Abstract
BackgroundAngiopoietin-2 (Ang-2) is associated with lung injury in ALI/ARDS. As endothelial activation by thrombin plays a role in the permeability of acute lung injury and Ang-2 may modulate the kinetics of thrombin-induced permeability by impairing the organization of vascular endothelial (VE-)cadherin, and affecting small Rho GTPases in human pulmonary microvascular endothelial cells (HPMVECs), we hypothesized that Ang-2 acts as a sensitizer of thrombin-induced hyperpermeability of HPMVECs, opposed by Ang-1.Methodology/Principal FindingsPermeability was assessed by measuring macromolecule passage and transendothelial electrical resistance (TEER). Angiopoietins did not affect basal permeability. Nevertheless, they had opposing effects on the thrombin-induced permeability, in particular in the initial phase. Ang-2 enhanced the initial permeability increase (passage, P = 0.010; TEER, P = 0.021) in parallel with impairment of VE-cadherin organization without affecting VE-cadherin Tyr685 phosphorylation or increasing RhoA activity. Ang-2 also increased intercellular gap formation. Ang-1 preincubation increased Rac1 activity, enforced the VE-cadherin organization, reduced the initial thrombin-induced permeability (TEER, P = 0.027), while Rac1 activity simultaneously normalized, and reduced RhoA activity at 15 min thrombin exposure (P = 0.039), but not at earlier time points. The simultaneous presence of Ang-2 largely prevented the effect of Ang-1 on TEER and macromolecule passage.Conclusions/SignificanceAng-1 attenuated thrombin-induced permeability, which involved initial Rac1 activation-enforced cell-cell junctions, and later RhoA inhibition. In addition to antagonizing Ang-1, Ang-2 had also a direct effect itself. Ang-2 sensitized the initial thrombin-induced permeability accompanied by destabilization of VE-cadherin junctions and increased gap formation, in the absence of increased RhoA activity.
Highlights
Excessive and sustained activation of the pulmonary endothelium is central in the pathogenesis of the pulmonary inflammation and permeability of the life-threatening syndromes acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) [1]
After a second magnetic separation of human pulmonary microvascular endothelial cells (HPMVECs) and non-endothelial cells, the culture showed a purity of .99% as confirmed by the presence of endothelial cell markers vascular endothelial (VE-)cadherin, CD31, von Willebrand factor (VWF), Tie2 and endothelial nitric oxide synthase and the absence of smooth muscle cell (SMC) marker a-actin and epithelial cell marker pancytokeratin
Basal effects of angiopoietins Unexpectedly, neither Ang-2, nor Ang-1 affected the basal endothelial barrier function as determined by HRP passage (Figure 1a) or transendothelial electrical resistance (TEER) (Figure 1b). This was found with Ang-1 and 2 concentrations ranging from 5 to 400 ng/ml. To exclude that this was due to the short stimulation period (30 min), HPMVECs were stimulated with Ang-2 for 5 hours
Summary
Excessive and sustained activation of the pulmonary endothelium is central in the pathogenesis of the pulmonary inflammation and permeability of the life-threatening syndromes acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) [1]. ALI/ARDS was not appropriately modeled using those cell types, since endothelial cells from different vascular beds display remarkable heterogeneity in structure and function [22,23,24] It remains to be investigated in an in vitro model of ALI using human pulmonary microvascular endothelial cells (HPMVECs), whether Ang-2 modulates the thrombin-induced permeability and which pathways are involved. As endothelial activation by thrombin plays a role in the permeability of acute lung injury and Ang-2 may modulate the kinetics of thrombin-induced permeability by impairing the organization of vascular endothelial (VE-)cadherin, and affecting small Rho GTPases in human pulmonary microvascular endothelial cells (HPMVECs), we hypothesized that Ang-2 acts as a sensitizer of thrombin-induced hyperpermeability of HPMVECs, opposed by Ang-1
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