Oligodendrocyte precursor cell transplantation promotes angiogenesis and remyelination via Wnt/ β-catenin pathway in a mouse model of middle cerebral artery occlusion

Abstract

White matter injury is a critical pathological characteristic during ischemic stroke. Oligodendrocyte precursor cells participate in white matter repairing and remodeling during ischemic brain injury. Since oligodendrocyte precursor cells could promote Wnt-dependent angiogenesis and migrate along vasculature for the myelination during the development in the central nervous system, we explore whether exogenous oligodendrocyte precursor cell transplantation promotes angiogenesis and remyelination after middle cerebral artery occlusion in mice. Here, oligodendrocyte precursor cell transplantation improved motor and cognitive function, and alleviated brain atrophy. Furthermore, oligodendrocyte precursor cell transplantation promoted functional angiogenesis, and increased myelin basic protein expression after ischemic stroke. The further study suggested that white matter repairing after oligodendrocyte precursor cell transplantation depended on angiogenesis induced by Wnt/β-catenin signal pathway. Our results demonstrated a novel pathway that Wnt7a from oligodendrocyte precursor cells acting on endothelial β-catenin promoted angiogenesis and improved neurobehavioral outcomes, which facilitated white matter repair and remodeling during ischemic stroke.

Keywords: Angiogenesis; Wnt/β-catenin; ischemia; oligodendrocyte precursor cells; white matter.

Figure 1.

OPC isolation, identification and injection. (a) Morphology of cultured cells under phase contrast microscope. (b–c) Representative image of PDGFR-α (b) and NG2 (c) in cultured cells. (d–e) Survival of OPCs after injection. Green fluorescent OPCs (CFDA-SE stained) were located in the ischemic hemisphere at 14 and 28 days after tMCAO. Scale bar = 50 µm.

Figure 2.

OPC transplantation improved neurological outcomes and reduced brain atrophy volume in ischemic mice. (a–c) Quantifications of the neurological score, time stay on the rotarod and fear conditioning test in sham, PBS and OPC groups at 7 and 14 days after tMCAO. N = 9–11 per group. (d) Trace pictures and the scatter plots of step through test showed that the dark zone time and dark zone entries. N = 7–8 per group. (e) Cresyl violet staining showed the brain infarct volume at 3 days after tMCAO. N = 7–8 per group. (f) Cresyl violet staining showed the brain atrophy volume at 14 days after tMCAO. N = 8–9 per group. Data are mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

OPC transplantation promoted angiogenesis in ischemic mice. (a) Representative images of CD31⁺ microvessels in peri-infarct region and bar graphs of the CD31⁺ microvessels number and the vessel branch number in sham, PBS and OPC groups at 14 days after tMCAO. Scale bar = 100 µm, n = 4 per group. (b) Representative images of CD31 (red) and Ki67 double (green) positive cells in peri-infarct region at 14 days after tMCAO. Scale bar = 50 µm, n = 4 per group. (c) SR angiography showed microvessels in the peri-infarct region at 14 days after tMCAO. Scale bar = 500 µm, n = 3 per group. (d) IgG staining at 14 days after tMCAO. Bar graph showed the quantification of leaked IgG protein. Scale bar = 25 µm, n = 5 per group. Data are mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

OPC transplantation increased MBP expression in ischemic mice. (a) Immunofluorescent images of MBP (red) and bar graph of MBP quantification in peri-infarct region in sham, PBS and OPC groups at 14 days after tMCAO. Scale bar = 100 µm. N = 4 per group. (b) The Western blot result of MBP at 14 days after tMCAO. N = 4 per group. (c) The transplanted OPCs (green, CFDA-SE stained) did not express MBP (red) at 3 days after tMCAO while some transplanted OPCs (green) expressed MBP (red) at 14 days after tMCAO. The transplanted cells (green) still expressed NG2 (red) at 14 days after tMCAO. White arrows indicated the transplanted cells. Scale bar = 25 µm. (d) The transplanted OPCs (green) expressed MBP (red) at 28 days after tMCAO. And some transplanted cells (green) still expressed NG2 (red) at 28 days after tMCAO. White arrows indicated transplanted cells. Scale bar = 100 µm. (e) The percentage of CFDA-SE cell expressing MBP and the percentage of CFDA-SE cell expressing NG2. Data are mean±SD, **p < 0.01.

Figure 5.

Wnt/β-catenin pathway was involved in angiogenesis induced by OPC transplantation after tMCAO. (a) Western blot of Wnt7a expression in sham, PBS and OPC groups at 14 days after tMCAO. (b) Western blot of β-catenin expression. (c) Western blot of VEGF expression. (d) Immunofluorescent images of β-catenin (green)/GFAP (red), β-catenin (green)/NeuN (red) and β-catenin (green)/CD31 (red). White arrows indicated β-catenin expression. Scale bar = 50 µm. Data are mean ± SD, n = 4 per group, *p < 0.05, **p < 0.01, NS>0.05.

Figure 6.

Endothelial β-catenin inhibition blocked beneficial role of OPCs in HUVECs. (a) Western blot of β-catenin expression of HUVECs in CM, CM of OPC, CM of NC, CM of SI, CM of OPC+XAV939 and CM+Wnt7a groups. Data are mean $\pm$ SD, n = 3 per group, **p < 0.01. CON: control, only with cultured medium; CM of OPC: conditioned medium of OPC; CM of NC: conditioned medium of OPC transfected by negative control siRNA; CM of SI: conditioned medium of OPC transfected by Wnt7a siRNA; CM of OPC+XAV939: conditioned medium of OPC+XAV939; Wnt7a: cultured medium+Wnt7a protein. (b) Representative images and bar graph of Ki67 (red)/CD31 (green)/DAPI (blue) staining showed the proliferation of HUVECs. Scale bar = 50 µm. Data are mean ± SD, n = 7 per group, *p < 0.05. (c) Representative images and bar graphs showed the tube-formation of HUVECs. Scale bar = 200 µm. Data are mean±SD, n = 9 per group, *p < 0.05. (d) The migration ability of OPCs was assessed by transwell in vitro. Images showed the migrated OPCs during 12 and 24 hours. Scale bar = 50 µm, n = 4 per group. Data are mean±SD, *p < 0.05, **p < 0.01, as compared to the CON, CM of HUVEC+XAV-939 and XAV-939 groups. CON: control, cultured medium without OPCs; CM of HUVEC: conditioned medium of HUVECs; CM of HUVEC+XAV-939: conditioned medium of HUVECs treated with XAV-939; XAV-939: cultured medium only with XAV-939.

Figure 7.

Inhibition of β-catenin reversed the beneficial role of OPCs in vivo. (a) Quantification of the neurological score in the OPC and OPC+XAV-939 groups at 7 and 14 days after tMCAO. N = 6-8 per group. (b) Quantification of the time stay on the rotarod in the OPC and OPC+XAV-939 groups at 7 and 14 days after tMCAO. N = 6-8 per group. (c) Representative images of CD31+ microvessels in the peri-infarct region and bar graphs of the CD31+ microvessels number in the OPC and OPC+XAV-939 groups at 14 days after tMCAO. Scale bar = 100 µm. N = 4 per group. Data are mean±SD, *p < 0.05.