title: Opposing effects of the angiopoietins on the thrombin-induced permeability of human pulmonary microvascular endothelial cells.
abstract: BACKGROUND
  Angiopoietin-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.
  
  
  RESULTS
  Permeability 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
  Ang-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.
authors.affiliation: Department of Intensive Care, Institute for Cardiovascular Research, VU University Medical Centre, Amsterdam, The Netherlands.
authors.forename: Melanie
authors.initials: M
authors.lastname: van der Heijden
authors.forename: Geerten P
authors.initials: GP
authors.lastname: van Nieuw Amerongen
authors.forename: Jan
authors.initials: J
authors.lastname: van Bezu
authors.forename: Marinus A
authors.initials: MA
authors.lastname: Paul
authors.forename: A B Johan
authors.initials: AB
authors.lastname: Groeneveld
authors.forename: Victor W M
authors.initials: VW
authors.lastname: van Hinsbergh
mesh_terms: D042683:Angiopoietin-1 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D042702:Angiopoietin-2 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D042682:Angiopoietins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015703:Antigens, CD / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D003029:Coagulants / Q000494:pharmacology; D017097:Electric Impedance; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D006801:Humans; D015151:Immunoblotting; D008168:Lung / Q000098:blood supply; D010766:Phosphorylation / Q000187:drug effects; D042787:Receptor, TIE-2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D013997:Time Factors; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D000099152:Cadherin 5
chemical_list: D042683:Angiopoietin-1; D042702:Angiopoietin-2; D042682:Angiopoietins; D015703:Antigens, CD; D015820:Cadherins; D003029:Coagulants; D042787:Receptor, TIE-2; D011994:Recombinant Proteins; D013917:Thrombin; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; D000099152:Cadherin 5