Caveolin-1 and Rac regulate endothelial capillary-like tubular formation and fenestral contraction in sinusoidal endothelial cells.
OBJECTIVE
Rho guanidine triphosphatases (GTPases) are major regulators of cell migration. We investigated the cytoskeleton and Rho GTPases during cell migration and morphogenesis processes in isolated rat liver sinusoidal endothelial cells (LSECs) cultured on Matrigel while stimulated by the vascular endothelial growth factor (VEGF).


METHODS
To obtain primary monolayers, LSECs were cultured on Matrigel for 5-17 h with or without VEGF. Sinusoidal endothelial fenestrae (SEF) morphology was observed using scanning electron microscopy and transmission electron microscopy. RhoA, Rac1 and phosphorylated myosin light-chain kinase, Rho-binding domain of Rhotekin and the p21-binding domain of p21-activated protein kinase were analysed using Western blotting.


RESULTS
The LSECs showed cellular protrusions and or cords of aligned cells resembling primitive capillary-like structures, with SEF contraction. Time course analyses of Rac1 activation matched specific morphological changes. Rac1 activity increased progressively to 17 h in cells cultured without VEGF, but markedly increased at 7 h in the presence of VEGF. RhoA activity was slightly elevated at 5 h. The levels of endogenous caveolin-1 (CAV-1) expression increased in a time-dependent manner, reaching a peak at 7 h. CAV-1 expression occurred immediately before the formation of the capillary-like tube. Moreover, treatment with VEGF regulated CAV-1 expression in LSECs.


CONCLUSIONS
Spatial activation of Rac1 is involved in the formation of a capillary-like tubular network accompanying SEF contraction in LSECs, implying that endothelial migration and adhesion are necessary for LSECs tubular formation in the liver. CAV-1 might play an important positive role in the regulation of LSEC tubular formation.
Yokomori
Hiroaki
H
Department of Internal Medicine, Kitasato Institute Medical Center Hospital, Kitasato University, Saitama, Japan. yokomori@insti.kitasato-u.ac.jp
Oda
Masaya
M
Yoshimura
Kazunori
K
Nagai
Toshihiro
T
Fujimaki
Kayo
K
Watanabe
Shuichi
S
Hibi
Toshifumi
T
D000818:Animals; D015153:Blotting, Western; D051242:Caveolin 1 / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D042783:Endothelial Cells / Q000502:physiology* / Q000648:ultrastructure; D008099:Liver / Q000098:blood supply*; D008297:Male; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D018919:Neovascularization, Physiologic / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D051242:Caveolin 1; C107953:RAC1 protein, human; D042461:Vascular Endothelial Growth Factor A; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein