Unfractionated Heparin Attenuates Endothelial Barrier Dysfunction via the Phosphatidylinositol-3 Kinase/Serine/Threonine Kinase/Nuclear Factor Kappa-B Pathway
Acute lung injury (ALI) is a critical condition characterized by excessive inflammation in the lungs, leading to high morbidity and mortality. Despite extensive research, effective therapies for ALI remain limited. A key feature of ALI is the disruption of the pulmonary endothelial barrier, which results in vascular hyperpermeability and pulmonary edema. Vascular endothelial cadherin (VE-cadherin)-based adherens junctions play a crucial role in regulating endothelial permeability. Lipopolysaccharide (LPS), a component of gram-negative bacteria, induces endothelial hyperpermeability by disrupting VE-cadherin-mediated cell-cell junctions. Unfractionated heparin (UFH), a commonly used anticoagulant, has been shown to improve endothelial barrier function, but the underlying mechanisms are not fully understood. This study investigates the protective effects of UFH on LPS-induced endothelial barrier dysfunction and explores the involvement of the phosphatidylinositol-3 kinase (PI3K)/serine/threonine kinase (Akt)/nuclear factor kappa-B (NF-κB) signaling pathway.
Methods
Animal Studies
Male C57BL/6 mice were divided into three groups: vehicle, LPS, and LPS + UFH. LPS (30 mg/kg) was administered intraperitoneally to induce sepsis. Mice in the LPS + UFH group received a subcutaneous injection of 8 U UFH 30 minutes before LPS administration. Lung tissue was collected six hours post-LPS injection to assess lung injury using the lung wet/dry (W/D) weight ratio and histological analysis. Bronchoalveolar lavage fluid (BALF) was analyzed for protein concentration, total cell count, polymorphonuclear neutrophil (PMN) percentage, and tumor necrosis factor-alpha (TNF-α) levels.
Cell Culture and Treatment
Human pulmonary microvascular endothelial cells (HPMECs) were cultured and treated with LPS (10 µg/mL) or TNF-α (10 ng/mL) to induce endothelial barrier dysfunction. UFH (10 U/mL) was added 30 minutes before LPS or TNF-α stimulation. Transendothelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran (FITC-dextran) permeability assays were performed to measure endothelial barrier function. Immunofluorescence staining and Western blot analysis were used to assess the expression of VE-cadherin, p120-catenin, phosphorylated myosin light chain (p-MLC), and F-actin remodeling. The activation of the PI3K/Akt/NF-κB pathway was evaluated by measuring the phosphorylation of Akt, IκB kinase (IKK), and nuclear translocation of NF-κB.
Results
UFH Attenuates LPS-Induced Endothelial Barrier Dysfunction In Vivo
Histopathological analysis revealed that LPS induced significant lung injury, including neutrophil infiltration, erythrocyte effusion, alveolar collapse, and septal thickening. UFH pretreatment markedly reduced these pathological changes. The lung W/D ratio, a measure of lung edema, was significantly lower in the LPS + UFH group compared to the LPS group (5.05 ± 0.18 vs. 6.93 ± 0.20, P = 0.0022). UFH also decreased protein concentration (0.32 ± 0.04 vs. 0.57 ± 0.04 mg/mL, P = 0.0092), total cell count (3.65 ± 0.78 vs. 9.57 ± 1.23 × 10^5/mL, P = 0.0155), PMN percentage (22.20% ± 3.92% vs. 88.05% ± 2.88%, P = 0.0002), and TNF-α levels (189.33 ± 14.19 vs. 460.33 ± 23.48 pg/mL, P = 0.0006) in BALF. Western blot analysis showed that UFH prevented the LPS-induced decrease in membrane expression of VE-cadherin and p120-catenin in lung tissue.
UFH Reduces LPS- or TNF-α-Induced Hyperpermeability In Vitro
LPS and TNF-α significantly reduced TEER and increased FITC-dextran permeability in HPMECs, indicating endothelial barrier dysfunction. UFH pretreatment increased TEER (LPS + UFH: 15.84 ± 1.09 vs. LPS: 8.90 ± 0.66 V·cm², P = 0.0056; TNF-α + UFH: 18.15 ± 0.98 vs. TNF-α: 11.28 ± 0.64 V·cm², P = 0.0042) and decreased FITC-dextran permeability (LPS + UFH: 39.70 ± 1.98 vs. LPS: 56.25 ± 1.51, P = 0.0027; TNF-α + UFH: 36.51 ± 1.20 vs. TNF-α: 55.42 ± 1.42, P = 0.0005). Immunofluorescence staining and Western blot analysis revealed that UFH prevented the LPS- or TNF-α-induced decrease in membrane expression of VE-cadherin and p120-catenin, reduced p-MLC expression, and inhibited F-actin remodeling.
UFH Inhibits LPS-Induced Activation of the PI3K/Akt/NF-κB Pathway
LPS stimulation increased the phosphorylation of Akt and IKK and promoted nuclear translocation of NF-κB in HPMECs. UFH pretreatment significantly reduced the expression of p-Akt (LPS + UFH: 0.466 ± 0.035 vs. LPS: 0.977 ± 0.081, P = 0.0045), p-IKK (LPS + UFH: 0.578 ± 0.044 vs. LPS: 1.023 ± 0.070, P = 0.0060), and NF-κB nuclear translocation (LPS + UFH: 0.503 ± 0.065 vs. LPS: 1.003 ± 0.077, P = 0.0078). Similar effects were observed with the PI3K inhibitor wortmannin, suggesting that UFH’s protective effects are mediated through the PI3K/Akt/NF-κB pathway.
Discussion
This study demonstrates that UFH attenuates LPS-induced endothelial barrier dysfunction by stabilizing VE-cadherin and inhibiting the PI3K/Akt/NF-κB signaling pathway. VE-cadherin-based adherens junctions are critical for maintaining endothelial barrier integrity. LPS and TNF-α disrupt these junctions by inducing VE-cadherin internalization, leading to endothelial hyperpermeability. UFH prevents this disruption by maintaining the membrane expression of VE-cadherin and p120-catenin, reducing p-MLC expression, and inhibiting F-actin remodeling.
The PI3K/Akt/NF-κB pathway plays a central role in regulating endothelial barrier function. LPS activates this pathway, leading to increased phosphorylation of Akt and IKK, and nuclear translocation of NF-κB, which promotes inflammation and endothelial damage. UFH inhibits this pathway, thereby reducing endothelial hyperpermeability and inflammation. The findings are consistent with previous studies showing that UFH attenuates LPS-induced interleukin-8 (IL-8) secretion and endothelial barrier dysfunction via the PI3K/Akt/NF-κB pathway.
In conclusion, UFH protects against LPS-induced endothelial barrier dysfunction by stabilizing VE-cadherin and inhibiting the PI3K/Akt/NF-κB pathway. These findings suggest that UFH may have therapeutic potential for ALI and other conditions characterized by endothelial barrier disruption. Future studies should explore the effects of UFH in clinical settings and investigate its potential as a treatment for ALI.
doi.org/10.1097/CM9.0000000000000905
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