Macrophages-derived NRG-1 promotes angiogenesis after ischemic stroke via the Akt-mTOR pathway

Figures

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  Figure 1. Serum NRG-1 levels are increased in patients with acute ischemic stroke and are related to neurological dysfunction severity. (A) Serum NRG-1 levels in patients with AIS and healthy controls. (B) Serum NRG-1 levels in patients with AIS with an NIHSS score ≥ 6 or an NIHSS score < 6 at admission. (C) Serum NRG-1 levels in patients with and without HT. (D) Long-term post-ischemia outcomes in patients with AIS with mRS ≤ 1 and those with mRS > 1. (E) Serum NRG-1 level is negatively correlated with 3-month mRS score (r = −0.449, P = 0.047). Data are indicated as mean ± SEM. Statistical significance was determined using Student’s t-test and Spearman correlation analysis; *P < 0.05; **P < 0.01; ***P < 0.001. HC: Healthy control; HT: hemorrhagic transformation; mRS: modified Rankin Scale; NIHSS: National Institutes of Health Stroke Scale; NRG-1: Neuregulin-1; ns: not significant.

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  Figure 2. Single-cell transcriptomics identification of NRG-1+ macrophage subtypes from peripheral blood samples and clots from patients undergoing endovascular therapy. (A) Unsupervised clustering and tSNE identified nine major immune cell clusters from 14,163 cells in three samples. (B) Heatmap showing the relative expression of marker genes across CD45+ cell clusters. (C) NRG-1 is relatively high expressed in macrophages. (D, E) Macrophages were further re-clustered into six subtypes (Macro1–Macro6). (F) NRG-1 is highly expressed in the Macro2 subtype. (G) Volcano plot of transcriptomic differences between NRG-1-expressing and NRG-1-non-expressing cell subtypes. (H, I) Kyoto Encyclopedia of Genes and Genomes pathway analysis of expression differences between the Macro2 cluster and the other clusters. (J) Immunohistochemical staining for NRG-1 in clots from patients with acute ischemic stroke who received endovascular treatment. Scale bar: 1 μm. NRG-1: Neuregulin-1; t-SNE: t-distributed stochastic neighbor embedding.

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  Figure 3. Bioinformatics detection of NRG-1 expression in infiltrating myeloid cells in post-ischemic mice. (A) Unsupervised clustering and tSNE identified 17 major cell clusters. (B) Violin plot showing that Nrg1 is mainly expressed in the fourth and ninth cell subtypes. (C) Nrg1 expression was increased in both cluster 4 and cluster 9 post-cerebral ischemic. (D) Cluster 4 was identified as astrocytes, based on the marker gene Gfap, and cluster 9 was identified as myeloid cells, based on the marker gene LYZ. NRG-1 expression increased post-ischemic stroke in both astrocytes and myeloid cells, especially in infiltrating myeloid cells. (E) FACS sorting of infiltrating cerebral myeloid cells post-ischemic stroke. (F) Quantitative reverse transcription–polymerase chain reaction analysis showing high expression levels of Nrg1 in infiltrating myeloid cells. Data are represented as mean ± SEM. Statistical significance was identified using Student’s t-test. *P < 0.05. FACS: Fluorescence-activated cell sorting; FSC-H: forward scatter - height; NRG-1: Neuregulin-1; ns: not significant; tSNE: t-distributed stochastic neighbor embedding.

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  Figure 4. Macrophage-derived NRG-1 lowers cerebral infarction volume and improves neurological deficits in mice with early-stage stroke. (A) Macrophage-derived NRG-1 decreases infarct volume in mice subjected to tMCAO. White indicates the infarct region. (B) Macrophage-derived NRG-1 enhances behavioral prognosis in mice subjected to tMCAO, as assessed by Garcia and Longa tests. (C) Open field testing indicated that NRG-1 improves motor function in mice subjected to tMCAO. (D) Mean velocity of mouse locomotion in the open field test. Data are indicated as mean ± SEM. Statistical significance was determined using one-way analysis of variance with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. NRG-1: Neuregulin-1; ns: not significant; tMCAO: transient middle cerebral artery occlusion.

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  Figure 5. Macrophage-derived NRG-1 alleviates neuroinflammation and tissue damage in mice. (A) Experimental timeline. (B) Nissl staining of mouse brain sections. (C) Infarct volume. (D) Electron microscopy images of mouse brain myelin. Scale bars: 1 μm. (E) G-ratio of myelin in each group. (F) Western blot and quantitative reverse transcription–polymerase chain reaction analyses showing that expression of the anti-neuroinflammatory factors IL-4 and IL-10 was increased in mice subjected to tMCAO and treated with NRG-1. (G) Western blot analysis showing that macrophage-derived NRG-1 alleviated neuroinflammation by reducing TNF-α and IL-1β levels after tMCAO. (H, I) Quantitative western blot and quantitative reverse transcription–polymerase chain reaction analyses of the anti-neuroinflammatory effects of NRG-1 after tMCAO. Data are indicated as mean ± SEM. Statistical significance was determined using one-way analysis of variance with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; IL: interleukin; NRG-1: Neuregulin-1; tMCAO: transient middle cerebral artery occlusion; TNF-α: tumor necrosis factor-α.

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  Figure 6. Macrophage-derived NRG-1 exerts protective effects by promoting angiogenesis in the peri-infarct area after tMCAO. (A) Laser speckle imaging of each group. (B) Quantification of CBF in each group. (C) Immunohistochemistry showing neovascularization in each group. (D) Quantification of the neovascularization shown in C. (E) Immunofluorescence showing CD31- and BrdU-positive areas and quantification of CD31+BrdU+ endothelial cells. (F) Western blot analysis showing VEGF-A expression in each group and quantitative reverse transcription–polymerase chain reaction analysis indicating that VEGF-A expression was elevated after administration of NRG-1. Data are presented as mean ± SEM. Statistical significance was identified using one-way analysis of variance with Tukey’s post hoc test. *P < 0.05; **P < 0.01. BrdU: 5-Bromo-2′-deoxyuridine; CBF: cerebral blood flow; CL: contralateral; DAPI: 4′,6-diamidino-2-phenylindole; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; IL: ipsilateral; NRG-1: neuregulin-1; ns: not significant; tMCAO: transient middle cerebral artery occlusion; VEGF: vascular endothelial growth factor.

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  Figure 7. Macrophage-derived NRG-1 promotes post-ischemic angiogenesis through the Akt/mTOR pathway. (A–C) Western blot analyses showing ErbB4, Akt, and mTOR expression and phosphorylation in each group. (D–F) Quantification of the western blot data shown in A–C. (G) Quantitative reverse transcription–polymerase chain reaction analysis indicating ErbB4-Akt-mTOR signaling pathway activation in each group. Each group included at least three samples, with over three exposures for each sample. Data are denoted as mean ± SEM. Statistical significance was determined using one-way analysis of variance with Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; NRG-1: neuregulin-1; tMCAO: transient middle cerebral artery occlusion.