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• W2029315212 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 αvβ5 and tyrosine phosphorylation of the cytoskeletal components paxillin and p130 cas. 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 (∼70%), adhesion (∼82%), and migration (∼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. 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 αvβ5 and tyrosine phosphorylation of the cytoskeletal components paxillin and p130 cas. 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 (∼70%), adhesion (∼82%), and migration (∼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. The blood-brain barrier (BBB) 1The abbreviations used are: BBB, blood-brain barrier; ECM, extracellular matrix; FAK, focal adhesion kinase; HBMEC, human brain microvascular endothelial cell; KDR, kinase insert domain-containing receptor; RAFTK, related adhesion focal tyrosine kinase; VEGF, vascular endothelial growth factor; WT, wild type; GFP, green fluorescent protein; PBS, phosphate-buffered saline; VE, vascular endothelial.1The abbreviations used are: BBB, blood-brain barrier; ECM, extracellular matrix; FAK, focal adhesion kinase; HBMEC, human brain microvascular endothelial cell; KDR, kinase insert domain-containing receptor; RAFTK, related adhesion focal tyrosine kinase; VEGF, vascular endothelial growth factor; WT, wild type; GFP, green fluorescent protein; PBS, phosphate-buffered saline; VE, vascular endothelial. is formed by a complex cellular system of endothelial cells, astroglia, pericytes, perivascular macrophages, and basal lamina (1de Vries H.E. Kuiper J. de Boer A.G. Van Berkel T.J. Breimer D.D. Pharmacol. Rev. 1997; 49: 143-155PubMed Google Scholar). The microvasculature responds dynamically to the flow of substances, free radical exposure, and cytokine generation associated with focal ischemia. During focal cerebral ischemia, with the fall in tissue oxygenation, microvascular permeability barriers are lost (1de Vries H.E. Kuiper J. de Boer A.G. Van Berkel T.J. Breimer D.D. Pharmacol. Rev. 1997; 49: 143-155PubMed Google Scholar, 2Barone F.C. Feuerstein G.Z. J. Cereb. Blood Flow Metab. 1999; 19: 819-834Crossref PubMed Scopus (831) Google Scholar, 3del Zoppo G.J. Ann. N. Y. Acad. Sci. 1997; 823: 132-147Crossref PubMed Scopus (116) Google Scholar, 4del Zoppo G. Ginis I. Hallenbeck J.M. Iadecola C. Wang X. Feuerstein G.Z. Brain Pathol. 2000; 10: 95-112Crossref PubMed Scopus (564) Google Scholar), and the expression of VEGF is significantly enhanced in the ischemic core and penumbra of the ischemic brain (5Yang Z.J. Qiu M.H. Zhang L.M. Lu S.D. Huang Y.L. Sun F.Y. J. Neurosci. Res. 2002; 70: 140-149Crossref PubMed Scopus (57) Google Scholar). Angiogenesis, the formation of new blood capillaries, is an important component of normal physiological processes such as wound healing and development (6Folkman J. Shing Y.Angiogenesis. J. Biol. Chem. 1992; 267: 10931-10934Abstract Full Text PDF PubMed Google Scholar). Angiogenesis is also a complex process involving endothelial cell movement and proliferation and endothelial cell-mediated degradation of the extracellular matrix. VEGF is a key regulator of endothelial cell functions (6Folkman J. Shing Y.Angiogenesis. J. Biol. Chem. 1992; 267: 10931-10934Abstract Full Text PDF PubMed Google Scholar, 7Folkman J. J. Natl. Cancer Inst. 1990; 82: 4-6Crossref PubMed Scopus (4360) Google Scholar, 8Yoshiji H. Gomez D.E. Shibuya M. Thorgeirsson U.P. Cancer Res. 1996; 56: 2013-2016PubMed Google Scholar, 9Fava R.A. Olsen N.J. Spencer-Green G. Yeo K.T. Yeo T.K. Berse B. Jackman R.W. Senger D.R. 