Unfractionated Heparin Alleviates Sepsis-Induced Acute Lung Injury by Protecting Tight Junctions.

BACKGROUND: Unfractionated heparin (UFH) has been shown to ameliorate lung edema and lung vascular leakage in lipopolysaccharide-induced lung injury. Impaired tight junction (TJ) function is a sign of sepsis-induced acute respiratory distress syndrome (ARDS) and acute lung injury (ALI), which is closely related to the downregulated expression of TJ-specific proteins or the upregulated expression of inflammatory cytokines. Because UFH has been intensively studied in modulating inflammation, we hypothesize that UFH may play a positive role in treating sepsis-induced ARDS/ALI by protecting TJs. MATERIAL AND METHODS: Rat sepsis-induced lung injury was induced by cecal ligation and puncture and treated with UFH. Hematoxylin and eosin staining, lung wet/dry weight (W/D) ratio, and pulmonary microvascular leakage were evaluated to assess lung injury. Cytokines in bronchoalveolar lavage fluid were detected to determine lung inflammation. A transendothelial electrical resistance assay, a Transwell permeability assay, and transmission electron microscopy were used to study endothelial TJs in human lung microvascular endothelial cells. TJ protein expression was measured by western blotting or immunohistochemistry. RESULTS: UFH treatment alleviated lung injury in vivo by reducing IL-6 in bronchoalveolar lavage fluid and protecting TJs in LMVECs. UFH also protected TJs against lipopolysaccharide-stimulated damage and functioned upstream by inhibiting the ERK1/2 MAPK pathway to attenuate endothelial hyperpermeability and downregulating the expression of TJ proteins such as claudin-5, occludin, and ZO-1. CONCLUSIONS: These findings suggested that UFH has therapeutic potential for sepsis-induced ARDS or ALI through protecting TJs in LMVECs.

Unfractionated heparin ameliorates pulmonary microvascular endothelial barrier dysfunction via microtubule stabilization in acute lung injury.

BACKGROUND: Endothelial barrier dysfunction is central to the pathogenesis of sepsis-associated acute lung injury (ALI). Microtubule (MT) dynamics in vascular endothelium are crucial for the regulation of endothelial barrier function. Unfractionated heparin (UFH) possesses various biological activities, such as anti-inflammatory activity and endothelial barrier protection during sepsis. METHODS: Here, we investigated the effects and underlying mechanisms of UFH on lipopolysaccharide (LPS)-induced endothelial barrier dysfunction. C57BL/6 J mice were randomized into vehicle, UFH, LPS and LPS + UFH groups. Intraperitoneal injection of 30 mg/kg LPS was used to induce sepsis. Mice in the LPS + UFH group received intravenous UFH 0.5 h prior to LPS injection. Human pulmonary microvascular endothelial cells (HPMECs) were cultured for analyzing the effects of UFH on LPS-induced and nocodazole-induced hyperpermeability, F-actin remodeling, and LPS-induced p38 MAPK activation. RESULTS: UFH pretreatment significantly attenuated LPS-induced pulmonary histopathological changes, and increased the lung W/D ratio and Evans blue accumulation in vivo. Both in vivo and in vitro studies showed that UFH pretreatment blocked the LPS-induced increase in guanine nucleotide exchange factor (GEF-H1) expression and myosin phosphatase target subunit 1 (MYPT1) phosphorylation, and microtubule (MT) disassembly in LPS-induced ALI mouse model and human pulmonary microvascular endothelial cells (HPMECs). These results suggested that UFH ameliorated LPS-induced endothelial barrier dysfunction by inhibiting MT disassembly and GEF-H1 expression. In addition, UFH attenuated LPS-induced hyperpermeability of HPMECs and F-actin remodeling. In vitro, UFH pretreatment inhibited LPS-induced increase in monomeric tubulin expression and decrease in tubulin polymerization and acetylation. Meanwhile, UFH ameliorates nocodazole-induced MTs disassembly and endothelial barrier dysfunction.Additionally, UFH decreased p38 phosphorylation and activation, which was similar to the effect of the p38 MAPK inhibitor, SB203580. CONCLUSIONS: UFH exert its protective effects on pulmonary microvascular endothelial barrier dysfunction via microtubule stabilization and is associated with the p38 MAPK pathway.

