Abstract
Pro-angiogenic factors are key regulators of angiogenesis. Here we report that highly migratory cells patrol the area of capillary formation in chick embryo chorioallantoic membrane. These cells deposit migrasomes on their migration tracks, creating migrasome-enriched areas. Single-cell sequencing identified these cells as monocytes. Depletion of monocytes impairs capillary formation. Quantitative mass spectrometry analysis reveals that monocyte migrasomes are enriched with pro-angiogenic factors. Purified migrasomes promote capillary formation and monocyte recruitment in vivo, and endothelial cell tube formation and monocyte chemotaxis in vitro. Knockdown or knockout of TSPAN4 reduces migrasome formation and impairs capillary formation and monocyte recruitment. Mechanistically, monocytes promote angiogenesis via VEGFA and CXCL12 in migrasomes. In summary, monocytes deposit migrasomes enriched in pro-angiogenic factors to promote angiogenesis.
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Data availability
RNA-seq data that support the findings of this study have been deposited in the National Center for Biotechnology Information Sequence Read Archive under the accession code SAMN30722995. The mass spectrometry proteomics data have been deposited in ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier or the primary accession code PXD036647. The statistical source data for all figures and extended data figures have been provided as numerical source data. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Further information and requests for resources and reagents should be directed to and will be fulfilled by L.Y. Source data are provided with this paper.
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Acknowledgements
We are grateful to the members of the Yu groups for their helpful discussions and suggestions. This research was supported by the Ministry of Science and Technology of the People’s Republic of China (grant no. 2017YFA0503404), the National Natural Science Foundation of China (grant nos. 31790401, 92054301 and 32030023) and the Beijing Municipal Science & Technology Commission (grant nos. Z201100005320019 and Z211100003321002) and internal grant from Tsinghua university (2030 plan). We thank the State Key Laboratory of Membrane Biology for facility support and assistance with spinning disk microscopy. We thank the Protein Chemistry Facility at the Centre for Biomedical Analysis of Tsinghua University for mass spectrometry sample analysis. We acknowledge the assistance of the Imaging Core Facility, Technology Center for Protein Sciences, Tsinghua University for assistance with Imaris analysis. We acknowledge the assistance of SLSTU-Nikon Biological Imaging Center for assistance with using the NIKON A1RSiHD25 laser scanning confocal microscope and software. We thank the Core Facility, Center of Biomedical Analysis, Tsinghua University for technical support with flow cytometry and data analysis.
Author information
Authors and Affiliations
Contributions
L.Y. conceived the project. L.Y. and C.Z. designed and C.Z. performed in vitro and in vivo experiments and analysed data. L.Y. and C.Z. wrote the manuscript. M.G., Y.L. and M.S. performed migrasome 3D reconstruction experiments. S.Y. and H.H. performed TSPAN4-KO and monocyte depletion experiments. T.L. and J.W. designed and performed the single-cell sequencing experiment. D.J. performed migrasome purification experiments. All authors discussed the manuscript, commented on the project and contributed to the preparation of the paper.
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L.Y. is the scientific founder of Migrasome therapeutics Ltd. All other authors declare no conflicts of interest.
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Nature Cell Biology thanks Rajendra Apte, Michael Sixt and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Morphology of WGAhigh/low cell.
(a) Migration of WGAhigh and WGAlow cells was monitored by time-lapse confocal microscopy. Scale bar, 5 µm. (b) Cells from (a) were quantified for migration speed. Data are presented as means ± SEM; n = 21 cells per group pooled from three independent experiments. (c) Cells from (a) were quantified for size. Data are presented as means ± SEM; n = 21 cells per group pooled from three independent experiments. P values were calculated using a two-tailed, unpaired t-test, P < 0.0001. Source numerical data are available in source data.
Extended Data Fig. 2 Monocytes depletion by alendronate impairs angiogenesis.
(a) After treatment with control liposomes or alendronate liposomes, CAM9d were stained by CD115-Alexa488 to detect monocytes inside and outside the blood vessels. Inside vessels: CD115-Alexa488 was microinjected into the CAM vasculature. Outside vessels: CD115-Alexa488 was add onto the CAM surface. Images were taken 30 minutes after CD115-Alexa488 staining. Scale bar, 50 μm. (b-c) The numbers of monocytes inside or outside vessels were quantified according to the CD115 signal. Data are presented as mean ± SEM; n = 32 samples from three independent experiments; P values were calculated using a two-tailed, unpaired t-test, P < 0.0001. (d) CAMs treated with control liposomes and alendronate liposomes were visualized with a stereomicroscope (left panel) or a confocal microscope after dextran-FITC microinjection (right panel). Scale bar, left panel, 500 μm; right panel, 50 μm. (e) The number of sprouting capillaries from (d) was quantified according to the intensity of the dextran signal. Data are presented as mean ± SEM; n = 32 samples from three independent experiments; P values were calculated using a two-tailed, unpaired t-test, P < 0.0001. Source numerical data are available in source data.
Extended Data Fig. 3 mRNA levels of TGFB3 by single-cell sequencing.
(a) Violin plots showing the mRNA levels of TGFB3 from single-cell sequencing analysis of monocyte and endothelium cells.
Extended Data Fig. 4 VEGFA/CXCL12 are enriched in migrasomes from CAM or mouse monocytes.
(a) CAM9d were stained with WGA and the indicated antibodies and visualized by confocal microscopy. Scale bar, 20 μm. Immunofluorescence in CAMs was visualized by confocal z-stack imaging and presented as the maximum intensity projection. (b) Migrasomes from mouse monocytes are enriched with VEGFA and CXCL12. Mouse bone marrow monocytes (BMMs) acquired from WT C57BL/6 mice were stained with WGA and the indicated antibodies and visualized by confocal microscopy. Scale bar, 10 μm. Enlarged images of migrasomes are shown in the white boxes. Scale bar, 2.5μm.
Extended Data Fig. 5 Migrasome delivery promotes EC proliferation and sprouting.
(a) Dissected CAMs from E8d were mounted on the top of a collagen I-Matrigel matrix mix, and soft agarose with migrasomes (+Mig) or without migrasomes (-Mig) was added on opposite sides of the CAM. 2, 4 and 6 days after mounting, the samples were visualized by confocal microscopy. Scale bar, 200 µm. (b) The proliferation of ECs and sprouting progression from (a) was quantified by the tube length from the edge of CAM to the angiogenic front. Data are presented as mean ± SEM; n =30 fields of CAM leaflets from five independent experiments; P values were calculated using a two-way ANOVA unpaired multiple comparisons, N.S: no significance (P=0.9681); P < 0.0001. Source numerical data are available in source data.
Extended Data Fig. 6 Migrasome triggers VEGFR2 and AKT phosphorylation.
(a) Cells were treated with migrasome or VEGF165 for 24 hours and the Lysates of cell were analysed by western blot using indicated antibody. 3 repeats were shown (repeat 1 is figure 5q). (b) The relative fold change of indicated signal in the control, VEGF165 and migrasome treated cells was quantified. Data are presented as mean ± SEM; n = 3 from three independent experiments; P values were calculated using a one-way ANOVA unpaired multiple comparisons. VEGFR2 protein level: (N.S: no significance; P=0.9981, Control/control vs. VEGF/control; P=0.9725, Control/control vs. Mig/control). p-VEGFR2 protein level: (P=0.0325, Control/control vs. VEGF/control; P=0.0037, Control/control vs. Mig/control). AKT protein level: (N.S: no significance; P=0.6451, Control/control vs. VEGF/control; P=0.2196, Control/control vs. Mig/control). p-AKT protein level: (P=0.0263, Control/control vs. VEGF/control; P=0.0263, Control/control vs. Mig/control). Source numerical data and unprocessed blots are available in source data.
Extended Data Fig. 7 Migrasomes rescue TSPAN4 KD induced capillarization and monocyte recruitment defects.
(a) Migrasomes were delivered to CAM9d by mixing them with Matrigel. After 48 h, CAMs were visualized by stereomicroscopy. Scale bar, 500 µm. (b) Migrasomes were delivered to CAM9d by mixing them with low-melting-point agarose. After 48 h, CAMs were visualized by stereomicroscopy. Scale bar, 500 µm. (c) CAMs from (a) were quantified for the number of newly formed capillaries. Data are presented as mean ± SEM; n =15 fields from three independent experiments; P values were calculated using a two-tailed, unpaired t-test, P < 0.0001. (d) CAMs from (b) were quantified for the number of sprouting capillaries. Data are presented as mean ± SEM; n =15 fields from three independent experiments; P values were calculated using a two-tailed, unpaired t-test, P < 0.0001. (e) 48 h after transfection with control of TSPAN4 (T4) siRNA, migrasomes embedded in low-melting-point agarose were added to CAMs. 48 h later, CAMs were visualized by stereomicroscopy. Scale bar, 500 μm. (f) CAMs from (e) were quantified for the relative change in the in the number of sprouting capillaries. Data are presented as means ± SEM, n = 7 fields from three independent experiments, P values were calculated using a two way ANOVA unpaired multiple comparisons, N.S: no significance (P=0.0640), P < 0.0001. Source numerical data are available in source data.
Extended Data Fig. 8 TSPAN4 KO impairs migrasome formation.
(a) Diagram showing the strategy for knocking out TSPAN4 and knocking in mCherry in chick embryos. The mCherry coding sequence was inserted into TSPAN4 at the position targeted by the sgRNA. Thus, an mCherry-positive signal indicates that native TSPAN4 gene expression was silenced simultaneously. (b) Time-lapse imaging of migration and migrasome formation by WGAhigh cells isolated from WT or TSPAN4-KO CAM8d. Cells were stained by WGA and observed by spinning disk microscopy. Scale bar, 5 µm. (c) CAMs from Fig. 6k and 6l were stained for WGA and observed by spinning disk microscopy. Scale bar, 30 µm. (d) CAMs from Fig. 6k and 6l were quantified for the number of WGAhigh cells. Data are presented as mean ± SEM; n = 15 cells from three independent experiments; P values were calculated using a two-tailed, unpaired t-test, P < 0.0001. Source numerical data are available in source data.
Extended Data Fig. 9 Migrasomes rescue VEGFA/CXCL12 KD induced capillarization and monocyte recruitment defects.
(a) CAMs were treated with VEGFA siRNA. 72 h after transfection, the CAMs were harvested, and the VEGFA knockdown efficiency in the CAMs was determined using q–PCR. Data are presented as mean ± SEM; n = 16 pieces from three independent experiments; P values were calculated using a two-tailed, unpaired t-test, P < 0.0001. (b) CAMs treated with CXCL12 siRNA for 72 h were harvested, and CXCL12 knockdown efficiency in CAM pieces was detected by q–PCR. Data are presented as mean ± SEM; n = 16 pieces from three independent experiments; P values were calculated using a two-tailed, unpaired t-test, P < 0.0001. (c) VEGFA and CXCL12 knockdown efficiencies in siRNA-treated CAM pieces were determined by western blot analysis using then indicated antibodies. (d) CAMs were transfected with the indicated siRNAs. After 48 h, migrasomes embedded in low-melting-point agarose were added. After another 48 h, CAMs were visualized by stereomicroscopy. Scale bar, 1 mm. (e) CAMs treated with VEGF siRNA (top) or CXCL12 siRNA (bottom) from (d) were quantified for the change in the number of sprouting capillaries. Data are presented as mean ± SEM; n = 9 fields from three independent experiments; in siVEGFA group, P values were calculated using a two way ANOVA unpaired multiple comparisons, N.S: no significance (P=0.7626, 96h/0h, siNC+Migrasome vs. siVEGFA+Migrasome); P = 0.0003(96h/0h, siNC+Agarose vs. siNC+Migrasome); P < 0.0001; in siCXCL12 group, P values were calculated using a two way ANOVA unpaired multiple comparisons, P=0.0028 (96h/0h, siNC+Migrasome vs. siCXCL12+Migrasome); P=0.0012 (96h/0h, siCXCL12+Agarose vs. siCXCL12+Migrasome); P<0.0001. Source numerical data and unprocessed blots are available in source data.
Supplementary information
Supplementary Information
Unprocessed western blots for Figs. 4c,h and 5q and Extended Data Figs. 6a and 9c. Video and table legends.
Supplementary Table 1
Oligonucleotide sequences for all TSPAN4, VEGFA, CXCL12 KD siRNA, TSPAN4 knockout gRNA and primers for sequences identification. All sequences shown from 5′ to 3′.
Supplementary Video 1
Migrasome formation in CAM9d in vivo level.
Source data
Source Data Fig. 1
Numerical source data for Fig. 1 statistic results are provided.
Source Data Fig. 2
Numerical source data for Fig. 2 statistic results are provided.
Source Data Fig. 3
Numerical source data for Fig. 3 statistic results are provided.
Source Data Fig. 4
Unprocessed western blots for Fig. 4.
Source Data Fig. 5
Numerical source data for Fig. 5 statistic results are provided.
Source Data Fig. 5
Unprocessed western blots for Fig. 5.
Source Data Fig. 6
Numerical source data for Fig. 6 statistic results are provided.
Source Data Fig. 7
Numerical source data for Fig. 7 statistic results are provided.
Source Data Extended Data Fig. 1
Numerical source data for Extended Data Fig. 1 statistic results are provided.
Source Data Extended Data Fig. 2
Numerical source data for Extended Data Fig. 2 statistic results are provided.
Source Data Extended Data Fig. 5
Numerical source data for Extended Data Fig. 5 statistic results are provided.
Source Data Extended Data Fig. 6
Unprocessed western blots for Extended Data Fig. 6.
Source Data Extended Data Fig. 6
Numerical source data for Extended Data Fig. 6 statistic results are provided.
Source Data Extended Data Fig. 7
Numerical source data for Extended Data Fig. 7 statistic results are provided.
Source Data Extended Data Fig. 8
Numerical source data for Extended Data Fig. 8 statistic results are provided.
Source Data Extended Data Fig. 9
Unprocessed western blots for Extended Data Fig. 9.
Source Data Extended Data Fig. 9
Numerical source data for Extended Data Fig. 9 statistic results are provided.
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Zhang, C., Li, T., Yin, S. et al. Monocytes deposit migrasomes to promote embryonic angiogenesis. Nat Cell Biol 24, 1726–1738 (2022). https://doi.org/10.1038/s41556-022-01026-3
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DOI: https://doi.org/10.1038/s41556-022-01026-3
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