Paracrine and Autocrine Functions of Neuronal Vascular Endothelial Growth Factor (VEGF) in the Central Nervous System

Recent data have demonstrated that vascular endothelial growth factor (VEGF) is expressed by subsets of neurons, coincident with angiogenesis within the developing cerebral cortex. Here we investigate the characteristics of VEGF expression by neurons and test the hypothesis that VEGF may serve both paracrine and autocrine functions in the developing central nervous system. To begin to address these questions, we assayed expression of VEGF and one of its potential receptors, Flk-1 (VEGFR-2), in the embryonic mouse forebrain and embryonic cortical neurons grown in vitro. Both VEGF and Flk-1 are present in subsets of post-mitotic neurons in vivo and in vitro. Moreover, VEGF levels are up-regulated in neuronal cultures subjected to hypoxia, consistent with our previous results in vivo. While the abundance of Flk-1 is unaffected by hypoxia, the receptor exhibits a higher level of tyrosine phosphorylation, as do downstream signaling kinases, including extracellular signal-regulated protein kinase, p90RSK and STAT3a, demonstrating activation of the VEGF pathway. These same signaling components also exhibited higher tyrosine phosphorylation levels in response to exogenous addition of rVEGFA165. This activation was diminished in the presence of specific inhibitors of Flk-1 function and agents that sequester VEGF, resulting in a dose-dependent increase in apoptosis in these neuronal cultures. Further, inhibition of MEK resulted in increased apoptosis, while inhibition of phosphatidylinositol 3-kinase had no appreciable affect. In addition to the novel function for VEGF that we describe in neuronal survival, neuronal VEGF also affected the organization and differentiation of brain endothelial cells in a three-dimensional culture paradigm, consistent with its more traditional role as a vascular agent. Thus, our in vitro data support a role for neuronal VEGF in both paracrine and autocrine signaling in the maintenance of neurons and endothelia in the central nervous system. Recent data have demonstrated that vascular endothelial growth factor (VEGF) is expressed by subsets of neurons, coincident with angiogenesis within the developing cerebral cortex. Here we investigate the characteristics of VEGF expression by neurons and test the hypothesis that VEGF may serve both paracrine and autocrine functions in the developing central nervous system. To begin to address these questions, we assayed expression of VEGF and one of its potential receptors, Flk-1 (VEGFR-2), in the embryonic mouse forebrain and embryonic cortical neurons grown in vitro. Both VEGF and Flk-1 are present in subsets of post-mitotic neurons in vivo and in vitro. Moreover, VEGF levels are up-regulated in neuronal cultures subjected to hypoxia, consistent with our previous results in vivo. While the abundance of Flk-1 is unaffected by hypoxia, the receptor exhibits a higher level of tyrosine phosphorylation, as do downstream signaling kinases, including extracellular signal-regulated protein kinase, p90RSK and STAT3a, demonstrating activation of the VEGF pathway. These same signaling components also exhibited higher tyrosine phosphorylation levels in response to exogenous addition of rVEGFA165. This activation was diminished in the presence of specific inhibitors of Flk-1 function and agents that sequester VEGF, resulting in a dose-dependent increase in apoptosis in these neuronal cultures. Further, inhibition of MEK resulted in increased apoptosis, while inhibition of phosphatidylinositol 3-kinase had no appreciable affect. In addition to the novel function for VEGF that we describe in neuronal survival, neuronal VEGF also affected the organization and differentiation of brain endothelial cells in a three-dimensional culture paradigm, consistent with its more traditional role as a vascular agent. Thus, our in vitro data support a role for neuronal VEGF in both paracrine and autocrine signaling in the maintenance of neurons and endothelia in the central nervous system. VEGF, 1The abbreviations used are: VEGFvascular endothelial growth factorSTATsignal transducers and activators of transcriptionEembryonic dayTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end labeling: MEK, mitogen-activated protein kinaseERKextracellular signal-regulated protein kinasePIphosphatidylinositol 1The abbreviations used are: VEGFvascular endothelial growth factorSTATsignal transducers and activators of transcriptionEembryonic dayTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end labeling: MEK, mitogen-activated protein kinaseERKextracellular signal-regulated protein kinasePIphosphatidylinositol a hypoxia-inducible endothelial cell mitogen, has been characterized as a potent vascular permeability factor and a critical factor in vasculo- and angiogenesis (1.Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar, 2.Jakeman L.B. Armanini M. Phillips H.S. Ferrara N. 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Oncogene. 1998; 16: 359-367Crossref PubMed Scopus (146) Google Scholar, 8.Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3118) Google Scholar, 9.Shalaby F. Rossant J. Yamaguchi E.A. Nature. 1995; 37: 62-66Crossref Scopus (3308) Google Scholar). Both receptors are critical for the proper differentiation and organization of endothelial cells into vascular beds. In the central nervous system, a cellular response to hypoxic exposure is increased VEGF production by glial cells that invest cerebral vessels (10.Kuo N.T. Benhayon D. Pryzbylski R.J. Martin R.J. Lamanna J.C. J. Appl. Physiol. 1999; 86: 260-264Crossref PubMed Scopus (82) Google Scholar, 11.Ogunshola O.O. Stewart W.B. Mihalcik V. Solli T. Madri J.A. Ment L.R. Dev. Brain Res. 2000; 119: 139-153Crossref PubMed Scopus (217) Google Scholar, 12.Sheiki D. Neeman M. Itin A. Keshet E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 768-772Crossref PubMed Scopus (534) Google Scholar), which results in increased angiogenesis and changes in vessel homeostasis. vascular endothelial growth factor signal transducers and activators of transcription embryonic day terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling: MEK, mitogen-activated protein kinase extracellular signal-regulated protein kinase phosphatidylinositol vascular endothelial growth factor signal transducers and activators of transcription embryonic day terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling: MEK, mitogen-activated protein kinase extracellular signal-regulated protein kinase phosphatidylinositol More recently, in the peripheral nervous system VEGF has been shown to be a neurotropic factor, which stimulates axonal outgrowth, enhances cell survival, and increases Schwann cell proliferation in cultured superior cervical and dorsal root ganglia from adult mice. Co-expression of VEGF and Flk-1 in many neurons in these superior cervical ganglia cultures was also noted (13.Sondell M. Lundborg G. Kanje M. Brain Res. 1999; 846: 219-228Crossref PubMed Scopus (169) Google Scholar, 14.Sondell M. Lundborg G. Kanje M. J. Neurosci. 1999; 19: 5731-5740Crossref PubMed Google Scholar, 15.Sondell M. Sundler F. Kanje M. Eur. J. Neursci. 2000; 12: 4243-4254Crossref PubMed Scopus (334) Google Scholar). In addition, VEGF expression has been localized to subpopulations of neurons in the developing and mature central nervous system (14.Sondell M. Lundborg G. Kanje M. J. Neurosci. 1999; 19: 5731-5740Crossref PubMed Google Scholar, 15.Sondell M. Sundler F. Kanje M. Eur. J. Neursci. 2000; 12: 4243-4254Crossref PubMed Scopus (334) Google Scholar, 16.Hayashi T. Abe K. Suzuki H. Itoyama Y. Stroke. 1997; 28: 2039-2044Crossref PubMed Scopus (296) Google Scholar, 17.Issa R. Krupinski J. Bujny T. Kuman S. Kaluza J. Kuman P. Lab. Invest. 1999; 79: 417-425PubMed Google Scholar, 18.Jin K.L. Mao X.O. Nagayama T. Goldsmith P.C. Greenberg D.A. Neuroscience. 2000; 99: 577-585Crossref PubMed Scopus (155) Google Scholar). Jin et al. demonstrated VEGF gene and protein expression in neurons of the cortex and hippocampus following a model for global cerebral ischemia in the adult rat brain, and the application of exogenous VEGF was shown to promote survival of rat cerebral neurons in culture and rescue HN33 hippocampal cells from death by serum withdrawal (18.Jin K.L. Mao X.O. Nagayama T. Goldsmith P.C. Greenberg D.A. Neuroscience. 2000; 99: 577-585Crossref PubMed Scopus (155) Google Scholar). Furthermore, our previous work has demonstrated that VEGF expression by neurons of the developing cerebral cortex coincides both spatially and temporally with angiogenesis and that levels of VEGF in cortical neurons increase following hypoxic exposure (11.Ogunshola O.O. Stewart W.B. Mihalcik V. Solli T. Madri J.A. Ment L.R. Dev. Brain Res. 2000; 119: 139-153Crossref PubMed Scopus (217) Google Scholar). Thus, this previously unknown source of endogenous VEGF production in the brain presents a novel paradigm for examining VEGF function. Interest in VEGF as a therapeutic molecule in a wide number of pathological conditions such as stroke and peripheral nerve damage led us to test the hypothesis that neuronal VEGF secretion may have specific signaling functions in the central nervous system (17.Issa R. Krupinski J. Bujny T. Kuman S. Kaluza J. Kuman P. Lab. Invest. 1999; 79: 417-425PubMed Google Scholar,19.Ferrara N. Curr. Opin. Biotechnol. 2000; 11: 617-624Crossref PubMed Scopus (351) Google Scholar, 20.Hayashi T. Abe K. Itoyama Y. J. Cereb. Blood Flow Metab. 1998; 18: 887-895Crossref PubMed Scopus (300) Google Scholar, 21.Hobson M.I. Green C.J. Terenghi G. J. Anat. (Lond). 2000; 197: 591-605Crossref PubMed Google Scholar). Here we report that neuronal VEGF and its receptor Flk-1 are expressed by cortical neurons of E15 embryos in vitro and in vivo. Moreover, use of an in vitro culture model demonstrates that neuronal VEGF expression is hypoxia-inducible, with the resulting VEGF capable of supporting tube formation in a brain microvascular endothelial cell line. Co-localization of VEGF and its Flk-1 receptor, as well as changes in tyrosine phosphorylation of both Flk-1 and downstream signaling molecules, including mitogen-activated protein kinase, p90RSK, and STAT family members, suggests that VEGF mediates both auto- and paracrine signaling functions in central nervous system neurons. Finally, inhibition of either VEGF function or Flk-1 activity results in increased cell apoptosis, indicating that endogenously produced VEGF acts as a neuronal survival factor. Sections of embryonic day 15 (E15) mouse brains or cortical neurons derived from them and grown in vitro (11.Ogunshola O.O. Stewart W.B. Mihalcik V. Solli T. Madri J.A. Ment L.R. Dev. Brain Res. 2000; 119: 139-153Crossref PubMed Scopus (217) Google Scholar) were fixed and subjected to immunohistochemistry. The antibodies used follow: polyclonal anti-VEGF (Neomarkers Inc., 1:250), anti-Flk-1 (Santa Cruz Biotechnology Inc., 1:250), monoclonal anti-neuron-specific class III β-tubulin Tuj-1 (Berkeley Antibody Co., Richmond, CA., 1:500), anti-Erk-2 (Santa Cruz Biotechnology Inc., 1:1000), anti-phospho-Erk (Santa Cruz Biotechnology Inc., 1:1000), anti-STAT1, anti-STAT3a, and anti-p-STAT3a, (Chemicon, International, Inc., Temecula, CA; 1:1000), anti-PI 3-kinase (Upstate Biotechnology, Lake Placid, NY; 1:1000), anti-p90RSK and anti-p90RSK PY (Cell Signaling Technology, Inc., New England Biolabs, Beverly, MA, 1:1000), and anti Cleaved Caspase 3 (D175) (Cell Signaling, Beverly, MA, 1:500). Wortmannin, LY294002, and PD98059 were purchased from Sigma. TUNEL labeling was performed as recommended by the manufacturer (Roche Diagnostics, Indianapolis, IN). The dorsal telencephalon was dissected from E15 mice (Charles River Laboratories), as previously described (22.Sestan N. Artavanis-Tsakonas S. Rakic P. Science. 1999; 286: 741-746Crossref PubMed Scopus (491) Google Scholar). Cells were plated on either glass coverslips or plastic Petri dishes coated with poly-l-ornithine and laminin. Cells were incubated for 6 days in normal atmosphere with 5% CO2 or in hypoxic conditions consisting of a mixture of 5% CO2, 10% O2, 85% N2 (BOC Gases, North Haven, CT). In some experiments cultures were incubated with the following: (ε)-3(3,5-diisoproply-4-hydroxyphenyl)-2-3-phenyl-n-propyl)amino-carbonylacrylonitrile (SU1498) (23.Strawn L.M. McMahon G. App H. Schreck R. Kuchler W.R. Longhi M.P. Hui T.H. Tang C. Levitzki A. Gazit A. Chen I. Keri G. Orfi L. Risau W. Flamme W. Ullrich A. Hirth K.P. Shawver L.K. Cancer Res. 1996; 56: 3540-3545PubMed Google Scholar), a potent and selective inhibitor of Flk-1 kinase; 4-94′-chloro-2′-fluoro)phenylamino-6,7-dimethoxyquinazoline (CB676475) (24.Hennequin L.F. Thomas A.P. Johnstone C. Stokes E.S. Ple P.A. Lohmann J.J. Ogilvie D.J. Dukes M. Wedge S.R.J.O.C. Kendrew J. Lambert-van der Brempt C. J. Med. Chem. 1999; 42: 5369-5389Crossref PubMed Scopus (251) Google Scholar), a potent and selective inhibitor of VEGFR1 and 2 tyrosine kinase activity; an inhibitor of the Flk-1 kinase or a neutralizing antibody directed against VEGF-A (R recombinant VEGF-A165 (Vendor); a recombinant soluble Flt-1 (Flt (1.Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar, 2.Jakeman L.B. Armanini M. Phillips H.S. Ferrara N. Endocrinology. 1993; 133: 848-859Crossref PubMed Scopus (192) Google Scholar, 3.Leung D.W. Cachianes G. Kuang W.-J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4396) Google Scholar)-IgG, a truncated Flt 1–3 Fc fusion protein), a generous gift of Dr. Napoleon Ferrara (Genentech; San Francisco, CA) (25.van Bruggen N. Thibodeaux H. Palmer J.T. Lee W.P. Fu L. Cairns B. Tumas D. Gerlai R. Williams S.P. van Lookeren Campagne M. Ferrara N. J. Clin. Invest. 1999; 104: 1613-1620Crossref PubMed Scopus (386) Google Scholar); or PD98059, a MEK inhibitor, or Wortmannin and LY24002, inhibitors of PI 3-K, (Sigma). Western blotting was carried out on lysates of E15 neurons and E15-conditioned media as previously described. (11.Ogunshola O.O. Stewart W.B. Mihalcik V. Solli T. Madri J.A. Ment L.R. Dev. Brain Res. 2000; 119: 139-153Crossref PubMed Scopus (217) Google Scholar) Antisera directed against VEGF, Flk-1, PI 3-kinase, p90RSK, and were was carried out with exposure to was carried out on and Systems, San CA) the on a experiments were performed was performed cultures were with and and into was carried out as previously described N. S. Madri J.A. J. Sci. 1999; PubMed Google Scholar). experiments were performed brain microvascular endothelial cells F. N. M. P. J. A. P. J. Physiol. PubMed Scopus Google Scholar) were cultured as previously described J.A. J. Biol. PubMed Scopus Google Scholar). The were cultured in in either or hypoxic E15 media for 6 days in 5% CO2 with media on day 6 days the cultures were fixed and on for were carried out were performed were a with and a to performed to VEGF and Flk-1 in E15 forebrain a neuronal to neuronal that VEGF was expressed by neurons the of the developing Flk-1 was also expressed by neurons of the E15 forebrain and and levels as both VEGF and Flk-1 were present within vascular to the cortical a and To cells in vitro expressed VEGF and Flk-1, we cultures of E15 cortical neurons and performed immunohistochemistry. that of cells in culture expressed VEGF and of cells expressed Flk-1 Furthermore, of cells that expressed VEGF also expressed Flk-1 To expression cells were grown in we assayed levels of VEGF and Flk-1 in neurons, as well as cells grown in Both of cells expressed VEGF and and Flk-1 and and the VEGF produced by these neurons was into the media E15 cortical neurons Flk-1 as cells or cells grown in vitro and These results demonstrated that neurons in culture neurons in the brain in to and In addition, have a receptor with which to this factor. we the activation of Flk-1 in cortical neuronal cultures and that the Flk-1 was indicating that a of the Flk-1 receptors is in an Furthermore, an anti-Flk-1 of and consistent with these PI 3-kinase, and known signaling of Flk-1 D. R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). To VEGF and Flk-1 levels are by hypoxia, we performed Western on neuronal culture lysates derived from and hypoxic cultures. While protein levels of Flk-1 levels of VEGF in both lysates and media were up-regulated as by Flk-1 protein levels in response to hypoxia, Flk-1 tyrosine phosphorylation levels were to increase in response to Further, to signaling components to Flk-1 activation are to hypoxic we levels of protein and protein for p90RSK, and PI of p90RSK, and protein cells grown in hypoxic In levels of p90RSK, and were increased following hypoxia, indicating that are and phosphorylation levels hypoxic of and PI 3-kinase were also increased by is a known for VEGF we were in addition of exogenous the changes in signaling components that we following culture in of for 6 we that levels of p90RSK, and protein cells grown and with results with hypoxic levels of p90RSK, and were increased following addition of indicating activation of a signaling and as in hypoxic phosphorylation levels and and PI 3-kinase expression levels were by VEGF To the effects of signaling in cortical neurons two were we VEGF receptor tyrosine kinase activity a potent and selective Flk-1 tyrosine kinase inhibitor (23.Strawn L.M. McMahon G. App H. Schreck R. Kuchler W.R. Longhi M.P. Hui T.H. Tang C. Levitzki A. Gazit A. Chen I. Keri G. Orfi L. Risau W. Flamme W. Ullrich A. Hirth K.P. Shawver L.K. Cancer Res. 1996; 56: 3540-3545PubMed Google Scholar), or a VEGF receptor tyrosine kinase inhibitor (24.Hennequin L.F. Thomas A.P. Johnstone C. Stokes E.S. Ple P.A. Lohmann J.J. Ogilvie D.J. Dukes M. Wedge S.R.J.O.C. Kendrew J. Lambert-van der Brempt C. J. Med. Chem. 1999; 42: 5369-5389Crossref PubMed Scopus (251) Google Scholar). was to increased apoptosis by TUNEL in a dose-dependent to E15 neuronal cultures exhibited two The was to sequester endogenously expressed VEGF with either a neutralizing VEGF antibody or a recombinant soluble Flt-1 In both cultures either of these agents exhibited increased apoptosis as by TUNEL labeling To the specific signaling downstream of the inhibition of apoptosis a was inhibitors of either MEK and PI 3-kinase and LY24002, were from day one to day of culture of the E15 neurons. in cells were by 3 of cultures and cultures incubated with either of the PI 3-kinase inhibitors or Wortmannin In cultures incubated with of the MEK inhibitor PD98059 exhibited increased apoptosis as by the increase in 3 To neuronal VEGF is capable of endothelial cell organization and we the effects of media by cortical cultures on brain microvascular endothelial cells cultured in three-dimensional S. L. and J. A. Brain Res. Dev. Brain Res. that media and differentiation of rat brain endothelial resulting in the formation of The to tube formation with levels of VEGF in the as no in cultures with media tube formation is in cultures with media from neuronal cultures and tube formation in cultures that were with media from hypoxic neuronal cultures Thus, produced by cortical neurons in neuronal VEGF has the to endothelial function and that embryonic cortical neurons, in vivo and in VEGF and one of its receptors, Flk-1 was in these Moreover, Flk-1 is in cultured neurons and to signaling via a that kinase, and the p90RSK, and hypoxic cortical neurons levels of VEGF and Flk-1 activation is supporting the that a response to by cortical neurons is to increase VEGF production and Flk-1 response was exogenous that neurons are capable of to both endogenous and exogenous VEGF of VEGF either agents to Flk-1 kinase activity or that sequester endogenous VEGF, results in increased apoptosis, that VEGF may function as an autocrine and paracrine neuronal survival factor. Further, inhibitors of PI 3-kinase and MEK we data consistent with the that the is in for neuronal survival in this culture the PI to be in the of neuronal apoptosis in our in vitro model and Finally, media by both and hypoxic cortical neurons in vitro with levels of VEGF with the of tube previous that neurons in the brain VEGF are by these (11.Ogunshola O.O. Stewart W.B. Mihalcik V. Solli T. Madri J.A. Ment L.R. Dev. Brain Res. 2000; 119: 139-153Crossref PubMed Scopus (217) Google the maintenance of expression of VEGF by neurons in culture demonstrates that this expression is to these neurons, in the of proper cellular and Furthermore, the that VEGF and Flk-1 are the that neuronal VEGF in the brain as an autocrine as well as a paracrine factor for neurons and In addition to its more role in VEGF is in of the of P.A. S. J.L. J. Neurosci. 2000; PubMed Google Scholar). In the peripheral nervous system, the and effects of exogenous VEGF in of the Flk-1 receptor as well as increased survival of peripheral neurons in that functions may (14.Sondell M. Lundborg G. Kanje M. J. Neurosci. 1999; 19: 5731-5740Crossref PubMed Google Scholar, 15.Sondell M. Sundler F. Kanje M. Eur. J. Neursci. 2000; 12: 4243-4254Crossref PubMed Scopus (334) Google Scholar). In the central nervous system, exogenous VEGF neuronal cell death in an in vitro model of cerebral ischemia (18.Jin K.L. Mao X.O. Nagayama T. Goldsmith P.C. Greenberg D.A. Neuroscience. 2000; 99: 577-585Crossref PubMed Scopus (155) Google Scholar, K.L. Mao X.O. Greenberg D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, K.L. Mao X.O. Greenberg D.A. J. Neurosci. 2000; PubMed Google Scholar). In this we a role to the endogenous production of VEGF by neuronal cells and that VEGF from exogenous as and endothelial is for neuronal to the of that have been to that increased cell death results from a of endogenous VEGF signaling indicating the of both VEGF and the Flk-1 receptor in neuronal that signaling are to of endogenous VEGF levels in cortical neurons to hypoxic conditions in vitro. to by Jin et which the of PI signal system in in HN33 cells K.L. Mao X.O. Greenberg D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google K.L. Mao X.O. Greenberg D.A. J. Neurosci. 2000; PubMed Google Scholar), we that the is to the survival of cortical neurons in vitro. This be to in as well as in the cell both autocrine and paracrine Flk-1 p90RSK, and STAT phosphorylation levels are either in abundance or in activity in cortical neurons hypoxic conditions or exposure to These with the of phosphorylation, the that VEGF a number of downstream some of which have been our data suggests that neurons a source of VEGF to survival a response that on the of the this of VEGF into the may survival of cells that are angiogenesis is and novel of neurons to changes in VEGF expression and are to be that media differentiation and tube formation in a three-dimensional model of we the presence of in the our suggests that neuronal VEGF is signal to endothelial and a to angiogenesis and in response to and Furthermore, media tube consistent with our previous that neuronal VEGF a to angiogenesis in the developing cortex. cortical neurons are and we that this is in in be in the to the effects of VEGF on cortical is also to that may be a of cortical neurons VEGF receptors, which may be a some cells are more to and In addition, the of levels of Flk-1 signaling hypoxic conditions cells from the apoptosis are to is an and as may have for the of cortical Finally, our results that to be a in which and angiogenesis In the we the that VEGF in neuronal and the potential of VEGF in neuronal within the developing brain and the that neurons and the vessels that Dr. Napoleon San Francisco, for the generous gift of truncated Flt 1–3 Fc fusion and Dr. F. F. for the generous gift of rat brain endothelial