Unfractionated heparin attenuates endothelial barrier dysfunction via the phosphatidylinositol-3 kinase/serine/threonine kinase/nuclear factor kappa-B pathway.

BACKGROUND: Vascular endothelial dysfunction is considered a key pathophysiologic process for the development of acute lung injury. In this study, we aimed at investigating the effects of unfractionated heparin (UFH) on the lipopolysaccharide (LPS)-induced changes of vascular endothelial-cadherin (VE-cadherin) and the potential underlying mechanisms. METHODS: Male C57BL/6 J mice were randomized into three groups: vehicle, LPS, and LPS + UFH groups. Intraperitoneal injection of 30 mg/kg LPS was used to induce sepsis. Mice in the LPS + UFH group received subcutaneous injection of 8 U UFH 0.5 h before LPS injection. The lung tissue of the mice was collected for assessing lung injury by measuring the lung wet/dry (W/D) weight ratio and observing histological changes. Human pulmonary microvascular endothelial cells (HPMECs) were cultured and used to analyze the effects of UFH on LPS- or tumor necrosis factor-alpha (TNF-α)-induced vascular hyperpermeability, membrane expression of VE-cadherin, p120-catenin, and phosphorylated myosin light chain (p-MLC), and F-actin remodeling, and on the LPS-induced activation of the phosphatidylinositol-3 kinase (PI3K)/serine/threonine kinase (Akt)/nuclear factor kappa-B (NF-κB) signaling pathway. RESULTS: In vivo, UFH pretreatment significantly attenuated LPS-induced pulmonary histopathological changes (neutrophil infiltration and erythrocyte effusion, alveolus pulmonis collapse, and thicker septum), decreased the lung W/D, and increased protein concentration (LPS vs. LPS + UFH: 0.57 ±â€Š0.04 vs. 0.32 ±â€Š0.04 mg/mL, P = 0.0092), total cell count (LPS vs. LPS + UFH: 9.57 ±â€Š1.23 vs. 3.65 ±â€Š0.78 × 10/mL, P = 0.0155), polymorphonuclear neutrophil percentage (LPS vs. LPS + UFH: 88.05% ±â€Š2.88% vs. 22.20% ±â€Š3.92%, P = 0.0002), and TNF-α (460.33 ±â€Š23.48 vs. 189.33 ±â€Š14.19 pg/mL, P = 0.0006) in the bronchoalveolar lavage fluid. In vitro, UFH pre-treatment prevented the LPS-induced decrease in the membrane expression of VE-cadherin (LPS vs. LPS + UFH: 0.368 ±â€Š0.044 vs. 0.716 ±â€Š0.064, P = 0.0114) and p120-catenin (LPS vs. LPS + UFH: 0.208 ±â€Š0.018 vs. 0.924 ±â€Š0.092, P = 0.0016), and the LPS-induced increase in the expression of p-MLC (LPS vs. LPS + UFH: 0.972 ±â€Š0.092 vs. 0.293 ±â€Š0.025, P = 0.0021). Furthermore, UFH attenuated LPS- and TNF-α-induced hyperpermeability of HPMECs (LPS vs. LPS + UFH: 8.90 ±â€Š0.66 vs. 15.84 ±â€Š1.09 Ω·cm, P = 0.0056; TNF-α vs. TNF-α + UFH: 11.28 ±â€Š0.64 vs. 18.15 ±â€Š0.98 Ω·cm, P = 0.0042) and F-actin remodeling (LPS vs. LPS + UFH: 56.25 ±â€Š1.51 vs. 39.70 ±â€Š1.98, P = 0.0027; TNF-α vs. TNF-α + UFH: 55.42 ±â€Š1.42 vs. 36.51 ±â€Š1.20, P = 0.0005) in vitro. Additionally, UFH decreased the phosphorylation of Akt (LPS vs. LPS + UFH: 0.977 ±â€Š0.081 vs. 0.466 ±â€Š0.035, P = 0.0045) and I kappa B Kinase (IKK) (LPS vs. LPS + UFH: 1.023 ±â€Š0.070 vs. 0.578 ±â€Š0.044, P = 0.0060), and the nuclear translocation of NF-κB (LPS vs. LPS + UFH: 1.003 ±â€Š0.077 vs. 0.503 ±â€Š0.065, P = 0.0078) in HPMECs, which was similar to the effect of the PI3K inhibitor, wortmannin. CONCLUSIONS: The protective effect of UFH against LPS-induced pulmonary endothelial barrier dysfunction involves VE-cadherin stabilization and PI3K/Akt/NF-κB signaling.

