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-alpha)-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-kappa B) 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 (LPSvs. LPS + UFH: 0.57 +/- 0.04vs. 0.32 +/- 0.04 mg/mL,P = 0.0092), total cell count (LPSvs. LPS + UFH: 9.57 +/- 1.23vs. 3.65 +/- 0.78 x 10(5)/mL,P = 0.0155), polymorphonuclear neutrophil percentage (LPSvs. LPS + UFH: 88.05% +/- 2.88%vs. 22.20% +/- 3.92%,P = 0.0002), and TNF-alpha (460.33 +/- 23.48vs. 189.33 +/- 14.19 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 (LPSvs. LPS + UFH: 0.368 +/- 0.044vs. 0.716 +/- 0.064,P = 0.0114) and p120-catenin (LPSvs. LPS + UFH: 0.208 +/- 0.018vs. 0.924 +/- 0.092,P = 0.0016), and the LPS-induced increase in the expression of p-MLC (LPSvs. LPS + UFH: 0.972 +/- 0.092vs. 0.293 +/- 0.025,P = 0.0021). Furthermore, UFH attenuated LPS- and TNF-alpha-induced hyperpermeability of HPMECs (LPSvs. LPS + UFH: 8.90 +/- 0.66vs. 15.84 +/- 1.09 omega center dot cm(2),P = 0.0056; TNF-alpha vs. TNF-alpha + UFH: 11.28 +/- 0.64vs. 18.15 +/- 0.98 omega center dot cm(2),P = 0.0042) and F-actin remodeling (LPSvs. LPS + UFH: 56.25 +/- 1.51vs. 39.70 +/- 1.98,P = 0.0027; TNF-alpha vs. TNF-alpha + UFH: 55.42 +/- 1.42vs. 36.51 +/- 1.20,P = 0.0005)in vitro. Additionally, UFH decreased the phosphorylation of Akt (LPSvs. LPS + UFH: 0.977 +/- 0.081vs. 0.466 +/- 0.035,P = 0. 0045) and I kappa B Kinase (IKK) (LPSvs. LPS + UFH: 1.023 +/- 0.070vs. 0.578 +/- 0.044,P = 0.0060), and the nuclear translocation of NF-kappa B (LPSvs. LPS + UFH: 1.003 +/- 0.077vs. 0.503 +/- 0.065,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-kappa B signaling.