An in vitro model to study the role of endothelial rho GTPases during leukocyte transendothelial migration.
Tissue injury induces release of cytokines that stimulate the expression of adhesion receptors in the endothelial wall of neighboring vessels. These endothelial receptors recruit leukocytes from the bloodstream and facilitate their transendothelial migration (TEM) toward the inflamed area. The molecules involved in leukocyte-endothelium interaction and TEM have been studied in vivo and in vitro over the past 20 years. Human umbilical vein endothelial cells (HUVECs) are a popular in vitro model to analyze TEM, and have been used to investigate the role of Rho GTPases in this process. Here we describe methods to activate HUVECs, to investigate Rho GTPase-activation by the endothelial adhesion receptor ICAM-1, and to inhibit or activate Rho GTPases using C3 transferase or adenoviruses coding for dominant negative or constitutive active Rho GTPases. Finally, we describe how to image and quantitate leukocyte TEM by digital time-lapse microscopy.
Ludwig Institute for Cancer Research, University College London, UK.
Jaime
J
Millán
Lynn
L
Williams
Anne J
AJ
Ridley
D036002:ADP Ribose Transferases / Q000494:pharmacology; D001905:Botulinum Toxins / Q000494:pharmacology; D002465:Cell Movement / Q000502:physiology*; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000502:physiology; D007962:Leukocytes / Q000187:drug effects / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology; D020741:rho GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein