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• W2034328033 abstract "Brain-derived neurotrophic factor (BDNF) is expressed by endothelial cells. We investigated the characteristics of BDNF expression by brain-derived endothelial cells and tested the hypothesis that BDNF serves paracrine and autocrine functions affecting the vasculature of the central nervous system. In addition to expressing TrkB and p75NTR and BDNF under normoxic conditions, these cells increased their expression of BDNF under hypoxia. While the expression of TrkB is unaffected by hypoxia, TrkB exhibits a base-line phosphorylation under normoxic conditions and an increased phosphorylation when BDNF is added. TrkB phosphorylation is decreased when endogenous BDNF is sequestered by soluble TrkB. Exogenous BDNF elicits robust angiogenesis and survival in three-dimensional cultures of these endothelial cells, while sequestration of endogenous BDNF caused significant apoptosis. The effects of BDNF engagement of TrkB appears to be mediated via the phosphatidylinositol (PI) 3-kinase-Akt pathway. Modulation of BDNF levels directly correlate with Akt phosphorylation and inhibitors of PI 3-kinase abrogate the BDNF responses. BDNF-mediated effects on endothelial cell survival/apoptosis correlated directly with activation of caspase 3. These endothelial cells also express p75NTR and respond to its preferred ligand, pro-nerve growth factor (pro-NGF), by undergoing apoptosis. These data support a role for neurotrophins signaling in the dynamic maintenance/differentiation of central nervous system endothelia. Brain-derived neurotrophic factor (BDNF) is expressed by endothelial cells. We investigated the characteristics of BDNF expression by brain-derived endothelial cells and tested the hypothesis that BDNF serves paracrine and autocrine functions affecting the vasculature of the central nervous system. In addition to expressing TrkB and p75NTR and BDNF under normoxic conditions, these cells increased their expression of BDNF under hypoxia. While the expression of TrkB is unaffected by hypoxia, TrkB exhibits a base-line phosphorylation under normoxic conditions and an increased phosphorylation when BDNF is added. TrkB phosphorylation is decreased when endogenous BDNF is sequestered by soluble TrkB. Exogenous BDNF elicits robust angiogenesis and survival in three-dimensional cultures of these endothelial cells, while sequestration of endogenous BDNF caused significant apoptosis. The effects of BDNF engagement of TrkB appears to be mediated via the phosphatidylinositol (PI) 3-kinase-Akt pathway. Modulation of BDNF levels directly correlate with Akt phosphorylation and inhibitors of PI 3-kinase abrogate the BDNF responses. BDNF-mediated effects on endothelial cell survival/apoptosis correlated directly with activation of caspase 3. These endothelial cells also express p75NTR and respond to its preferred ligand, pro-nerve growth factor (pro-NGF), by undergoing apoptosis. These data support a role for neurotrophins signaling in the dynamic maintenance/differentiation of central nervous system endothelia. Angiogenesis is a tightly controlled process in which new vessels form from those pre-existing. This process occurs in a regulated fashion during development and growth as well as in response to physiological and pathological stimuli. Angiogenesis as been shown to be a receptor- and ligand-regulated process, with a still growing, diverse number of soluble factors and their cognate receptors being involved in the different phases of the angiogenic process (1Yancopoulos G.D. Klagsbrun M. Folkman J. Cell. 1998; 93: 661-664Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). In the developing brain, angiogenesis has been shown to be regulated by factors secreted by neuronal and glial cell populations in an orderly, spatiotemporal fashion (2Ogunshola O.O. Stewart W.B. Mihalcik V. Solli T. Madri J.A. Ment L.R. Brain Res. Dev. Brain Res. 2000; 119: 139-153Crossref PubMed Scopus (219) Google Scholar). In recent studies we and others have (3Ogunshola O.O. Antic A. Donoghue M.J. Fan S.Y. Kim H. Stewart W.B. Madri J.A. Ment L.R. J. Biol. Chem. 2002; 277: 11410-11415Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 4Sondell M. Sundler f. Kanje M. Eur. J. Neurosci. 