title: Involvement of local lamellipodia in endothelial barrier function.
abstract: Recently we observed that endothelial cells cultured in tightly confluent monolayers display frequent local lamellipodia, and that thrombin, an agent that increases endothelial permeability, reduces lamellipodia protrusions. This led us to test the hypothesis that local lamellipodia contribute to endothelial barrier function. Movements of subcellular structures containing GFP-actin or VE-cadherin-GFP expressed in endothelial cells were recorded using time-lapse microscopy. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function. Changes in both lamellipodia dynamics and TER were assessed during baseline and after cells were treated with either the barrier-disrupting agent thrombin, or the barrier-stabilizing agent sphingosine-1-phosphate (S1P). The myosin II inhibitor blebbistatin was used to selectively block lamellipodia formation, and was used to test their role in the barrier function of endothelial cell monolayers and isolated, perfused rat mesenteric venules. Myosin light chain (MLC) phosphorylation was assessed by immunofluorescence microscopy. Rac1 and RhoA activation were evaluated using G-LISA assays. The role of Rac1 was tested with the specific inhibitor NSC23766 or by expressing wild-type or dominant negative GFP-Rac1. The results show that thrombin rapidly decreased both TER and the lamellipodia protrusion frequency. S1P rapidly increased TER in association with increased protrusion frequency. Blebbistatin nearly abolished local lamellipodia protrusions while cortical actin fibers and stress fibers remained intact. Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules. Both thrombin and S1P increased MLC phosphorylation and activation of RhoA. However, thrombin and S1P had differential impacts on Rac1, correlating with the changes in TER and lamellipodia protrusion frequency. Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity. Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.
authors: Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, Florida, United States of America.
authors: Jerome W
authors: JW
authors: Breslin
authors: Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, Florida, United States of America.
authors: Xun E
authors: XE
authors: Zhang
authors: Department of Pharmacology, School of Medicine, Louisiana State University Health Sciences Center, New Orleans, Louisiana, United States of America.
authors: Rebecca A
authors: RA
authors: Worthylake
authors: Department of Physiology, School of Medicine, Louisiana State University Health Sciences Center, New Orleans, Louisiana, United States of America.
authors: Flavia M
authors: FM
authors: Souza-Smith
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008246:Lysophospholipids / Q000494:pharmacology; D008297:Male; D008643:Mesentery / Q000098:blood supply; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011554:Pseudopodia / Q000502:physiology*; D011743:Pyrimidines / Q000494:pharmacology; D017207:Rats, Sprague-Dawley; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D059008:Time-Lapse Imaging / Q000379:methods; D014699:Venules / Q000187:drug effects / Q000502:physiology; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism; D000099152:Cadherin 5
chemical_list: D000199:Actins; D000634:Aminoquinolines; D015703:Antigens, CD; D015820:Cadherins; D049452:Green Fluorescent Proteins; D006576:Heterocyclic Compounds, 4 or More Rings; D008246:Lysophospholipids; D018994:Myosin Light Chains; D011743:Pyrimidines; D013110:Sphingosine; D013917:Thrombin; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; D000099152:Cadherin 5; C487513:NSC 23766; C472645:blebbistatin; C060506:sphingosine 1-phosphate