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. 2010 Mar 22;5(3):e9767.
doi: 10.1371/journal.pone.0009767.

Murine neural stem/progenitor cells protect neurons against ischemia by HIF-1alpha-regulated VEGF signaling

Kate M Harms  1 , Lu Li, Lee Anna Cunningham
Affiliations

Murine neural stem/progenitor cells protect neurons against ischemia by HIF-1alpha-regulated VEGF signaling

Kate M Harms et al. PLoS One. .

Abstract

Focal cerebral ischemia following middle cerebral artery occlusion (MCAO) stimulates a robust cytogenic response from the adult subventricular zone (SVZ) that includes massive proliferation of neural stem/progenitor cells (NSPCs) and cellular migration into the injury area. To begin to explore beneficial roles of NSPCs in this response, we investigated the ability of embryonic and postnatal NSPCs to promote neuronal survival under conditions of in vivo and in vitro ischemia. Intracerebral transplantation of NSPCs attenuated neuronal apoptosis in response to focal ischemia induced by transient MCAO, and prevented neuronal cell death of cortical neurons in response to oxygen-glucose deprivation (OGD) in culture. NSPC-mediated neuroprotection was blocked by the pharmacological inhibitors of vascular endothelial growth factor (VEGF), SU1498 and Flt-1Fc. Embryonic and postnatal NSPCs were both intrinsically resistant to brief OGD exposure, and constitutively expressed both hypoxia-inducible factor 1alpha (HIF-1alpha) transcription factor and its downstream target, VEGF. Genomic deletion of HIF-1alpha by Cre-mediated excision of exon 2 in NSPC cultures resulted in >50% reduction of VEGF production and ablation of NSPC-mediated neuroprotection. These findings indicate that NSPCs promote neuronal survival under ischemic conditions via HIF-1alpha-VEGF signaling pathways and support a role for NSPCs in promotion of neuronal survival following stroke.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. NSPCs protect against 30 minute MCAO.
(A–D) Coronal histological sections through the ischemic striatum 3 days following MCAO, stained for TUNEL (A,B) or NeuN (C,D). Mice received intrastriatal injections of exogenous PBS (A,C) or EGFP+NSPCs (B,D,E) 72 hr prior to MCAO. Inset in (B) shows 40× magnified view of the injection site. (E) Monochrome conversion of image shown in B to demonstrate concentric ring structures used for Sholl analysis. (F) Quantification of TUNEL+ cells using Sholl analysis performed on fluorescent images. *p<0.05, n = 5 mice per group. Scale bar: A,B  =  20 µm; C,D  =  10 µm.
Figure 2
Figure 2. NSPCs provide neuroprotection against OGD and are resistant to in vitro ischemia.
(A) Diagram of transwell coculture. Micrograph depicts cortical neurons in lower compartment immunofluorescently labeled for MAP-2 (red) and GFAP (green). NSPCs in upper compartment are immunofluorescent for nestin (red). Nuclei are labeled with DAPI (blue). (B) Neuronal survival at 24 hrs following 2 hr exposure to OGD in the absence (left) or presence (right) of NSPCs. (C) Survival of NSPCs grown as monocultures in upper compartment. Data were acquired using the MTT colorimetric viability assay as described in methods section. *p<0.01, Student's t-test, n = 4 cultures/group.
Figure 3
Figure 3. HIF-1α and VEGF expression in eNSPC cultures under control vs. OGD conditions.
(A) HIF-1α protein levels in cell lysates from eNSPCs at 24 hrs following a 2 hr exposure to OGD or control conditions. (B) VEGF within neuronal or NSPC conditioned media following 2 hr exposure to control or OGD conditions. Media was conditioned for 24 hrs following exposure to OGD or control conditions. *p<0.05, Student's t-test, n = 4 cultures/group.
Figure 4
Figure 4. Pharmacological inhibition of VEGF signaling impairs the neuroprotective effects of eNSPCs.
(A,B) Neuronal viability 24 hrs following exposure to 2 hr OGD vs. control conditions in the presence or absence of NSPCs and the VEGFR kinase inhibitor, SU1498 (A) or the VEGF receptor decoy receptor, Flt-1-Fc (B). (C) Neuronal viability in the presence or absence of NSPC-conditioned media and SU1498 or Flt-1Fc. *p<0.05, n = 4 cultures/group.
Figure 5
Figure 5. Gene deletion of HIF-1α impairs neuroprotective ability of eNSPC.
(A) PCR amplification products from genomic DNA isolated from wild-type and HIF-1αfl/fl NSPCs exposed to 50 MOI Ad-CMV-Cre for 48 hrs. Excision of exon 2 from HIF-1α gene is indicated by the shorter band at 250 bp, present after Ad-CMV-Cre exposure of HIF-1α fl/fl but not wild-type NSPCs. (B) Transcriptional activity of HIF-1α in wild-type vs. HIF-1αΔ/Δ NSPCs assayed by HRE binding assay. (C) VEGF in 24 hr conditioned media from wild-type vs. HIF-1αΔ/Δ NSPCs. (D) Viability of cortical cultures 24 hr following 2 hr OGD in the presence or absence of wild-type vs. HIF-1αΔ/Δ NSPCs CM. *p<0.05, n =  4 cultures/group.
Figure 6
Figure 6. Postnatal NSPCs are neuroprotective and resistant to ischemia.
(A) HIF-1α protein levels in cell lysates and (B) VEGF concentration in conditioned medium from pNSPCs at 24 hr following 2 hr exposure to control or OGD conditions. (C) Viability of pNSPCs at 24 hr post-OGD. (D) Viability of cortical neuronal cultures 24 hr following 2 hr OGD in the presence of neuronal vs. pNSPC conditioned medium. *p<0.05, n = 4 cultures/group.
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