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Brain endothelial cells are linked to each other by gap, tight, and adherens-type junctions and are linked to the extracellular matrix by a variety of integrin and other adhesion molecules. VEGF activates endothelial cells, in part, by stimulating signal transduction pathways that regulate the enzymatic components of adhesion complexes. VEGF induced the tyrosine phosphorylation of vascular endothelial (VE) cadherins, molecules important in endothelial cell migration (15Esser S. Lampugnani M.G. Corada M. Dejana E. Risau W. J. Cell Sci. 1998; 111: 1853-1865Crossref PubMed Google Scholar), and also induced phosphorylation of the tight junction proteins occludin and occludin-1 (16Kevil C.G. Payne D.K. Mire E. Alexander J.S. J. Biol. Chem. 1998; 273: 15099-15103Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 17Antonetti D.A. Barber A.J. Hollinger L.A. Wolpert E.B. Gardner T.W. J. Biol. Chem. 1999; 274: 23463-23467Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). VEGF enhanced the expression of α1β1 and α2β2 integrins, and neutralizing antibodies to αvβ1 integrins blocked growth factor-induced neovascularization (18Senger D.R. Claffey K.P. Benes J.E. Perruzzi C.A. Sergiou A.P. Detmar M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13612-13617Crossref PubMed Scopus (451) Google Scholar, 19Friedlander M. Brooks P.C. Shaffer R.W. Kincaid C.M. Varner J.A. Cheresh D.A. Science. 1995; 270: 1500-1502Crossref PubMed Scopus (1214) Google Scholar, 20Soldi R. Mitola S. Strasly M. Defilippi P. Tarone G. Bussolino F. EMBO J. 1999; 18: 882-892Crossref PubMed Scopus (523) Google Scholar). VEGF exhibits high affinity binding to two distinct receptor tyrosine kinases, the Fms-like tyrosine kinase Flt-1 and the Flk-1/KDR (21Takahashi T. Shibuya M. Oncogene. 1997; 14: 2079-2089Crossref PubMed Scopus (271) Google Scholar, 22de Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1875) Google Scholar). Flk-1/KDR and not Flt-1 is able to mediate the mitogenic and chemotactic effects of VEGF in endothelial cells. VEGF stimulates the tyrosine phosphorylation of phospholipase Cγ, mitogen-activated protein kinase, phosphatidylinositol 3-kinase, FAK, and paxillin in human umbilical vein endothelial cells and of phospholipase Cγ, p120GAP, and NCK in bovine aortic endothelial cells (23Abedi H. Zachary I. J. Biol. Chem. 1997; 272: 15442-15451Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 24Guo D. Jia Q. Song H.Y. Warren R.S. Donner D.B. J. Biol. Chem. 1995; 270: 6729-6733Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 25Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar, 26Stoletov K.V. Ratcliffe K.E. Spring S.C. Terman B.I. J. Biol. Chem. 2001; 276: 22748-22755Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). However, the key targets that mediate the diverse biological functions of VEGF in endothelial cells remain incompletely understood for both VEGF receptors. 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. Signaling through focal adhesions regulates a variety of cellular processes including cell growth, migration, and apoptosis (27Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1639) Google Scholar). Focal adhesions are dynamic structures, and thus, their formation and breakdown are regulated by many different extracellular stimuli. 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. Formation of these complexes has been shown to require the activity of the small GTP-binding protein Rho (30Richardson A. Parsons T. Nature. 1996; 380: 538-540Crossref PubMed Scopus (450) Google Scholar, 31Richardson A. Malik R.K. Hildebrand J.D. Parsons J.T. Mol. Cell. Biol. 1997; 17: 6906-6914Crossref PubMed Scopus (287) Google Scholar). Activation of tyrosine kinases is a prerequisite for focal adhesion assembly because inhibitors of tyrosine kinase activity block cell adhesion and focal adhesion formation (28Parsons J.T. Curr. Opin. Cell Biol. 1996; 8: 146-152Crossref PubMed Scopus (277) Google Scholar). Clustering of integrins appears sufficient to recruit FAK and tensin and results in the autophosphorylation and activation of FAK (29Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1113) Google Scholar). FAK is recruited to clustered integrins by a direct interaction of the N-terminal domain of FAK and the cytoplasmic domain of the β-integrin subunits. Subsequent tyrosine phosphorylation of FAK at Tyr397 (proximal to the kinase domain) creates a binding site for Src, resulting in the formation of a complex