Abstract
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.
Highlights
Vascular endothelial growth factor (VEGF) plays a significant role in blood-brain barrier breakdown and angiogenesis after brain injury
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
To analyze whether VEGF can induce focal adhesion formation in brain microvascular endothelial cells and to determine the specific role of integrins in VEGF-mediated focal adhesion assembly, HBMECs were grown overnight on coverslips coated with fibronectin (20 g/ml) or gelatin (0.2%)
Summary
Attachment of cells to the extracellular matrix (ECM; e.g. laminin, fibronectin, and collagen) is mediated by structures called “focal adhesions,” which connect the extracellular matrix with the plasma membrane and the underlying actin cytoskeletal network. Attachment of cells to the ECM results in the clustering of integrin receptors and initiates the recruitment of numerous cytoplasmic proteins to the focal adhesion complex. These proteins include both structural and catalytically active signaling proteins. Clustering of integrin subunits by ECM, growth factor stimulation (platelet-derived growth factor and epidermal growth factor), and signaling through certain G protein-coupled receptors (such as the receptor for lysophosphatidic acid) have been shown to result in focal adhesion formation.
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