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Expression of vascular endothelial growth factor and its receptors in rat neural stem cells
Neuroscience Letters 344 (2003) 165–168 www.elsevier.com/locate/neulet
Expression of vascular endothelial growth factor and its receptors in rat neural stem cells Martin H. Maurer*, Wolf K.C. Tripps, Robert E. Feldmann Jr., Wolfgang Kuschinsky Department of Physiology and Pathophysiology, University of Heidelberg, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany Received 12 March 2003; received in revised form 24 March 2003; accepted 25 March 2003
Abstract Neural stem cells serve neurogenesis in several areas of the adult mammalian brain. The present study investigates an additional feature, i.e. the expression of vascular endothelial growth factor (VEGF) and its receptors in cultured neurospheres from hippocampus, subventricular zone, and olfactory bulb in adult rats. RT-PCR showed that all three lines expressed VEGF mRNA, but different sets of VEGF receptor mRNAs. Stimulation by either 50 ng/ml VEGF-A165 or by 24 h of anoxia resulted in an altered pattern of receptor expression for VEGF-R2 (Flk-1), VEGF-R3 (Flt-4), neuropilin-1, and neuropilin-2, whereas VEGF-R1 (Flt-1) remained unexpressed. The basal expression of VEGF and of several of its receptor mRNAs indicates a hitherto unknown angiogenic potential of neural stem cells. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vascular endothelial growth factor; Neural stem cell; Angiogenesis; Neurogenesis; Cell culture; Anoxia; Rat
Neural stem cells have been isolated from various regions of the adult brain [8]. Neurogenesis in these regions can be induced by various stimuli, among them hormones, neurotransmitters, and growth and transcription factors (for review see Ref. [1]). When activated by such factors, neural stem cells are able to replace and repair compromised brain tissue [3,8,12]. When new cells in the brain originate from neural stem cells, it appears likely that new vessels may be formed for adequate blood supply. Therefore, neovascularization or angiogenesis may be necessary for remodeling cellular networks by migration and differentiation of angioblasts [4,5,17]. A major angiogenic factor is the vascular endothelial growth factor (VEGF). The VEGF family consists of six members, VEGFA, -B, -C, -D, -E and the placenta-derived growth factor (PlGF). Concerning VEGF-A, multiple splice variants are known. The VEGF molecules bind to receptors known as VEGF-R1 (Flt-1), VEGF-R2 (KDR, Flk-1), VEtformat 1 1 GF-R3 (Flt-4), neuropilin-1, neuropilin-2, and heparan sulfate proteoglycans. Signal transduction mediated by these receptors serves various functions in cell proliferation and survival [6,15,18]. In addition, direct neurotrophic effects have been described for VEGF [11,19]. An increase * Corresponding author. Tel.: þ49-6221-54-8604; fax: þ 49-6221-544561. E-mail address: [email protected] (M.H. Maurer).
in VEGF concentration and an altered VEGF receptor expression pattern have been found in response to anoxia in hippocampal neurons in vitro and in mouse brain, kidney, testis, lung, heart, and liver in vivo [11,13,19]. This study tests the following questions: (1) Is VEGF expressed in neural stem cells? (2) Does a regional diversity in the expression pattern of VEGF and its receptors exist in the three types of adult neural stem cells isolated from the regions of the rat brain with known spontaneous neurogenesis, i.e. hippocampus, subventricular zone, and olfactory bulb? (3) Can this expression pattern be changed by exogenous VEGF and/or anoxic stimuli? Neural stem cells were isolated from the brain areas with known spontaneous neurogenesis, i.e. hippocampus, olfactory bulb, and subventricular zone, of 4– 6-week-old male Wistar rats as described [16]. Protocols are concordant with the policy on the use of animals, as endorsed by the National Research Council of the USA, and fulfill the requirements of German law. Briefly, animals were anesthetized by isoflurane inhalation and sacrificed by decapitation. The brains were removed and washed in 50 ml ice-cold Dulbecco’s phosphate buffered saline (DPBS) supplemented with 4.5 g/l glucose (DPBS/Glc). Hippocampus, olfactory bulb, and subventricular zone from six animals were dissected, washed in 10 ml DPBS/Glc and centrifuged for 5 min at 1600 £ g at 4 8C. After removal of the supernatant, the tissue was homogenized with scissors and
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00407-5
60 60 58 60 60 60 60 50 -CCGAATAGCGAGCAGATTTC-30 50 -TTCCATCCGGAACAAATCTC-30 50 -TGAGCTCTGAGAACTGTGCA-30 50 -ATGTCGGGAACTCTGATTGG-30 50 -CGGATCCTGATGAAACGAGT-30 50 -AAATGCTTTCTCCGCTCTGA-30 50 -GGATGGAATTGTGAGGGAGA-30 50 -TTCCGGACTTTCAACACCTC-30 50 -CGATGTCTCCTCCATCGTTT-30 50 -TAAGGTGTACACCACGCAGA-30 50 -GGAGCTACTGGGCTGTGAAG-30 50 -ACACAAGGAGCCATTTCCAG-30 50 -CTCACCAAAGCCAGCACATA-30 50 -GAGGACCAGGTTGTCTCCTG-30 VEGF-R1 (Flt-1) VEGF-R2 (KDR, Flk-1) VEGF-R3 (Flt-4) Neuropilin-1 Neuropilin-2 VEGF-A (all splice variants) GAPDH
D28498 NM_013062 NM_053652 AF018957 NM_030869 NM_031836 NM_017008
Reverse primer Forward primer GenBank accession number
scalpels. The tissue pieces were washed with DPBS/Glc medium for 5 min at 800 £ g, and the three pellets were resuspended in 0.01% (w/v) papain, 0.1% (w/v) dispase II (neutral protease), 0.01% (w/v) DNase I, and 12.4 mM manganese sulfate in Hank’s balanced salt solution (HBSS). The tissue was triturated with plastic pipette tips and incubated for 40 min at room temperature, but every 10 min the solution was mixed well. The suspension was centrifuged at 800 £ g for 5 min at 4 8C and pellets were washed three times in 10 ml DMEM-Ham’s F-12 medium supplemented with 2 mM L -glutamine, 100 units/ml penicillin and 100 units/ml streptomycin. Then, the cell pellets were resuspended in 1 ml Neurobasal medium supplemented with B27 (Invitrogen, Carlsbad, CA), 2 mM L -glutamine, 100 units/ml penicillin, 100 units/ml streptomycin, 20 ng/ml endothelial growth factor (EGF), 20 ng/ml fibroblast growth factor-2 (FGF-2), and 2 mg/ml heparin. Cells were plated under sterile conditions in six-well dishes at 25,000 –100,000 cells/ml. The dishes were incubated at 37 8C in 5% CO2. Cell culture media were changed once a week, where about two-thirds of the media were replaced. Experiments were performed after 6 – 8 weeks in vitro. For stimulation with exogenous VEGF, human recombinant VEGF-A165 (Chemicon, Temecula, CA) was added to the cell culture medium in a final concentration of 50 ng/ml which has a maximum effect on hematopoietic stem cells [11]. Total RNA was extracted from cultured neural stem cells isolated from hippocampus, subventricular zone, and olfactory bulb using the RNeasy total RNA extraction kit following the manufacturer’s instructions (Qiagen, Santa Clarita, CA). RNA concentration was determined photometrically, the quality of the total RNA extract was controlled by agarose gel electrophoresis, and RNA was stored at 2 80 8C until use. Reverse transcription was performed on 5 mg of the total RNA extracts of hippocampus, subventricular zone, and olfactory bulb by using the SuperScriptII RNase H2 Reverse Transcriptase (Invitrogen-Life Technologies, Carlsbad, CA) with an oligo(dT)12-18 primer for first strand cDNA synthesis. For amplification, specific PCR primer pairs (Table 1) and Taq DNA polymerase (Promega, Madison, WI) were used in a protocol of 5 min initial denaturation at 94 8C, 35 cycles of amplification with 30 s denaturation at 94 8C, 50 s annealing at 55 8C, and 60 s extension at 72 8C, followed by a final extension for 5 min. Amplification products were visualized by agarose gel electrophoresis. GAPDH was used as internal control. For anoxia, cell culture dishes were incubated in a modular incubator chamber (Billups-Rothenberg, Del Mar, CA) at 37 8C for 24 h in a humidified 5% CO2 and 95% N2 atmosphere as described [11]. Viable cells were detected by Trypan Blue exclusion. The cell suspension was diluted 1:10 in Trypan Blue and viable cells excluding the dye were set in relation to the total number of cells. For quantitation of cell survival, the CellTiter 96
Melting temperature (8C)
M.H. Maurer et al. / Neuroscience Letters 344 (2003) 165–168
Table 1 Specific PCR primer pairs for VEGF and its receptors
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M.H. Maurer et al. / Neuroscience Letters 344 (2003) 165–168
AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) was used, based on a colorimetric tetrazolium salt method. The mean ^ SD of eight experiments each for the normoxia, normoxia þ VEGF, anoxia, and anoxia þ VEGF groups were compared by Student’s t-test with corrections for multiple testing. To determine whether neural stem cells produce VEGFA or any of its receptors, RT-PCR was performed on total RNA isolated from neurosphere cultures. During normoxia, strong signals were found for VEGF in each of the three cell types taken from hippocampus, subventricular zone, and olfactory bulb (Fig. 1, row 1). In parallel, all three types of stem cells expressed mRNA encoding for VEGF-R2. Neuropilin-1 mRNA was only expressed in hippocampal neural stem cells whereas neuropilin-2 mRNA was expressed in hippocampal and olfactory bulb stem cells. No signal was found for VEGF-R1 and VEGF-R3 under normoxic conditions. When stimulated by 50 ng/ml exogenous VEGF-A, all three cell types expressed mRNA for neuropilin-1 and neuropilin-2 (Fig. 1, row 2). The signal for VEGF-R2 diminished in all three cell types, whereas VEGF expression was not suppressed. The incubation under severe anoxic conditions consisting of 5% CO2 and 95% N2 at 37 8C for 24 h changed the expression pattern: the expression of VEGF-R2 mRNA in the cells from subventricular zone and olfactory bulb vanished, but it persisted in hippocampal neural stem cells. Additionally, the expression of neuropilin-1 and neuropilin-2 diminished in these cells, whereas it persisted in the hippocampal cells. On the other hand, VEGF-3 mRNA was induced in the neural stem cells from hippocampus (Fig. 1, row 3). The combination of anoxia and VEGF showed expression of VEGF-R2 in
Fig. 1. Regional heterogeneity in the expression pattern of VEGF and its receptors in neural stem cells. Agarose gel electrophoresis of RT-PCR products shows high expression levels of mRNA for VEGF, VEGF-R2, neuropilin-1, and neuropilin-2 in several types of neural stem cells isolated from hippocampus (H), subventricular zone (S), and olfactory bulb (B). Row 1, normoxic expression pattern; row 2, normoxic expression pattern with the addition of 50 ng/ml exogenous VEGF; row 3, anoxic expression pattern; row 4, anoxic expression pattern with the addition of 50 ng/ml exogenous VEGF. Eight repetitions were performed for each condition.
167
hippocampal, neuropilin-1 in olfactory bulb, and neuropilin2 in all three types of neural stem cells (Fig. 1, row 4). Anoxia caused a decreased cell viability after 24 h down to 15 – 20% of surviving cells. The addition of 50 ng/ml exogenous VEGF in the anoxia experiments resulted in a doubled survival rate compared to hypoxia alone (Fig. 2). VEGF expression has not yet been investigated in adult neural stem cells, but recently VEGF expression has been reported in hematopoietic stem cells [9]. The authors of this study [9] postulated an internal autocrine loop mechanism, by which VEGF could regulate cell survival when coexpressed with its receptors. Our study is in accordance with this hypothesis: VEGF mRNA is expressed in all neural stem cell lines from hippocampus, subventricular zone, and olfactory bulb. Neither anoxia nor exogenous VEGF could suppress its expression, indicating that the intrinsic loop of VEGF stimulation in brain-derived stem cells is effective. Moreover, this autocrine intrinsic loop is independent of the receptor expression, as the altered VEGF receptor pattern indicates. Interestingly, the highest mRNA expression for VEGF was found in vivo in the regions with spontaneous neurogenesis [13]. Thus our finding of VEGF expression in neural stem cells indicates that these cells may contribute to neovascularization and angiogenesis as a migratory stimulus for endothelial precursors or by direct angiogenic effects. A comprehensive analysis of the expression patterns of the different VEGF receptors hat not yet been performed in neural stem cells. Our study shows a regional heterogeneity of VEGF receptor expression in neural stem cells isolated from different brain structures, i.e. from hippocampus, subventricular zone and olfactory bulb, indicating a heterogeneous developmental potential of these cells. A recent
Fig. 2. Cell viability assays for the four experimental conditions. Tetrazolium salt color development and Trypan Blue exclusion were used to quantify surviving cells. Twenty-four hour anoxia decreased cell survival dramatically. Stimulation with 50 ng/ml exogenous VEGF doubled the amount of surviving cells during anoxia in all three neural stem cell lines compared to anoxia alone. Data show that exogenous VEGF rescues neural stem cells from anoxia. Eight repetitions were performed for each condition.
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study found that VEGF-R2 is expressed in cortical cell cultures from mouse embryos and that VEGF-R2 expression increased in vivo in brains stimulated by administration of exogenous VEGF [10]. The diverse expression pattern of the VEGF receptors might be linked to the neuroprotective effect of VEGF, which has been described recently [11,14,19]. In neurons, the neuroprotective effects are mediated by VEGF receptors and neuropilins which were originally specified as semaphorin receptors but then were found also to bind VEGF [15]. Similarly, our study shows protective effects of VEGF towards anoxia in neural stem cells. This protective effect might be related to VEGF-R2 expression which is known to mediate cell proliferation and survival. Initially, the neuropilins had been regarded as pure co-receptors for VEGF-R2, but two recent studies showed a VEGFindependent up-regulation of neuropilins after cerebral ischemia [7,20]. The induction of neuropilins upon exogenous VEGF stimuli could indicate a growth limiting signal for proliferative neural cells. Neuropilins are mediating negative axon guidance for neurons by inhibiting the direction of axon growth due to the induction of apoptosis [2]. Our findings suggest that neuropilins might also have growth limiting abilities in neural progenitors in regions where new vasculature should be provided. The loss of VEGF-R2 in two of the stem cell lines in reaction to anoxia might indicate an alternative protective mechanism, since VEGF-R2 is mainly responsible for cell proliferation and survival. Proliferation and mitosis are processes with an increased cellular vulnerability, when cells are extremely susceptible for DNA damage. Thus, a reduced proliferation by down-regulating the responsible receptor indicates an additional protective mechanism. Of note, VEGF-R3 was induced by anoxia in hippocampal cells, which also showed the highest rate of survival of the three lines, indicating a yet unknown function of this receptor in cell survival. Previous studies only had shown that VEGF-R3 is necessary for lymphangiogenesis [6,18]. In conclusion, the present study shows three major results: (1) VEGF is expressed in cultivated adult neural stem cells isolated from the regions of the rat brain with known spontaneous neurogenesis, i.e. hippocampus, subventricular zone, and olfactory bulb; (2) a regional diversity in the expression pattern of VEGF receptors exists in the three types of adult neural stem cells; and (3) the expression pattern can be changed by exogenous VEGF and/or anoxic stimuli.
Acknowledgements We thank Mrs Inge Keller and Maria Harlacher for technical assistance. This study was supported by the German Federal Ministry of Education and Research (BMBF, Kompetenznetz Schlaganfall, Projektgruppe B2 Heidelberg).
References [1] K. Abe, Therapeutic potential of neurotrophic factors and neural stem cells against ischemic brain injury, J. Cereb. Blood Flow Metab. 20 (2000) 1393–1408. [2] D. Bagnard, C. Vaillant, S.T. Khuth, N. Dufay, M. Lohrum, A.W. Puschel, M.F. Belin, J. Bolz, N. Thomasset, Semaphorin 3A-vascular endothelial growth factor-165 balance mediates migration and apoptosis of neural progenitor cells by the recruitment of shared receptor, J. Neurosci. 21 (2001) 3332–3341. [3] A. Bjo¨rklund, O. Lindvall, Self-repair in the brain, Nature 405 (2000) 892 –895. [4] P. Carmeliet, Mechanisms of angiogenesis and arteriogenesis, Nat. Med. 6 (2000) 389–395. [5] E.M. Conway, D. Collen, P. Carmeliet, Molecular mechanisms of blood vessel growth, Cardiovasc. Res. 49 (2001) 507 –521. [6] N. Ferrara, VEGF: an update on biological and therapeutic aspects, Curr. Opin. Biotechnol. 11 (2000) 617 –624. [7] H. Fujita, B. Zhang, K. Sato, J. Tanaka, M. Sakanaka, Expressions of neuropilin-1, neuropilin-2 and semaphorin 3A mRNA in the rat brain after middle cerebral artery occlusion, Brain Res. 914 (2001) 1–14. [8] F.H. Gage, Mammalian neural stem cells, Science 287 (2000) 1433–1438. [9] H.P. Gerber, A.K. Malik, G.P. Solar, D. Sherman, X.H. Liang, G. Meng, K. Hong, J.C. Marsters, N. Ferrara, VEGF regulates haematopoietic stem cell survival by an internal autocrine loop mechanism, Nature 417 (2002) 954–958. [10] K. Jin, Y. Zhu, Y. Sun, X.O. Mao, L. Xie, D.A. Greenberg, Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo, Proc. Natl. Acad. Sci. USA 99 (2002) 11946–11950. [11] K.L. Jin, X.O. Mao, D.A. Greenberg, Vascular endothelial growth factor: direct neuroprotective effect in in vitro ischemia, Proc. Natl. Acad. Sci. USA 97 (2000) 10242–10247. [12] H.G. Kuhn, T.D. Palmer, E. Fuchs, Adult neurogenesis: a compensatory mechanism for neuronal damage, Eur. Arch. Psychiatry Clin. Neurosci. 251 (2001) 152–158. [13] H.H. Marti, W. Risau, Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors, Proc. Natl. Acad. Sci. USA 95 (1998) 15809– 15814. [14] H. Matsuzaki, M. Tamatani, A. Yamaguchi, K. Namikawa, H. Kiyama, M.P. Vitek, N. Mitsuda, M. Tohyama, Vascular endothelial growth factor rescues hippocampal neurons from glutamate-induced toxicity: signal transduction cascades, FASEB J. 15 (2001) 1218–1220. [15] G. Neufeld, T. Cohen, S. Gengrinovitch, Z. Poltorak, Vascular endothelial growth factor (VEGF) and its receptors, FASEB J. 13 (1999) 9–22. [16] J. Ray, D.A. Peterson, M. Schinstine, F.H. Gage, Proliferation, differentiation, and long-term culture of primary hippocampal neurons, Proc. Natl. Acad. Sci. USA 90 (1993) 3602– 3606. [17] W. Risau, Mechanisms of angiogenesis, Nature 386 (1997) 671 –674. [18] C.J. Robinson, S.E. Stringer, The splice variants of vascular endothelial growth factor (VEGF) and their receptors, J. Cell Sci. 114 (2001) 853–865. [19] B. Svensson, M. Peters, H.G. Konig, M. Poppe, B. Levkau, M. Rothermundt, V. Arolt, D. Kogel, J.H. Prehn, Vascular endothelial growth factor protects cultured rat hippocampal neurons against hypoxic injury via an antiexcitotoxic, caspase-independent mechanism, J. Cereb. Blood Flow Metab. 22 (2002) 1170–1175. [20] Z.G. Zhang, W. Tsang, L. Zhang, C. Powers, M. Chopp, Upregulation of neuropilin-1 in neovasculature after focal cerebral ischemia in the adult rat, J. Cereb. Blood Flow Metab. 21 (2001) 541 –549.
Expression of vascular endothelial growth factor and its receptors in rat neural stem cells Martin H. Maurer*, Wolf K.C. Tripps, Robert E. Feldmann Jr., Wolfgang Kuschinsky Department of Physiology and Pathophysiology, University of Heidelberg, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany Received 12 March 2003; received in revised form 24 March 2003; accepted 25 March 2003
Abstract Neural stem cells serve neurogenesis in several areas of the adult mammalian brain. The present study investigates an additional feature, i.e. the expression of vascular endothelial growth factor (VEGF) and its receptors in cultured neurospheres from hippocampus, subventricular zone, and olfactory bulb in adult rats. RT-PCR showed that all three lines expressed VEGF mRNA, but different sets of VEGF receptor mRNAs. Stimulation by either 50 ng/ml VEGF-A165 or by 24 h of anoxia resulted in an altered pattern of receptor expression for VEGF-R2 (Flk-1), VEGF-R3 (Flt-4), neuropilin-1, and neuropilin-2, whereas VEGF-R1 (Flt-1) remained unexpressed. The basal expression of VEGF and of several of its receptor mRNAs indicates a hitherto unknown angiogenic potential of neural stem cells. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vascular endothelial growth factor; Neural stem cell; Angiogenesis; Neurogenesis; Cell culture; Anoxia; Rat
Neural stem cells have been isolated from various regions of the adult brain [8]. Neurogenesis in these regions can be induced by various stimuli, among them hormones, neurotransmitters, and growth and transcription factors (for review see Ref. [1]). When activated by such factors, neural stem cells are able to replace and repair compromised brain tissue [3,8,12]. When new cells in the brain originate from neural stem cells, it appears likely that new vessels may be formed for adequate blood supply. Therefore, neovascularization or angiogenesis may be necessary for remodeling cellular networks by migration and differentiation of angioblasts [4,5,17]. A major angiogenic factor is the vascular endothelial growth factor (VEGF). The VEGF family consists of six members, VEGFA, -B, -C, -D, -E and the placenta-derived growth factor (PlGF). Concerning VEGF-A, multiple splice variants are known. The VEGF molecules bind to receptors known as VEGF-R1 (Flt-1), VEGF-R2 (KDR, Flk-1), VEtformat 1 1 GF-R3 (Flt-4), neuropilin-1, neuropilin-2, and heparan sulfate proteoglycans. Signal transduction mediated by these receptors serves various functions in cell proliferation and survival [6,15,18]. In addition, direct neurotrophic effects have been described for VEGF [11,19]. An increase * Corresponding author. Tel.: þ49-6221-54-8604; fax: þ 49-6221-544561. E-mail address: [email protected] (M.H. Maurer).
in VEGF concentration and an altered VEGF receptor expression pattern have been found in response to anoxia in hippocampal neurons in vitro and in mouse brain, kidney, testis, lung, heart, and liver in vivo [11,13,19]. This study tests the following questions: (1) Is VEGF expressed in neural stem cells? (2) Does a regional diversity in the expression pattern of VEGF and its receptors exist in the three types of adult neural stem cells isolated from the regions of the rat brain with known spontaneous neurogenesis, i.e. hippocampus, subventricular zone, and olfactory bulb? (3) Can this expression pattern be changed by exogenous VEGF and/or anoxic stimuli? Neural stem cells were isolated from the brain areas with known spontaneous neurogenesis, i.e. hippocampus, olfactory bulb, and subventricular zone, of 4– 6-week-old male Wistar rats as described [16]. Protocols are concordant with the policy on the use of animals, as endorsed by the National Research Council of the USA, and fulfill the requirements of German law. Briefly, animals were anesthetized by isoflurane inhalation and sacrificed by decapitation. The brains were removed and washed in 50 ml ice-cold Dulbecco’s phosphate buffered saline (DPBS) supplemented with 4.5 g/l glucose (DPBS/Glc). Hippocampus, olfactory bulb, and subventricular zone from six animals were dissected, washed in 10 ml DPBS/Glc and centrifuged for 5 min at 1600 £ g at 4 8C. After removal of the supernatant, the tissue was homogenized with scissors and
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00407-5
60 60 58 60 60 60 60 50 -CCGAATAGCGAGCAGATTTC-30 50 -TTCCATCCGGAACAAATCTC-30 50 -TGAGCTCTGAGAACTGTGCA-30 50 -ATGTCGGGAACTCTGATTGG-30 50 -CGGATCCTGATGAAACGAGT-30 50 -AAATGCTTTCTCCGCTCTGA-30 50 -GGATGGAATTGTGAGGGAGA-30 50 -TTCCGGACTTTCAACACCTC-30 50 -CGATGTCTCCTCCATCGTTT-30 50 -TAAGGTGTACACCACGCAGA-30 50 -GGAGCTACTGGGCTGTGAAG-30 50 -ACACAAGGAGCCATTTCCAG-30 50 -CTCACCAAAGCCAGCACATA-30 50 -GAGGACCAGGTTGTCTCCTG-30 VEGF-R1 (Flt-1) VEGF-R2 (KDR, Flk-1) VEGF-R3 (Flt-4) Neuropilin-1 Neuropilin-2 VEGF-A (all splice variants) GAPDH
D28498 NM_013062 NM_053652 AF018957 NM_030869 NM_031836 NM_017008
Reverse primer Forward primer GenBank accession number
scalpels. The tissue pieces were washed with DPBS/Glc medium for 5 min at 800 £ g, and the three pellets were resuspended in 0.01% (w/v) papain, 0.1% (w/v) dispase II (neutral protease), 0.01% (w/v) DNase I, and 12.4 mM manganese sulfate in Hank’s balanced salt solution (HBSS). The tissue was triturated with plastic pipette tips and incubated for 40 min at room temperature, but every 10 min the solution was mixed well. The suspension was centrifuged at 800 £ g for 5 min at 4 8C and pellets were washed three times in 10 ml DMEM-Ham’s F-12 medium supplemented with 2 mM L -glutamine, 100 units/ml penicillin and 100 units/ml streptomycin. Then, the cell pellets were resuspended in 1 ml Neurobasal medium supplemented with B27 (Invitrogen, Carlsbad, CA), 2 mM L -glutamine, 100 units/ml penicillin, 100 units/ml streptomycin, 20 ng/ml endothelial growth factor (EGF), 20 ng/ml fibroblast growth factor-2 (FGF-2), and 2 mg/ml heparin. Cells were plated under sterile conditions in six-well dishes at 25,000 –100,000 cells/ml. The dishes were incubated at 37 8C in 5% CO2. Cell culture media were changed once a week, where about two-thirds of the media were replaced. Experiments were performed after 6 – 8 weeks in vitro. For stimulation with exogenous VEGF, human recombinant VEGF-A165 (Chemicon, Temecula, CA) was added to the cell culture medium in a final concentration of 50 ng/ml which has a maximum effect on hematopoietic stem cells [11]. Total RNA was extracted from cultured neural stem cells isolated from hippocampus, subventricular zone, and olfactory bulb using the RNeasy total RNA extraction kit following the manufacturer’s instructions (Qiagen, Santa Clarita, CA). RNA concentration was determined photometrically, the quality of the total RNA extract was controlled by agarose gel electrophoresis, and RNA was stored at 2 80 8C until use. Reverse transcription was performed on 5 mg of the total RNA extracts of hippocampus, subventricular zone, and olfactory bulb by using the SuperScriptII RNase H2 Reverse Transcriptase (Invitrogen-Life Technologies, Carlsbad, CA) with an oligo(dT)12-18 primer for first strand cDNA synthesis. For amplification, specific PCR primer pairs (Table 1) and Taq DNA polymerase (Promega, Madison, WI) were used in a protocol of 5 min initial denaturation at 94 8C, 35 cycles of amplification with 30 s denaturation at 94 8C, 50 s annealing at 55 8C, and 60 s extension at 72 8C, followed by a final extension for 5 min. Amplification products were visualized by agarose gel electrophoresis. GAPDH was used as internal control. For anoxia, cell culture dishes were incubated in a modular incubator chamber (Billups-Rothenberg, Del Mar, CA) at 37 8C for 24 h in a humidified 5% CO2 and 95% N2 atmosphere as described [11]. Viable cells were detected by Trypan Blue exclusion. The cell suspension was diluted 1:10 in Trypan Blue and viable cells excluding the dye were set in relation to the total number of cells. For quantitation of cell survival, the CellTiter 96
Melting temperature (8C)
M.H. Maurer et al. / Neuroscience Letters 344 (2003) 165–168
Table 1 Specific PCR primer pairs for VEGF and its receptors
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M.H. Maurer et al. / Neuroscience Letters 344 (2003) 165–168
AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) was used, based on a colorimetric tetrazolium salt method. The mean ^ SD of eight experiments each for the normoxia, normoxia þ VEGF, anoxia, and anoxia þ VEGF groups were compared by Student’s t-test with corrections for multiple testing. To determine whether neural stem cells produce VEGFA or any of its receptors, RT-PCR was performed on total RNA isolated from neurosphere cultures. During normoxia, strong signals were found for VEGF in each of the three cell types taken from hippocampus, subventricular zone, and olfactory bulb (Fig. 1, row 1). In parallel, all three types of stem cells expressed mRNA encoding for VEGF-R2. Neuropilin-1 mRNA was only expressed in hippocampal neural stem cells whereas neuropilin-2 mRNA was expressed in hippocampal and olfactory bulb stem cells. No signal was found for VEGF-R1 and VEGF-R3 under normoxic conditions. When stimulated by 50 ng/ml exogenous VEGF-A, all three cell types expressed mRNA for neuropilin-1 and neuropilin-2 (Fig. 1, row 2). The signal for VEGF-R2 diminished in all three cell types, whereas VEGF expression was not suppressed. The incubation under severe anoxic conditions consisting of 5% CO2 and 95% N2 at 37 8C for 24 h changed the expression pattern: the expression of VEGF-R2 mRNA in the cells from subventricular zone and olfactory bulb vanished, but it persisted in hippocampal neural stem cells. Additionally, the expression of neuropilin-1 and neuropilin-2 diminished in these cells, whereas it persisted in the hippocampal cells. On the other hand, VEGF-3 mRNA was induced in the neural stem cells from hippocampus (Fig. 1, row 3). The combination of anoxia and VEGF showed expression of VEGF-R2 in
Fig. 1. Regional heterogeneity in the expression pattern of VEGF and its receptors in neural stem cells. Agarose gel electrophoresis of RT-PCR products shows high expression levels of mRNA for VEGF, VEGF-R2, neuropilin-1, and neuropilin-2 in several types of neural stem cells isolated from hippocampus (H), subventricular zone (S), and olfactory bulb (B). Row 1, normoxic expression pattern; row 2, normoxic expression pattern with the addition of 50 ng/ml exogenous VEGF; row 3, anoxic expression pattern; row 4, anoxic expression pattern with the addition of 50 ng/ml exogenous VEGF. Eight repetitions were performed for each condition.
167
hippocampal, neuropilin-1 in olfactory bulb, and neuropilin2 in all three types of neural stem cells (Fig. 1, row 4). Anoxia caused a decreased cell viability after 24 h down to 15 – 20% of surviving cells. The addition of 50 ng/ml exogenous VEGF in the anoxia experiments resulted in a doubled survival rate compared to hypoxia alone (Fig. 2). VEGF expression has not yet been investigated in adult neural stem cells, but recently VEGF expression has been reported in hematopoietic stem cells [9]. The authors of this study [9] postulated an internal autocrine loop mechanism, by which VEGF could regulate cell survival when coexpressed with its receptors. Our study is in accordance with this hypothesis: VEGF mRNA is expressed in all neural stem cell lines from hippocampus, subventricular zone, and olfactory bulb. Neither anoxia nor exogenous VEGF could suppress its expression, indicating that the intrinsic loop of VEGF stimulation in brain-derived stem cells is effective. Moreover, this autocrine intrinsic loop is independent of the receptor expression, as the altered VEGF receptor pattern indicates. Interestingly, the highest mRNA expression for VEGF was found in vivo in the regions with spontaneous neurogenesis [13]. Thus our finding of VEGF expression in neural stem cells indicates that these cells may contribute to neovascularization and angiogenesis as a migratory stimulus for endothelial precursors or by direct angiogenic effects. A comprehensive analysis of the expression patterns of the different VEGF receptors hat not yet been performed in neural stem cells. Our study shows a regional heterogeneity of VEGF receptor expression in neural stem cells isolated from different brain structures, i.e. from hippocampus, subventricular zone and olfactory bulb, indicating a heterogeneous developmental potential of these cells. A recent
Fig. 2. Cell viability assays for the four experimental conditions. Tetrazolium salt color development and Trypan Blue exclusion were used to quantify surviving cells. Twenty-four hour anoxia decreased cell survival dramatically. Stimulation with 50 ng/ml exogenous VEGF doubled the amount of surviving cells during anoxia in all three neural stem cell lines compared to anoxia alone. Data show that exogenous VEGF rescues neural stem cells from anoxia. Eight repetitions were performed for each condition.
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study found that VEGF-R2 is expressed in cortical cell cultures from mouse embryos and that VEGF-R2 expression increased in vivo in brains stimulated by administration of exogenous VEGF [10]. The diverse expression pattern of the VEGF receptors might be linked to the neuroprotective effect of VEGF, which has been described recently [11,14,19]. In neurons, the neuroprotective effects are mediated by VEGF receptors and neuropilins which were originally specified as semaphorin receptors but then were found also to bind VEGF [15]. Similarly, our study shows protective effects of VEGF towards anoxia in neural stem cells. This protective effect might be related to VEGF-R2 expression which is known to mediate cell proliferation and survival. Initially, the neuropilins had been regarded as pure co-receptors for VEGF-R2, but two recent studies showed a VEGFindependent up-regulation of neuropilins after cerebral ischemia [7,20]. The induction of neuropilins upon exogenous VEGF stimuli could indicate a growth limiting signal for proliferative neural cells. Neuropilins are mediating negative axon guidance for neurons by inhibiting the direction of axon growth due to the induction of apoptosis [2]. Our findings suggest that neuropilins might also have growth limiting abilities in neural progenitors in regions where new vasculature should be provided. The loss of VEGF-R2 in two of the stem cell lines in reaction to anoxia might indicate an alternative protective mechanism, since VEGF-R2 is mainly responsible for cell proliferation and survival. Proliferation and mitosis are processes with an increased cellular vulnerability, when cells are extremely susceptible for DNA damage. Thus, a reduced proliferation by down-regulating the responsible receptor indicates an additional protective mechanism. Of note, VEGF-R3 was induced by anoxia in hippocampal cells, which also showed the highest rate of survival of the three lines, indicating a yet unknown function of this receptor in cell survival. Previous studies only had shown that VEGF-R3 is necessary for lymphangiogenesis [6,18]. In conclusion, the present study shows three major results: (1) VEGF is expressed in cultivated adult neural stem cells isolated from the regions of the rat brain with known spontaneous neurogenesis, i.e. hippocampus, subventricular zone, and olfactory bulb; (2) a regional diversity in the expression pattern of VEGF receptors exists in the three types of adult neural stem cells; and (3) the expression pattern can be changed by exogenous VEGF and/or anoxic stimuli.
Acknowledgements We thank Mrs Inge Keller and Maria Harlacher for technical assistance. This study was supported by the German Federal Ministry of Education and Research (BMBF, Kompetenznetz Schlaganfall, Projektgruppe B2 Heidelberg).
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