heparitin-sulfate and Respiratory-Distress-Syndrome

To investigate the role and possible pathogenesis of high mobility group protein B1 (HMGB1) in lipopolysaccharide (LPS)-induced acute lung injury/acute respiratory distress syndrome (ALI/ARDS).. (1) In vivo, 24 SPFC57BL/6 male mice were randomly divided into normal control group, ALI/ARDS model group, ethyl pyruvate (EP) treatment group and EP control group, with 6 mice in each group. The ALI/ARDS model was established by intraperitoneal injection of 20 mg/kg LPS. Mice in normal control group and EP control group were intraperitoneally injected with the same amount of sterile normal saline. Then, mice in the EP treatment group and EP control group were intraperitoneally injected with 40 mg/kg HMGB1 inhibitor EP. After 6 hours, the mice were sacrificed and lung tissues were collected. The expressions of heparan sulfate (HS), syndecans-1 (SDC-1), heparanase (HPA) and matrix metalloproteinases-9 (MMP-9) in lung tissues were detected by immunofluorescence technique. Orbital blood of mice was collected and serum was extracted to detect the content of HMGB1 by enzyme linked immunosorbent assay (ELISA). (2) In vitro, human umbilical vein endothelial cells (HUVECs) were randomly divided into 6 groups: normal control group, HUVECs damage group (treated with 1 mg/L LPS for 6 hours), HMGB1 group (treated with 1 μmol/L recombinant HMGB1 for 6 hours), HMGB1+EP group (treated with recombinant HMGB1 for 1 hour and then added 1 μmol/L EP for 6 hours), LPS+EP group (treated with LPS for 1 hour and then added 1 μmol/L EP for 6 hours), EP group (treated with 1 μmol/L EP for 6 hours). The expressions of HS, SDC-1, HPA and MMP-9 in endothelial cells were detected by immunofluorescence technique.. (1) In vivo, light microscopy showed that the alveolar space was thickened after LPS stimulation, and there were a large number of inflammatory cells infiltrating in the alveolar space. Compared with ALI/ARDS model group, the expressions of HS and SDC-1 in lung tissue of EP treatment group were significantly increased [HS (fluorescence intensity): 0.80±0.20 vs. 0.53±0.02, SDC-1 (fluorescence intensity): 0.72±0.02 vs. 0.51±0.01, both P < 0.05], and the expressions of HPA and MMP-9 were significantly decreased [HPA (fluorescence intensity): 2.36±0.05 vs. 3.00±0.04, MMP-9 (fluorescence intensity): 2.55±0.13 vs. 3.26±0.05, both P < 0.05]; there were no significant changes of the above indexes in EP control group. Compared with ALI/ARDS model group, the content of serum HMGB1 in EP treatment group decreased significantly (μg/L: 131.88±16.67 vs. 341.13±22.47, P < 0.05); there was no significant change in the EP control group. (2) In vitro, compared with HMGB1 group, the expressions of HS and SDC-1 in HMGB1+EP group were significantly higher [HS (fluorescence intensity): 0.83±0.07 vs. 0.56±0.03, SDC-1 (fluorescence intensity): 0.80±0.01 vs. 0.61±0.01, both P < 0.05], and the expressions of HPA and MMP-9 were significantly lower [HPA (fluorescence intensity): 1.30±0.02 vs. 2.29±0.05, MMP-9 (fluorescence intensity): 1.55±0.04 vs. 2.50±0.06, both P < 0.05]; the expression of HS, SDC-1, HPA and MMP-9 had no significant changes in EP group.. HMGB1 participates in LPS-induced injury of endothelial cell glycocalyx, leading to increased lung permeability, and inhibition of HMGB1 can alleviate lung injury.

Topics: Acute Lung Injury; Animals; Heparitin Sulfate; HMGB1 Protein; Human Umbilical Vein Endothelial Cells; Humans; Lipopolysaccharides; Lung; Male; Matrix Metalloproteinase 9; Mice; Pyruvates; Respiratory Distress Syndrome; Saline Solution; Syndecans