[Effect of heparin pretreatment on the level of neutrophil extracellular traps of serum and lung tissue in septic mice].

OBJECTIVE: To investigate the influence of heparin pretreatment on serum and lung tissue level of neutrophil extracellular traps (NETs) in septic mice model and its molecular mechanism. METHODS: Ninety male C57BL/6J mice were randomly divided into control group (n = 30), lipopolysaccharides (LPS) group (n = 30, 30 mg/kg LPS in 100 ?L normal saline was intraperitoneally injected) and LPS+heparin group (n = 30, 8 U of heparin in 20 ?L normal saline was subcutaneously injected 30 minutes before the injection of LPS). Six hours later of LPS injection, blood was collected and lung tissue was harvested. Enzyme linked immunosorbent assay (ELISA) was used to assess the concentration of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and histones 2AX (H2AX), neutrophil elastase (NE), which reflected NETs concentration. PicoGreen fluorescent dyes was used to detect serum circulating free DNA (cf-DNA/NETs) concentration. The protein expression levels of H2AX and NE in lung tissue were examined by Western Blot. RESULTS: The serum concentrations of TNF-α, IL-6, H2AX, NE, cf-DNA/NETs, and the protein expression levels of H2AX and NE in lung tissue of septic mice were significantly higher than those of control group [TNF-α (ng/L): 133.0±14.1 vs. 2.7±1.0, IL-6 (ng/L): 3 911.2±189.2 vs. 298.9±52.5, H2AX (ng/L): 545.5±40.0 vs. 21.9±8.3, NE (µg/L): 6.48±0.12 vs. 0.47±0.15, cf-DNA/NETs (µg/L): 846.3±137.5 vs. 152.7±36.4, H2AX protein (gray value): 1.14±0.09 vs. 0.68±0.04, NE protein (gray value): 0.56±0.03 vs. 0.32±0.04, all P < 0.05]. After heparin pretreatment, levels of serum TNF-α, H2AX, NE, cf-DNA/NETs, and protein expression levels of H2AX and NE in lung tissue were significantly reduced [TNF-α (ng/L): 83.2±7.6 vs. 133.0±14.1, H2AX (ng/L): 435.0±39.0 vs. 545.5±40.0, NE (µg/L): 4.26±0.17 vs. 6.48±0.12, cf-DNA/NETs (µg/L): 606.5±73.9 vs. 846.3±137.5, H2AX protein (gray value): 0.91±0.03 vs. 1.14±0.09, NE protein (gray value): 0.42±0.03 vs. 0.56±0.03, all P < 0.05], but no significant change was found in IL-6 (ng/L: 3 919.9±166.6 vs. 3 911.2±189.2, P > 0.05). CONCLUSIONS: Heparin pretreatment could significantly decrease the level of NETs in serum and lung tissue, and can be the potential mechanism of its organ protection in sepsis.