Rho and ROCK signaling in VEGF-induced microvascular endothelial hyperpermeability.
OBJECTIVE
Vascular endothelial growth factor (VEGF) plays an important role in the regulation of microvascular permeability under various physiological and pathological conditions. The authors tested the hypothesis that the small GTPase Rho and its downstream effector ROCK (Rho-associated coiled-coil-containing protein kinase) mediate VEGF-induced increases in venular permeability. They also investigated myosin light chain (MLC) phosphorylation and actin polymerization, two well-characterized targets of the Rho-ROCK pathway that are implicated in the regulation of endothelial barrier function.


METHODS
The apparent permeability coefficient of albumin (P(a)) was measured in intact isolated porcine coronary venules and in cultured coronary venular endothelial cell (CVEC) monolayers. RhoA activation was determined using a Rhotekin-agarose pull down assay. MLC phosphorylation was evaluated by immunoblotting with phospho-specific antibodies, and endothelial cellular F-actin was viewed using fluorescence microscopy.


RESULTS
VEGF increased P(a) in both isolated coronary venules and CVEC monolayers. The hyperpermeability response occurred in a similar time course to that of Rho activation, MLC phosphorylation, and actin stress fiber formation. Selective blockage of ROCK with Y27632 dose-dependently inhibited VEGF-induced venular hyperpermeability. Moreover, inhibition of either Rho with exoenzyme C3 or ROCK with Y-27632 attenuated VEGF-induced increases in permeability, MLC phosphorylation, and actin-stress fiber formation in CVEC monolayers.


CONCLUSIONS
Collectively, these findings suggest that the Rho-ROCK signal pathway contributes to VEGF-induced hyperpermeability. Myosin light-chain phosphorylation and actin stress fiber formation occur concomitantly with the increase in permeability upon VEGF stimulation.
Sun
Hengrui
H
Department of Surgery, University of California at Davis School of Medicine, Sacramento, California, USA.
Breslin
Jerome W
JW
Zhu
Jun
J
Yuan
Sarah Y
SY
Wu
Mack H
MH
D000418:Albumins / Q000493:pharmacokinetics; D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects*; D002417:Cattle; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D022502:Stress Fibers / Q000378:metabolism; D013552:Swine; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*; D014699:Venules / Q000378:metabolism / Q000502:physiology; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D000418:Albumins; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D042461:Vascular Endothelial Growth Factor A; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein