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±0.04 vs.0.32±0.04 mg/mL,P=0.0092),total cell count(LPS vs.LPS+UFH:9.57±1.23 vs.3.65±0.78×105/mL,P=0.0155),polymorphonuclear neutrophil percentage(LPS vs.LPS+UFH:88.05%±2.88%vs.22.20%±3.92%,P=0.0002),and TNF-α(460.33±23.48 vs.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(LPS vs.LPS+UFH:0.368±0.044 vs.0.716±0.064,P=0.0114)and p120-catenin(LPS vs.LPS+UFH:0.208±0.018 vs.0.924±0.092,P=0.0016),and the LPS-induced increase in the expression of p-MLC(LPS vs.LPS+UFH:0.972±0.092 vs.0.293±0.025,P=0.0021).Furthermore,UFH attenuated LPS-and TNF-α-induced hyperpermeability of HPMECs(LPS vs.LPS+UFH:8.90±0.66 vs.15.84±1.09Ω·cm 2,P=0.0056;TNF-αvs.TNF-α+UFH:11.28±0.64 vs.18.15±0.98Ω·cm 2,P=0.0042)and F-actin remodeling(LPS vs.LPS+UFH:56.25±1.51 vs.39.70±1.98,P=0.0027;TNF-αvs.TNF-α+UFH:55.42±1.42 vs.36.51±1.20,P=0.0005)in vitro.Additionally,UFH decreased the phosphorylation of Akt(LPS vs.LPS+UFH:0.977±0.081 vs.0.466±0.035,P=0.0045)and I kappa B Kinase(IKK)(LPS vs.LPS+UFH:1.023±0.070 vs.0.578±0.044,P=0.0060),and the nuclear translocation of NF-κB(LPS vs.LPS+UFH:1.003±0.077 vs.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-κB signaling.