2000; 12: 4243-4254Crossref PubMed Scopus (336) Google Scholar) shown that selected angiogenic factors, VEGF 1The abbreviations used are: VEGF, vascular endothelial factor; VEGFR, VEGF receptor; BDNF, brain-derived neurotrophic factor; rBDNF, recombinant BDNF; NT, neurotrophin; NGF, nerve growth factor; PI, phosphatidylinositol; HA, hemagglutinin; FACS, fluorescence-activated cell sorter; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. 1The abbreviations used are: VEGF, vascular endothelial factor; VEGFR, VEGF receptor; BDNF, brain-derived neurotrophic factor; rBDNF, recombinant BDNF; NT, neurotrophin; NGF, nerve growth factor; PI, phosphatidylinositol; HA, hemagglutinin; FACS, fluorescence-activated cell sorter; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. in particular, are capable of not only affecting a variety of endothelial behaviors but also are capable of affecting neuronal behavior in a receptor-specific fashion. Interestingly, recent studies have demonstrated that neurotrophins expressed by endothelia and are capable of influencing several endothelial cell functions including endothelial cell survival and vessel stabilization (5Leventhal C. Rafii S. Rafii D. Shahar A. Goldman S.A. Mol. Cell. Neurosci. 1999; 13: 450-464Crossref PubMed Scopus (342) Google Scholar, 6Nakahashi T. Fujimura H. Altar C.A. Li J. Kambayashi J. Tandon N.N. Sun B. FEBS Lett. 2000; 470: 113-117Crossref PubMed Scopus (336) Google Scholar, 7Donovan M.J. Lin M.I. Wiegn P. Ringstedt T. Kraemer R. Hahn R. Wang S. Ibanez C.F. Rafii S. Hempstead B.L. Development (Camb.). 2000; 127: 4531-4540PubMed Google Scholar) and that endothelial cells may express neurotrophin receptors (8Ricci A. Greco S. Amenta F. Bronzetti E. Felici L. Rossodivata I. Sabbatini M. Mariotta S. J. Vasc. Res. 2000; 37: 355-363Crossref PubMed Scopus (30) Google Scholar). Neurotrophins form a large family of dimeric polypeptides that include nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5, NT-6, and NT-7 (9Leibrock J. Lottspeich F. Hohn A. Hofer M. Hengerer B. Masiakowski P. Thoenen H. Barde Y.A. Nature. 1989; 341: 149-152Crossref PubMed Scopus (1231) Google Scholar, 10Hofer M. Pagliusi S.R. Hohn A. Leibrock J. Barde Y.A. EMBO J. 1990; 9: 2459-2464Crossref PubMed Scopus (840) Google Scholar, 11Hohn A. Leibrock J. Bailey K. Barde Y.A. Nature. 1990; 22: 339-341Crossref Scopus (943) Google Scholar, 12Kolbeck R. Jungbluth S. Barde Y.A. Eur. J. Biochem. 1994; 225: 995-1003Crossref PubMed Scopus (68) Google Scholar). They are known to promote the growth, survival, and differentiation of developing neurons in the central and peripheral nervous systems (13Barde Y.A. Prog. Growth Factor Res. 1990; 2: 237-248Abstract Full Text PDF PubMed Scopus (265) Google Scholar, 14Lindsay R.M. Thoenen H. Barde Y.A. Dev. Biol. 1985; 112: 319-328Crossref PubMed Scopus (317) Google Scholar, 15Sobue G. Yamamoto M. Doyu M. Li M. Yasuda T. Mitsuma T. Neurochem. Res. 1998; 23: 821-829Crossref PubMed Scopus (65) Google Scholar, 16Tucker K.L. Meyer M. Barde Y.A. Nat. Neurosci. 2001; 4: 29-37Crossref PubMed Scopus (428) Google Scholar, 17Sofroniew M.V. Howe C.L. Mobley W.C. Annu. Rev. Neurosci. 2001; 24: 1217-1281Crossref PubMed Scopus (1092) Google Scholar, 18Huang E.J. Reichardt L.F. Annu. Rev. Neurosci. 