The main pathophysiological mechanism of acute respiratory distress syndrome (ARDS) invovles the increase in alveolar barrier permeability that is primarily caused by epithelial glycocalyx and tight junction (TJ) protein destruction. This study was performed to explore the effects of the alveolar epithelial glycocalyx on the epithelial barrier, specifically on TJ proteins, in ARDS. We used C57BL/6 mice and human lung epithelial cell models of lipopolysaccharide (LPS)-induced ARDS. Changes in alveolar permeability were evaluated via pulmonary histopathology analysis and by measuring the wet/dry weight ratio of the lungs. Degradation of heparan sulfate (HS), an important component of the epithelial glycocalyx, and alterations in levels of the epithelial TJ proteins (occludin, zonula occludens 1, and claudin 4) were assessed via ELISA, immunofluorescence analysis, and western blotting analysis. Real-time quantitative polymerase chain reaction was used to detect the mRNA of the TJ protein. Changes in glycocalyx and TJ ultrastructures in alveolar epithelial cells were evaluated through electron microscopy. In vivo and in vitro, LPS increased the alveolar permeability and led to HS degradation and TJ damage. After LPS stimulation, the expression of the HS-degrading enzyme heparanase (HPA) in the alveolar epithelial cells was increased. The HPA inhibitor N-desulfated/re-N-acetylated heparin alleviated LPS-induced HS degradation and reduced TJ damage. In vitro, recombinant HPA reduced the expression of the TJ protein zonula occludens-1 (ZO-1) and inhibited its mRNA expression in the alveolar epithelial cells. Taken together, our results demonstrate that shedding of the alveolar epithelial glycocalyx aggravates the epithelial barrier and damages epithelial TJ proteins in ARDS, with the underlying mechanism involving the effect of HPA on ZO-1.

Topics: A549 Cells; Alveolar Epithelial Cells; Animals; Blood-Air Barrier; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Glycocalyx; Heparitin Sulfate; Humans; Male; Mice, Inbred C57BL; Permeability; Respiratory Distress Syndrome; Tight Junctions; Zonula Occludens-1 Protein

The lung epithelial glycocalyx is a carbohydrate-enriched layer lining the pulmonary epithelial surface. Although epithelial glycocalyx visualization has been reported, its composition and function remain unknown. Using immunofluorescence and mass spectrometry, we identified heparan sulfate (HS) and chondroitin sulfate within the lung epithelial glycocalyx. In vivo selective enzymatic degradation of epithelial HS, but not chondroitin sulfate, increased lung permeability. Using mass spectrometry and gel electrophoresis approaches to determine the fate of epithelial HS during lung injury, we detected shedding of 20 saccharide-long or greater HS into BAL fluid in intratracheal LPS-treated mice. Furthermore, airspace HS in clinical samples from patients with acute respiratory distress syndrome correlated with indices of alveolar permeability, reflecting the clinical relevance of these findings. The length of HS shed during intratracheal LPS-induced injury (≥20 saccharides) suggests cleavage of the proteoglycan anchoring HS to the epithelial surface, rather than cleavage of HS itself. We used pharmacologic and transgenic animal approaches to determine that matrix metalloproteinases partially mediate HS shedding during intratracheal LPS-induced lung injury. Although there was a trend toward decreased alveolar permeability after treatment with the matrix metalloproteinase inhibitor, doxycycline, this did not reach statistical significance. These studies suggest that epithelial HS contributes to the lung epithelial barrier and its degradation is sufficient to increase lung permeability. The partial reduction of HS shedding achieved with doxycycline is not sufficient to rescue epithelial barrier function during intratracheal LPS-induced lung injury; however, whether complete attenuation of HS shedding is sufficient to rescue epithelial barrier function remains unknown.

Topics: Animals; Capillary Permeability; Endothelium, Vascular; Glycocalyx; Heparitin Sulfate; Lipopolysaccharides; Lung Injury; Mice; Respiratory Distress Syndrome; Syndecans

The endothelial glycocalyx is a glycosaminoglycan-enriched endovascular layer that, with the development of novel fixation and in vivo microscopy techniques, has been increasingly recognized as a major contributor to vascular homeostasis. Sepsis-associated degradation of the endothelial glycocalyx mediates the onset of the alveolar microvascular dysfunction characteristic of sepsis-induced lung injury (such as the Acute Respiratory Distress Syndrome, ARDS). Emerging evidence indicates that processes of glycocalyx reconstitution are necessary for endothelial repair and, as such, are promising therapeutic targets to accelerate lung injury recovery. This review discusses what has been learned about the homeostatic and pathophysiologic role of the pulmonary endothelial glycocalyx during lung health and injury, with the goal to identify promising new areas for future mechanistic investigation.

Topics: Endothelium, Vascular; Glucuronidase; Glycocalyx; Heparitin Sulfate; Humans; Lung; Lung Injury; Respiratory Distress Syndrome; Sepsis

Topics: Animals; Glycocalyx; Heparitin Sulfate; Humans; Lung; Respiratory Distress Syndrome; Sepsis; Syndecan-1

Acute respiratory distress syndrome (ARDS) is a syndrome of acute respiratory failure characterized by major pathologic mechanisms of increased microvascular permeability and inflammation. The glycocalyx lines on the endothelial surface, which determines the vascular permeability, and heparanase play pivotal roles in the degradation of heparan sulfate (HS). HS is the major component of the glycocalyx. The aim of this study is to examine the effects of Ulinastatin (UTI) on vascular permeability and pulmonary endothelial glycocalyx dysfunction induced by lipopolysaccharide (LPS). In our study, C57BL/6 mice and human umbilical vein endothelial cells were stimulated with LPS to induce injury models. After 6 h of LPS stimulation, pulmonary pathological changes, pulmonary edema, and vascular permeability were notably attenuated by UTI. UTI inhibited LPS-induced endothelial glycocalyx destruction and significantly decreased the production of HS as determined by ELISA and immunofluorescence. UTI also reduced the active form of heparanase (50 kDa) expression and heparanase activity. Moreover, lysosome pH was investigated because heparanase (65 kDa) can be reduced easily in its active form at 50 kDa in a low pH environment within lysosome. Results showed that UTI could inhibit LPS-induced pH elevation in lysosome. In conclusion, UTI protects pulmonary endothelial glycocalyx integrity and inhibits heparanase activity during LPS-induced ARDS.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Capillary Permeability; Cell Survival; Endothelium; Gene Expression; Glucuronidase; Glycocalyx; Glycoproteins; Heparitin Sulfate; Human Umbilical Vein Endothelial Cells; Humans; Lipopolysaccharides; Lung; Lysosomes; Male; Mice; Mice, Inbred C57BL; Respiratory Distress Syndrome

Systemic inflammatory mediators, including high mobility group box 1 (HMGB1), play an important role in the development of sepsis. Anticoagulants, such as danaparoid sodium (DA), may be able to inhibit sepsis-induced inflammation, but the mechanism of action is not well understood. We hypothesised that DA would act as an inhibitor of systemic inflammation and prevent endotoxin-induced acute lung injury in a rat model.. We used male Wistar rats. Animals in the intervention arm received a bolus of 50 U/kg of DA or saline injected into the tail vein after lipopolysaccharide (LPS) administration. We measured cytokine (tumour necrosis factor (TNF)alpha, interleukin (IL)-6 and IL-10) and HMGB1 levels in serum and lung tissue at regular intervals for 12 h following LPS injection. The mouse macrophage cell line RAW 264.7 was assessed following stimulation with LPS alone or concurrently with DA with identification of HMGB1 and other cytokines in the supernatant.. Survival was significantly higher and lung histopathology significantly improved among the DA (50 U/kg) animals compared to the control rats. The serum and lung HMGB1 levels were lower over time among DA-treated animals. In the in vitro study, administration of DA was associated with decreased production of HMGB1. In the cell signalling studies, DA administration inhibited the phosphorylation of IkappaB.. DA decreases cytokine and HMGB1 levels during LPS-induced inflammation. As a result, DA ameliorated lung pathology and reduces mortality in endotoxin-induced systemic inflammation in a rat model. This effect may be mediated through the inhibition of cytokines and HMGB1.

Topics: Animals; Chondroitin Sulfates; Dermatan Sulfate; Endotoxins; Heparitin Sulfate; HMGB1 Protein; I-kappa B Proteins; Interleukin-10; Interleukin-6; Male; Mice; NF-kappa B; Rats; Rats, Wistar; Respiratory Distress Syndrome; Survival Rate; Systemic Inflammatory Response Syndrome; Tumor Necrosis Factor-alpha

Acute lung injury syndromes have many characteristics including protein-rich alveolar edema, hyaline membranes, and abnormal surface tension at the alveolar air-liquid interface. Increased surface tension can occur because of a relative surfactant deficiency and/or dysfunction. It has been previously demonstrated that surfactant dysfunction occurs when plasma protein inhibitors leak into the alveolar space during the induction of the lung injury and edema formation. The present study investigated whether inhibitors that would be generated during the stage of repair from lung injury could impair surfactant function. We determined whether fibrinogen degradation products (FDP) which would be released during lysis of the fibrin(ogen)-containing alveolar exudate and hyaline membranes, and components of the lungs' ground substance could inhibit the in vitro function of a lipid extract surfactant preparation. FDP were prepared by incubating human fibrinogen with plasmin or neutrophil elastase for 4 min to 60 h and were characterized by SDS-PAGE. Early (fragment X and Y) and late (fragment D and E) plasmin-derived FDP (MW greater than 40,000) inhibited surfactant function as assessed by a bubble surfactometer. The early elastase-derived FDP also inhibited surfactant, but the later and much smaller fragments (MW less than 15,000) did not affect surfactant function. Laminin also inhibited surfactant in a dose-dependent manner. Neither hyaluronic acid nor heparan sulfate affected surfactant performance in vitro. We conclude that plasmin-induced lysis of intraalveolar fibrinogen and hyaline membranes will result in prolonged generation (i.e. days) of surfactant inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cattle; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Fibrin Fibrinogen Degradation Products; Fibrinolysin; Heparitin Sulfate; Humans; Hyaline Membrane Disease; Hyaluronic Acid; In Vitro Techniques; Infant, Newborn; Laminin; Lung; Pancreatic Elastase; Pulmonary Surfactants; Respiratory Distress Syndrome; Surface Tension