervention group. HUVEC apoptosis was determined by flow cytometry, VE-Cadherin expression in endothelial cells was determined by Western blot, and the integrity of adhesion connections between endothelial cells was evaluated with confocal fluorescence microscopic images. Male C57BL/6 mice aged 6-8 weeks and weighing 22-25 g were used for the in vivo experiment. Then, the mice were given cecal ligation and puncture (CLP) as well as histone injection at 50 mg/kg via the tail vein for sepsis modeling. The experimental animals were divided into 6 groups: 1) the blank control group, 2) the blank control+TAK-242 intervention group, 3) the CLP model group, 4) the CLP+TAK-242 intervention group, 5) the histone model group, and 6) the histone+TAK-242 intervention group. After 24 h, the concentrations of serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined using ELISA kits. Western blot was performed to determine the expression of vascular endothelial (VE)-cadherin in the lung tissue. Hematoxylin and eosin (HE) staining was performed to observe the pathological changes in the lung tissue of the mice. Evans Blue was injected via the tail vein 30 min before the mice were sacrificed. Lung tissue was collected after the mice were sacrificed. Then, the concentrations of Evans blue dye per unit mass in the lung tissue from mice of different groups were evaluated, the rates of pulmonary endothelial leakage were calculated, and the integrity of the pulmonary endothelial barrier was evaluated. The results of the in vitro experiment showed that, compared with those of the control group, HUVEC apoptosis was significantly increased under histone stimulation (P<0.05), the expression of VE-cadherin was decreased (P<0.05), and the integrity of adherens junctions between endothelial cells was damaged. TAK-242 can significantly inhibit histone-induced HUVEC apoptosis and VE-cadherin expression reduction and maintain the integrity of adherens junctions between endothelial cells. According to the findings from the in vivo experiments, in mice with CLP-induced and histone-induced sepsis, TAK-242 effectively alleviated the increase in serum concentrations of IL-6 and TNF-α, reduced the downregulation of VE-cadherin expression in the lung tissue (P<0.05), decreased endothelial permeability of the lung vessels, and improved pathological injury in the lung tissue. By binding to TLR-4, histone decreases VE-cadherin expression on the surface of vascular endothelial cells, disrupts the integrity of intercellular adherens junctions, and triggers pathological damage to lung tissue. Using TLR-4 inhibitors can prevent sepsis-induced ARDS in histone-induced sepsis.

Objective To evaluate the effects of different ratios of medicine dosage for isoflurane and propofol on GABAA receptor(GABAAR)α1 subunit proteostasis during hypoxia injury to hippocampal neurons of rats. Methods The hippocampal neurons isolated from fetal rats obtained from Wistar rats were primarily cultured and divided into 6 groups(n=60 each)using a random number table: control group(group C), hypoxia group(group H), isoflurane group(group I), propofol group(group P)and different ratios of medicine dosage for isoflurane and propofol groups(group IP1 and group IP2). The cells were subjected to hypoxia for 6 h in group H. Cells were incubated for 3 h with 1.9 % isoflurane and with 22.4 μmol/L propofol after being subjected to hypoxia for 6 h in I and P groups, respectively.Cells were incubated for 3 h with 1.0% isoflurane and 6.7 μmol/L propofol and with 1.4 % isoflurane and 3.4 μmol/L propofol after being subjected to hypoxia for 6 h in IP1 and IP2 groups, respectively.Then the culture medium was replaced with plain culture medium.At 24 h of incubation, the cells were collected for measurement of cell viability by CCK-8 assay, GABAAR α1 mRNA expression(by quantitative polymerase chain reaction), GABAAR α1 expression in the cytomembrane(by Western blot), level of GABAAR α1 subunit endoplasmic reticulum-associated degradation(ERAD) (by immunoprecipitation and Western blot)and CCAAT/enhancer-binding protein homologous protein(CHOP)expression (by immunofluorescence). Results Compared with group C, the cell viability was significantly decreased, the expression of GABAAR α1 mRNA and GABAAR α1 in cytomembrane was down-regulated, the expression of CHOP was up-regulated, and the level of GABAAR α1 subunit ERAD was increased in the other five groups(P<0.05). Compared with group H, the cell viability was significantly decreased, the expression of GABAAR α1 mRNA and GABAAR α1 in cytomembrane was down-regulated, the expression of CHOP was up-regulated, and the level of GABAAR α1 subunit ERAD was increased in I, P and IP2 groups(P <0.05), and no significant change was found in the parameters mentioned above in group IP1(P<0.05). Compared with group I or group P, the cell viability was significantly increased, the expression of GABAAR α1 mRNA and GABAAR α1 in cytomembrane was up-regulated, the expression of CHOP was down-regulated, and the level of GABAAR α1 subunit ERAD was decreased in IP1 and IP2 groups(P<0.05). Compared with group IP1, the cell viability was significantly decreased, the expression of GABAAR α1 mRNA and GABAAR α1 in cytomembrane was down-regulated, the expression of CHOP was up-regulated, and the level of GABAAR α1 subunit ERAD was increased in group IP2(P<0.05). Conclusion Combination of 1.0% isoflurane and 6.7 μmol/L propofol does not aggravate hypoxia-induced destruction of GABAAR α1 subunit proteostasis in hippocampal neurons of rats. Key words: Propofol; Isoflurane; Receptors, GABA-A; Hypoxia-ischemia, brain

Objective To observe the expression of axon guidance cues Slit2 and Robo4 in lung tissue of rat with acute lung injury （ALI） and explore the function of Slit2 and Robo4 in ALI. MethodsForty-eight Sprague-Dawley rats were randomly （random number） divided into control group （n = 24） and ALI group （n = 24）. ALI model was reproduced by cecum ligation and puncture （CLP）. The control group only experienced a simulated operation without CLP. Both groups were further divided into 3 subgroups with 8 rats in each subgroup： 12 h, 24 h, and 48 h subgroups, artery blood gas analysis, lung tissue wet/dry weight （W/D） ratio, lung histopathologic changes, pulmonary microvascular permeability were observed. The serum tumor nocrosis factor-α （TNF-α） was measured with enzyme linked immunosorbent assay （ELISA）. The expression of Slit2 and Robo4 mRNA were detected by reverse transcription-polymerase chain reaction （RT-PCR）. The expression of Slit2 and Robo4 protein in lung tissues was assessed by immunohistochemistry. Date were analyzed by one-way ANOVA with SPSS version 13.0 software. Statistical significance was established at a P value of less than 0. 05. Results Compared with the control group, in ALI rats at different time points, partial pressure of oxygen in arterial blood （PaO2） decreased significantly, lung W/D weight ratio and pulmonary microvascular permeability, the serum TNF-α increased significantly （ all P 〈 0. 05 ）, histopathology of lung revealed signs of injury. The expression of Slit2 mRNA in lung tissues was decreased markedly after CLP compared with control group [ （0.56±0.13）vs. （0.87±0.05）, F=41.39, P〈0.05, （0.42±0.10） vs. （0.85±0.07）, F= 93.54, P〈0.05, （0.26±0.08） vs. （0.89±0.09）, F=227.05, P〈0.05]. but there were no significant difference in expression of Robo4 mRNA in lung tissue between ALI group and control group [ （0.86±0.07） vs. （0.83±0.05）, F=0.695, P〉0.05, （0.82±0.05） vs. （0.89±0.08）, F= 2. 061, P 〉 0.05, （0. 86 ± 0.08） vs. （0. 86 ± 0. 05）, F = 0. 035, P 〉 0. 05 ]. Immunohistoehemistry study showed Slit2 protein was mainly expressed on the extracellular surface of vascular endothelial cells, while lung epithelial cell nuclei and endoehylema. Robo4 protein was only expressed on the extracellular surface of vascular endothelial cells. Compared with the control group, expression of Slit2 protein in lung tissue in ALl group decreased markedly [ （0.37±0.05） vs. （0.45±0.07）, F=6.82, P〈0.05, （0.32±0.06） vs. （0.47±0.09）, F=23.54, P〈0.05, （0.28±0.07） vs. （0.46±0.06）, F= 28.01, P 〈 0. 05 ]. As good as RT-PCR, there were no significant difference in expression of Robo4 protein in lung tissue between two groups [ （0. 53 ±0.04） vs. （0. 52 ±0.05）, F =0. 155, P 〉0. 05, （0. 53 ± 0.09） vs. （0.50±0.05）, F=0.498, P〉0.05, （0.55±0.06） vs. （0.56±0.07）, F=0.073, P 〉 0. 05 ]. Conclusions Lung tissues of control group rats express Slit2 and Robo4. The decreased Slit2 mRNA and protein expressions in the lung tissue of rat with ALl caused by CLP may be associated with the occurrence of ALl. Key words: Slit2; Robo4; Acute lung injury; Sepsis; Endothelial permeability; Cecum ligation and puncture; TNF-α; Axon guidance cues

Blood flow modulates endothelial cell (EC) functions through specific signaling events. Previous data show that flow stimulates SHP2 translocation to cell membranes and binding to phosphotyrosine proteins. Flow-induced ERK1/2 phosphorylation depends on SHP2 phosphatase activity and SHP2 binding to phospho-PECAM1 (platelet endothelial adhesion molecule 1), suggesting that SHP2 forms a signaling module with PECAM1. We hypothesized that flow induces assembly of the multi-protein complexes with SHP2 that are required for downstream signaling. ECs were exposed to flow for 10 min, and endogenous SHP2 was immunoprecipitated. SHP2-associated proteins were analyzed by SDS-PAGE and identified by mass spectrometry. Tie2 and several known SHP2-binding proteins were identified in flow-induced SHP2 complexes. Flow significantly increased tyrosine phosphorylation of both Tie2 and PECAM1 and their association with SHP2. To evaluate their functional roles, ECs were treated with Tie2 or PECAM1 small interfering RNA (siRNA). Tie2 and PECAM1 expression decreased >80% after siRNA treatment, and flow-stimulated phosphorylation of ERK1/2, Akt, and endothelial nitric oxide synthase was significantly inhibited by Tie2 and PECAM1 siRNA. Tie2 phosphorylation by flow was significantly inhibited by PECAM1 siRNA treatment. These results establish Tie2 transactivation via PECAM1 as an early event in flow-mediated mechanotransduction and suggest an important role for a PECAM1-SHP2-Tie2 pathway in flow-mediated signal transduction.

Background: Pulmonary endothelial injury is a critical process in the pathogenesis of acute lung injury (ALI) during sepsis. Heat shock protein A12B (HSPA12B) is mainly expressed in endothelial cells and protects against several harmful factors. However, the effects of HSPA12B in sepsis-induced ALI and its potential mechanisms of action remain unclear. Methods: For in vivo experiments, C57BL/6 mice were randomly divided into four groups (n=15): a sham operation group, a cecal ligation and puncture (CLP) group, a HSPA12B siRNA-CLP group and a negative control (NC) siRNA-CLP group. The mice were treated by nasal inhalation of 2-OMe-modified HSPA12B siRNA or NC siRNA. Sepsis was induced by CLP. Samples were harvested 24 and 48 hours post-CLP surgery. Pathological changes and scoring of lung tissue samples were monitored using hematoxylin and eosin staining. Levels of pro-inflammatory cytokines (e.g., interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6) and myeloperoxidase activity in bronchoalveolar lavage fluid were analyzed by ELISA. Pulmonary edema was assessed using a wet-to-dry weight ratio. Neutrophils and alveolar macrophages were counted using flow cytometry. Pulmonary endothelial cell apoptosis was detected by TUNEL staining. Expression levels of MAPK family signaling molecules and caspase-3 were measured by Western blot analysis. In addition, 7-day survival was recorded. For in vitro experiments, human umbilical vein endothelial cells were pre-transfected with HSPA12B siRNA or pIRES2-EGFP-HSPA12B-Flag plasmid and treated with lipopolysaccharide; subsequently, the expression levels of MAPK family signaling molecules and caspase-3 were measured by Western blotting. Results: Nasal inhalation of nano-polymer-encapsulated HSPA12B siRNA specifically downregulated mRNA and protein expression levels of HSPA12B in lung tissues. The administration of HSPA12B siRNA aggravated lung pathological injury, upregulated pro-inflammatory cytokine (e.g., IL-1β, TNF-α, and IL-6) expression, and increased myeloperoxidase activity, neutrophil infiltration, pulmonary edema, and pulmonary endothelial cell apoptosis. Additionally, HSPA12B knockdown worsened survival after CLP surgery. The potential protective mechanisms of HSPA12B may involve the inhibition of ERK phosphorylation and caspase-3 activation in vivo and in vitro. Conclusion: HSPA12B protected against sepsis-induced ALI. The potential mechanism may be partly due to the inhibition of ERK phosphorylation and caspase-3 activation. These findings provide a potential therapeutic target for treating sepsis.

a randomized clinical trial by the Acute Respiratory Distress Syndrome Network ([1][1]) demonstrated a 22% reduction in mortality in patients by reducing the tidal volume for mechanical ventilation from the conventional setting of 12 ml/kg to a lower setting of 6 ml/kg. This dramatic decrease in

In this report, sphingosine-1-phosphate (S1P), a serum-borne bioactive lipid, is shown to activate tight-junction-associated protein Zonula Occludens-1 (ZO-1), which in turn plays a critical role in regulating endothelial chemotaxis and barrier integrity. After S1P stimulation, ZO-1 was redistributed to the lamellipodia and cell-cell junctions via the S1P1/G(i)/Akt/Rac pathway. Similarly, both endothelial barrier integrity and cell motility were significantly enhanced in S1P-treated cells through the G(i)/Akt/Rac pathway. Importantly, S1P-enhanced barrier integrity and cell migration were abrogated in ZO-1 knockdown cells, indicating ZO-1 is functionally indispensable for these processes. To investigate the underlying mechanisms, we demonstrated that cortactin plays a critical role in S1P-induced ZO-1 redistribution to the lamellipodia. In addition, S1P significantly induced the formation of endothelial tight junctions. ZO-1 and alpha-catenin polypeptides were colocalized in S1P-induced junctional structures; whereas, cortactin was not observed in these regions. Together, these results suggest that S1P induces the formation of two distinct ZO-1 complexes to regulate two different endothelial functions: ZO-1/cortactin complexes to regulate chemotactic response and ZO-1/alpha-catenin complexes to regulate endothelial barrier integrity. The concerted operation of these two ZO-1 complexes may coordinate two important S1P-mediated functions, i.e. migration and barrier integrity, in vascular endothelial cells.

Acute ethanol intoxication suppresses pentraxin 3 expression in a mouse sepsis model involving cecal ligation and puncture.

Dynamics of Hepatic Gene Expression Profile in a Rat Cecal Ligation and Puncture Model

Inhibitory effects of three diketopiperazines from marine-derived bacteria on endothelial protein C receptor shedding in human endothelial cells and mice

Objective To evaluate the effect of lovastatin on shedding of heparan sulfate proteoglycan (HSPG) and syndecan-1(SDC-1) in the lung tissues of rats with sepsis-induced acute lung injury. Methods One-hundred and twenty male Wistar rats aged 8-12 weeks, weighing 325-425 g, were randomly divided into 3 groups (n =40 each) using a random number table: sham operation group (group S), cecal ligation and puncture (CLP) group and lovastatin group (group L). Lovastatin 4 mg/kg was injected once a day for 5 consecutive days in S and L groups, while the equal volume of 0.5% CMC (the solvent) was given in CLP group. Sepsis was produced by CLP on 5th day of administration in CLP and L groups. The left lung was lavaged at 24 h after operation. The broncho-alveolar lavage fluid (BALF) was collected for determination of protein concentrations, white blood cell (WBC) count and percentage of neutrophils. Blood samples were collected for determination of the concentrations of HSPG and SDC-1 in serum (by ELISA). Evans blue was injected at 24 h after operation in the remaining 20 rats of each group. The lungs were removed for examination of the pathological changes and for measurement of HSPG and SDC-1 mRNA and protein expression (using Western blot and PCR), and Evans blue content (reflecting pulmonary capillary permeability) in the lung tissue. Results Compared with group S, the protein concentrations, WBC count and percentage of neutrophils in BALF, Evans blue content in lung tissues and the concentrations of HSPG and SDC-1 in serum were significantly increased, and HSPG and SDC-1 mRNA and protein expression was down-regulated in CLP and L groups. Compared with group CLP, the protein concentrations, WBC count and percentage of neutrophils in BALF, Evans blue content in lung tissues and the concentrations of HSPG and SDC-1 in serum were significantly decreased, and HSPG and SDC-1 mRNA and protein expression was up-regulated in group L. The pathological changes of lungs were significantly attenuated in group L as compared with group CLP. Conclusion The mechanism by which lovastatin attenuates acute lung injury induced by sepsis may be related to reduced shedding of HSPG and SDC-1 in lung tissues and improved function of pulmonary vascular endothelium in rats. Key words: Lovastatin; Sepsis; Respiratory distress syndrome, adult; Heparan sulfateproteoglycans; Syndecan-1

Angiogenesis is an essential step for many physiological and pathological processes. Tumor necrosis factor (TNF) superfamily cytokines are increasingly recognized as key modulators of angiogenesis. In this study, we tested whether TNF-related activation-induced cytokine (TRANCE), a new member of the TNF superfamily, possesses angiogenic activity in vitro and in vivo. TRANCE stimulated DNA synthesis, chemotactic motility, and capillary-like tube formation in primary cultured human umbilical vein endothelial cells (HUVECs). Both Matrigel plug assay in mice and chick chorioallantoic membrane assay revealed that TRANCE potently induced neovascularization in vivo. TRANCE had no effect on vascular endothelial growth factor (VEGF) expression in HUVECs and TRANCE-induced angiogenic activity was not suppressed by VEGF-neutralizing antibody, implying that TRANCE-induced angiogenesis may be the result of its direct action on endothelial cells. TRANCE evoked a time- and dose-dependent activation of the mitogen-activated protein kinases ERK1/2 and focal adhesion kinase p125(FAK) in HUVECs, which are closely linked to angiogenesis. These signaling events were blocked by the Src inhibitor PP1 or the phospholipase C (PLC) inhibitor. Furthermore, these inhibitors and the Ca(2+) chelator BAPTA-AM suppressed TRANCE-induced HUVEC migration. These results indicate that the angiogenic activity of TRANCE is mediated through the Src-PLC-Ca(2+) signaling cascade upon receptor engagement in endothelial cells, suggesting the role of TRANCE in neovessel formation under physiological and pathological conditions.

