The angiogenic factor angiopoietin-1 is a proneurogenic peptide on subventricular zone stem/progenitor cells

Abstract

In the adult mammalian brain, the subventricular zone (SVZ) hosts stem cells constantly generating new neurons. Angiopoietin-1 (Ang-1) is an endothelial growth factor with a critical role in division, survival, and adhesion of endothelial cells via Tie-2 receptor activity. Expression of Tie-2 in nonendothelial cells, especially neurons and stem cells, suggests that Ang-1 may be involved in neurogenesis. In the present work, we investigated the putative role of Ang-1 on SVZ neurogenesis. Immature cells from SVZ-derived neurospheres express Ang-1 and Tie-2 mRNA, suggesting a role for the Ang-1/Tie-2 system in the neurogenic niche. Moreover, we also found that Tie-2 protein expression is retained on differentiation in neurons and glial cells. Ang-1 triggered proliferation via activation of the ERK1/2 (extracellular signal-regulated kinase 1/2) mitogen-activated protein kinase (MAPK) kinase pathway but did not induce cell death. Accordingly, coincubation with an anti-Tie-2 neutralizing antibody prevented the pro-proliferative effect of Ang-1. Furthermore, Ang-1 increased the number of NeuN (neuronal nuclear protein)-positive neurons in cultures treated for 7 d, as well as the number of functional neurons, as assessed by monitoring [Ca(2+)](i) rises after application of specific stimuli for neurons and immature cells. The proneurogenic effect of Ang-1 is mediated by Tie-2 activation and subsequent mTOR (mammalian target of rapamycin kinase) mobilization. In agreement, neuronal differentiation significantly decreased after exposure to an anti-Tie-2 neutralizing antibody and to rapamycin. Moreover, Ang-1 elicited the activation of the SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase) MAPK, involved in axonogenesis. Our work shows a proneurogenic effect of Ang-1, highlighting the relevance of blood vessel/stem cell cross talk in health and disease.

Figure 1.

SVZ cells express both Ang-1 and Tie-2. A, RT-PCR detection of Ang-1 and Tie-2 mRNAs in SVZ neurospheres. Lanes 1 and 2 correspond to the negative control (nontemplate negative control) and positive control (total mRNAs from mouse placental tissue), respectively. Lane 3 corresponds to SVZ neurospheres. Ang-1, ∼101 bp; Tie-2, ∼313 bp. B, Detection of Tie-2 and Ang-1 proteins by Western blotting in SVZ neurospheres. Lane 1 corresponds to the positive control (total proteins form mouse placenta), and lane 2 corresponds to SVZ proliferating cells. C, D, Representative fluorescent confocal digital images depicting Tie-2 and Ang-1 immunoreactivity in SVZ neurospheres (red staining for Tie-2 and Ang-1; green staining for nestin, as a marker of immature cells) c1 and c2, and d1 and d2 are magnifications of squares in C and D, respectively. E, The Tie-2 receptor is maintained in SVZ cells migrating out of a neurosphere 2 d after plating, and it is expressed in neuroblasts (red staining for Tie-2; green staining for DCX, a marker of immature neurons). F, The Tie-2 receptor is also maintained in SVZ cell-derived neurons after 7 d of differentiation (red staining for Tie-2; green staining for the Tau protein, an axonal marker). G, H, Tie-2 expression is retained in astrocytes (red staining for Tie-2; green staining for GFAP) and oligodendrocyte progenitor cells (red staining for Tie-2; green staining for NG2 chondroitin sulfate proteoglycan, a marker of oligodendrocyte progenitors), respectively. The arrows indicate regions of Tie-2 labeling. Hoechst 33342 staining (blue) was used to visualize cell nuclei. Scale bars, 20 μm.

Figure 2.

Ang-1 modulates cell proliferation in mouse SVZ cell cultures, an effect mediated by the Tie-2 receptor. A, B, Representative fluorescent confocal digital images of BrdU (red nuclei) and Hoechst 33342 staining (blue nuclei) in a control culture and in a culture treated with 500 ng/ml Ang-1, respectively. C, Bar graph depicts the number of BrdU-positive cells, expressed as percentages of the total number of counted nuclei, in control cultures and in cultures exposed to 500 ng/ml Ang-1 and/or 5 μg/ml anti-Tie-2 neutralizing antibody for 48 h. Data are expressed as mean ± SEM. ***p < 0.001, using the unpaired Student t test for comparison with SVZ control cultures. ⁺⁺⁺p < 0.001, using the unpaired Student t test for comparison with Ang-1-treated SVZ cultures. D, E, Representative z-stack confocal digital images of control SVZ cultures showing BrdU-positive cells (in red) positive for Tie-2 (white staining) and DCX (green staining) (D) or nestin (green staining) (E). The arrows indicate areas of triple labeling. Hoechst 33342 staining (blue) was used to visualize cell nuclei. Scale bars, 20 μm.

Figure 3.

Ang-1 modulates cell proliferation in mouse SVZ cell cultures, an effect mediated by the MAPK/ERK kinase pathway. Bar graph depicts the number of BrdU-positive cells, expressed as percentages of the total number of nuclei per culture, in control cultures and in cultures exposed to 500 ng/ml Ang-1 and/or 20 μm U0126, a highly selective inhibitor of MEK1 and -2, for 48 h. Data are expressed as a mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05, using the unpaired Student t test for comparison with SVZ control cultures. ⁺⁺⁺p < 0.001, using the unpaired Student t test for comparison with Ang-1-treated SVZ cultures.

Figure 4.

Ang-1 induces neuronal differentiation in mouse SVZ cell cultures via Tie-2 activation. A, B, Representative fluorescent photomicrographs of NeuN-positive neurons (red nuclei) and Hoechst 33342 staining (blue nuclei) in control SVZ cultures (A) and in cultures treated with Ang-1 (B). Scale bar, 20 μm. C, Bar graph depicts the number of NeuN-positive cells, expressed as percentages of the total number of cells per culture, in control cultures and in cultures treated with 500 ng/ml Ang-1 and/or 5 μg/ml anti-Tie-2 neutralizing antibody for 7 d. Data are expressed as mean ± SEM. ***p < 0.001, using the unpaired Student t test for comparison with SVZ control cultures. ⁺p < 0.05, using the unpaired Student t test for comparison with SVZ cultures treated with Ang-1. D, E, Representative Western blots for βIII tubulin (D) and GFAP (E)—the β-actin blots are provided as loading controls—of total protein extract from SVZ cells treated for 7 d in the absence (control) or the presence of 500 ng/ml Ang-1. F, G, Representative confocal digital images of SVZ cell cultures treated for 7 d in the absence (control) (F) or the presence of 500 ng/ml Ang-1 (G) and stained for βIII tubulin (green staining) and GFAP (red staining). Hoechst 33342 staining (blue) was used to visualize cell nuclei. Scale bars, 50 μm.

Figure 5.

Ang-1 increases the generation of neuronal-like responding cells in mouse SVZ cell cultures via Tie-2 activation. A, SVZ cultures were perfused continuously with Krebs' solution for 15 min and stimulated for 2 min (from minute 5 to minute 7) with 50 mm KCl and for 2 min (from minute 10 to minute 12) with 100 μm histamine. B–D Shown are representative single-cell calcium imaging profiles of response of 20 cells in a control culture (B), in a 500 ng/ml Ang-1-exposed culture (C), and in a culture treated with both 500 ng/ml Ang-1 and 5 μg/ml anti-Tie-2 neutralizing antibody (D). E, Bar graph depicts the percentages of neuronal-like responding cells in SVZ control cultures and in cultures exposed to Ang-1 and/or anti-Tie-2 for 7 d. Data are expressed as mean ± SEM. **p < 0.01, using the unpaired Student t test for comparison with SVZ control cultures. ⁺p < 0.05, using the unpaired Student t test for comparison with SVZ cultures treated with Ang-1.

Figure 6.

Ang-1 increases the generation of neuronal-like responding cells in mouse SVZ cell cultures via mTOR. SVZ cultures were perfused continuously with Krebs' solution for 15 min and stimulated for 2 min (from minute 5 to minute 7) with 50 mm KCl and for 2 min (from minute 10 to minute 12) with 100 μm histamine. The bar graph depicts the percentages of neuronal-like responding cells in SVZ control cultures and in cultures exposed to 500 ng/ml Ang-1 and/or 20 nm rapamycin for 7 d. Data are expressed as mean ± SEM. ***p < 0.001, using the unpaired Student t test for comparison with SVZ control cultures. ⁺⁺⁺p < 0.001, using the unpaired Student t test for comparison with SVZ cultures treated with Ang-1.

Figure 7.

Ang-1 induces activation of SAPK/JNK pathway on growing axons. A, Representative fluorescent confocal digital images depicts the P-SAPK/JNK (green), Tau protein (red), and Hoechst 33342 staining (blue nuclei) in cultures treated with 500 ng/ml Ang-1 for 6 h. Growing axons (double labeled for P-SAPK/JNK and Tau) are indicated by arrowheads. Scale bar, 20 μm. B, C, Bar graphs depict the total length (in micrometers) of P-JNK-positive ramifications and the number of ramifications per neurosphere. Data are expressed as mean ± SEM. Measurements were done in ∼20 nonoverlapping fields in each coverslip from two independent culture preparations using digital images (magnification, 20×). **p < 0.01; ***p < 0.001, using the unpaired Student t test for comparison with SVZ control cultures. ⁺p < 0.05, ⁺⁺⁺p < 0.001 using the unpaired Student t test for comparison with SVZ cultures treated with Ang-1.

Figure 8.

Tie-2 is expressed in neurons along the SVZ, the RMS, and the olfactory bulb. A, B, Representative z-stack confocal digital images of the SVZ showing BrdU-positive stem/progenitor cells (red nuclear staining for BrdU and green staining for EGFR) (A) and BrdU-positive neuroblasts (red nuclear staining for BrdU and green staining for DCX) (B), both cell types expressing Tie-2 (white staining). C, Representative z-stack confocal digital image of the RMS showing a DCX neuroblast (green staining) expressing Tie-2 (red staining). D, Representative z-stack confocal digital image of TH-expressing periglomerular cells (green staining) expressing Tie-2 (red staining). The arrows indicate regions of triple labeling. Hoechst 33342 staining (blue) was used to visualize cell nuclei. Scale bars, 20 μm.