Role of the small GTPase Rap1 for integrin activity regulation in endothelial cells and angiogenesis.
Ras-associated protein 1 (Rap1), a small GTPase, attracted attention because of its involvement in several aspects of cell adhesion, including integrin- and cadherin-mediated adhesion. Yet, the role of Rap1 genes and of Rap1 effectors for angiogenesis has not been investigated. Human umbilical vein endothelial cells (HUVECs) express Rap1a and Rap1b mRNA. To determine the contribution of Rap1 activity for angiogenesis, we overexpressed Rap1GAP1, a GTPase-activating protein that inhibits Rap1 activity. Overexpression of Rap1GAP1 significantly blocked angiogenic sprouting and tube-forming activity of HUVECs as well as migration and integrin-dependent adhesion. Silencing of Rap1a, Rap1b, or both significantly blocked HUVECs sprouting under basal and basic fibroblast growth factor-stimulated conditions and reduced HUVEC migration and integrin-dependent adhesion. We found that Rap1a and Rap1b are essential for the conformational activation of beta(1)-integrins in endothelial cells. Furthermore, silencing of Rap1a and Rap1b prevented phosphorylation of tyrosine 397 in focal adhesion kinase (FAK) and vascular endothelial growth factor-induced Akt1-activation. Rap1a(-/-)-deficient and Rap1a(+/-) heterozygote mice displayed reduced neovascularization after hind limb ischemia compared with wild-type mice. Silencing of RAPL significantly blocked the Rap1-induced sprouting of HUVECs, suggesting that the angiogenic activity of Rap1 is partly mediated by RAPL. Our data demonstrate a critical role of Rap1 in the regulation of beta(1)-integrin affinity, adhesion, and migration in endothelial cells and in postnatal neovascularization.
Carmona
Guillaume
G
Molecular Cardiology, Department of Internal Medicine III, J. W. Goethe University Frankfurt, Frankfurt.
Göttig
Stephan
S
Orlandi
Alessia
A
Scheele
Jürgen
J
Bäuerle
Tobias
T
Jugold
Manfred
M
Kiessling
Fabian
F
Henschler
Reinhard
R
Zeiher
Andreas M
AM
Dimmeler
Stefanie
S
Chavakis
Emmanouil
E
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D002448:Cell Adhesion / Q000502:physiology; D002465:Cell Movement / Q000502:physiology*; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D005346:Fibroblast Growth Factors / Q000378:metabolism; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism; D020868:Gene Silencing; D006614:Hindlimb / Q000098:blood supply / Q000201:enzymology; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism*; D007511:Ischemia / Q000201:enzymology; D051379:Mice; D018345:Mice, Knockout; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000502:physiology*; D010766:Phosphorylation / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt; D014471:Umbilical Veins / Q000166:cytology / Q000201:enzymology*; D020668:rap GTP-Binding Proteins / Q000378:metabolism*; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D005346:Fibroblast Growth Factors; D051417:Focal Adhesion Kinase 1; D020690:GTPase-Activating Proteins; D019012:Integrin beta1; D020559:Monomeric GTP-Binding Proteins; D051057:Proto-Oncogene Proteins c-akt; D020668:rap GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins; C430803:RAP1A protein, human; C484658:RAP1GAP protein, human; C454042:RASSF5 protein, human; C496050:PTK2 protein, human; C494918:AKT1 protein, human; C116753:RAP1B protein, human; C492136:Rap1b protein, mouse; C455831:Rassf5 protein, mouse