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Cited by 110Congenital heart defects researchCardiac Fibrosis and RemodelingAngiogenesis and VEGF in Cancer

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Nogo-B promotes angiogenesis and improves cardiac repair after myocardial infarction via activating Notch1 signaling

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Zheng, Yanjun, Lin, Jingrong, Liu, Dingsheng, Wan, Guoqing, Gu, Xuefeng, Ma, Jian·2022·Cell Death & Disease
DOI 10.1038/s41419-022-04754-4PMC PMC8983727
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Abstract

Nogo-B (Reticulon 4B) is reportedly a regulator of angiogenesis during the development and progression of cancer. However, whether Nogo-B regulates angiogenesis and post-myocardial infarction (MI) cardiac repair remains elusive. In the present study, we aimed to explore the role and underlying mechanisms of Nogo-B in cardiac repair during MI. We observed an increased expression level of Nogo-B in the heart of mouse MI models, as well as in isolated cardiac microvascular endothelial cells (CMECs). Moreover, Nogo-B was significantly upregulated in CMECs exposed to oxygen-glucose deprivation (OGD). Nogo-B overexpression in the endothelium via cardiotropic adeno-associated virus serotype 9 (AAV9) with the mouse endothelial-specific promoter Tie2 improved heart function, reduced scar size, and increased angiogenesis. RNA-seq data indicated that Notch signaling is a deregulated pathway in isolated CMECs along the border zone of the infarct with Nogo-B overexpression. Mechanistically, Nogo-B activated Notch1 signaling and upregulated Hes1 in the MI hearts. Inhibition of Notch signaling using a specific siRNA and γ-secretase inhibitor abolished the promotive effects of Nogo-B overexpression on network formation and migration of isolated cardiac microvascular endothelial cells (CMECs). Furthermore, endothelial Notch1 heterozygous deletion inhibited Nogo-B-induced cardioprotection and angiogenesis in the MI model. Collectively, this study demonstrates that Nogo-B is a positive regulator of angiogenesis by activating the Notch signaling pathway, suggesting that Nogo-B is a novel molecular target for ischemic disease.

