Abstract

The clearance of damaged myelin sheaths is critical to ensure functional recovery from neural injury. Here we show a previously unidentified role for microvessels and their lining endothelial cells in engulfing myelin debris in spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE). We demonstrate that IgG opsonization of myelin debris is required for its effective engulfment by endothelial cells and that the autophagy–lysosome pathway is crucial for degradation of engulfed myelin debris. We further show that endothelial cells exert critical functions beyond myelin clearance to promote progression of demyelination disorders by regulating macrophage infiltration, pathologic angiogenesis and fibrosis in both SCI and EAE. Unexpectedly, myelin debris engulfment induces endothelial-to-mesenchymal transition, a process that confers upon endothelial cells the ability to stimulate the endothelial-derived production of fibrotic components. Overall, our study demonstrates that the processing of myelin debris through the autophagy–lysosome pathway promotes inflammation and angiogenesis and may contribute to fibrotic scar formation.

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All data supporting the findings of the current study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank C. Badland for the artwork of Supplemental Fig. 13. We thank W. Lin and J. Wang for assisting with animal experiments. We thank D. Meckes for providing reagents. We thank R. Nowakowski and G. Hammel for editing the manuscript and thank F. Lin for help with some statistics. This work was supported by a visiting student scholarship granted to T.Z. from China Scholarship Council, Hong Kong Health and Medical Research Fund (03142036) and the National Basic Research Program of China (2014CB502200) to W.W., National Institutes of Health (R01GM072611-4) and National Science Foundation (DMS-1662139) to J.F. and National Science Foundation (DMS-0714589, DMS-1661727) to Y.R.

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Author notes

  1. These authors contributed equally: Tian Zhou, Yiming Zheng.

Affiliations

  1. Key Laboratory of Biorheological Science & Technology, Ministry of Education, State & Local Joint Engineering Laboratory for Vascular Implants, College of Bioengineering, Chongqing University, Chongqing, China

    • Tian Zhou
    •  & Guixue Wang
  2. Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL, USA

    • Tian Zhou
    • , Yiming Zheng
    • , Li Sun
    • , Yuanhu Jin
    • , Yang Liu
    • , Alyssa J. Rolfe
    • , Zhijian Cheng
    • , Zhaoshuai Huang
    • , Na Zhao
    • , Choogon Lee
    • , Timothy L. Megraw
    •  & Yi Ren
  3. School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China

    • Smaranda Ruxandra Badea
    •  & Haitao Sun
  4. Department of Immunology, Guizhou Medical University, Guiyang, China

    • Yang Liu
    •  & Yi Ren
  5. Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou, China

    • Haitao Sun
  6. Institute of Neurosciences, the Fourth Military Medical University, Xi’an, China

    • Xi Wang
  7. Institute of Inflammation and Diseases, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China

    • Zhaoshuai Huang
    • , Na Zhao
    •  & Yi Ren
  8. Guangdong-Hong Kong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China

    • Xin Sun
    •  & Wutian Wu
  9. Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia

    • Jinhua Li
  10. Statistical Laboratory, Princeton University, Princeton, NJ, USA

    • Jianqing Fan
  11. Re-Stem Biotechnology Co., Ltd, Suzhou, China

    • Wutian Wu
  12. School of Biomedical Sciences, The University of Hong Kong, Hong Kong, China

    • Wutian Wu

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Contributions

Y.R. and Y.Z conceived the study. Y.R., Y.Z., and T.Z. designed the experiments and analyzed the data. T.Z. and Y.Z. executed most of the experiments. L.S. performed flow cytometry, guided MBP ELISA and led the sample preparation for RNA sequencing. S.R.B., H.S. and W.W. established the mouse EAE model and provided EAE spinal cord samples. Y.J. and C.L. designed the CRISPR–Cas9 knockout of Atg5. Y.L. performed spinal cord injection and some SCI. A.J.R. analyzed the RNA-sequencing data. X.W. provided the preliminary phenotype on in vivo microvessel angiogenesis after SCI. Z.C. provided some preliminary SCI tissues. Z.H. assisted in tissues collection and cryosectioning. N.Z. assisted in western blot assay and some quantifications. X.S., J.L., J.F. and W.W. analyzed some data. T.L.M. helped design some of the experiments, contributed to discussion and edited the manuscript and figures. J.L., J.F., C.L., W.W. and G.W. contributed to discussion. Y.Z., Y.R. and T.Z. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Yiming Zheng or Guixue Wang or Yi Ren.

Integrated supplementary information

  1. Supplementary Figure 1 Internalization of myelin debris (MBP, green) by microvessels (CD31, red) in injured and uninjured regions of spinal cords from different time points after SCI.

    The x-y and x-z views show myelin debris was internalized by microvessels in injured regions of spinal cords from 1-day, 3-day and 5-day SCI mice. The staining was performed in three mice with similar results. Scale bar, 20 μm.

  2. Supplementary Figure 2 MOG-induced EAE disease phases and behavioral assessment.

    (a) The graph shows disease progression through the 3 stages described. Arrows indicate the number of animals used for the assessment up until the specified sacrifice date. Following MOG35–55/CFA immunization but before onset, C57BL/6J mice do not display any motor deficits (pre-onset stage), however several days later, at the onset of disease the tail becomes weak or flaccid and the mice start showing hind limb weakness (onset stage). After disease onset, the mice progressively lose hind limb function and may even develop forelimb weakness consequent to hind limb paralysis (disease stage), with a disease peak occurring relatively soon after onset. EAE score is shown as means ± s.e.m. The number of mice collected for EAE score analysis is: n =14 for 7 days post induction; n =8 for 11 days post induction; n = 4 for 15 days post induction. (b) Immunostaining for MBP (red) and nucleus (blue) spinal cord lumbar segment and thoracic segment after 15-day EAE. The arrowheads indicate the demyelinated plaques. Lumbar segment has more severe demyelination than thoracic segment. The staining was performed in two mice with similar results. Scale bar, 500 μm.

  3. Supplementary Figure 3 BMDMϕ engulf myelin debris rapidly.

    Detection of engulfed CFSE-myelin debris (green) in BMDMϕ (F4/80, red) exposed to myelin debris for 0, 15 min, 1 hr and 3 hr, respectively. This staining was performed more than five times with similar results. Scale bar, 5 μm.

  4. Supplementary Figure 4 CR3 or Mac2 is involved in BMDMϕ engulfment of myelin debris, but not involved in endothelial cell engulfment of myelin debris.

    (a, b) Detection of myelin debris (green) engulfment by BMDMϕ (a) and BMECs (b) that were pre-treated with the indicated neutralizing antibodies for 3 hr, followed by myelin debris treatment for 3 hr and 72 hr, respectively. A combined use of CR3 and Mac2 neutralizing antibodies blocked BMDMϕ uptake of myelin debris, but had no effect on BMEC uptake of myelin debris. This staining was performed three times with similar results. Scale bar, 100 μm.

  5. Supplementary Figure 5 Deficiency of MBP, a proposed ligand for LRP-1 receptor, appears to not affect myelin debris uptake by BMECs.

    Detection of engulfed CFSE-myelin debris (CFSE, green or MBP, red) in BMECs (CD31, purple) exposed to myelin debris (isolated from wild-type mice) and MBP-deficient myelin debris (isolated from Shiverer mice) for 72 hr. This staining was performed more than five times with similar results. Scale bar, 20 μm.

  6. Supplementary Figure 6 Serum-dependent engulfment of myelin debris by BMECs.

    Detection of engulfed CFSE-myelin debris (green) in BMECs after 48 hr exposure to myelin debris in the presence of 0%, 1% and 5% FBS. This staining was performed more than five times with similar results. Scale bar, 100 μm.

  7. Supplementary Figure 7 Generation and characterization of Atg5-knockout BMEC cell line.

    (a) Diagram showing Atg5 gene locus and guide RNA targeting sequence. (b) Verification of genomic editing of Atg5 by CRISPR-Cas9 system using T7E1 assay. T7E1 assay was run in acrylamide gel, which caused inaccurate indication of 100bp DNA ladder. The PCR product band is 440bp. Two digestion bands are 228 and 212bp, which are indistinguishable from each other in the gel. This assay was performed twice with similar results. (c) Functional verification of Atg5-/-BMECs by immunostaining of poly-ubiquitin (green) and p62 (red). The staining was performed twice with similar results. Scale bar, 20 μm. (d) Cell death analysis of Atg5-/- BMECs and drugs-treated wild-type BMECs by propidium iodide (PI) staining. Drug dose and incubation period are corresponding to Fig. 4k. H2O2 treatment was included as positive control. Data are shown as means ± s.e.m. (n = 3 independent experiments). p<0.00001 (****) by unpaired two-sided Student’s t-test.

  8. Supplementary Figure 8 Additional data on in vivo angiogenesis.

    (a) Microvessels (CD31, red) in normal spinal cord from uninjured mice and injured spinal cords from mice at 1 week or 6 weeks after SCI. The insets showed the enlarged images. Quantitative data are shown in Fig. 5b. Scale bar, 200 μm; 20 μm in inset images. (b) Representative images and quantification of Ki-67 positive (green) and CD31 (red) microvessels after the indicated injections into normal spinal cords in mice. Scale bar, 100 μm; 50 μm in zoomed images. Data are shown as means ± s.e.m. from 3 mice. Myelin vs PBS, p = 0.6779 (ns); naïve ECs vs PBS, p = 0.0474 (*); myelin-ECs vs PBS, p = 0.0004 (***); myelin-ECs vs naïve ECs, p = 0.0005 (***); myelin-ECs (IgG-inactivated) vs myelin-ECs, p = 0.0005 (***) by unpaired two-sided Student’s t-test.

  9. Supplementary Figure 9 The cellular consequences of necrotic neuronal cells or zymosan engulfment by BMECs and BMDMϕ.

    (a) Characteristic morphology of neuronal cells that were differentiated from N2A cells, a mouse neuroblastoma cell line. Neuronal differentiation assay was repeated at least five times. Scale bar, 50 μm. (b) BMECs (red) engulfment of necrotic neuronal cell (green) for 72 hr. BMEC nuclei (Hoechst staining) were shown in pseudo white. Arrowhead indicated the squeezed BMEC nucleus by the engulfed necrotic cell. The staining was performed four times with similar results. Scale bar, 10 μm. (c) Quantification of the percentage of BMDMϕ and BMECs that contained necrotic cells after 6 hr and 72 hr engulfment, respectively. Data are shown as means ± s.e.m. from 3 biologically independent replicates. p = 0.0006 (***) by unpaired two-sided Student’s t-test. (d, e, f) BMECs were treated with or without necrotic cells. (d) Proliferation assessed by counting BMEC nuclei, p = 0.0418 (*). (e) Mcp-1 gene expression detected by q-PCR analysis, p = 0.9510 (ns). (f) α-SMA+/CD31+ cells were counted, p = 0.5369 (ns). Data are shown as means ± s.e.m. from 3 biologically independent cultures by unpaired two-sided Student’s t-test. (g, h) Detection of zymosan engulfment by BMDMϕ and BMECs. Zymosan was labelled by CFSE in green. The staining was performed three times with same results. Scale bar, 10 μm.

  10. Supplementary Figure 10 Analysis of astrocytes’ effects on BMECs using a co-culture system.

    (a) Primary mouse astrocytes were isolated and characterized by GFAP staining (green). (b) Diagram showing astrocyte-BMEC co-culture in a transwell. (c) BMEC proliferation after co-cultured with the indicated astrocytes. Myelin-treated BMECs were included as positive control. Data are shown as means ± s.e.m. from 3 biologically independent cultures. Control astrocytes vs medium, p = 0.0712 (ns); LPS-treated astrocytes vs medium, p = 0.1566 (ns); myelin debris-treated ECs vs medium, p = 0.0466 (*) by unpaired two-sided Student’s t-test.

  11. Supplementary Figure 11 Additional data on inflammation.

    (a) Quantification of number of GFP+ BMDCs associated with microvessels is shown in Fig. 6a. Data are shown as means ± s.e.m. (n = 3 mice). p = 0.0043(**), p = 0.0003 (***), p<0.00001 (****) by unpaired two-sided Student’s t-test. (b) Quantification of number of Iba1+ cells associated with one microvessel with normal size or increased size in Fig. 6b. Data are shown as means ± s.e.m. (n = 3 mice). p = 0.0114 (*) by paired two-sided Student’s t-test. (c) Representative images showing adhered BMDMϕ (Mac-2, red) on BMECs with the indicated treatments. Quantitative data are shown in Fig. 6c’. Scale bar, 100 μm. (d) Representative images of Iba-1 (green) and GFAP staining (red) after the indicated injections into normal spinal cords in mice. Scale bar, 100 μm; 10 μm in inset images. (e) Quantification of Iba-1 fluorescent intensity shown in d and Fig. 6h. Data are shown as means ± s.e.m. (n = 3 injected mice). Myelin debris vs PBS, p = 0.0527 (ns); naïve ECs vs PBS, p = 0.1942 (ns); myelin-ECs vs PBS, p = 0.0038 (**); myelin-ECs vs naïve ECs, p = 0.0286 (*); myelin-ECs vs myelin debris, p = 0.0067 (**); myelin-ECs (IgG-inactivated) vs myelin-ECs, p = 0.0279 (*) by unpaired two-sided Student’s t-test. (f) Quantification of GFAP fluorescent intensity is shown in d. Data are shown as means ± s.e.m. (n = 3 injected mice). Myelin debris vs PBS, p = 0.1523 (ns); naïve ECs vs PBS, p = 0.3266 (ns); myelin-ECs vs PBS, p = 0.0183 (*); myelin-ECs vs naïve ECs, p = 0.0349 (*); myelin-ECs vs myelin debris, p = 0.044 (*); myelin-ECs (IgG-inactivated) vs myelin-ECs, p = 0.0336 (*) by unpaired two-sided Student’s t-test.

  12. Supplementary Figure 12 Schematic diagram depicting the novel function of microvascular endothelial cells in engulfment and autophagic processing of myelin debris, which in turn boosts secondary injury by promoting inflammation, microvessel dilation, and fibrotic scar formation.

    (a) In the injured spinal cord, one of the immediate responses to SCI is the fragmentation of myelin sheaths into myelin debris, which can be quickly cleared by professional phagocytes such as BMDMϕ and microglia. We demonstrated that microvessels and the lining microvascular endothelial cells are able to engulf myelin debris which promote chronic inflammation and pathological healing. The microvessels in injury core are dilated (not depicted), in close vicinity to BMDMϕ, deposit high levels of pro-fibrotic components like collagen and fibronectin. These changes in the injury core, all of which could be stemmed as a result of microvascular engulfment of myelin debris, are closely associated with the progression of SCI. (b) Mechanisms for myelin debris entry, processing and actions in microvascular ECs. IgG opsonization of myelin debris is engulfed by endothelial cells through Fc receptor. Engulfed myelin debris is delivered through autophagosomes to lysosomes for myelin degradation into neutral lipids. Functionally, myelin debris uptake promotes endothelial cells proliferation/angiogenesis, endothelial inflammation and endothelial deposition of pro-fibrotic components, which could contribute to the dilation of microvessels, chronic inflammation and fibrotic scar formation seen in injury core.

  13. Supplementary Figure 13 Full, uncropped immunoblots.

    Full, uncropped immunoblots for Figs. 4f, 4g, 4i, 6d, 7i, and 8f.

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https://doi.org/10.1038/s41593-018-0324-9