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. 2002 Sep 3;99(18):11946-50.
doi: 10.1073/pnas.182296499. Epub 2002 Aug 14.

Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo

Kunlin Jin  1 , Yonghua Zhu, Yunjuan Sun, Xiao Ou Mao, Lin Xie, David A Greenberg
Affiliations

Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo

Kunlin Jin et al. Proc Natl Acad Sci U S A. .

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.

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Figures

Figure 1
Figure 1
VEGF stimulates BrdUrd incorporation in murine cortical cultures in vitro. (A) Immunodetection of BrdUrd labeling (red) in control (Left), and 10 ng/ml VEGF-treated (Right) cortical cultures at 24 h in vitro. (Original magnification, 20×; Insets, 40×.) (B) Quantification of VEGF-stimulated BrdUrd incorporation by ELISA, expressed as a percentage of BrdUrd incorporation in control cultures. (C) Cell counts (number of DAPI-stained nuclei) in VEGF-treated cultures, expressed as a percentage of cell counts in control cultures. (D) Colocalization of BrdUrd labeling (red) with immunoreactivity for the immature neuronal marker ENCAM (green, Left) and the neuroepithelial precursor cell marker nestin (green, Right). Data are representative fields (A and D) or mean values ± SEM (B and C) from at least three experiments per panel.
Figure 2
Figure 2
Association of VEGF-stimulated BrdUrd incorporation with VEGFR2/Flk-1 receptors in murine cortical cultures in vitro. (A) Immunocolocalization of VEGFR2/Flk-1 (green, Top) but not VEGFR1/Flt-1 (green, Bottom) receptors with BrdUrd labeling (red). (Original magnification, 40×.) (B) Inhibition of VEGF (10 ng/ml for 24 h)-stimulated BrdUrd incorporation into cortical cultures by the VEGFR2/Flk-1 receptor tyrosine kinase inhibitor SU1498. Data are representative fields (A) or mean values ±SEM (B) from at least three experiments per panel.
Figure 3
Figure 3
VEGF stimulates BrdUrd incorporation into neuroproliferative zones of rat brain in vivo. aCSF (Left) or 10 μg/ml of VEGF (Right) was infused i.c.v. at 1 μl/h and BrdUrd 5′-monophosphate (50 mg/kg) was given i.p. twice daily for 3 days. Four days later, BrdUrd was localized by immunohistochemistry in hippocampus (A) and rostral SVZ (B) (Original magnification, 4×; high-power views of SVZ, 20×). Note that BrdUrd incorporation is not confined to neuroproliferative zones, but extends, for example, into the dentate hilus in (A) consistent with the labeling of both neuronal and non-neuronal (e.g., astroglial) cells (see Fig. 6). Data are representative fields from at least three experiments per panel.
Figure 4
Figure 4
VEGF receptor expression in neuroproliferative zones of rat brain in vivo. Brain sections through SVZ (A and B) and SGZ (C and D) were stained with antibodies against VEGFR2/Flk-1 (A and C) and VEGFR1/Flt-1 (B and D). (Original magnification, 4×.) VEGFR2/Flk-1, but not VEGFR1/Flt-1, immunostaining was observed in SVZ and SGZ (arrows), as well as in ependymal cells (ciliated cells at far left in A). VEGFR2/Flk-1 expression (green) was colocalized with Dcx (red) in SGZ (E) and SVZ (F). Data are representative fields from at least three experiments per panel.
Figure 5
Figure 5
Caspase-3 cleavage in SGZ (A) and SVZ (B) from control (A Top; B Left) and VEGF-treated (A Bottom; B Right) rat brains in vivo. Brain sections through SGZ were stained with an antibody against the 17 to 20-kDa cleavage product of caspase-3. (Original magnification, 4×; high-power views, 20×.) Data are representative fields from at least three experiments per panel.
Figure 6
Figure 6
VEGF stimulates incorporation of BrdUrd into neurons, astrocytes, and endothelial cells in vivo. Rats were given VEGF and BrdUrd as described in the legend to Fig. 3. Brain sections through SGZ (A) and SVZ (B) were stained with antibodies against the immature neuronal marker Dcx (green) and against BrdUrd (red). (C) Sections were stained with antibodies against BrdUrd (purple) and either the mature neuronal marker NeuN (Left), the astroglial marker GFAP (Center), or the endothelial cell marker vWF (Right; brown). Note that BrdUrd and NeuN are nuclear markers, whereas Dcx, GFAP, and vWF are extranuclear. In C Left, all cells shown stain for NeuN, and all except the cell at far left also stain for BrdUrd. In C Center, a single cell is seen, with GFAP-positive processes and BrdUrd-positive nucleus. In C Right, the vessel running diagonally from top left to bottom right stains for vWF, and the four associated nuclei stain for BrdUrd. Data are representative fields from at least three experiments per panel.
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