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

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 ␣ v ␤ 5 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 (ϳ70%), adhesion (ϳ82%), and migration (ϳ65%). 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.

HIV-1 Tat-Mediated Effects on Focal Adhesion Assembly and Permeability in Brain Microvascular Endothelial Cells

“…Tat induced the formation of focal adhesions in HBMECs when seeded on fibronectin, and pretreatment with Tat-specific Abs or with the specific inhibitor SU-1498, which inhibits VEGFR-2 receptor phosphorylation, blocked the ability of Tat to stimulate focal adhesion assembly. These effects of Tat on focal adhesion assembly were very similar to those observed with VEGF (28). This suggests that Tat mimics VEGF-mediated effects in brain endothelium via its binding to VEGFR-2, leading to VEGFR-2 activation and VEGFR-2 mediated signaling in BMEC.…”

“…Our previous studies have shown that VEGF regulates focal adhesion assembly in HBMECs through activation of FAK and related adhesion focal tyrosine kinase (28). Because Tat is known to induce the expression of some cytokines and growth factors in ECs, we examined whether the effects of Tat on focal adhesion assembly in HBMECs are mediated via VEGF induction.…”

“…The migration assay used a modified Boyden chamber and was performed essentially as previously described (28). Cells were grown to 50% confluence and then either untreated or infected with adenoviral constructs as described below for 2 h. Cells were lifted from the dish using nonenzymatic cell dissociation solution (Sigma-Aldrich), centrifuged for 2 min at 1000 rpm, and resuspended in medium 199 containing 0.5% BSA.…”

Mechanical Strain on Osteoblasts Activates Autophosphorylation of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 Tyrosine Sites Involved in ERK Activation

“…It was shown that, during cell adhesion, clustering of integrins leads to the recruitment of FAK and PYK2 to the newly formed focal adhesion sites and activates the phosphorylation of a variety of downstream pathways (71). As for PYK2 activation mechanisms, it has been shown that this tyrosine kinase displays an integrin-dependent phosphorylation in lymphocyte B, CMK cells, and osteoclasts (52,53,62).…”

Cardiovascular gene delivery: The good road is awaiting☆

“…Focal adhesion kinase (FAK) is a potential target for this approach because it mediates both integrin and growth factor signaling in the activation of SMC proliferation and migration, as well as the expression of the matrix metalloproteinases that mediate migration and vascular remodeling [141][142][143][144][145][146][147]. Upon integrin activation via ECM or growth factor stimulation, FAK localizes intracellularly to focal adhesion complexes with its carboxyterminal FAT domain, autophosphorylates itself, and iniiates various cell survival, migration, and proliferation signaling cascades [148].…”