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. 2008 Sep;28(9):1530-42.
doi: 10.1038/jcbfm.2008.38. Epub 2008 May 14.

Neural stem/progenitor cells promote endothelial cell morphogenesis and protect endothelial cells against ischemia via HIF-1alpha-regulated VEGF signaling

Tamara Roitbak  1 , Lu Li, Lee Anna Cunningham
Affiliations

Neural stem/progenitor cells promote endothelial cell morphogenesis and protect endothelial cells against ischemia via HIF-1alpha-regulated VEGF signaling

Tamara Roitbak et al. J Cereb Blood Flow Metab. 2008 Sep.

Abstract

Vascular cells provide a neural stem/progenitor cell (NSPC) niche that regulates expansion and differentiation of NSPCs within the germinal zones of the embryonic and adult brain under both physiologic and pathologic conditions. Here, we examined the NSPC-endothelial cell (NSPC/EC) interaction under conditions of ischemia, both in vitro and after intracerebral transplantation. In culture, embryonic mouse NSPCs supported capillary morphogenesis and protected ECs from cell death induced by serum starvation or by transient oxygen and glucose deprivation (OGD). Neural stem/progenitor cells constitutively expressed hypoxia-inducible factor 1alpha (HIF-1alpha) transcription factor and vascular endothelial growth factor (VEGF), both of which were increased approximately twofold after the exposure of NSPCs to OGD. The protective effects of NSPCs on ECs under conditions of serum starvation and hypoxia were blocked by pharmacological inhibitors of VEGF signaling, SU1498 and Flt-1-Fc. After intracerebral transplantation, NSPCs continued to express HIF-1alpha and VEGF, and promoted microvascular density after focal ischemia. These studies support a role for NSPCs in stabilization of vasculature during ischemia, mediated via HIF-1alpha-VEGF signaling pathways, and suggest therapeutic application of NSPCs to promote revascularization and repair after brain injury.

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Figures

Figure 1
Figure 1. NSPCs support ECs morphogenesis and survival in serum-free conditions
Micrographs show ECs and NSPC/EC co-cultures plated on Matrigel matrix. GFP- positive NSPCs are shown using the green channel. A) Confocal DIC images, Bar=50 μm. B) Confocal images demonstrating immunofluorescence for the endothelial cell marker CD31/PECAM-1 (red), Bar=20 μm. ECs were plated either in the presence of serum (A and B, left panels) or absence of serum (A and B, middle panels), or in the presence of NSPCs in serum-free, growth factor-free cell medium (A and B, right panels). C) Quantification of endothelial cell morphogenesis, n=3 coverslips per group.
Figure 2
Figure 2. NSPCs support morphology and survival of ECs following OGD
Direct NSPC/EC co-cultures and EC cultures (both grown in serum- and growth factor-free medium) were subjected to 3-hour oxygen-glucose deprivation (OGD) and analyzed at 7 days following OGD (middle and bottom panels). Control cultures (top panel), grown in the same experimental conditions, were not subjected to OGD. EC morphology was assessed using DIC confocal microscopy imaging and immunostaining with the antibody against CD31/PECAM-1 (bottom panels). NSPCs are shown on the green channel. Bars= 50 μm.
Figure 3
Figure 3. Phenotypic analysis of NSPCs in co-culture with ECs
Neural (doublecortin for immature neurons, Tuj-1 for mature neurons, and GFAP for astrocytes), stem cell (nestin) and endothelial cell (VE-cadherin, CD31) markers were used to characterize the phenotype of NSPCs co-cultured with ECs. NSPCs (green) were co-cultured with ECs for 3 days (a and b) or 10 days (c, d, e, and f) and immunostained for doublecortin (a, red), nestin (b, red), Tuj1 (c and d, red) and GFAP (c and d, blue); VE-cadherin (e, red) and CD31/PECAM-1 (f, red). The VE-cadherin and CD31-positive endothelial were not captured, since the NSPCs were imaged in a different focal plan. However ECs were clearly visible at Z-stack composite confocal images (not shown on the figure). Cells were imaged on a Zeiss LSM510-META confocal imaging system. Bars = 20 μm (a-c), 50 μm (d ), and 5μm (e and f ).
Figure 4
Figure 4. NSPCs constitutively express both HIF-1α and VEGF in culture, and upregulate expression following exposure to OGD
A) Western blot analysis of intracellular VEGF (top) and HIF-1α (bottom) in NSPC and EC lysates. B) NSPC conditioned media and cell lysates were assayed for VEGF and HIF-1α, respectively, under conditions of normoxia (open bars) or 3 days following exposure to 3 h OGD (filled bars). One-way ANOVA and Tukey’s Multiple Comparison post hoc analysis were performed for statistical analysis. ** - p<0.01; *** - p<0.001, n=3 samples per group assayed in triplicate. (C,D) Cultured NSPCs maintained under normoxic conditions were immunostained with the antibodies against VEGF (C, red channel) and HIF-1α (D, red channel). Nuclear localization was verified by colocalization with DAPI staining (blue, arrow). Cells were imaged on a Zeiss LSM510-META confocal imaging system. Bars=10 μm.
Figure 5
Figure 5. Inhibition of VEGF signaling impairs NSPC-mediated survival and morphogenesis of ECs following serum deprivation and OGD
Open Bars, Left) Viability of ECs following 3 days of culture in the presence or absence of serum, NSPC conditioned medium (NSPC-CM), or the VEGF inhibitors, SU1498 or Flt-1-Fc. Filled Bars, Right) Viability of ECs exposed to 3 hr of OGD, followed by 3 days of incubation with or without serum, NSPC-CM or VEGF inhibitors. Percent viability in each culture condition was compared to the viability of EC cultures grown under normoxic conditions in the presence of serum, which was set to 100% (far left open bar = control). NSPC-CM represents media conditioned for 3 days by monocultures of NSPCs grown in serum- and growth factor-free media. Data were acquired using the MTT colorimetric viability assay as described under Methods, * * - p< 0.01; *** - p< 0.0001, One way ANOVA with Tukey’s Multiple Comparison post hoc analysis, n=3 cultures per group, assayed in triplicate.
Figure 6
Figure 6. NSPCs support brain revascularization following ischemia
a and b) Coronal sections through the lesioned striatum of EGFP-NSPC recipients (b) or recipients of control PBS injections (a) immunostained for the endothelial cell marker, Glut-1 (red). Transplanted EGFP-NSPCs are visible as green cells. c,d,e) Double labeling of endothelial cells (Glut-1+, red) and proliferating cells (Ki67+, blue). EGFP-NSPC grafts are shown on green channel. The presence of Ki67+ nuclei within Glut-1+ blood vessels was assessed using confocal microscopy viewed in 3-dimensional (d) and orthogonal (e) image projections. The images were acquired using Zeiss LSM510- META confocal imaging system. Bars=20 μm (a-c), 10μm (d) and 2μm (e). Graph) Percentage of striatal area covered by Glut-1 immunoreactive vessels in PBS control mice (open bars) vs. EGFP-NSPC recipients (filled bars). Image analysis was performed over entire striatal area within three histological sections containing grafted cells or PBS injection sites on both lesioned (ipsilateral) and non-lesioned (contralateral) sides.
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