Opposing Effects of the Angiopoietins on the Thrombin-Induced Permeability of Human Pulmonary Microvascular Endothelial Cells

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.

The Temporal Kinetics of Circulating Angiopoietin Levels in Children With Sepsis

“…In a recent study, angpt-2 was shown to enhance thrombin-induced cell permeability in cultured endothelial cells via the disruption of vascular endothelial (VE)-cadherins. Further supporting the concept that the angpt-2/1 ratio is likely more important than absolute levels, the addition of angpt-1 attenuated this response (37). …”

MARCKS protein mediates hydrogen peroxide regulation of endothelial permeability

“…The phosphorylation of MARCKS appears to be the fundamental molecular mechanism that determines both its subcellular localization and its interactions with key structural and signaling molecules. Agents that increase endothelial permeability, such as thrombin and diacylglycerol (37)(38)(39), also induce MARCKS phosphorylation in endothelial cells (40,41), suggesting a link between MARCKS phosphorylation and permeability. We have characterized MARCKS phosphorylation pathways in BAECs treated with H 2 O 2 and have found that H 2 O 2 induces MARCKS phosphorylation and translocation from membrane to cytosol (Fig.…”

Angiopoietin-1, Angiopoietin-2 and Myocardial Ischemia-Reperfusion Injury

“…Ang-1 has been shown to inhibit vascular permeability and exert anti-inflammatory effect via the Tie-2 receptor. Ang-1 attenuates thrombin-induced permeability in human pulmonary microvascular endothelial cells (HPMVECs) by enforcing the VE-cadherin organization [10]. Ang-1 could counteract the inflammatory effects of vascular endothelial growth factor (VEGF) on endothelial cells [11], ameliorate ROS-induced acute lung injury [12] and lipopolysaccharide-induced acute kidney injury [13] by reducing the induction of ICAM-1 and VCAM-1, causing a reduction in leukocyte adhesion and subsequent inflammation.…”

“…While Ang-1 has been shown to have a protective effect against inflammation, Ang-2 has been shown to have the opposite effect. Ang-2 could sensitize thrombin-induced permeability in HPMVECs by destabilizing VEcadherin junctions and increasing gap formation [10]. Overexpression of Ang-2 also sensitizes ICAM-1 and VCAM-1 expression in diabetic mouse hearts [14].…”