Vascular endothelial growth factor (VEGF) stimulates neurogenesis <em>in vitro</em> and <em>in vivo</em>
Abstract
Vascular endothelial growth factor (VEGF) is an angiogenic protein with neurotrophic and neuroprotective effects. Because VEGF promotes the proliferation of vascular endothelial cells, we examined the possibility that it also stimulates the proliferation of neuronal precursors in murine cerebral cortical cultures and in adult rat brain in vivo. VEGF (>10 ng/ml) stimulated 5-bromo-2′-deoxyuridine (BrdUrd) incorporation into cells that expressed immature neuronal marker proteins and increased cell number in cultures by 20–30%. Cultured cells labeled by BrdUrd expressed VEGFR2/Flk-1, but not VEGFR1/Flt-1 receptors, and the effect of VEGF was blocked by the VEGFR2/Flk-1 receptor tyrosine kinase inhibitor SU1498. Intracerebroventricular administration of VEGF into rat brain increased BrdUrd labeling of cells in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG), where VEGFR2/Flk-1 was colocalized with the immature neuronal marker, doublecortin (Dcx). The increase in BrdUrd labeling after the administration of VEGF was caused by an increase in cell proliferation, rather than a decrease in cell death, because VEGF did not reduce caspase-3 cleavage in SVZ or SGZ. Cells labeled with BrdUrd after VEGF treatment in vivo include immature and mature neurons, astroglia, and endothelial cells. These findings implicate the angiogenesis factor VEGF in neurogenesis as well.
Vascular endothelial growth factor (VEGF) is a hypoxia-inducible secreted protein that interacts with receptor tyrosine kinases on endothelial cells to promote angiogenesis. Recent evidence indicates that VEGF can also act directly on neurons to produce neurotrophic and neuroprotective effects. For example, VEGF stimulates axonal outgrowth and improves the survival of cultured superior cervical and dorsal root ganglion neurons (1, 2), enhances the survival of mesencephalic neurons in organotypic explant cultures (3), protects HN33 (mouse hippocampal neuron × neuroblastoma) cells from death induced by serum withdrawal (4), reduces hypoxic death of HN33 cells and cultured cerebral cortical neurons (5), and protects cultured hippocampal neurons from glutamate toxicity (6). Conversely, inhibition of VEGF signaling leads to apoptosis in cortical neuron cultures (7), and deletion of the hypoxia-response element from the VEGF promoter causes motor-neuron degeneration in mice (8), perhaps because of loss of a direct neurotrophic effect of VEGF.
Neurogenesis, the process through which precursor cells differentiate toward a mature neuronal phenotype, persists in discrete regions of the adult brain, including the rostral subventricular zone (SVZ) (9, 10) and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) (11). Comparatively little is known regarding the possible role of VEGF in adult neurogenesis, although other growth factors, including epidermal growth factor (EGF) (12), fibroblast growth factor-2 (FGF-2) (13), brain-derived neurotrophic factor (BDNF) (14), and erythropoietin (15), have been implicated. In addition, the VEGF receptor Flk-1 is expressed in neural progenitor cells of the mouse retina (16), and its activation stimulates their differentiation into amacrine neurons and photoreceptor cells (17). Finally, neurogenesis in the adult SGZ appears to occur in intimate association with angiogenesis, suggesting that common factors, such as VEGF, might be involved in both processes (18).
To investigate the possibility that VEGF is a neurogenic as well as an angiogenic factor, we examined its effects in in vitro and in vivo models of neurogenesis, by using the cell-proliferation marker 5-bromodeoxyuridine (BrdUrd). The in vitro model employs primary cultures of embryonic rat cortical neurons, and has been used to identify growth factors involved in hypoxia-induced neurogenesis, including heparin-binding EGF (19). The in vivo model involves intracerebroventricular (i.c.v.) administration of growth factors in the adult rat, and has been used to study the induction of neurogenesis in the brain in vivo by factors such as EGF, FGF-2, and BDNF (20–22).
Acknowledgments
This work was supported by National Institutes of Health Grant NS37695 (to D.A.G.).
Abbreviations
aCSF | artificial cerebrospinal fluid |
BrdUrd | 5-bromo-2′-deoxyuridine |
DAPI | 4′,6-diamidino-2-phenylindole |
Dcx | doublecortin |
EGF | epidermal growth factor |
ENCAM | embryonic nerve cell adhesion molecule |
GFAP | glial fibrillary acidic protein |
i.c.v. | intracerebroventricular |
MTT | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide |
NeuN | neuronal nuclear antigen |
SGZ | subgranular zone |
SVZ | subventricular zone |
VEGF | vascular endothelial growth factor |
vWF | von Willebrand factor |
Footnotes
This paper was submitted directly (Track II) to the PNAS office.
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