Vascular endothelial growth factor regulates focal adhesion assembly in human brain microvascular endothelial cells through activation of the focal adhesion kinase and related adhesion focal tyrosine kinase.
Vascular endothelial growth factor (VEGF) plays a significant role in blood-brain barrier breakdown and angiogenesis after brain injury. VEGF-induced endothelial cell migration is a key step in the angiogenic response and is mediated by an accelerated rate of focal adhesion complex assembly and disassembly. In this study, we identified the signaling mechanisms by which VEGF regulates human brain microvascular endothelial cell (HBMEC) integrity and assembly of focal adhesions, complexes comprised of scaffolding and signaling proteins organized by adhesion to the extracellular matrix. We found that VEGF treatment of HBMECs plated on laminin or fibronectin stimulated cytoskeletal organization and increased focal adhesion sites. Pretreating cells with VEGF antibodies or with the specific inhibitor SU-1498, which inhibits Flk-1/KDR receptor phosphorylation, blocked the ability of VEGF to stimulate focal adhesion assembly. VEGF induced the coupling of focal adhesion kinase (FAK) to integrin alphavbeta5 and tyrosine phosphorylation of the cytoskeletal components paxillin and p130cas. Additionally, FAK and related adhesion focal tyrosine kinase (RAFTK)/Pyk2 kinases were tyrosine-phosphorylated by VEGF and found to be important for focal adhesion sites. Overexpression of wild type RAFTK/Pyk2 increased cell spreading and the migration of HBMECs, whereas overexpression of catalytically inactive mutant RAFTK/Pyk2 markedly suppressed HBMEC spreading ( approximately 70%), adhesion ( approximately 82%), and migration ( approximately 65%). Furthermore, blocking of FAK by the dominant-interfering mutant FRNK (FAK-related non-kinase) significantly inhibited HBMEC spreading and migration and also disrupted focal adhesions. Thus, these studies define a mechanism for the regulatory role of VEGF in focal adhesion complex assembly in HBMECs via activation of FAK and RAFTK/Pyk2.
Avraham
Hava Karsenty
HK
Division of Experimental Medicine and Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02115, USA. savraham@bidmc.harvard.edu
Lee
Tae-Hee
TH
Koh
Youngho
Y
Kim
Tae-Aug
TA
Jiang
Shuxian
S
Sussman
Mark
M
Samarel
Allen M
AM
Avraham
Shalom
S
D000256:Adenoviridae / Q000235:genetics; D015153:Blotting, Western; D001921:Brain / Q000098:blood supply*; D002460:Cell Line; D002465:Cell Movement; D002478:Cells, Cultured; D050719:Crk-Associated Substrate Protein; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology*; D004789:Enzyme Activation; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions / Q000378:metabolism*; D005799:Genes, Dominant; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D016023:Integrins / Q000378:metabolism; D007797:Laminin / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008833:Microcirculation / Q000378:metabolism*; D018613:Microscopy, Confocal; D008954:Models, Biological; D009154:Mutation; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins; D019030:Receptors, Vitronectin / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D050717:Retinoblastoma-Like Protein p130; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism / Q000502:physiology*
C493841:BCAR1 protein, human; D050719:Crk-Associated Substrate Protein; D003598:Cytoskeletal Proteins; D005353:Fibronectins; D016023:Integrins; D007797:Laminin; D008164:Luminescent Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011506:Proteins; D019030:Receptors, Vitronectin; D011994:Recombinant Proteins; D050717:Retinoblastoma-Like Protein p130; D042461:Vascular Endothelial Growth Factor A; C064660:integrin alphaVbeta5; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human