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. Author manuscript; available in PMC: 2009 Sep 15.
Published in final edited form as: J Cereb Blood Flow Metab. 2007 Oct 31;28(4):764–771. doi: 10.1038/sj.jcbfm.9600573

Coupling of angiogenesis and neurogenesis in cultured endothelial cells and neural progenitor cells after stroke

Hua Teng 1, Zheng Gang Zhang 1, Lei Wang 1, Rui Lan Zhang 1, Li Zhang 1, Dan Morris 2, Sara R Gregg 1, Zhenhua Wu 3, Angela Jiang 1, Mei Lu 4, Berislav V Zlokovic 5, Michael Chopp 1,6
PMCID: PMC2744583  NIHMSID: NIHMS127047  PMID: 17971789

Abstract

Angiogenesis and neurogenesis are coupled processes. Using a coculture system, we tested the hypothesis that cerebral endothelial cells activated by ischemia enhance neural progenitor cell proliferation and differentiation, while neural progenitor cells isolated from the ischemic subventricular zone promote angiogenesis. Coculture of neural progenitor cells isolated from the subventricular zone of the adult normal rat with cerebral endothelial cells isolated from the stroke boundary substantially increased neural progenitor cell proliferation and neuronal differentiation and reduced astrocytic differentiation. Conditioned medium harvested from the stroke neural progenitor cells promoted capillary tube formation of normal cerebral endothelial cells. Blockage of vascular endothelial growth factor receptor 2 suppressed the effect of the endothelial cells activated by stroke on neurogenesis as well as the effect of the supernatant obtained from stroke neural progenitor cells on angiogenesis. These data suggest that angiogenesis couples to neurogenesis after stroke and vascular endothelial growth factor likely mediates this coupling.

Keywords: angiogenesis, neurogenesis, stroke, rat, VEGF

Introduction

Endothelial cells release factors that stimulate the self-renewal of both embryonic and adult neural stem cells, inhibit their differentiation, and promote their production of neurons (Shen et al, 2004). In the adult rodent brain, neural progenitor cells are localized adjacent to endothelial cells in the subventricular zone (SVZ) and the dentate gyrus (Capela and Temple, 2002; Palmer et al, 2000).

Cerebral ischemia increases neurogenesis, and the newly generated neurons in the SVZ migrate toward the ischemic boundary where angiogenesis takes place (Arvidsson et al, 2002; Jin et al, 2001; Parent et al, 2002; Zhang et al, 2001, 2004). Blockage of stromal derived factor 1α (SDF-1α) and angiopoietin 1 in remodeling cerebral vessels attenuates neuroblast migration toward the cortical ischemic boundary (Ohab et al, 2006). These data suggest that angiogenesis affects neurogenesis and neuroblast migration in the adult ischemic brain.

Adult SVZ neural progenitor cells express many genes involved in angiogenesis. Ischemic stroke upregulates angiogenic gene expression in SVZ neural progenitor cells (Liu et al, 2007). Thus, it is possible that, in addition to their roles in neural progenitor cell biology, angiogenic genes in the neural progenitor cells promote angiogenesis. Accordingly, using a coculture system, the present study tested the hypothesis that cerebral endothelial cells activated by ischemia enhance neural progenitor cell proliferation and differentiation while neural progenitor cells isolated from the ischemic SVZ promote angiogenesis.

Materials and methods

All experimental procedures were approved by the Institutional Animals Care and Use Committee of Henry Ford Hospital.

Animal Model

The middle cerebral artery (MCA) of male Wistar rats (3 to 6months) was occluded by placement of an embolus at the origin of the MCA (Zhang et al, 1997). In this model, occlusion of the MCA evokes a peak increase of neurogenesis in the SVZ and upregulates VEGF and angiopoietin family genes 7 days after stroke (Zhang et al, 2001, 2002, 2004). Therefore, all rats were killed 7 days after middle cerebral artery occlusion (MCAo).

Experimental Protocols

The effect of rat brain endothelial cells activated by stroke on neural progenitor cell proliferation and differentiation

  1. To investigate the effect of rat brain endothelial cells (RBECs) activated by stroke on SVZ cell proliferation, RBECs derived from stroke rats were cocultured with normal SVZ cells in the SVZ growth medium for 4 days. To label proliferating SVZ cells, bromodeoxyuridine (BrdU, 10 μg/mL; Sigma, St Louis, MO, USA) was added 16 h before termination of experiments. The number of BrdU-positive SVZ cells was counted.

  2. To measure the effect of RBECs activated by stroke on SVZ cell differentiation, RBECs derived from stroke rats were cocultured with normal SVZ cells in the SVZ growth medium for 4days. Transwells containing RBECs were discarded and basic fibroblast growth factor (bFGF) as well as epidermal growth factor was withdrawn from the medium. The SVZ cells were further cultured in the differentiation medium for another 7 days. Genotypes and phenotypes of cell fate were determined with real-time PCR and immunostaining, respectively.

Normal SVZ cells cocultured with RBECs harvested from normal rats were used as control groups.

The effect of soluble proteins secreted by stroke neural progenitor cells on RBECs

  1. To measure whether soluble proteins secreted by stroke SVZ cells affect angiogenesis, a capillary-like tube formation assay was performed. The supernatant was collected from normal and stroke SVZ cells (1 × 106 cells/mL cultured in a growth medium containing low bFGF (10 ng/mL) for 2 days). RBECs were seeded on 96-well plates (coated with 70% matrigel and 30% DMEM) and incubated with the supernatant collected from normal or stroke SVZ cells. The capillary tube length was measured 3 to 4 h after incubation.

  2. To measure soluble proteins secreted by stroke SVZ cells, levels of VEGF in the supernatant were measured using ELISA. To investigate whether VEGF in the supernatant promotes capillary-like tube formation, RBCEs were cultured with the supernatant in the presence of a specific VEGF receptor 2 antagonist (SU1498, 5 μmol/L; LC Laboratories, Woburn, MA, USA).

Culture of Rat Brain Microvascular Endothelial Cells

Cerebral microvascular endothelial cells were isolated from normal adult rats (n = 9) and from rats subjected to 7 days of embolic MCA occlusion (n = 12), according to published protocols (Wu et al, 2003). Briefly, rats were killed and their brains were collected in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 1% penicillin and streptomycin (Invitrogen). Cerebella, white matter, meninges, and visible blood vessels of the brain were removed under a microscope. The cerebral cortex and subcortex of normal rats and the ischemic boundary of cortex and subcortex of stroke rats were cut into small pieces and homogenized. Homogenates were suspended in 15% dextran (Sigma, St Louis, MO, USA) and centrifuged at 6,000g for 15 mins at 4°C. Pellets were resuspended and digested with 0.1% collagenase/dispase (Roche Applied Science, Penzberg, Germany) and 2% fetal bovine serum albumin (Invitrogen) in RPMI1640. Digested microvessels were separated with 45% Percoll (Sigma, St Louis, MO, USA) (20,000 g, 10 mins, 4°C) and plated into Collagen I (BD Biosciences, Bedford, MA, USA) coated plates. Cultures were maintained in endothelial growth medium described by Wu et al (2003). Passage 2 to 4 endothelial cells were employed in the present study.

Culture of Neural Progenitor Cells

Subventricular zone (SVZ) cells were isolated from normal rats (n = 9) and from rats subjected to 7 days of embolic MCAo (n = 12), as previously reported (Morshead et al, 1994; Wang et al, 2004b). The cells were plated at a density of 2 × 104 cells/ml in DMEM-F-12 medium (Invitrogen) containing 20 ng/ml of epidermal growth factor and bFGF (R&D System, Minneapolis, MN, USA). This medium was termed as SVZ growth medium in the present study. The generated neurospheres (primary sphere) were passaged by mechanical dissociation and reseeded as single cells at a density of 20 cells/μL in the growth medium (passage 1 cells) (Wang et al, 2004b). Passage 1 to 3 SVZ cells were used in the present experiments. Differentiation of SVZ cells was induced in a differentiation medium containing 2% fetal bovine serum (Invitrogen) but without bFGF and epidermal growth factor.

Coculture of Endothelial Cells with Neural Progenitor Cells

Coculture system was set up according to published protocols with minor modification (Shen et al, 2004). Briefly, the day before coculturing with SVZ cells, endothelial cells (1 × 105) were plated into 0.4 μm transwell membrane inserts (Becton Dickinson Labware, Franklin Lakes, NJ, USA) in endothelial growth medium for 24 h. After that, transwells were rinsed and placed above SVZ cells (1 × 105) plated freshly in six-well plate containing the SVZ growth medium.

Measurement of Caspase-3 Activity

Caspase-3 activity in live SVZ cells was measured with a NucView 488 Caspase-3 Assay Kit for Live Cells according to the manufacturer's instructions (Biotium, Hayward, CA, USA). The NucView™ 488 Caspase-3 Substrate is a true enzyme substrate that detects caspase-3 in live cells without interfering with the enzyme activity (Biotium, Hayward, CA, USA). Briefly, the cells were grown on coverslips (n = 4/group), media were aspirated, and cells were incubated with 5 μmol/L NucView™ 488 Caspase-3 substrate in phosphate-buffered saline for 30 mins. Five fields per section were digitized with a × 20 objective under a fluorescence microscope (Olympus IX71, Olympus Optical Co. Ltd, Tokyo, Japan) attached to a color CoolSnap CCD camera (Roper Scientific Photometrics, Tucson, AZ, USA) using MetaMorph Software (Universal Imaging Co., Downingtown, PA, USA). The number of caspase-3-positive cells and the total number of cells per field were counted and the percentage of caspase-3-positive cells per field was determined.

Terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling assay

Apoptotic cells were detected by an ApopTag Fluorescein In Situ Apoptosis Detection Kit, according to the manufacturer's instructions (Chemicon International Inc., Temecula, CA, USA). Briefly, the cultured cells on coverslips were fixed with paraformaldehyde and incubated with TdT enzyme. Cell nuclei were stained with 4′,6′-diamidino-2-phenylindole (1:10,000; Vector Laboratories, Burlingame, CA, USA). The number of terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling-positive cells and nuclei were counted. The data are presented as percentage of total cells.

Capillary Tube Formation Assay

Ninety-six-well plates were coated with 100 μL solution containing 70% matrigel (BD Biosciences, Bedford, MA, USA) and 30% DMEM (Invitrogen)). RBECs (2 × 104 cells/well) were then seeded in the coated 96-well plates and incubated with 100 μL supernatant from normal or stroke SVZ cells for 3 to 4 h at 37°C. Formation of capillary-like networks was recorded under a × 10 objective (Olympus IX71, Olympus Optical Co. Ltd) via a color CoolSnap CCD camera (Roper Scientific Photometrics, Tucson, AZ, USA) and the total length of tube formation was measured by means of the microcomputer imaging device (Imaging Research, St Catharines, ON, Canada) (Wang et al, 2004a; Zhang et al, 2003). Data are presented as the length in mm/mm2.

Real-Time PCR

Quantitative real-time PCR was performed according to published methods (Liu et al, 2006; Wang et al, 2004b, 2006). Briefly, total RNA from cocultured SVZ cells was isolated using the Absolutely RNA Microprep Kit (Stratagene, La Jolla, CA, USA) and was followed by reverse transcription in accordance with the manufacturer's procedure. Real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on an ABI 7000 PCR instrument (Applied Biosystems, Foster City, CA, USA). Three-stage program parameters provided by the manufacturer were employed as follows: 2 mins at 50°C, 10 mins at 95°C, and then 40 cycles of 15 secs at 95°C and 1 min at 60°C. The specificity of PCR product was verified by performing dissociation reaction plots. Each sample was tested in triplicate, and data obtained from three independent experiments were used to quantify the relative gene expression by the 2−ΔΔCt method (Livak and Schmittgen, 2001).

Using a Primer Express software (Applied Biosystems, Foster City, CA, USA), we designed the following primers used in the present study: β-actin (forward, 5′-CCATCATGAAGTGTGACGTTG-3′; reverse, 5′-CAATGATCTTGATCTTCATGGTG-3′), β-III tubulin (forward, 5′-CCCGAGGGCTCAAGATGTC-3′; reverse, 5′-CGCTTGAACAGCTCCTGGAT-3′), glial fibrillary acidic protein (GFAP) (forward, 5′-TGGAACTGACACGTTGTGTTCA-3′; reverse, 5′-CAGCTTCCGAGAGGGTACACTAA-3′), Sox2 (forward, 5′-CACAACTCGGAGATCAGCAA-3′; reverse, 5′-CTCCGGGAAGCGTGTACTTA-3′), and Hes6 (forward, 5′-GGCTTGTATGGTAACCCTGATG-3′, reverse, 5′-CCCTTCTTTCTGTAGTGTCCTCC-3′).

Immunofluorescent Cytochemistry and Image Analysis

Immunofluorescent staining was performed as described previously (Wang et al, 2004b; Zhang et al, 2004). Briefly, cells were fixed in 4% (w/v) paraformaldehyde, and nonspecific binding was blocked by a block solution containing of 1% (w/v) bovine serum albumin and 0.3% Triton-100 (v/v) for 1 h at room temperature. Cells were incubated with primary antibodies for 1 h at room temperature followed by incubation with CY3-conjugated secondary antibodies. The following primary antibodies were employed: mouse anti-PECAM-1 (CD31, a marker of endothelial cells, 1:100; Chemicon International Inc., Temecula, CA, USA); mouse anti-ED1(a marker of pericytes, 1:100; Serotec Immunological Excellence, Oxford, UK); rabbit anti-ZO-1(a marker of tight junction, 1:200; Zymed Laboratories Inc., South San Francisco, CA, USA); mouse anti-BrdU (a marker of proliferating cells, 1:100; Boehringer Mannheim, Indianapolis, IN, USA); mouse anti-β-tubulin III (Tuj-1, a marker of neurons, 1:1,000; Covance the Development Services Company, Berkeley, CA, USA); rabbit anti-GFAP (a marker of astrocytes, 1:500; Dako Cytomation California Inc., Carpinteria, CA, USA). Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (1:10,000; Vector Laboratories). For Dil-Ac-LDL labeling, cells were incubated with 10 μg/ml Dil-Ac-LDL (Biomedical Technologies Inc., Stoughton, MA, USA) at 37°C for 4 h. The labeled cells were washed with phosphate-buffered saline twice and observed under a fluorescence microscope (Olympus IX71, Roper Scientific Photometrics, Tucson, AZ, USA).

Seven fields of view (670 × 460 mm) per section immunostained with different antibodies were digitized using a × 20 objective of a fluorescent microscope (Axiophot2, Carl Zeiss Inc., Thornwood, NY, USA) via a digital camera (Hamamatsu Photonics, K.K., Hamamatsu City, Japan) equipped with a microcomputer imaging device analysis system (Imaging Research). The number of TuJ-1-, GFAP-, or BrdU-positive cells and the total number of 4′,6′-diamidino-2-phenylindole cells per field were counted and the percentage of each cell type per field was determined (Wang et al, 2004b; Zhang et al, 2004).

Western Blot Analysis

Western blot was performed according to published methods (Wang et al, 2006). Briefly, Equal amounts of protein (40 μg/lane) for each sample were electrophoresed through a 10% SDS–PAGE gel (Invitrogen) and subsequently electrotransferred to nitrocellulose membranes. Membranes were probed with the primary antibodies phospho-VEGF receptor 2 (1:1,000; Cell Signaling Technology, Beverly, MA, USA), and β-actin (1:10,000; Abcam, Cambridge, MA, USA) for 16 h at 4°C. For detection, horseradish peroxidase-conjugated secondary antibodies were used (1:2000) followed by enhanced chemiluminescence development (Pierce, Rockford, IL, USA).

ELISA For VEGF

Non-stroke or stroke SVZ cells (1 × 106/ml) were cultured in growth medium containing 10 ng/ml of bFGF for 48 h, and the supernatants were collected. VEGF levels in the supernatants were measured using a commercial mouse VEGF ELISA kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA) (Wang et al, 2004a).

Statistical Analysis

For each experiment, one-way analysis of variance was conducted with Tukey's test for group comparisons adjusting for multiplicities. A significant difference was detected if P < 0.05.

Results

Characterization of Rat Cerebral Microvascular Endothelial Cells

Cells isolated from cerebral microvessels formed a confluent monolayer and exhibited cobblestone morphology (Figure 1A). More than 90% of the cells were labeled with DiI-Ac-LDL (Figure 1B). Immunofluorescent staining showed that these cells were CD31 positive (Figure 1C), a marker of endothelial cells, and ZO-1 positive (Figure 1D), a tight junction protein expressed in brain capillary endothelial cells. Few cells were GFAP or ED1 positive, markers of astrocytes or pericytes, respectively, and real-time RT-PCR analysis did not detect GFAP mRNA. Collectively, our data indicate that these cells are primarily cerebral microvascular endothelial cells, and we refer to them as RBECs.

Figure 1.

Figure 1

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Characterization of rat cerebral microvascular endothelial cells (RBECs). Cells isolated from cerebral microvessels formed a confluent monolayer and exhibited cobblestone morphology (A), and these cells were labeled with DiI-Ac-LDL (B, red). Immunofluorescent staining showed that these cells were CD31 (C, red) and ZO-1 (D, red) positive. Bar = 100 μm.

Endothelial Cells Activated by Stroke Increase Proliferation and Neuronal Differentiation of SVZ Cells

We then employed RBECs to investigate the effect of cerebral endothelial cells on SVZ neural progenitor cells. Coculture of normal SVZ cells with RBECs harvested from normal brain in the growth medium significantly increased the number of BrdU+ SVZ cells (47.8% ± 1.3%) compared with the number in normal SVZ cells-alone group (40.8% ± 3.0%; Figures 2A, 2B, and 2E). When the normal SVZ cells were cocultured with RBECs isolated from the stroke boundary in the growth medium, the number of BrdU+ SVZ cells further increased (56.5% ± 4.4%, P < 0.05; Figures 2C and 2E) compared with the number in SVZ cells cocultured with RBECs isolated from normal brain (Figures 2B and 2E). When transwells containing stroke or normal RBECs were removed 4 days after coculture, SVZ cells differentiated into neurons and glia in the differentiation medium. Coculture of normal SVZ cells with stroke but not normal RBECs significantly increased mRNA levels of β-III tubulin (Figure 2Q) and the number of Tuj1+ SVZ cells (Figures 2H, 2I, 2O, and 2Q) compared with that in cultured SVZ cells without RBECs (Figures 2G, 2O, and 2Q). Interestingly, coculture of normal SVZ cells with normal or stroke RBECs significantly reduced GFAP mRNA levels (Figure 2Q) and the number of GFAP+ cells (Figures 2K to M and 2P). In parallel, SVZ cells cocultured with stroke RBECs exhibited a reduction in the levels of Sox2 mRNA, a gene involved in the maintenance of neural progenitor cell identity (Bani-Yaghoub et al, 2006; Graham et al, 2003), and an increase in the levels of Hes6 mRNA, a gene that promotes neuronal differentiation but inhibits astrocyte differentiation (Gratton et al, 2003; Jhas et al, 2006) (Figure 3). However, when normal SVZ cells were cocultured with stroke RBECs in the growth or differentiation medium in the presence of SU1498, a VEGFR2 antagonist, the number of BrdU+ (Figures 2C to E) and Tuj1+ (Figures 2I, 2J, and 2O) cells was significantly reduced, while the number of GFAP+ cells did not significantly change (26.8% ± 1.8% versus 23.5% ± 4.0% in the SU1498 group, Figures 2M, 2N, and 2P). Western blot analysis revealed that stroke RBECs induced phosphorylation of VEGFR2 in normal SVZ cells, which was substantially suppressed by SU1498 (Figure 2F), indicating that SU1498 inactivates VEGFR2 triggered by stroke RBECs. These data suggest that VEGF signaling mediates the effect of activated endothelial cells on proliferation and differentiation of neural progenitor cells.

Figure 2.

Figure 2

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Coculture of normal subventricular zone (SVZ) cells with RBECs isolated from the ischemic boundary increases neural progenitor cell proliferation. SVZ cells were immunostained with antibody against BrdU, and the number of BrdU-positive cells (red) was counted. Panels A to D show representative images of BrdU-positive cells (red) in normal SVZ cells alone (A, n = 4), normal SVZ cells cocultured with normal (B, n = 5) or stroke (C, n = 6) RBECs, and normal SVZ cells cocultured with stroke RBECs in the presence of SU 1498, a specific VEGFR2 antagonist (D, n = 4). Panel E shows quantitative data of BrdU-positive SVZ cells. Panel F is a representative Western blot image showing phosphorylated VEGFR2 in normal SVZ cells cocultured with stroke RBECs (MCAo), normal SVZ cells cocultured with stroke RBECs in the presence of SU1498 (MCAo + SU), and SVZ cells only (SVZ only). Coculture of normal SVZ cells with RBECs isolated from the ischemic boundary promotes neural progenitor cell differentiation into neurons and represses glial differentiation. Panels G to N show representative images of Tuj1- (red, G to J) and GFAP- (red, K to N) positive cells 7 days after the culture in normal SVZ cells alone (G, n = 4; K, n = 4), normal SVZ cells cocultured with normal (H, n = 5; L, n = 4) or stroke RBECs (I, n = 5; M, n = 5), and normal SVZ cells cocultured with stroke RBECs in the presence of SU 1498 (J, n = 4; N, n = 5). Panels O and P show quantitative data of Tuj1- (O) and GFAP- (P) positive cells. Panel Q (n = 3) shows quantitative data of β-III tubulin and GFAP mRNA levels measured by real-time RT-PCR. Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (blue). *P < 0.05 versus the SVZ cell-alone group (none), #P < 0.05 versus the normal RBEC group (normal), +P < 0.05 versus the stroke RBEC group (MCAo). MCAo + SU represents normal SVZ cells cocultured with stroke RBECs in the presence of SU 1498. Bar = 100 μm.

Figure 3.

Figure 3

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Sox2 and Hes6 mRNA levels. Coculture of normal SVZ cells with RBECs isolated from the ischemic boundary decreased Sox 2 and increased Hes6 mRNA levels measured with real-time RT-PCR. *P < 0.05 versus the normal RBEC group (n = 3).

VEGF mediates neural stem cell survival (Wada et al, 2006). To examine whether increases in the number of BrdU+ and Tuj1+ cells observed after the coculture are due to reduction of neural progenitor cell apoptosis by VEGF, we examined the number of caspase-3-positive cells in live SVZ cells by means of NucView™ 488 Caspase-3 Substrate. When SVZ cells were cultured in the growth medium, 2.0% ± 0.5% (n = 4), 1.9% ± 0.6% (n = 4), and 2.1% ± 0.6% (n = 4) cells were caspase-3 positive in the normal SVZ cells cocultured with normal endothelial cells, stroke endothelial cells, and stroke endothelial cells in the presence of SU1498, respectively (P > 0.05, among the groups). The number of caspase-3-positive cells among the three groups was also not significantly different in normal SVZ cells cultured in the differentiation medium (1.9% ± 0.7%, n = 4 for normal endothelial cells; 2.0% ± 0.9%, n = 4 for stroke endothelial cells; and 2.2% ± 1.1%, n = 4 for stroke endothelial cells in the presence of SU1498, P > 0.05). In parallel, the number of terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling-positive cells was not significantly changed among the three groups (3.0% ± 0.5% for normal endothelial cells; 2.3% ± 0.7% for stroke endothelial cells; and 2.5% ± 0.8% for stroke endothelial cells in the presence of SU1498, P > 0.05). These data suggest that the effect of VEGF on neural progenitor cells is unlikely to result from reduction of cell apoptosis.

Activated Neural Progenitor Cells Stimulate Angiogenesis In Cultured Endothelial Cells via Secretion of VEGF

Neuroblasts in the SVZ are closely associated with cerebral vessels when they migrate toward the ischemic boundary (Zhang et al, 2004). To examine whether soluble proteins secreted by stroke SVZ cells promote angiogenesis, we performed a capillary-like tube formation assay. Incubation of RBECs isolated from normal rats with the supernatant obtained from stroke SVZ cells significantly (P < 0.05) increased tube formation (70 ± 2.6 mm/mm2; Figures 4B and 4D) compared with RBECs cultured with supernatant collected from normal SVZ cells (47 ± 5.2 mm/mm2; Figures 4A and 4D). ELISA analysis revealed that VEGF levels were significantly (P < 0.05) higher in the supernatant collected from stroke SVZ cells (507 ± 29 pg/mg, n = 4) than the levels in the supernatant obtained from normal SVZ cells (458 ± 13 pg/mg, n = 4). Blockage of VEGFR2 with SU1498 significantly (P < 0.05) attenuated capillary-like tube formation induced by the supernatant of stroke SVZ cells (48 ± 5.6 mm/mm2; Figures 4C and 4D), suggesting that VEGF secreted by stroke SVZ cells promotes capillary-like tube formation.

Figure 4.

Figure 4

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Stroke neural progenitor cells stimulate in vitro angiogenesis via VEGF. Panels A to C show representative images of capillary-like tube formation of endothelial cells incubated with the supernatant harvested from normal SVZ cells (A), stroke SVZ cells (B), or stroke SVZ cells in the presence of SU 1498 (C). Panel D shows quantitative data of capillary lengths. *P < 0.05 versus the supernatant from the normal SVZ group (normal); #P < 0.05 versus the supernatant from the stroke SVZ group (MCAo) (n = 15). MCAo + SU and Normal + SU represent supernatants from stroke and normal SVZ cells, respectively, in the presence of SU 1498. Bar = 100 μm.

Discussion

The present study demonstrates that cerebral endothelial cells activated by stroke enhanced neural progenitor cell proliferation and neuronal differentiation, while neural progenitor cells isolated from stroke SVZ promoted capillary tube formation. Both the effects were blocked by the VEGFR2 antagonist. These data suggest that angiogenesis couples to neurogenesis after stroke, and VEGF likely mediates this coupling.

Endothelial cells derived from cell lines stimulate the self-renewal of adult neural stem cells and promote their production of neurons (Shen et al, 2004). To closely mimic the in vivo condition, we isolated RBECs and SVZ cells from adult normal and stroke rat brains. Consistent with previous reports (Shen et al, 2004), the present study showed that coculture of normal SVZ cell with normal RBECs enhanced (15%) neural progenitor cell proliferation but did not increase the number of neurons. However, RBECs activated by stroke not only increased neural progenitor cell proliferation by 28% but also augmented neuronal population by 46%, indicating that activated cerebral endothelial cells promote neurogenesis. In contrast to the effect of normal RBECs on neuronal differentiation, stroke RBECs substantially reduced neural progenitor cell differentiation into astrocytes. Concurrently, stroke RBECs down and upregulated Sox2 and Hes6 expression, respectively. Sox2 is required to maintain the neural progenitor pool, and overexpression of Sox2 promotes neural progenitor cell differentiation into astrocytes but inhibits neurogenesis (Bani-Yaghoub et al, 2006; Graham et al, 2003). Hes6 promotes neuronal fate and suppresses astrocyte differentiation (Gratton et al, 2003; Jhas et al, 2006). Therefore, increased neurogenesis from stroke RBEC cocultured neural progenitor cells could be at the expense of gliogenesis. Collectively, our data provide the first direct evidence that cerebral endothelial cells activated by stroke enhance neurogenesis by stimulating neural progenitor cell proliferation and promoting neural progenitor cell differentiation into neurons while blocking the progenitor cell differentiation into astrocytes.

In addition to its role in angiogenesis, VEGF mediates neurogenesis by augmenting proliferation and neuronal differentiation of neural progenitor cells (Jin et al, 2002; Meng et al, 2006). Our data are consistent with and extend published studies by demonstrating that blockage of VEGFR2, which is a primary receptor of VEGF, attenuated the effect of stroke RBECs on neural progenitor cell proliferation and neuronal differentiation but did not block the effect of stroke RBECs on astrocyte fate, suggesting that VEGF secreted by stroke endothelial cells promotes neurogenesis, while suppression of astrocyte differentiation could be regulated by other factors released by stroke endothelial cells. Moreover, coculture of SVZ cells with stroke RBECs did not alter the number of capase-3-positive SVZ cells, and inhibition of VEGFR2 did not increase apoptosis, suggesting that VEGF signaling is not involved in cell survival in our coculture system. However, using neural stem cells derived from VEGFR2 knockout mice, a recent study showed that VEGF mediates neural stem cell survival but does not regulate neural stem cell proliferation and differentiation (Wada et al, 2006). Exogenous VEGF did not increase endothelial cell-induced neurogenesis when mouse brain endothelial cells were cocultured with SVZ cells (Shen et al, 2004). We do not know the exact causes of the discrepancy other than the different sources of endothelial cells, with primary brain endothelial cells employed in the present study, and different experimental conditions used in the present study.

The effect of neural progenitor cells on cerebral endothelial cells has not been extensively investigated. During brain development, the ventricular neuroectoderm produces VEGF, which promotes vessel growth (Breier et al, 1992; Raab et al, 2004). Adult SVZ neural progenitor cells express many angiogenic factors, including VEGF and its receptors (Liu et al, 2007; Maurer et al, 2003; Rafuse et al, 2005; Tonchev et al, 2007). Stroke upregulates angiogenic genes in SVZ cells (Liu et al, 2007). The present study demonstrates that stroke SVZ cells secreted high levels of VEGF, which promoted capillary tube formation, and inhibition of VEGF signaling with VEGFR2 antagonist completely abolished SVZ supernatant-induced angiogenesis. These in vitro data provide direct evidence that adult neural progenitor cells could promote angiogenesis via VEGF signaling. However, it cannot be ruled out that other angiogenic factors released by stroke neural progenitor cells might also promote angiogenesis.

In summary, cerebral endothelial cells activated by stroke promote neurogenesis while stroke SVZ cells augment angiogenesis. VEGF mediates coupling of angiogenesis and neurogenesis. These in vitro data underlie in vivo findings that neuroblasts in the SVZ are closely associated with cerebral vessels when they migrate toward the ischemic boundary.

Acknowledgments

We thank Ying Wang, Toh Yier, and Yvonne LeTourneau for technical assistance.

This work was supported by NINDS Grants PO1 NS23393, PO1 NS42345, RO1NS38292, and RO1HL 64766.

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