title: Localized RhoA GTPase activity regulates dynamics of endothelial monolayer integrity.
abstract: OBJECTIVE
  Endothelial cells (ECs) control vascular permeability by forming a monolayer that is sealed by extracellular junctions. Various mediators modulate the endothelial barrier by acting on junctional protein complexes and the therewith connected F-actin cytoskeleton. Different Rho GTPases participate in this modulation, but their mechanisms are still partly resolved. Here, we aimed to elucidate whether the opening and closure of the endothelial barrier are associated with distinct localized RhoA activities at the subcellular level.
  
  
  RESULTS
  Live fluorescence resonance energy transfer (FRET) microscopy revealed spatially distinct RhoA activities associated with different aspects of the regulation of endothelial monolayer integrity. Unstimulated ECs were characterized by hotspots of RhoA activity at their periphery. Thrombin receptor activation in the femoral vein of male wistar rats and in cultured ECs enhanced RhoA activity at membrane protrusions, followed by a more sustained RhoA activity associated with cytoplasmic F-actin filaments, where prolonged RhoA activity coincided with cellular contractility. Unexpectedly, thrombin-induced peripheral RhoA hotspots were not spatially correlated to the formation of large inter-endothelial gaps. Rather, spontaneous RhoA activity at membrane protrusions coincided with the closure of inter-endothelial gaps. Electrical impedance measurements showed that RhoA signalling is essential for this protrusive activity and maintenance of barrier restoration.
  
  
  CONCLUSIONS
  Spontaneous RhoA activity at membrane protrusions is spatially associated with closure, but not formation of inter-endothelial gaps, whereas RhoA activity at distant contractile filaments contributes to thrombin-induced disruption of junctional integrity. Thus, these data indicate that distinct RhoA activities are associated with disruption and re-annealing of endothelial junctions.
authors.affiliation: Department for Physiology, VU University Medical Center, Institute for Cardiovascular Research, van der Boechorststraat 7, Amsterdam 1108 BH, The Netherlands.
authors.forename: Robert
authors.initials: R
authors.lastname: Szulcek
authors.forename: Cora M L
authors.initials: CM
authors.lastname: Beckers
authors.forename: Jasmina
authors.initials: J
authors.lastname: Hodzic
authors.forename: Jelle
authors.initials: J
authors.lastname: de Wit
authors.forename: Zhenlong
authors.initials: Z
authors.lastname: Chen
authors.forename: Tim
authors.initials: T
authors.lastname: Grob
authors.forename: Rene J P
authors.initials: RJ
authors.lastname: Musters
authors.forename: Richard D
authors.initials: RD
authors.lastname: Minshall
authors.forename: Victor W M
authors.initials: VW
authors.lastname: van Hinsbergh
authors.forename: Geerten P
authors.initials: GP
authors.lastname: van Nieuw Amerongen
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002199:Capillary Permeability / Q000502:physiology*; D042783:Endothelial Cells / Q000201:enzymology* / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007365:Intercellular Junctions / Q000502:physiology; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D000199:Actins; C066737:RHOA protein, human; D013917:Thrombin; D020742:rhoA GTP-Binding Protein
keywords: Cytoskeleton; Endothelial function; FRET microscopy; Rho GTPase; Vasoactive agents
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