抄録
Background
Sepsis has been a leading cause of death worldwide. Microvascular dysfunction is a hallmark of sepsis and is thought to promote the neutrophil adhesion to vessel walls, coagulation abnormalities, microvascular leakage and hypoperfusion associated with sepsis-induced multiorgan failure. Therefore, recovery and maintenance of the endothelial barrier function is critical to survival in sepsis. Histidine-rich glycoprotein (HRG) is a 75kDa plasma protein which can regulate angiogenesis, coagulation, fibrinolysis and immune response. We previously demonstrated that supplementary treatment with HRG can improve the survival rate of septic mice and inhibit the adhesion of neutrophils to vascular endothelial cells. In the present study, we aim to investigate the effects of HRG on the LPS/TNF-α-induced barrier dysfunction of vascular endothelial cells (VECs) in vitro and in vivo. Rearrangement of cytoskeleton, cell-cell adhesion and cell-matrix adhesion all participate in the regulation of endothelial cell morphology, migration ability and the permeability control. Therefore, we focus on the effects of HRG on the barrier dysfunction, cytoskeleton rearrangement and intercellular adherent junctions of VECs after stimulation with LPS/TNF-α to further clarify the definitive roles of HRG in sepsis.
Methods
EA.hy 926 endothelial cells were pretreated with HRG or human serum albumin (80μg/ml) for 30min before stimulation with LPS/TNF-α for different time point. The immunostaining of F-actin cytoskeleton and adherent junctions VE-cadherin/β-catenin were performed and observed under confocal microscope. LPS-induced focal adhesion kinase (FAK) cleavage was quantified with western-blot. Moreover, the cell-matrix detachemnt experiments were analyzed by crystal violet staining. Finally, in vitro permeabilty of VECs was measured with FITC-dextran transwell assay and in vivo permeabilty was evaluated with Evans blue leakage assay.
Results
HRG can maintain the endothelial monolayer integrity by inhibiting the cytoskeleton reorganization, loss of VE-cadherin/β-catenin, FAK cleavage and cell detachment after stimulation with LPS/TNF-α. Furthermore, HRG effectively prevents the LPS/TNF-α-induced increase in capillary permeability both in vitro and in vivo.
Conclusion
Taken together, HRG has protective effects on vascular barrier function which finally results in the relief of the uncontrolled inflammatory responses in septic condition.
