title: Hypoxia-reoxygenation-induced endothelial barrier failure: role of RhoA, Rac1 and myosin light chain kinase.
abstract: Hypoxia-reoxygenation induces loss of endothelial barrier function and oedema formation, which presents a major impediment for recovery of the organ. The integrity of the endothelial barrier is highly dependent on its contractile machinery and actin dynamics, which are precisely regulated by Rho GTPases. Perturbed activities of these Rho-GTPases under hypoxia-reoxygenation lead to derangement of the actin cytoskeleton and therefore may affect the integrity of the endothelial barrier. The aim of the present study was to analyse the role of these GTPases in regulating endothelial barrier function during hypoxia-reoxygenation in cultured porcine aortic endothelial cells and isolated perfused rat hearts. Hypoxia-reoxygenation induced an increase in albumin permeability of endothelial monolayers accompanied by an activation of the endothelial contractile machinery, derangement of the actin cytoskeleton and loss of VE-cadherin from cellular junctions. Inhibition of contractile activation with ML-7 partially protected against hypoxia-reoxygenation-induced hyperpermeability. Likewise, reoxygenation caused an increase in RhoA and a reduction in Rac1 activity accompanied by enhanced stress fibre formation and loss of peripheral actin. Inhibition of RhoA/rho kinase (Rock) signalling with RhoA or Rock inhibitors led to a complete depolymerisation and derangement of the actin cytoskeleton and worsened hypoxia-reoxygenation-induced hyperpermeability. Activation of Rac1 using a cAMP analogue, 8-CPT-O-Me-cAMP, which specifically activates Epac/Rap1 signalling, restored peripheral localisation of actin and VE-cadherin at cellular junctions and abrogated reoxygenation-induced hyperpermeability. Similar results were reproduced in isolated saline-perfused rat hearts. These data show that activation of Rac1 but not the inhibition of RhoA preserves endothelial integrity against reoxygenation-induced loss of barrier function.
authors.affiliation: Physiologisches Institut, Justus-Liebig-Universität, Giessen, Germany. muhammad.aslam@physiologie.med.uni-giessen.de
authors.forename: Muhammad
authors.initials: M
authors.lastname: Aslam
authors.forename: Klaus-Dieter
authors.initials: KD
authors.lastname: Schluter
authors.forename: Susanne
authors.initials: S
authors.lastname: Rohrbach
authors.forename: Amir
authors.initials: A
authors.lastname: Rafiq
authors.forename: Sabiha
authors.initials: S
authors.lastname: Nazli
authors.forename: Hans Michael
authors.initials: HM
authors.lastname: Piper
authors.forename: Thomas
authors.initials: T
authors.lastname: Noll
authors.forename: Rainer
authors.initials: R
authors.lastname: Schulz
authors.forename: Dursun
authors.initials: D
authors.lastname: Gündüz
mesh_terms: D000199:Actins / Q000378:metabolism; D022005:Adherens Junctions / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001011:Aorta / Q000166:cytology / Q000502:physiology; D015820:Cadherins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D015687:Cell Hypoxia; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D042783:Endothelial Cells / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D066298:In Vitro Techniques; D009131:Muscle, Smooth, Vascular / Q000502:physiology*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010539:Permeability; D051381:Rats; D015398:Signal Transduction; D022502:Stress Fibers / Q000378:metabolism; D013552:Swine; D014661:Vasoconstriction; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*; D000099152:Cadherin 5
chemical_list: D000199:Actins; D015703:Antigens, CD; D015820:Cadherins; D002118:Calcium; D000242:Cyclic AMP; D020662:Guanine Nucleotide Exchange Factors; D009219:Myosin-Light-Chain Kinase; D020830:rac1 GTP-Binding Protein; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; D000099152:Cadherin 5; C473217:8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate