Vascular endothelial growth factor activates neural stem cells through epidermal growth factor receptor signal after spinal cord injury

Abstract

Aims: Neural stem cells (NSCs) in the adult mammalian spinal cord are activated in response to spinal cord injury (SCI); however, mechanisms modulating this process are not clear. Here, we noticed SCI elevated expression of vascular endothelial growth factor (VEGF) and we aimed to validate the roles of VEGF in NSCs activation after SCI and investigated the related signals during the process.

Methods: In vitro we detected whether VEGF promoted spinal cord NSCs proliferation and investigated the involved signals; In vivo, we injected VEGF into rat spinal cord to check the NSCs activation.

Results: In vitro, VEGF triggered spinal cord NSCs proliferation and maintained self-renewal. Further investigations demonstrated VEGF transactivated epidermal growth factor receptor (EGFR) through VEGF receptor 2 (VEGFR2) to promote spinal cord NSCs proliferation. In vivo, we injected VEGF into spinal cord by laminectomy to confirm the roles of VEGF-VEGFR2-EGFR signals in NSCs activation. VEGF significantly elevated the number of activated NSCs and increased EGFR phosphorylation. In contrast, intraspinal injection of specific inhibitors targeting EGFR and VEGFR2 decreased NSCs activation after SCI. Our results demonstrate that VEGF-VEGFR2-EGFR axis is important for NSCs activation after SCI, providing new insights into the mechanisms of spinal cord NSCs activation postinjury.

Keywords: VEGF receptor 2; epidermal growth factor receptor; neural stem cells activation; spinal cord injury; vascular endothelial growth factor.

Figure 1

Endogenous spinal cord NSCs are activated and accompanied by elevated VEGF expression after SCI (n = 5 rats/group). A, Endogenous spinal cord NSCs are activated after SCI. Few nestin⁺ NSCs in 0 dpi group. SCI, spinal cord injury. dpi, days postinjury. Scale bar represents 500 µm. B, Immunofluorescence shows increased VEGF expression after SCI. Scale bar represents 500 µm. C, Western blot shows expression of both nestin and VEGF is increased after SCI (3 dpi). The lesion sites are outlined by dashed lines in (A) and (B). 0 dpi group represents models without spinal cord injury but with laminectomy

Figure 2

VEGF promotes the proliferation of spinal cord‐derived neurospheres in vitro. A, Different concentrations of VEGF promote the formation of spinal cord‐derived neurospheres. Scale bar represents 200 µm. B, Average diameters of spheres cultured for 3 and 5 d. P = 0.023, 0.0046, and 0.0071 for VEGF 20, 50, and 100 ng/mL, compared with control on day 3, respectively. P = 0.030, 0.013, and 0.0068 for VEGF 20, 50, and 100 ng/mL compared with control on day 5, respectively. Control group indicates 0 ng/mL VEGF. C, EdU staining showing that VEGF promotes the proliferation of spinal cord‐derived neurospheres. Scale bar represents 100 µm. D, Quantification of percentage of EdU⁺ cells under different concentrations of VEGF. VEGF 100 ng/mL shows the highest percentage of EdU⁺ cells (P = 0.00010). *P ≤ 0.05 and **P ≤ 0.01. E, VEGF promotes cyclin D1 expression in the neurospheres. The right shows the region in the red box after secondary exposure

Figure 3

VEGF‐induced spinal cord‐derived neurospheres have self‐renewal ability and differentiation potential similar to NSCs. A, Neurospheres cultured for 5 d can form secondary spheres after being passaged in the presence of VEGF. Scale bar represents 100 µm. B, Average diameters of secondary spheres. VEGF 100 ng/mL shows the most proliferation compared with control (P = 0.0011). *P ≤ 0.05 and **P ≤ 0.01. Control, 0 ng/mL VEGF. C and D, Neurospheres induced by VEGF express NSCs markers. Nestin (green); BLBP (red); vimentin (red). Scale bar represents 200 µm. E, Neurospheres induced by VEGF differentiate into Tuj1⁺ neurons (green) and GFAP⁺ astrocytes (green). Scale bar represents 100 µm. EGF + FGF group is used as the positive control in (C‐E)

Figure 4

VEGF promotes the proliferation of spinal cord NSCs through EGFR phosphorylation. A, VEGF activates EGFR phosphorylation in spinal cord NSCs in vitro. EGF is used as the positive control. EGF, 20 ng/mL; VEGF, 100 ng/mL. Scale bar represents 200 µm. B, Western blotting shows that VEGF activates EGFR. VEGF, 100 ng/mL. 10 minutes for VEGF stimulation. C, Inhibitor of pEGFR, gefitinib, blocks VEGF‐induced EGFR phosphorylation. Gefitinib (dissolved in DMSO), 1 µM; VEGF, 100 ng/mL. D and E, Gefitinib decreases the proliferation of spinal cord NSCs. Gefitinib, 1 µM; VEGF, 100 ng/mL. Scale bar represents 200 µm. P = 0.026

Figure 5

VEGFR2‐EGFR signaling maintains the self‐renewal ability of spinal cord NSCs. A, Spinal cord NSCs express VEGFR2 but not VEGFR1 in vitro. Scale bar represents 200 µm. B, VEGF at 20 and 100 ng/mL phosphorylate VEGFR2 and EGFR. C, Inhibitor of pVEGFR2, cabozantinib, blocks the phosphorylation of VEGFR2 and EGFR. Cabozantinib (dissolved in DMSO), 1 µM. D, Cabozantinib decreases the diameters of spinal cord neurospheres. Cabozantinib, 1 µM P = 0.019. E, Inhibitors of EGFR and VEGFR2 block the self‐renewal of spinal cord NSCs. Both cabozantinib and gefitinib are used at 1 µM. F, Calcium‐chelating agent EGTA inhibits VEGF‐induced pEGFR signaling. 10 min for VEGF stimulation. VEGF, 100 ng/mL. EGTA, 1.5 mM

Figure 6

VEGF activates spinal cord NSCs through VEGFR2‐EGFR signaling on day 3 after injection in vivo (n = 5 rats/group). A, Model of the spinal cord intraspinal injection. The needle is about 25 gauge, at a 45° angle to the spinal cord (see Materials and Methods). The zigzag region indicates the injection site (T8‐T9). B, Injection of cabozantinib and gefitinib decreases the number of nestin⁺/Ki67⁺ spinal cord NSCs in the central canal. Injection of combination of PBS and DMSO as control. The results show that injection induced NSCs activation in central canal. Scale bar represents 200 µm. C, Nestin (green) and pEGFR (red) staining at the site of injection. Scale bar represents 100 µm. D, Nestin (green) and pVEGFR2 (red) staining at the site of injection. Scale bar represents 100 µm. A number of activated NSCs aggregate around the injection site (B‐D). E, Cabozantinib and gefitinib reduce nestin⁺/Ki67⁺ NSCs after SCI. Left, whole spinal section. Right, central canal; dpi, days postinjury. Nestin (green), Ki67 (red). Scale bar represents 500 µm in left and 200 µm in right panel