2001; 24: 677-736Crossref PubMed Scopus (3333) Google Scholar). BDNF, given peripherally, accelerates the regenerative sprouting of injured adult spinal motor neurons and axotomized retinal ganglion cells (19Novikov L. Novikova L. Kellerth J.O. Neuroscience. 1997; 79: 765-774Crossref PubMed Scopus (175) Google Scholar). Therefore, BDNF appears to be involved in peripheral sensory and motor neuron regeneration at the site of nerve injury. Neurotrophins mediate their action on responsive neurons by binding to two classes of cell surface receptor (20Huang E.J. Reichardt L.F. Annu. Rev. Biochem. 2003; 72: 609-642Crossref PubMed Scopus (1953) Google Scholar). TrkA, TrkB, and TrkC selectively bind brain growth factor, BDNF, and NT-3 (21Snider W.D. Cell. 1994; 77: 627-638Abstract Full Text PDF PubMed Scopus (1310) Google Scholar). In addition, the neurotrophins can interact with another low affinity neurotrophin receptor, p75NTR, which has been shown to initiate an apoptotic signal in neurons when engaged by pro-NGF (22Roux P.P. Barker P.A. Prog. Neurobiol. 2002; 67: 203-233Crossref PubMed Scopus (593) Google Scholar, 23Lee R. Kermani P. Teng K.K. Hempstead B.L. Science. 2001; 294: 1945-1948Crossref PubMed Scopus (1377) Google Scholar). TrkB and BDNF are expressed at high levels not only in central and peripheral nervous tissue (24Ernfors P. Lee K.F. Jaenisch R. Nature. 1994; 368: 147-150Crossref PubMed Scopus (899) Google Scholar, 25Jones K.R. Farinas I. Backus C. Reichardt L.F. Cell. 1994; 76: 989-999Abstract Full Text PDF PubMed Scopus (901) Google Scholar, 26Schwartz P.M. Borghesani P.R. Levy R.L. Pomeroy S.L. Segal R.A. Neuron. 1997; 19: 269-291Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar), but also in several nonneuronal tissues, including muscle, heart, and the vasculature at levels comparable with those of the brain (27Scarisbrick I.A. Jones E.G. Isackson P.J. J. Neurosci. 1993; 13: 875-893Crossref PubMed Google Scholar, 28Timmusk T. Belluardo N. Metsis M. Persson H. Eur. J. Neurosci. 1993; 5: 605-613Crossref PubMed Scopus (230) Google Scholar, 29Donovan M.J. Hahn R. Tessarollo L. Hempstead B.L. Nat. Genet. 1996; 14: 210-213Crossref PubMed Scopus (159) Google Scholar, 30Hiltunen J.O. Arumae U. Moshnyakov M. Saarma M. Circ. Res. 1996; 79: 930-939Crossref PubMed Scopus (53) Google Scholar). In pathologic states, BDNF and TrkB expression are induced in neointimal vascular smooth muscle cells of the adult rodent and human aorta following vascular injury (31Donovan M.J. Miranda R.C. Kraemer R. McCaffrey T.A. Tessarollo L. Mahadeo D. Sharif S. Kaplan D.R. Tsoulfas P. Parada L. Toran-Allerand C.D. Hajjar D.P. Hempstead B.L. Am. J. Pathol. 1995; 147: 309-324PubMed Google Scholar). These studies suggest that there may be a complex and dynamically regulated cross-talk between neuronal cells and endothelial cells during development, growth, and in response to pathological stimuli in the brain and prompted us to investigate these potential interactions. In this report we have demonstrated the expression of BDNF by brain-derived endothelial cells and the expression and activation of the neurotrophin receptors TrkB and p75NTR in these brain-derived endothelial cells. In addition, we have shown that engagement of either TrkB or p75NTR (by BDNF and pro-NGF, respectively) results in distinct endothelial behaviors, survival and angiogenesis in the case of BDNF activation of TrkB and apoptosis in the case of pro-NGF activation of p75NTR. Furthermore, the importance of these findings in the control of neurovascular development and responses to chronic sublethal hypoxic injury is discussed. Recombinant NGF and Pro-NGF—Cleavage-resistant pro-NGF was purified from the media of cells stably expressing the construct, using nickel chromatography and imidazole elution as described (23Lee R. Kermani P. Teng K.K. Hempstead B.L. Science. 