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Fig. 1 Nogo-B was increased in CMECs after MI. A, B Time course of the relative mRNA level of Nogo-B measured by Q-PCR in cardiac tissues and CMECs in the infarct border zone after MI (n = 5). C Representative and quantitative Nogo-B protein level in CMECs in the infarct border zone after MI (n = 5). D Representative and quantitative analysis of scratch, transwell, and tube formation images under normoxic or OGD conditions (n = 5). E, F mRNA and protein levels of Nogo-B after normoxic or OGD treatment (n = 5). Data were expressed as mean ± SEM. ∗P < 0.05 versus sham or normoxic group. Unpaired Student’s t-test (D–F), one-way ANOVA (A–C).
Fig. 1 Nogo-B was increased in CMECs after MI. A, B Time course of the relative mRNA level of Nogo-B measured by Q-PCR in cardiac tissues and CMECs in the infarct border zone after MI (n = 5). C Representative and quantitative Nogo-B protein level in CMECs in the infarct border zone after MI (n = 5). D Representative and quantitative analysis of scratch, transwell, and tube formation images under normoxic or OGD conditions (n = 5). E, F mRNA and protein levels of Nogo-B after normoxic or OGD treatment (n = 5). Data were expressed as mean ± SEM. ∗P < 0.05 versus sham or normoxic group. Unpaired Student’s t-test (D–F), one-way ANOVA (A–C).CC BY · PubMed Central
Fig. 2 Nogo-B treatment improved cardiac functions. A The survival rate of sham and MI mice injected with AAV during the 4-week follow-up period. The log-rank test demonstrated significant differences between the two survival curves. B LVEF, LVFS, LVEDD, and LVEDS were measured by echocardiography (n = 8). C LVDP, LVEDP, LV maximum ascending rates of pressure (+dp/dt max), and LVESV measured by the pressure–volume loop at day 28 post-MI (n = 5). Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus AAV-NC group. #P < 0.05, ##P < 0.01, ###P < 0.001 versus AAV-NC-MI group. two-way ANOVA (B, C).
Fig. 2 Nogo-B treatment improved cardiac functions. A The survival rate of sham and MI mice injected with AAV during the 4-week follow-up period. The log-rank test demonstrated significant differences between the two survival curves. B LVEF, LVFS, LVEDD, and LVEDS were measured by echocardiography (n = 8). C LVDP, LVEDP, LV maximum ascending rates of pressure (+dp/dt max), and LVESV measured by the pressure–volume loop at day 28 post-MI (n = 5). Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus AAV-NC group. #P < 0.05, ##P < 0.01, ###P < 0.001 versus AAV-NC-MI group. two-way ANOVA (B, C).CC BY · PubMed Central
Fig. 3 Nogo-B decreased fibrosis and increased angiogenesis after MI. A The heart weight to body weight ratio (n = 8). B Representative cross-sectional images and quantitative data of hearts stained with Masson’s trichrome at day 28 post-MI (n = 5). C Representation and quantification of IHC staining for TUNEL + cardiomyocytes in the border zone of infarcted hearts at day 3 post-MI (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 versus AAV-NC group. #P < 0.05, ##P < 0.01, ###P < 0.001 versus AAV-NC-MI group. D The viability of NRCMs was measured by the Cell Counting Kit-8 (CCK8) in the medium (n = 3). E Fold changes of LDH activities in the NRCMs medium. *P < 0.05 versus Ad-NC group (n = 3). #P < 0.05, ##P < 0.01 versus Ad-NC-OGD group. Data were expressed as mean ± SEM. Unpaired Student’s t-test (B), two-way ANOVA (A and C–E).
Fig. 3 Nogo-B decreased fibrosis and increased angiogenesis after MI. A The heart weight to body weight ratio (n = 8). B Representative cross-sectional images and quantitative data of hearts stained with Masson’s trichrome at day 28 post-MI (n = 5). C Representation and quantification of IHC staining for TUNEL + cardiomyocytes in the border zone of infarcted hearts at day 3 post-MI (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 versus AAV-NC group. #P < 0.05, ##P < 0.01, ###P < 0.001 versus AAV-NC-MI group. D The viability of NRCMs was measured by the Cell Counting Kit-8 (CCK8) in the medium (n = 3). E Fold changes of LDH activities in the NRCMs medium. *P < 0.05 versus Ad-NC group (n = 3). #P < 0.05, ##P < 0.01 versus Ad-NC-OGD group. Data were expressed as mean ± SEM. Unpaired Student’s t-test (B), two-way ANOVA (A and C–E).CC BY · PubMed Central
Fig. 4 Nogo-B promoted angiogenesis after MI. A The mRNA level of VEGF at day 28 post-MI (n = 5). B The protein level of VEGF at day 28 post-MI in border (BZ) and remote zone (RZ) of infarcted hearts. C Immunochemistry staining for CD31 + endothelial cells and α-SMA + vessels at day 28 post-MI (n = 5). Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus AAV-NC group. two-way ANOVA (A–C).
Fig. 4 Nogo-B promoted angiogenesis after MI. A The mRNA level of VEGF at day 28 post-MI (n = 5). B The protein level of VEGF at day 28 post-MI in border (BZ) and remote zone (RZ) of infarcted hearts. C Immunochemistry staining for CD31 + endothelial cells and α-SMA + vessels at day 28 post-MI (n = 5). Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus AAV-NC group. two-way ANOVA (A–C).CC BY · PubMed Central
Fig. 5 Nogo-B promoted angiogenesis in vitro. A The expression efficiency of Ad-Nogo-B measured by Western blot (n = 4). B The downregulation efficiency of Ad-siNogo-B measured by Western blot (n = 4). C Transwell and scratch/wound assays and tube formation analysis demonstrated that Nogo-B promoted cellular migration of ECs (n = 5). D Effect of Ad-Nogo-B and Ad-shNogo-B on vessel outgrowth in aortic rings and ECs tube formation (n = 5). Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus Ad-NC group. #P < 0.05, ##P < 0.01 versus Ad-Scr group. one-way ANOVA (C, D).
Fig. 5 Nogo-B promoted angiogenesis in vitro. A The expression efficiency of Ad-Nogo-B measured by Western blot (n = 4). B The downregulation efficiency of Ad-siNogo-B measured by Western blot (n = 4). C Transwell and scratch/wound assays and tube formation analysis demonstrated that Nogo-B promoted cellular migration of ECs (n = 5). D Effect of Ad-Nogo-B and Ad-shNogo-B on vessel outgrowth in aortic rings and ECs tube formation (n = 5). Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus Ad-NC group. #P < 0.05, ##P < 0.01 versus Ad-Scr group. one-way ANOVA (C, D).CC BY · PubMed Central
Fig. 6 Nogo-B overexpression upregulated the Notch signaling pathway. A Heat map of genes related to the Notch pathway with RNA-seq for CMECs from AAV-NC or AAV-Nogo-B-infected mice after MI. B GSEA analyses of the gene set upregulated by Nogo-B overexpression in CMECs. C Protein levels of Notch and Hes1 in the AAV-Nogo-B and AAV-NC infected hearts after MI in border (BZ) and remote zone (RZ) of infarcted hearts (n = 5). *P < 0.05, **P < 0.01 versus sham group. D Protein levels of Notch and Hes1 in ECs treated with DAPT and siNotch (n = 5). *P < 0.05, **P < 0.01 versus Ad-NC group. ns, not significant. Data were expressed as mean ± SEM. Two-way ANOVA (C, D).
Fig. 6 Nogo-B overexpression upregulated the Notch signaling pathway. A Heat map of genes related to the Notch pathway with RNA-seq for CMECs from AAV-NC or AAV-Nogo-B-infected mice after MI. B GSEA analyses of the gene set upregulated by Nogo-B overexpression in CMECs. C Protein levels of Notch and Hes1 in the AAV-Nogo-B and AAV-NC infected hearts after MI in border (BZ) and remote zone (RZ) of infarcted hearts (n = 5). *P < 0.05, **P < 0.01 versus sham group. D Protein levels of Notch and Hes1 in ECs treated with DAPT and siNotch (n = 5). *P < 0.05, **P < 0.01 versus Ad-NC group. ns, not significant. Data were expressed as mean ± SEM. Two-way ANOVA (C, D).CC BY · PubMed Central
Fig. 7 Notch pathway inhibition abolished Nogo-B overexpression in ECs network formation and migration. A DAPT treatment on migration, invasion, and tube formation in Ad-NC and Ad-Nogo-B-infected ECs (n = 5). B DAPT treatment on the effect of Ad-Nogo-B and Ad-NC on vessel outgrowth in aortic rings and ECs tube formation (n = 5). Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01 versus Ad-NC group. #P < 0.05, ##P < 0.01 versus Ad-Nogo-B group. two-way ANOVA (A, B).
Fig. 7 Notch pathway inhibition abolished Nogo-B overexpression in ECs network formation and migration. A DAPT treatment on migration, invasion, and tube formation in Ad-NC and Ad-Nogo-B-infected ECs (n = 5). B DAPT treatment on the effect of Ad-Nogo-B and Ad-NC on vessel outgrowth in aortic rings and ECs tube formation (n = 5). Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01 versus Ad-NC group. #P < 0.05, ##P < 0.01 versus Ad-Nogo-B group. two-way ANOVA (A, B).CC BY · PubMed Central
Fig. 8 Notch pathway inhibition abolished the cardioprotective effect of Nogo-B in MI-injured hearts. A The vessel outgrowth in aortic rings of Nogo-B overexpressed and endothelial cell-specific Notch1 heterozygous (Notch1-EC+/−) mice or littermates (n = 3). *P < 0.05 versus Ad-NC group. #P < 0.05 versus Ad-Nogo-B group. B The survival rate of Notch1-EC+/− and Ctrl mice injected with AAV-Nogo-B and AAV-NC after MI. The log-rank test demonstrated significant differences between the two survival curves. C LVEF and LVFS were measured by echocardiography (n = 5). D Quantitative data of hearts stained with Masson’s trichrome at day 28 post-MI (n = 5). E Representation and quantification of IHC staining for TUNEL + cardiomyocytes in the border zone of infarcted hearts at day 3 post-MI (n = 3). F Immunochemistry staining for CD31 + endothelial cells and α-SMA + vessels at day 28 post-MI (n = 5). Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01 versus AAV-NC group. #P < 0.05, ##P < 0.01 versus AAV-Nogo-B group. two-way ANOVA (A and C–F).
Fig. 8 Notch pathway inhibition abolished the cardioprotective effect of Nogo-B in MI-injured hearts. A The vessel outgrowth in aortic rings of Nogo-B overexpressed and endothelial cell-specific Notch1 heterozygous (Notch1-EC+/−) mice or littermates (n = 3). *P < 0.05 versus Ad-NC group. #P < 0.05 versus Ad-Nogo-B group. B The survival rate of Notch1-EC+/− and Ctrl mice injected with AAV-Nogo-B and AAV-NC after MI. The log-rank test demonstrated significant differences between the two survival curves. C LVEF and LVFS were measured by echocardiography (n = 5). D Quantitative data of hearts stained with Masson’s trichrome at day 28 post-MI (n = 5). E Representation and quantification of IHC staining for TUNEL + cardiomyocytes in the border zone of infarcted hearts at day 3 post-MI (n = 3). F Immunochemistry staining for CD31 + endothelial cells and α-SMA + vessels at day 28 post-MI (n = 5). Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01 versus AAV-NC group. #P < 0.05, ##P < 0.01 versus AAV-Nogo-B group. two-way ANOVA (A and C–F).CC BY · PubMed Central

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Metadata

Authors
Zheng, Y., Lin, J., Liu, D., Wan, G., Gu, X., & Ma, J.
Affiliations
  • Shanghai University of Medicine and Health Sciences (CN)
  • Shanghai Jiao Tong University (CN)
Corresponding author
Ma, Jian
ORCID
Venue
Cell Death & Disease
Publisher
Springer Science and Business Media LLC
Published
2022-04-05
Volume
13
Issue
4
Pages
306-306
ISSN
2041-4889 (ISSN-L)
DOI
10.1038/s41419-022-04754-4
PMC
PMC8983727
OpenAlex
W4224325360
Cited by
110
References
52
Citations by year
2026: 20 · 2025: 53 · 2024: 21 · 2023: 14 · 2022: 1
FWCI
7.27
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Death and Disease (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Shanghai Key Laboratory of Molecular Imaging (18DZ2260400, 81970229, 81700354)
  • Ruijin Hospital
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Congenital heart defects research
  • Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Cardiac Fibrosis and Remodeling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Angiogenesis and VEGF in Cancer
Keywords
Nogo-B, angiogenesis, myocardial infarction, cardiac repair, Notch1 signaling, Hes1, cardiac microvascular endothelial cells, oxygen-glucose deprivation, gamma-secretase inhibition, AAV9 gene delivery, ischemic heart disease, endothelial cell migration
MeSH terms
Animals, Endothelium, Myocardial Infarction, Neovascularization, Pathologic, Signal Transduction, Endothelial Cells, Mice

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