consisting of the two tyrosine kinases. The formation of the FAK/Src complex results in the activation of Src and the subsequent activation of downstream signals (28Parsons J.T. Curr. Opin. Cell Biol. 1996; 8: 146-152Crossref PubMed Scopus (277) Google Scholar, 29Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1113) Google Scholar). The C terminus of FAK contains binding sites for a number of signaling molecules including phosphatidylinositol 3-kinase, Crk-associated substrate, GRB2, paxillin, and the GTPase regulator associated with FAK. In some cells, the C-terminal domain of FAK is expressed as a separate protein called FRNK (FAK-related non-kinase), whose overexpression results in inhibition of the rate of cell spreading and cell migration (30Richardson A. Parsons T. Nature. 1996; 380: 538-540Crossref PubMed Scopus (450) Google Scholar, 31Richardson A. Malik R.K. Hildebrand J.D. Parsons J.T. Mol. Cell. Biol. 1997; 17: 6906-6914Crossref PubMed Scopus (287) Google Scholar). These effects can be rescued by overexpression of FAK or Src, indicating that FRNK can act as a biological inhibitor of FAK signaling. We have previously cloned and characterized the related adhesion focal tyrosine kinase (RAFTK), also known as Pyk2, CAK-β, and CADTK. RAFTK is a nonreceptor tyrosine kinase that is related to FAK (32Avraham H. Price D.J. Methods. 1999; 17: 250-264Crossref PubMed Scopus (17) Google Scholar, 33Avraham S. London R. Fu Y. Ota S. Hiregowdara D. Li J. Jiang S. Pasztor L.M. White R.A. Groopman J.E. Avraham H. J. Biol. Chem. 1995; 270: 27742-27751Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 34Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1243) Google Scholar, 35Sasaki H. Nagura K. Ishino M. Tobioka H. Kotani K. Sasaki T. J. Biol. Chem. 1995; 270: 21206-21219Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 36Hiregowdara D. Avraham H. Fu Y. London R. Avraham S. J. Biol. Chem. 1997; 272: 10804-10810Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 37Salgia R. Avraham S. Pisick E. Li J.L. Raja S. Greenfield E.A. Sattler M. Avraham H. Griffin J.D. J. Biol. Chem. 1996; 271: 31222-31226Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 38Li J. Avraham H. Rogers R.A. Raja S. Avraham S. Blood. 1996; 88: 417-428Crossref PubMed Google Scholar, 39Raja S. Avraham S. Avraham H. J. Biol. Chem. 1997; 272: 10941-10947Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). It is implicated in the regulation of ion channel activity, stress responses, cell adhesion/cytoskeletal reorganization, vesicle trafficking, and nerve growth factor signaling (40Park S.Y. Avraham H. Avraham S. J. Biol. Chem. 2000; 275: 19768-19777Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). RAFTK, which regulates various cellular functions, is emerging as a critical “platform kinase” upon which a number of signaling molecules integrate (41Avraham H. Park S.Y. Schinkmann K. Avraham S. Cell Signalling. 2000; 12: 123-133Crossref PubMed Scopus (406) Google Scholar). RAFTK also associates with focal adhesion-like structures, participates in integrin-mediated signaling in megakaryocytes, and is tyrosine-phosphorylated following β1-integrin or B cell antigen receptor stimulation in B cells (42Astier A. Manie S.N. Avraham H. Hirai H. Law S.F. Zhang Y. Golemis E.A. Fu Y. Druker B.J. Haghayeghi N. Freedman A.S. Avraham S. J. Biol. Chem. 1997; 272: 19719-19724Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The assembly and disassembly of focal adhesions play a key role in the mechanism by which several extracellular stimuli regulate both cell morphology and movement. The assembly of focal adhesions in brain microvascular endothelial cells was reported (26Stoletov K.V. Ratcliffe K.E. Spring S.C. Terman B.I. J. Biol. Chem. 2001; 276: 22748-22755Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 43Shono T. Kanetake H. Kanda S. Exp. Cell Res. 2001; 264: 275-283Crossref PubMed Scopus (61) Google Scholar). In this study, we explored the cell signaling proteins that couple VEGF binding to its receptors with focal adhesion assembly in