VEGF is a chemoattractant for FGF-2-stimulated neural progenitors

Abstract

Migration of undifferentiated neural progenitors is critical for the development and repair of the nervous system. However, the mechanisms and factors that regulate migration are not well understood. Here, we show that vascular endothelial growth factor (VEGF)-A, a major angiogenic factor, guides the directed migration of neural progenitors that do not display antigenic markers for neuron- or glia-restricted precursor cells. We demonstrate that progenitor cells express both VEGF receptor (VEGFR) 1 and VEGFR2, but signaling through VEGFR2 specifically mediates the chemotactic effect of VEGF. The expression of VEGFRs and the chemotaxis of progenitors in response to VEGF require the presence of fibroblast growth factor 2. These results demonstrate that VEGF is an attractive guidance cue for the migration of undifferentiated neural progenitors and offer a mechanistic link between neurogenesis and angiogenesis in the nervous system.

Figure 1.

Morphological and immunocytochemical characterization of neural progenitors in culture. Neural progenitors were isolated and purified from the SVZ of newborn rat brains and cultured on matrigel-coated coverslips in the presence of 20 ng/ml FGF-2. (A and B) Phase-contrast images of neural progenitors at day 4 (A) and day 6 (B) in culture. (C) After 6 d in culture, the majority of cells are immunopositive for nestin, indicating that they are undifferentiated neural progenitors. (D) BrdU incorporation (red) showing that the majority of cells are proliferating. The rare cells that are positive for the neuronal marker (TuJ, green, arrow) are nonproliferative. (E and F) 5 d after the withdrawal of FGF-2, cells differentiate into GFAP-containing astrocytes (E, red), Tuj-positive neurons (E, green), and GalC-positive oligodendrocytes (F, green). Cell nuclei were counterstained with Hoechst 33342 in C, E, and F. Bar: (A and B) 80 μm; (C) 30 μm; (D) 19 μm; (E and F) 30 μm.

Figure 2.

VEGF stimulates chemotaxis of neural progenitors. (A) Schematic representation of Dunn chamber (top view) with the overlying coverslip, showing the position of the inner well, bridge, and outer well. (B) Cells over the annular bridge between the inner and outer wells of the chamber can be observed under phase-contrast optics. Arrow indicates the direction of the outer well of the Dunn chamber. Bar, 50 μm. Cell migration was recorded continuously by time-lapse frame grabbing, and the migration tracks were plotted in scatter diagrams (C–F) as described in Materials and methods. The starting point for each cell is the intersection between the X and Y axes (0,0), and data points indicate the final positions of individual cells at the end of the 2-h recording period. Chemotaxis was tested by placing 200 ng/ml VEGF (C) or FGF-2 (E) in the outer well. The direction of the gradient is vertically upwards. Note that neural progenitors undergo chemotaxis and display a clear directionality of migration in the presence of a VEGF (C) but not an FGF-2 gradient (E). For chemokinesis (D and F), equal amounts of VEGF (20 ng/ml) or FGF-2 (20 ng/ml) were added in both the inner and outer wells of the chamber.

Figure 3.

Migration tracks of neural progenitors. (A) Phase-contrast photos showing a representative cell (*) migrating up a VEGF gradient. Arrow indicates the source of VEGF. (B) Migration tracks of four representative cells in the presence of a VEGF concentration gradient. The starting point for each cell is the intersection between the X and Y axes (0,0), and the source of VEGF is at the top. (C) Phase-contrast photos showing a neural progenitor that randomly migrates in a uniform concentration of VEGF. Arrow indicates the outer well of the Dunn chamber. (D) Migration tracks of four representative cells that migrate randomly under conditions of uniform VEGF distribution. The starting point for each cell is the intersection between the X and Y axes (0,0).

Figure 4.

The migration speed (μm/hr) and FMI values under different conditions. (A) Cell migration speed was calculated for each time-lapse interval and the mean speed was derived for a period of 2 h. Data are shown as mean ± SEM from at least three independent experiments. FMI values (B), as described in Materials and methods, can be either positive or negative, depending on the direction in which the cells migrate. *, P < 0.01 by two-tailed unpaired t test, significantly different from chemokinesis or an FGF-2 gradient.

Figure 5.

VEGFR expression in neural progenitors. (A) Total cellular RNA was isolated and VEGFR mRNA expression was assessed by RNase protection analysis. Purified ³²P-labeled rat cRNA probes (Probe) were hybridized to hybridization mix (Probe + h.m.), yeast tRNA, or total RNA from cells grown in FGF-2 or starved of FGF-2 for 12 h. Rat acidic ribosomal phosphoprotein (P0) was used as an internal control. Rat lung was used as a positive control. (B) Quantitative analysis of VEGFR1 and VEGFR2 expression in cells cultured in the presence of FGF-2 or starved of FGF-2 for 12 h. Data are shown as the mean ± SEM from three independent experiments. *, P < 0.01 by two-tailed unpaired t test, significantly different from cells in FGF-2 (n = 3).

Figure 6.

VEGF stimulates chemotaxis of neural progenitors through VEGFR2. (A) Scatter plots showing the migation patterns of neural progenitors under control conditions or in the presence of VEGFR blockers. Cells treated with the VEGFR2-blocking Ab (DC101) lost the chemotactic response to VEGF. In contrast, the VEGFR1-blocking Ab (MF1) did not affect progenitor migration. (B) Speed and FMI under different migration conditions. Data are shown as the mean ± SEM from three independent experiments. *, P < 0.01 by two-tailed unpaired t test, significantly different from DC101-treated cells. (C and D) Migration tracks of representative cells (four for each condition) exposed to a VEGF concentration gradient, in the presence of either VEGFR2-blocking Ab (C) or control (polysialic acid blocking) Ab (D). The starting point for each cell is the intersection between the X and Y axes (0,0), and the source of VEGF is at the top in the gradient condition.

Figure 7.

FGF-2 is required for neural progenitors to chemotactically respond to a VEGF gradient. (A) Experimental protocol: in the first group of cultures, FGF-2 was withdrawn at day 5 for 12 h, and then cells were exposed to a VEGF gradient (B). The second group was further cultured in the presence of FGF-2 after the 12-h starvation period for 8 h, and then tested in a VEGF gradient (C). The final positions of the cells after 2 h of migration were indicated, with the starting point for each cell at (0,0), and the source of 200 ng/ml VEGF at the top. (D) Speed and FMI were calculated as described in Materials and methods. Data are shown as mean ± SEM from four independent experiments. After 12 h of FGF-2 starvation, cells lose their chemotactic response to the VEGF gradient. The starved neural progenitors resume their chemotactic response to VEGF upon readdition of FGF-2 to the cultures for 8 h (C). (E) VEGFR2 expression in neural progenitors cultured in FGF-2 or starved of FGF-2 for 12 h. Western blot analysis was performed on immunoprecipitates with an anti-VEGFR2 Ab. *, P < 0.01 by two-tailed unpaired t test.

Figure 8.

Effect of VEGF on neural progenitors migrating from SVZ explants. SVZ explants were cocultured with VEGF-secreting C₂C₁₂ cells and/or mock-transfected C₂C₁₂ cells in collagen gel matrices in the presence (A, B, D, E, and F) or absence (C) of FGF-2. (A) In the presence of FGF-2, neural progenitors migrate out of the SVZ explant in an asymmetric manner, with many more cells on the side of the VEGF-secreting C₂C₁₂ cells than on the side of control C₂C₁₂ cells. (B) Neural progenitors migrate out of the SVZ explant symmetrically when cultured with control C₂C₁₂ cells on both sides. (C) In the absence of FGF-2, few to no cells migrate out of the SVZ explant. (D) High-power photograph showing the SVZ explant on the side of control C₂C₁₂ cells. (E) High-power photograph showing many neural progenitors migrating out of the SVZ explant toward VEGF-secreting C₂C₁₂ cells. (F) Cells migrating out of the SVZ explant are positive for nestin, a marker for undifferentiated neural progenitors. Bars: (A–C) 700 μm; (D and E) 100 μm; (F) 50 μm.