Background: Dual specificity phosphatase 5 (DUSP5) is a member of the protein phosphatase subfamily that inactivate their target kinases by dephosphorylating both the phosphoserine/threonine and phosphotyrosine residues. DUSP5 regulates the mitogen activated protein (MAP) kinase ERK1/2, and although it has been shown to play a key role in embryonic vascular development, its role in post ischemic angiogenesis is not known. This study sort to investigate the role of DUSP5 is endothelial cell function and angiogenesis in ischemia. Methods: Hindlimb ischemia (HLI) was induced in mice and at day 3 post HLI, expression of DUSP5 was assessed in the ischemic hind limbs. We assessed DUSP5 expression in 3 major cell types found the ischemic hindlimb, endothelial cells or ECs (human umbilical vein endothelial cells, HUVECs), skeletal muscle myoblast (C2C12) and vascular smooth muscles cells (VSMC). Cells were exposed to simulated ischemia (2% oxygen with serum starvation) for 24 hours analyzed for DUSP5 mRNA and protein expression. The effect of loss of DUSP5 on EC function in ischemia was assessed by CRISPR/Cas9 mediated knock down of DUSP5 followed by analysis of EC proliferation, apoptosis and tube formation in simulated ischemia. Results: DUSP5 protein levels were significantly upregulated in mouse post ischemic hind limbs (ischemic (I) vs non-ischemic (NI); 1.63 ± 0.39 vs. 0.22 ± 0.02, p&lt;0.05, n=3). Post ischemic exposure, ECs and C2C12 showed significant upregulation of DUSP5 protein. (DUSP5/Tubulin, I vs NI, HUVEC: 0.43 ± 0.09 vs. 0.09 ± 0.03, p&lt;0.02; C2C12: 1.77 ± 0.10 vs. 0.42 ± 0.01, p&lt; 0.01, n=5). VSMCs showed no significant change in DUSP5 expression. Knock down of DUSP5 in HUVECs resulted in significant decrease in cell proliferation in ischemia (O.D 450 control vs knock-down : 0.46 ± 0.01 vs. 0.34 ± 0.01, p&lt;0.01, n=7) but no significant change in apoptosis (O.D 450 control vs knock-down: 0.13 ± 0.01 vs. 0.13 ± 0.00, p&gt;0.05, n=7). It also resulted in significant impairment in, in vitro angiogenesis (tube/sq cm: 28.6 ± 1.2 vs. 16.43 ± 1.6, p&lt;0.01, n=7). Conclusion: DUSP5 is highly upregulated in ischemic endothelial cells and plays an important role in EC proliferation and post ischemic angiogenesis.

Sepsis-induced blood vessel dysfunction is mainly caused by microvascular endothelial cell injury. However, the mechanism underlying sepsis-induced endothelial cell injury remains unclear. The present study hypothesized that sepsis-induced inflammatory injury of endothelial cells may be the first step of endothelial barrier dysfunction. Therefore, the present study aimed to uncover the mechanism underlying the inflammatory effects of sepsis. A rat model of cecal ligation and puncture-induced sepsis was established, and septic serum was collected. Subsequently, human umbilical vein endothelial cells (HUVECs) were treated with the isolated septic or normal serum. HUVEC viability was assessed using a Cell Count Kit-8 assay. Furthermore, transmission electron microscopy and reverse transcription-quantitative PCR (RT-qPCR) analysis were carried out to observe the cell morphology and determine the mRNA expression levels in septic serum-induced HUVECs. The protein expression levels were evaluated by western blot analysis, and the secretion of the inflammatory factors interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α was determined by ELISA. Additionally, reactive oxygen species (ROS) generation and nuclear factor (NF)-κB nuclear translocation were observed under a fluorescence microscope. The results of the present study demonstrated that HUVEC viability was significantly decreased following 12- or 24-h treatment with septic serum. In addition, chromatin condensation, mitochondrial vacuolization and endoplasmic reticulum degranulation were observed following treatment with septic serum. Furthermore, the secretion levels of IL-1β, IL-6 and TNF-α were increased in septic serum-stimulated HUVECs. Septic serum treatment also enhanced superoxide anion generation, promoted extra-cellular signal regulated kinase 1/2 (ERK1/2), N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) phosphorylation, and increased NF-κB levels in the nuclei of HUVECs. Finally, pre-treatment of HUVECs with the antioxidant N-acetylcysteine, the ERK1/2 inhibitor PD98059, the p38 inhibitor SB203580, the JNK inhibitor SP610025 or the NF-κB inhibitor pyrrolidine dithiocarbamate restored the septic serum-induced IL-1β, IL-6 and TNF-α expression. In conclusion, the results of the current study suggested that the septic serum-induced endothelial cell injury may be mediated by increasing ROS generation, activation of mitogen-activated protein kinases and NF-κB translocation.