HBMECs. Materials—Human recombinant VEGF165 and anti-human VEGF monoclonal antibody were obtained from Genentech, Inc. (San Francisco, CA). Monoclonal antibodies for αvβ5 (P1F6) were used for integrin immunoprecipitation, and rabbit polyclonal anti-β5 antibodies were used for immunoblotting. Monoclonal antibodies for p130 cas and paxillin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cell Culture—HBMECs were purchased from Cell Systems, Inc. (Kirkland, WA). The cells were seeded onto attachment factor-coated culture plates and maintained in CSC complete medium according to the protocol of the manufacturer. The HBMECs formed tubular-like networks on Matrigel and produced the endothelial-specific marker, von Willebrand factor, indicating that these cells maintained the general properties of endothelial cells. Interestingly, the in vitro resistance in both rat brain microvascular endothelial cells and human brain microvascular endothelial cells is very unique, and showed measurements of 1200–1900 ohm cm2 as compared with 10–20 ohm cm2 in peripheral endothelial cells, in accordance with previous reports (44Rubin L.L. Staddon J.M. Annu. Rev. Neurosci. 1999; 22: 11-28Crossref PubMed Scopus (828) Google Scholar). During the course of the experiment, the cells were routinely checked for expression of von Willebrand factor. The cells in this study were used until passages 4–6. HBMECs were maintained in F-10 medium supplemented with 4% fetal bovine serum, 5% penicillin/streptomycin, 1% glutamine, 1% heparin, 0.7% endothelial mitogen, and 15% horse serum, and the cells were grown at 37 °C with 5% CO2. Western Blotting—The cells were lysed in kinase lysis buffer (New England Biolabs). The proteins were separated by SDS-PAGE under reducing conditions and transferred onto nitrocellulose membrane (Millipore, Boston, MA). The membranes were blocked with 5% bovine serum albumin in PBS and subsequently incubated with primary antibody for overnight incubation at 4 °C. Bound antibodies were detected by horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence (Amersham Biosciences). Focal Adhesion Assay—HBMECs were seeded onto the indicated substrate-coated coverslips: fibronectin (20 μg/ml) and gelatin (0.2%). After 4 h of incubation in serum-free CSC medium, the cells were stimulated with VEGF (20 ng/ml) for the indicated times. The cells were then fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature, and treated with blocking buffer (0.1% goat serum and 0.1% bovine serum albumin in PBS). Polyclonal anti-FAK (1:500) antibodies or anti-RAFTK antibodies (1:1000) were generated in our lab as detailed previously (36Hiregowdara D. Avraham H. Fu Y. London R. Avraham S. J. Biol. Chem. 1997; 272: 10804-10810Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 38Li J. Avraham H. Rogers R.A. Raja S. Avraham S. Blood. 1996; 88: 417-428Crossref PubMed Google Scholar, 39Raja S. Avraham S. Avraham H. J. Biol. Chem. 1997; 272: 10941-10947Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and were used for this study. Monoclonal antibody for vinculin (1:250) or control antibody was added, followed by incubation with goat anti-rabbit IgG conjugated to fluorescein (1:200). For the labeling of F-actin, Texas Red-phalloidin (Molecular Probes, Eugene, OR) (1:1000) was used. The cells were then washed again and mounted in anti-fading compound (phenylenediamine; 1 mg/ml, 50% glycerol with PBS). The fluorescent signals were detected by optical sectioning using a Leica TCS-NT confocal laser scanning microscope. A minimum of 400 cells/condition were evaluated for the presence of focal adhesions. The cells that are positive usually have ∼15–30 plaques/cell. The cells with less than 5–7 plaques/cell were scored as negative. The experiments were repeated a minimum of three times. Cell Migration Assay and Cell Spreading—The migration assay utilized a modified Boyden chamber and was performed essentially as previously described. The cells were grown to 50% confluency and then either untreated or infected with adenoviral constructs for 2 h as described below. The cells were lifted from the dish using nonenzymatic cell dissociation solution (Sigma), centrifuged for 2 min at 1000 rpm, and resuspended in medium containing 0.5% bovine serum albumin. The cells were then plated on fibronectin-precoated Transwells (Costar Corp.) at a density of 105 cells/well. Medium with VEGF (10 ng/ml) was used as a chemoattractant in the lower wells. Cell migration assays were performed for 8 h at 37 °C. The cells that migrated through the Transwells were stained with chloromethylfluorescein diacetate dye (Sigma) and counted under a fluorescent microscope. Four different fields were counted for each experiment, and all of the samples were performed in triplicate. For cell spreading, the cells were detached with 1 mm EDTA and washed with PBS, and then 1 × 105 cells were plated on fibronectin-coated coverslips in serum-free medium. After the indicated times, the coverslips were fixed, stained, and analyzed by fluorescent microscopy. For quantification of the spreading cells, 150–200 cells were scored from each of three independent experiments. Adenovirus Constructs and Infections—Replication-deficient recombinant adenoviruses mediating overexpression have been previously described (45Melendez J. Welch S. Schaefer E. Moravec C.S. Avraham S. Avraham H. Sussman M.A. J. Biol. Chem. 2002; 277: 45203-45210Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). RAFTK/Pyk2 cDNA fragments encoding both wild type (Pyk2/RAFTK-WT) and a phosphorylation-deficient mutant (Pyk2/RAFTK-Y402) were used to create replication-defective recombinant adenoviruses with the AdEasy system (Microbix Biosystem Inc.) as directed by the manufacturer. The Tyr402 residue of RAFTK is the autophosphorylation site that also binds and activates Src. Mutation in this residue blocks autophosphorylation of RAFTK and its binding to Src, leading to the abrogation of Src phosphorylation. Subconfluent HBMEC cultures were infected with recombinant adenoviruses, including β-galactosidase expressing adenovirus as a control, for 2 h at a multiplicity of infection of 10, and then the medium was aspirated and replaced with maintenance medium. The lysates for the biochemical analyses were prepared ∼24–48 h after infection. A replication-defective adenovirus encoding GFP-tagged FRNK was also constructed as previously described (46Heidkamp M.C. Bayer A.L. Kalina J.A. Eble D.M. Samarel A.M. Circ. Res. 2002; 90: 1240-1242Crossref PubMed Scopus (111) Google Scholar). An adenovirus expressing GFP alone was used to control for the nonspecific effects of adenoviral infection. HBMECs were infected at a multiplicity of infection of 10 for 2 h at 25 °C with gentle agitation with replication-defective adenoviruses diluted in medium. The medium was then replaced with virus-free medium, and the cells were cultured for an additional 48 h before assaying for cell migration and focal adhesion assembly. Immunoprecipitation and Immunoblotting—For co-immunoprecipitation of FAK with integrin αvβ5, HBMECs were lysed in HNG buffer (50 mm Hepes, pH 7.4, 150 mm NaCl, 10% glycerol) containing 1% Brje. SDS-PAGE and immunoblotting were performed as previously described (47Lee T.H. Avraham H. Lee S.H. Avraham S. J. Biol. Chem. 2002; 277: 10445-10451Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 48Lee T.H. Avraham H.K. Jiang S. Avraham S. J. Biol. Chem. 2003; 278: 5277-5284Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Data Analysis—The results are expressed as the means ± S.D. Differences among the means were considered significant at p < 0.05. The data were analyzed using the SigmaStat statistical software package, version 1.0 (Jandel Scientific, San Rafael, CA). HBMECs as a Model System—We chose to carry out these studies in HBMECs because of their direct application as a model system for the in vivo system of the human BBB and for their high relevance to the studies of ischemia and stroke in humans. HBMECs formed tubular-like networks on Matrigel and had the ability to uptake acetylated low density lipoprotein (47Lee T.H. Avraham H. Lee S.H. Avraham S. J. Biol. Chem. 2002; 277: 10445-10451Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 49Radisavljevic Z. Avraham H. Avraham S. J. Biol. Chem. 2000; 275: 20770-20774Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) and to produce von Willebrand factor, indicating that these cells maintained the general properties of endothelial cells. HBMECs also produced γ-glutamyl transpeptidase, a brain endothelial marker. HBMECs maintained the unique characteristic of resistance greater than 1000–2000 ohm/cm2 as compared with the 10–20 ohm/cm2 characteristic of peripheral endothelial cells. HBMECs constitute the major component of the BBB and are therefore critical in maintaining its structural and functional integrity. VEGF Induced the Formation of Focal Adhesions—Cell adhesion to ECM is a highly dynamic process involving structures heterogeneous with respect to size, composition, and orientation to actin filaments (50O'Neill G.M. Fashena S.J. Golemis E.A. Trends Cell Biol. 2000; 10: 111-119Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 51Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2802) Google Scholar, 52Broday D.M. Bull. Math Biol. 2000; 62: 891-924Crossref PubMed Scopus (17) Google Scholar). The largest and tightest structures are usually referred to as focal adhesions, which link the actin cytoskeleton to ECM by integrin receptor complexes. Actin-binding proteins that co-localize with integrins in focal adhesions include actin, talin, vinculin, and tensin. Focal contacts were detected by staining the fixed cells either with the monoclonal antibody to vinculin, a cytoskeletal protein that localizes at focal adhesion contacts (52Broday D.M. Bull. Math Biol. 2000; 62: 891-924Crossref PubMed Scopus (17) Google Scholar, 53Girard P.R. Nerem R.M. J. Cell. Physiol. 1995; 163: 179-193Crossref PubMed Scopus (220) Google Scholar, 54Parsons J.T. Martin K.H. Slack J.K. Taylor J.M. Weed S.A. Oncogene. 2000; 19: 5606-5613Crossref PubMed Scopus (555) Google Scholar, 55Sieg D.J. Ilic D. Jones K.C. Damsky C.H. Hunter T. Schlaepfer D.D. EMBO J. 1998; 17: 5933-5947Crossref PubMed Scopus (286) Google Scholar), or with FAK monoclonal antibody (54Parsons J.T. Martin K.H. Slack J.K. Taylor J.M. Weed S.A. Oncogene. 2000; 19: 5606-5613Crossref PubMed Scopus (555) Google Scholar). 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 μg/ml) (integrin-dependent) or gelatin (0.2%) (integrin-independent). After 4 h of incubation in serum-free CSC medium, the cells were stimulated with VEGF (20 ng/ml) for 1 or 4 h as indicated. The cells were then fixed and immunostained for FAK- and RAFTK-specific antibodies. Actin stress fibers were also analyzed using Texas Red-phalloidin (Molecular Probes). Clear actin stress fibers that cross the cell body and condense at the cell periphery were observed in HBMECs (Fig. 1, red). No focal adhesion sites were observed when HBMECs were seeded on gelatin (0.2%). Upon VEGF stimulation for 1 h, we observed the dynamic redistribution of FAK with actin in the outer membrane, which was increased upon 4 h of stimulation of the HBMECs with VEGF. When HBMECs were seeded on fibronectin, upon 1 h of treatment with VEGF, a significant co-redistribution of FAK with actin to the outer side of the cells was enhanced (Fig. 1B). After 4 h of stimulation with VEGF, there was a significant increase in the dynamic co-redistribution of FAK and actin with the formation of focal contacts. Similarly, focal contacts immunostained with RAFTK/Pyk2 were detected after their seeding on fibronectin and stimulation with VEGF (Fig. 1C). These results suggest that VEGF plays a role in focal adhesion assembly in HBMECs and that FAK and RAFTK/Pyk2 are important components of these focal adhesion sites. To further analyze the effects of VEGF on focal adhesion assemb" @default.
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• W2029315212 date "2003-09-01" @default.
• W2029315212 modified "2023-10-16" @default.
• W2029315212 title "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" @default.
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