2001; 294: 1945-1948Crossref PubMed Scopus (1377) Google Scholar). Mature NGF or media from cells expressing the plasmid were used in parallel (23Lee R. Kermani P. Teng K.K. Hempstead B.L. Science. 2001; 294: 1945-1948Crossref PubMed Scopus (1377) Google Scholar). Cell Culture; RBE4 and bEnd-WT Cell Culture—Transformed rat brain endothelial (RBE4) cells were obtained from F. Roux (Hospital F. Widal, Paris, France). The RBE4 cells were cultured from passages 16-25 as described previously (32Chow J. Ogunshola O. Fan S.Y. Li Y. Ment L.R. Madri J.A. Res. Dev. Brain Res. 2001; 130: 123-132Crossref PubMed Scopus (87) Google Scholar). Immortalized mouse brain endothelial cells (bEnd-WT) were obtained from Dr. Britta Engelhardt (Max-Planck-Institute for Vascular Biology, Münster, Germany) and were cultured and passaged as described (33Graesser D. Solowiej A. Bruckner M. Osterweil E. Juedes A. Davis S. Ruddle N.H. Engelhardt B. Madri J.A. J. Clin. Invest. 2002; 109: 383-392Crossref PubMed Scopus (270) Google Scholar). For three-dimensional culture experiments, acid-soluble calf dermis type I collagen (ASC I) was prepared and solubilized in 10 mm acetic acid (2.5 mg/ml) as described previously (32Chow J. Ogunshola O. Fan S.Y. Li Y. Ment L.R. Madri J.A. Res. Dev. Brain Res. 2001; 130: 123-132Crossref PubMed Scopus (87) Google Scholar). RBE4 or bEnd-WT cells were added to the collagen to a final concentration of 2 × 105 cells/ml. Droplets of the cell-collagen suspension were spotted onto Petri dishes. Following polymerization, the droplets were overlaid with media (α-minimal essential medium and F-10 nutrient mixture with glutamine, basic fibroblast growth factor, Geneticin, and 10% fetal bovine serum) and incubated in 5% CO2 at 37 °C. For RBE4 and bEnd-WT culture experiments, recombinant BDNF at concentrations of 10 and 50 ng/ml, soluble, recombinant TrkB receptor bodies (R&D Systems, Minneapolis, MN) at a concentration of 2 μg/ml; pro-NGF at concentrations of 1, 5, and 10 ng/ml; and mature NGF at a concentration of 50 ng/ml were added. Wortmannin, LY294002, and PD98059 were purchased from Sigma. Cells were cultured for 6 days. Medium was changed, and recombinant proteins were added every 24 h. All hypoxia experiments were performed with cells incubated in a sealed, humidified chamber gassed with 10% O2, 5% CO2, 85% N2 at 37 °C as described (32Chow J. Ogunshola O. Fan S.Y. Li Y. Ment L.R. Madri J.A. Res. Dev. Brain Res. 2001; 130: 123-132Crossref PubMed Scopus (87) Google Scholar). Transfection of bEnd-WT Cells—bEnd-WT cells were infected with recombinant adenoviruses at ∼90% confluence. Cells were infected with adenovirus containing HA-tagged dominant negative Akt (Akt-AAA) with a marker of green fluorescent protein (a generous gift of Dr. William Sessa, Yale University) in serum-free Dulbecco's modified Eagle's medium medium for 1 h and then incubated for 24 h in complete growth medium as described (34Fujio Y. Walsh K. J. Biol. Chem. 1999; 274: 16349-16354Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar, 35Boo Y.C. Sorescu G. Boyd N. Shiojima I. Walsh K. Du J. Jo H. J. Biol. Chem. 2002; 277: 3388-3396Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 36Kim I. Oh J.L. Ryu Y.S. So J.N. Sessa W.C. Walsh K. Koh G.Y. FASEB J. 2002; 16: 126-128PubMed Google Scholar, 37Morales-Ruiz M. Fulton D. Sowa G. Languino L.R. Fujio Y. Walsh K. Sessa W.C. Cir. Res. 2000; 86: 892-896Crossref PubMed Scopus (349) Google Scholar) before the start of expereiments. Recombinant adenovirus encoding β-galactosidase was used as a control. Infection efficiency of bEnd-WT cells with recombinant adenoviruses at 40 multiplicity of infection was close to 100% as determined by the green fluorescent color observed in the cells and immunohistochemical staining of β-galactosidase. The expression and relative levels of endogenous and recombinant adenoviral Akt were confirmed by Western blotting. Matrigel Assay—BD Matrigel™ matrix was used to coat tissue culture dishes according to the manufacturer's instructions (BD Biosciences). Cells were plated onto the matrix at a density of 5 × 105 cells per 30-mm plate and allowed to grow for various times. At specific time points, light microscopy images were taken and analyzed, and cell lysates were prepared as described (38Solowiej A. Biswas P. Graesser D. Madri J.A. Am. J. Pathol. 2003; 162: 953-962Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Vessel Counting—Vessel counts were performed with three samples per condition. Three random fields were photographed per sample. Random digital images of cultures were taken using an inverted research microscope (Olympus Co.) equipped with Nikon coolpix 995 digital camera using Photoshop 5.0 software on a Macintosh G4 computer. NIH Image 1.62 or IP LabSpectrum software was used to select, measure, and analyze the images to determine aggregate tube length. Data were expressed in terms of pixel change (NIH Image) or microns (IP LabSpectrum) compared with normoxic (5% CO2 and room air (20% O2)) controls. Statistics (Student's t test and standard error) were calculated and graphically presented using Excel 2000 on a Macintosh G4 computer. Statistical significance was assumed for p values < 0.05. Immunoprecipitation and Western Blotting—Cell lysates and subsequent immunoprecipitation with anti-VEGFR-2/Flt-1 and Western blotting with anti-VEGFR-2/Flt-1 and anti-PY (PY 99) antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were performed as described (39Ilan N. Tucker A. Madri J.A. Lab. Invest. 2003; 83: 1105-1115Crossref PubMed Scopus (44) Google Scholar). Western blots were performed on lysates of RBE4 and bEnd-WT cells as described previously (3Ogunshola O.O. Antic A. Donoghue M.J. Fan S.Y. Kim H. Stewart W.B. Madri J.A. Ment L.R. J. Biol. Chem. 2002; 277: 11410-11415Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 32Chow J. Ogunshola O. Fan S.Y. Li Y. Ment L.R. Madri J.A. Res. Dev. Brain Res. 2001; 130: 123-132Crossref PubMed Scopus (87) Google Scholar). Lysates were made with Modified RIPA buffer (50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, leupeptin, 1 mm Na3VO4, 1 mm NaF). Antisera directed against BDNF (Santa Cruz Biotechnology, Inc., SC-546 at 1:200), TrkB (BD Biosciences, 610101 at 1:1,000 and Santa Cruz, SC-8316 at 1:1,000), pTrkB (Cell Signaling Technology, Inc., 9141 at 1:1,000), p75NTR (Santa Cruz Biotechnology, Inc., SC-8317 at 1:200), Flk-1 (VEGFR2) (Santa Cruz technology, SC-504 at 1:200), pERK (Cell Signaling Technology, Inc., 3191 at 1:1,000), ERK2 (Santa Cruz Biotechnology, Inc., SC-1647 at 1:10,000), pAkt (Cell Signaling Technology, Inc., 9171 at 1:1,000), Akt (Cell Signaling Technology, Inc., 9272 at 1:1,000), cleaved caspase 3 (Cell Signaling Technology, Inc., 9664 at 1:1,000) were used. Detection was carried out using Pierce supersignal detection reagent (Pierce) with membrane exposure to Hyperfilm reagent (Amersham Biosciences). Quantitation was performed on scanned images (Agfa Arcus II Scanner using Adobe Photoshop 5.0, Adobe Systems) using the BioMax Program (Eastman Kodak) on a Macintosh G4 computer. All experiments were performed at least three times. Statistical analysis was performed using Student's t test (p < 0.05). Immunocytochemistry—Cultured RBE4 cells were fixed with 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, and blocked with phosphate-buffered saline in 3% bovine serum albumin, 10% normal donkey serum, 0.1% Triton X-100. The primary antibodies used were anti-BDNF (Santa Cruz Biotechnology, Inc., SC-546 at 1:200) and anti-TrkB (Santa Cruz Biotechnology, Inc., SC-8316 at 1:200), anti-p75NTR (Santa Cruz Biotechnology, Inc., SC-8317 at 1:200). Primary antibodies were incubated overnight at 4 °C. CY3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc., Bar Harbor, ME) was used as secondary antibody. Images were taken using a Zeiss research microscope equipped with a SPOT camera. Images were collected using Photoshop 5.0 on a Macintosh G4 computer. FACS Analysis of Apoptosis—FACS analysis of cultured, transfected bEnd-WT cells was performed as described previously (33Graesser D. Solowiej A. Bruckner M. Osterweil E. Juedes A. Davis S. Ruddle N.H. Engelhardt B. Madri J.A. J. Clin. Invest. 2002; 109: 383-392Crossref PubMed Scopus (270) Google Scholar). Propidium iodide and Annexin V (BD Biosciences) were used to assess apoptotic cells. BD FACStation Software for Mac OSX was used to analyze the results, and Statview Software was used to determine significance. BDNF Expression Is Induced by Hypoxia in Vivo in the Microvasculature of the Central Nervous System and Is Expressed by RBE4 Cells and Induced in These Cells by Hypoxia in Vitro—Imunohistochemical staining of cortical sections of pups reared in normoxia (Nx) and hypoxia (Hx) revealed increased expression of BDNF protein, primarily in the microvasculature Fig. 1, A-D). We then performed immunofluorescence microscopy to assess the expression of BDNF on RBE4 cells. We also performed Western blotting on RBE4 cell lysates. We determined that RBE4 cells expressed BDNF under baseline (normoxic (Nx)) culture conditions. Interestingly, under hypoxic conditions (hypoxic (Hx)) BDNF expression was found to be increased in both RBE4 cells and astrocytes (data not shown) using both immunofluorescence and Western blotting methods (n = 5; p < 0.03) (Fig. 1, E-H). BDNF was localized in essentially all RBE4 cells, and its expression was noted to be significantly increased under hypoxic culture conditions. RBE4 Cells Form Tubes and Sprouts in Collagen Gels, Which Are Enhanced by Recombinant BDNF but Blocked by Soluble, Recombinant TrkB—RBE4 cells placed in three-dimensional matrices of collagen type I gel and cultured for 6 days cluster to form multicellular cysts, from which elongated tube-like processes extend (32Chow J. Ogunshola O. Fan S.Y. Li Y. Ment L.R. Madri J.A. Res. Dev. Brain Res. 2001; 130: 123-132Crossref PubMed Scopus (87) Google Scholar). Addition of exogenous recombinant BDNF stimulated increased tube formation in cultures (Fig. 2, compare A and B), while addition of recombinant, soluble TrkB (which sequesters BDNF) acted to inhibit the cyst and tube formation of RBE4 cells (Fig. 2, compare C with A and B). When cultured under hypoxic conditions, tube formation of RBE4 cells was reduced compared with that of normoxic cultures (Fig. 2, compare D and A). As noted in normoxic cultures, treatment with exogenous BDNF stimulated tube formation (Fig. 2E), and treatment with soluble, recombinant TrkB significantly reduced cyst and tube formation (Fig. 2F). Fig. 2G represents a quantitation analysis of these studies. Similar results were obtained when bEnd-WT cells were used. Representative fields of bEnd-WT cells cultured on Matrigel coatings under normoxic conditions in the absence or presence of 10 ng/ml rBDNF or 2.0 μg/ml rTrkB are illustrated in Fig. 2, H-J. Note the increased amount of tube formation in the presence of rBDNF and the decreased amount of tube formation in the presence of rTrkB. Quantitation of bEnd-WT cell tube formation (Fig. 2I) in normoxic conditions in the absence or presence of rBDNF or rTrkB revealed a robust increase in tube formation in the presence of rBDNF and a marked decrease in tube formation when rTrkB was added to the cultures. TrkB Is Expressed by and Activated by BDNF in RBE4 Cells—To determine whether TrkB is expressed on RBE4 cells, we performed Western blot analyses of lysates of RBE4 cells derived from normoxic and hypoxic cultures. Western blotting illustrated the presence of TrkB in RBE4 cell lysates, and overall, protein levels of TrkB remained unchanged in response to hypoxic stimulation (Fig. 3A). To determine whether the TrkB present in these brain-derive endothelial cells is activated, we performed Western bolts using anti-pTrkB followed by Western blotting analysis using anti-TrkB. We found that a fraction of TrkB was tyrosinephosphorylated under base-line culture conditions, and phosphorylated TrkB levels were increased following the addition of exogenous BDNF (Fig. 3B). Additionally, treatment of RBE4 cultures with recombinant, soluble TrkB resulted in a significant reduction of phosphorylated TrkB compared with the control (normoxic) cultures. Similar results were observed under hypoxic conditions. These results suggest that TrkB expressed on RBE4 cells is activated by endogenous and exogenous BDNF. Akt, but Not Mitogen-activated Protein Kinase, Activation Is Associated with BDNF-mediated RBE4 Cell Survival in Vitro—To elucidate the mechanisms involved in this BDNF-mediated endothelial cell survival and tube formation in this culture system, we assessed the activation states of Akt and ERK1/2, members of two signaling pathways known to be involved in mediating endothelial survival and tube formation (32Chow J. Ogunshola O. Fan S.Y. Li Y. Ment L.R. Madri J.A. Res. Dev. Brain Res. 2001; 130: 123-132Crossref PubMed Scopus (87) Google Scholar, 40Ilan N. Mahooti S. Madri J.A. J. Cell Sci. 1998; 111: 3621-3631Crossref PubMed Google Scholar). The level of phosphorylated ERK was significantly increased by hypoxia but not following treatment of exogenous BDNF or soluble, recombinant TrkB (Fig. 4B). In contrast, the level of serine-phosphorylated Akt was significantly increased following treatment with BDNF under normoxic and hypoxic conditions (Fig. 4A). In addition, treatment with soluble, recombinant TrkB reduced the levels of phosphorylated Akt in normoxic and hypoxic cultures (Fig. 4A). These results suggest that Akt is activated following BDNF engagement and activation of TrkB and this pathway may be in part responsible for mediating the survival of these endothelial cells. To confirm the role of Akt activation in mediating brain-derived endothelial survival bEnd-WT, cells were infected with a dominant negative HA-tagged Akt construct (Akt-AAA) or a β-galactosidase-containing vector and apoptotic levels determined following normal culture conditions, serum starvation, and BDNF treatment. While a base-line low apoptotic level was noted in cells infected with the β-galactosidase containing vector with and without addition of exogenous BDNF (approximately 10.6% ± 1.3%), high apoptotic levels were observed in the β-galactosidase vector-infected cells cultured under serum starvation (17.3%). In contrast, cells infected with the dominant negative Akt-AAA construct exhibited high apoptotic rates in the absence (approximately 17.3% ± 2%) and presence (approximately 20.5% ± 2%) of exogenous BDNF (Fig. 4C). These results lend additional support to the concept that Akt is activated following BDNF engagement, and activation of TrkB and this pathway may be in part responsible for mediating the survival of these endothelial cells. Exogenous BDNF Blunted Activation of Caspase 3, while Soluble, Recombinant TrkB Induced Activation of Caspase 3 in RBE4 Cells—Additional studies revealed that BDNF modulated caspase 3 cleavage. Exogenous BDNF induced tube formation and rescued the cells from hypoxic insult. Under normoxic conditions, cleaved caspase 3 expression was decreased significantly by addition of exogenous BDNF and was increased following treatment with recombinant soluble TrkB (Fig. 5). Culture of RBE4 cells under hypoxic conditions induced activation of caspase 3; however, cleaved caspase 3 levels were significantly decreased following the addition of exogenous BDNF to hypoxic cultures. As noted above, soluble, recombinant TrkB further increased the activation of caspase 3 in hypoxic cultures (Fig. 5). These results suggest that BDNF modulates apoptosis in these brain-derived endothelial cells in part by regulating caspase 3 activity. Modulation of Cleaved Caspase 3 Expression by PI 3-Kinase and MEK Inhibitors on BDNF-treated Endothelial Cells under Hypoxia—To further determine the specific signaling pathways involved in the BDNF-induced inhibition of apoptosis and inhibition of caspase 3 activation, a pharmacological approach was taken. Namely, chemical inhibitors of either MEK (PD98059, 20 μm) or PI 3-kinase (LY240002 and wortmannin, 20 μm (data not shown)) were added daily for 6 days to RBE4 cultures. Under normoxic conditions, 20 μm LY240002, but not PD98059, significantly increased the levels of cleaved caspase 3 in cultures treated with BDNF (Fig. 6). Under hypoxic conditions, these PI 3-kinase inhibitors, but not the MEK inhibitor, also increased the levels of cleaved caspase 3 significantly in cultures treated with BDNF (Fig. 6). These data suggest that the PI 3-kinase signaling pathway is involved in the BDNF-mediated modulation of RBE4 cell caspase activation and survival behavior. BDNF Treatment Increases VEGFR2 Expression on RBE4 Cells Cultured under Normoxic and Hypoxic Conditions—As we and others (3Ogunshola O.O. Antic A. Donoghue M.J. Fan S.Y. Kim H. Stewart W.B. Madri J.A. Ment L.R. J. Biol. Chem. 2002; 277: 11410-11415Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 32Chow J. Ogunshola O. Fan S.Y. Li Y. Ment L.R. Madri J.A. Res. Dev. Brain Res. 2001; 130: 123-132Crossref PubMed Scopus (87) Google Scholar) have reported, VEGF is a potent angiogenic and survival factor in the central nervous system, affecting both endothelial cells and neurons. In previous studies we determined that chronic hypoxia induces VEGF expression in rodent cerebral cortex, specifically by neurons and glia and by astrocytes and cortical neurons in culture (2Ogunshola O.O. Stewart W.B. Mihalcik V. Solli T. Madri J.A. Ment L.R. Brain Res. Dev. Brain Res. 2000; 119: 139-153Crossref PubMed Scopus (219) Google Scholar, 3Ogunshola O.O. Antic A. Donoghue M.J. Fan S.Y. Kim H. Stewart W.B. Madri J.A. Ment L.R. J. Biol. Chem. 2002; 277: 11410-11415Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 41Ment L.R. Stewart W.B. Scaramuzzino D. Madri J.A. In Vitro Cell Dev. Biol. Anim. 1997; 33: 684-691Crossref PubMed Scopus (25) Google Scholar, 42Ment L.R. Stewart W.B. Fronc R. Seashore C. Mahooti S. Scaramuzzino D. Madri J.A. Brain Res. Dev. Brain Res. 1997; 100: 52-61Crossref PubMed Scopus (68) Google Scholar). Considering that BD" @default.
• W2034328033 created "2016-06-24" @default.
• W2034328033 creator A5016134289 @default.
• W2034328033 creator A5020746135 @default.
• W2034328033 creator A5033091196 @default.
• W2034328033 creator A5086974646 @default.
• W2034328033 date "2004-08-01" @default.
• W2034328033 modified "2023-10-18" @default.
• W2034328033 title "Paracrine and Autocrine Functions of Brain-derived Neurotrophic Factor (BDNF) and Nerve Growth Factor (NGF) in Brain-derived Endothelial Cells" @default.
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