Involvement of Rho kinase in endothelial barrier maintenance.
OBJECTIVE
Rho kinase mediates vascular leakage caused by many vasoactive agents including thrombin. Enhanced Rho kinase activity induces endothelial barrier dysfunction by a contractile mechanism via inactivation of Myosin Phosphatase (MP). Here, we investigated the contribution of basal Rho kinase activity to the regulation of endothelial barrier integrity.


RESULTS
Using a phospho-specific antibody against the myosin phosphatase targeting subunit (Thr696-MYPT1) as a marker for Rho kinase activity, basal endothelial Rho kinase activity was observed at cell-cell contact sites, in vitro and in situ. Thrombin enhanced MYPT phosphorylation at F-actin stress fibers. Inhibition of basal Rho kinase activity for 24 hours or depletion of Rho kinase (ROCK-I and -II) by siRNA disrupted endothelial barrier integrity, opposite to the previously observed protection from the thrombin-enhanced endothelial permeability. This barrier dysfunction could not be explained by changes in RhoA, Rac1, eNOS, or apoptosis. Remarkably, basal Rho kinase activity was essential for proper expression of the adhesion molecule VE-cadherin.


CONCLUSIONS
Rho kinase has opposing activities in regulation of endothelial barrier function: (1) an intrinsic barrier-protective activity at the cell margins, and (2) an induced barrier-disruptive activity at contractile F-actin stress fibers. These findings may have implications for long-term antivascular leak therapy.
van Nieuw Amerongen
G P
GP
VU university Medical Center, Laboratory for Physiology, Institute for Cardiovascular Research, Amsterdam, The Netherlands. nieuwamerongen@vumc.nl
Beckers
C M L
CM
Achekar
I D
ID
Zeeman
S
S
Musters
R J P
RJ
van Hinsbergh
V W M
VW
D022005:Adherens Junctions / Q000502:physiology*; D015703:Antigens, CD / Q000378:metabolism*; D015820:Cadherins / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000502:physiology; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000502:physiology; D042783:Endothelial Cells / Q000201:enzymology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering; D014471:Umbilical Veins; D054460:rho-Associated Kinases; D000099152:Cadherin 5
D015703:Antigens, CD; D015820:Cadherins; D047908:Intracellular Signaling Peptides and Proteins; D043263:Myosin-Light-Chain Phosphatase; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D054460:rho-Associated Kinases; D000099152:Cadherin 5; C490372:PPP1R12A protein, human