Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Endothelial exocytosis of angiopoietin-2 resulting from CCM3 deficiency contributes to cerebral cavernous malformation

An Erratum to this article was published on 06 December 2016

This article has been updated

Abstract

Cerebral cavernous malformations (CCMs) are vascular malformations that affect the central nervous system and result in cerebral hemorrhage, seizure and stroke. CCMs arise from loss-of-function mutations in one of three genes: KRIT1 (also known as CCM1), CCM2 or PDCD10 (also known as CCM3). PDCD10 mutations in humans often result in a more severe form of the disease relative to mutations in the other two CCM genes, and PDCD10-knockout mice show severe defects, the mechanistic basis for which is unclear. We have recently reported that CCM3 regulates exocytosis mediated by the UNC13 family of exocytic regulatory proteins. Here, in investigating the role of endothelial cell exocytosis in CCM disease progression, we found that CCM3 suppresses UNC13B- and vesicle-associated membrane protein 3 (VAMP3)-dependent exocytosis of angiopoietin 2 (ANGPT2) in brain endothelial cells. CCM3 deficiency in endothelial cells augments the exocytosis and secretion of ANGPT2, which is associated with destabilized endothelial cell junctions, enlarged lumen formation and endothelial cell–pericyte dissociation. UNC13B deficiency, which blunts ANGPT2 secretion from endothelial cells, or treatment with an ANGPT2-neutralizing antibody normalizes the defects in the brain and retina caused by endothelial-cell-specific CCM3 deficiency, including the disruption of endothelial cell junctions, vessel dilation and pericyte dissociation. Thus, enhanced secretion of ANGPT2 in endothelial cells contributes to the progression of CCM disease, providing a new therapeutic approach for treating this devastating pathology.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Purchase on Springer Link

Instant access to full article PDF

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Endothelial-cell-inducible Pdcd10 deletion (Pdcd10ECKO) mice develop CCM lesions.
Figure 2: Pdcd10ECKO mice exhibit disrupted endothelial cell–pericyte and endothelial cell–endothelial cell junctions with increased ANGPT2 levels.
Figure 3: CCM3 restrains ANGPT2 release from endothelial cells and maintains endothelial junctions.
Figure 4: CCM3 maintains normal endothelial lumen formation and endothelial cell–pericyte association.
Figure 5: UNC13 deficiency rescues CCM phenotypes in Pdcd10ECKO mice.
Figure 6: ANGPT2-neutralizing antibody blunts CCM lesion progression in Pdcd10ECKO mice.

Similar content being viewed by others

Change history

  • 21 September 2016

    In the version of this article initially published, labels for micrographs in Figure 2d and Figure 5b were omitted, and two grants were not acknowledged in the Acknowledgments section. The errors have been corrected in the HTML and PDF versions of the article.

References

  1. Revencu, N. & Vikkula, M. Cerebral cavernous malformation: new molecular and clinical insights. J. Med. Genet. 43, 716–721 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Cavalcanti, D.D. et al. Cerebral cavernous malformations: from genes to proteins to disease. J. Neurosurg. 116, 122–132 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Tanriover, G. et al. Ultrastructural analysis of vascular features in cerebral cavernous malformations. Clin. Neurol. Neurosurg. 115, 438–444 (2013).

    Article  PubMed  Google Scholar 

  4. Riant, F., Bergametti, F., Ayrignac, X., Boulday, G. & Tournier-Lasserve, E. Recent insights into cerebral cavernous malformations: the molecular genetics of CCM. FEBS J. 277, 1070–1075 (2010).

    Article  CAS  PubMed  Google Scholar 

  5. Labauge, P., Denier, C., Bergametti, F. & Tournier-Lasserve, E. Genetics of cavernous angiomas. Lancet Neurol. 6, 237–244 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Sahoo, T. et al. Mutations in the gene encoding KRIT1, a Krev-1/rap1a binding protein, cause cerebral cavernous malformations (CCM1). Hum. Mol. Genet. 8, 2325–2333 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Liquori, C.L. et al. Mutations in a gene encoding a novel protein containing a phosphotyrosine-binding domain cause type 2 cerebral cavernous malformations. Am. J. Hum. Genet. 73, 1459–1464 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Bergametti, F. et al. Mutations within the programmed cell death 10 gene cause cerebral cavernous malformations. Am. J. Hum. Genet. 76, 42–51 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Gault, J., Shenkar, R., Recksiek, P. & Awad, I.A. Biallelic somatic and germ line CCM1 truncating mutations in a cerebral cavernous malformation lesion. Stroke 36, 872–874 (2005).

    Article  PubMed  Google Scholar 

  10. Akers, A.L., Johnson, E., Steinberg, G.K., Zabramski, J.M. & Marchuk, D.A. Biallelic somatic and germline mutations in cerebral cavernous malformations (CCMs): evidence for a two-hit mechanism of CCM pathogenesis. Hum. Mol. Genet. 18, 919–930 (2009).

    Article  CAS  PubMed  Google Scholar 

  11. Pagenstecher, A., Stahl, S., Sure, U. & Felbor, U. A two-hit mechanism causes cerebral cavernous malformations: complete inactivation of CCM1, CCM2 or CCM3 in affected endothelial cells. Hum. Mol. Genet. 18, 911–918 (2009).

    Article  CAS  PubMed  Google Scholar 

  12. McDonald, D.A. et al. A novel mouse model of cerebral cavernous malformations based on the two-hit mutation hypothesis recapitulates the human disease. Hum. Mol. Genet. 20, 211–222 (2011).

    Article  CAS  PubMed  Google Scholar 

  13. Chan, A.C. et al. Mutations in 2 distinct genetic pathways result in cerebral cavernous malformations in mice. J. Clin. Invest. 121, 1871–1881 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Cunningham, K. et al. Conditional deletion of Ccm2 causes hemorrhage in the adult brain: a mouse model of human cerebral cavernous malformations. Hum. Mol. Genet. 20, 3198–3206 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Boulday, G. et al. Tissue-specific conditional CCM2 knockout mice establish the essential role of endothelial CCM2 in angiogenesis: implications for human cerebral cavernous malformations. Dis. Model. Mech. 2, 168–177 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Maddaluno, L. et al. EndMT contributes to the onset and progression of cerebral cavernous malformations. Nature 498, 492–496 (2013).

    Article  CAS  PubMed  Google Scholar 

  17. Shenkar, R. et al. Exceptional aggressiveness of cerebral cavernous malformation disease associated with PDCD10 mutations. Genet. Med. 17, 188–196 (2015).

    Article  CAS  PubMed  Google Scholar 

  18. Whitehead, K.J. et al. The cerebral cavernous malformation signaling pathway promotes vascular integrity via Rho GTPases. Nat. Med. 15, 177–184 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Stockton, R.A., Shenkar, R., Awad, I.A. & Ginsberg, M.H. Cerebral cavernous malformations proteins inhibit Rho kinase to stabilize vascular integrity. J. Exp. Med. 207, 881–896 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhou, Z. et al. Cerebral cavernous malformations arise from endothelial gain of MEKK3–KLF2/4 signalling. Nature 532, 122–126 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Denier, C. et al. Genotype-phenotype correlations in cerebral cavernous malformations patients. Ann. Neurol. 60, 550–556 (2006).

    Article  PubMed  Google Scholar 

  22. Zheng, X. et al. CCM3 signaling through sterile 20-like kinases plays an essential role during zebrafish cardiovascular development and cerebral cavernous malformations. J. Clin. Invest. 120, 2795–2804 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Yoruk, B., Gillers, B.S., Chi, N.C. & Scott, I.C. Ccm3 functions in a manner distinct from Ccm1 and Ccm2 in a zebrafish model of CCM vascular disease. Dev. Biol. 362, 121–131 (2012).

    Article  CAS  PubMed  Google Scholar 

  24. Zhang, Y. et al. A network of interactions enables CCM3 and STK24 to coordinate UNC13D-driven vesicle exocytosis in neutrophils. Dev. Cell 27, 215–226 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Feldmann, J. et al. Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3). Cell 115, 461–473 (2003).

    Article  CAS  PubMed  Google Scholar 

  26. Jahn, R. & Südhof, T.C. Membrane fusion and exocytosis. Annu. Rev. Biochem. 68, 863–911 (1999).

    Article  CAS  PubMed  Google Scholar 

  27. Lowenstein, C.J., Morrell, C.N. & Yamakuchi, M. Regulation of Weibel-Palade body exocytosis. Trends Cardiovasc. Med. 15, 302–308 (2005).

    Article  CAS  PubMed  Google Scholar 

  28. Fiedler, U. et al. Angiopoietin-2 sensitizes endothelial cells to TNF-α and has a crucial role in the induction of inflammation. Nat. Med. 12, 235–239 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. Gaengel, K., Genové, G., Armulik, A. & Betsholtz, C. Endothelial-mural cell signaling in vascular development and angiogenesis. Arterioscler. Thromb. Vasc. Biol. 29, 630–638 (2009).

    Article  CAS  PubMed  Google Scholar 

  30. Eklund, L. & Olsen, B.R. Tie receptors and their angiopoietin ligands are context-dependent regulators of vascular remodeling. Exp. Cell Res. 312, 630–641 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. Maisonpierre, P.C. et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277, 55–60 (1997).

    Article  CAS  PubMed  Google Scholar 

  32. He, Y. et al. Stabilization of VEGFR2 signaling by cerebral cavernous malformation 3 is critical for vascular development. Sci. Signal. 3, ra26 (2010).

    PubMed  PubMed Central  Google Scholar 

  33. Fidalgo, M. et al. CCM3/PDCD10 stabilizes GCKIII proteins to promote Golgi assembly and cell orientation. J. Cell Sci. 123, 1274–1284 (2010).

    Article  CAS  PubMed  Google Scholar 

  34. Zhou, H.J. et al. AIP1 mediates vascular endothelial cell growth factor receptor-3-dependent angiogenic and lymphangiogenic responses. Arterioscler. Thromb. Vasc. Biol. 34, 603–615 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kluger, M.S., Clark, P.R., Tellides, G., Gerke, V. & Pober, J.S. Claudin-5 controls intercellular barriers of human dermal microvascular but not human umbilical vein endothelial cells. Arterioscler. Thromb. Vasc. Biol. 33, 489–500 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Nakatsu, M.N. & Hughes, C.C. An optimized three-dimensional in vitro model for the analysis of angiogenesis. Methods Enzymol. 443, 65–82 (2008).

    Article  CAS  PubMed  Google Scholar 

  37. Abraham, S. et al. A Rac/Cdc42 exchange factor complex promotes formation of lateral filopodia and blood vessel lumen morphogenesis. Nat. Commun. 6, 7286 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Felcht, M. et al. Angiopoietin-2 differentially regulates angiogenesis through TIE2 and integrin signaling. J. Clin. Invest. 122, 1991–2005 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Chang, W.G., Andrejecsk, J.W., Kluger, M.S., Saltzman, W.M. & Pober, J.S. Pericytes modulate endothelial sprouting. Cardiovasc. Res. 100, 492–500 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Varoqueaux, F. et al. Total arrest of spontaneous and evoked synaptic transmission but normal synaptogenesis in the absence of Munc13-mediated vesicle priming. Proc. Natl. Acad. Sci. USA 99, 9037–9042 (2002).

    Article  CAS  PubMed  Google Scholar 

  41. Holopainen, T. et al. Effects of angiopoietin-2-blocking antibody on endothelial cell-cell junctions and lung metastasis. J. Natl. Cancer Inst. 104, 461–475 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Gale, N.W. et al. Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1. Dev. Cell 3, 411–423 (2002).

    Article  CAS  PubMed  Google Scholar 

  43. Thomson, B.R. et al. A lymphatic defect causes ocular hypertension and glaucoma in mice. J. Clin. Invest. 124, 4320–4324 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Saharinen, P. et al. Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-cell and cell-matrix contacts. Nat. Cell Biol. 10, 527–537 (2008).

    Article  CAS  PubMed  Google Scholar 

  45. Fukuhara, S. et al. Differential function of Tie2 at cell-cell contacts and cell-substratum contacts regulated by angiopoietin-1. Nat. Cell Biol. 10, 513–526 (2008).

    Article  CAS  PubMed  Google Scholar 

  46. Daly, C. et al. Angiopoietin-2 functions as an autocrine protective factor in stressed endothelial cells. Proc. Natl. Acad. Sci. USA 103, 15491–15496 (2006).

    Article  CAS  PubMed  Google Scholar 

  47. Marchi, S. et al. Defective autophagy is a key feature of cerebral cavernous malformations. EMBO Mol. Med. 7, 1403–1417 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Kümpers, P. et al. Time course of angiopoietin-2 release during experimental human endotoxemia and sepsis. Crit. Care 13, R64 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Gingras, A.R., Liu, J.J. & Ginsberg, M.H. Structural basis of the junctional anchorage of the cerebral cavernous malformations complex. J. Cell Biol. 199, 39–48 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Cuttano, R. et al. KLF4 is a key determinant in the development and progression of cerebral cavernous malformations. EMBO Mol. Med. 8, 6–24 (2015).

    Article  CAS  PubMed Central  Google Scholar 

  51. Sako, K. et al. Angiopoietin-1 induces Kruppel-like factor 2 expression through a phosphoinositide 3-kinase/AKT-dependent activation of myocyte enhancer factor 2. J. Biol. Chem. 284, 5592–5601 (2009).

    Article  CAS  PubMed  Google Scholar 

  52. Zawistowski, J.S. et al. CCM1 and CCM2 protein interactions in cell signaling: implications for cerebral cavernous malformations pathogenesis. Hum. Mol. Genet. 14, 2521–2531 (2005).

    Article  CAS  PubMed  Google Scholar 

  53. Pouwels, J., Nevo, J., Pellinen, T., Ylänne, J. & Ivaska, J. Negative regulators of integrin activity. J. Cell Sci. 125, 3271–3280 (2012).

    Article  CAS  PubMed  Google Scholar 

  54. Brütsch, R. et al. Integrin cytoplasmic domain-associated protein-1 attenuates sprouting angiogenesis. Circ. Res. 107, 592–601 (2010).

    Article  CAS  PubMed  Google Scholar 

  55. Lampugnani, M.G. et al. CCM1 regulates vascular-lumen organization by inducing endothelial polarity. J. Cell Sci. 123, 1073–1080 (2010).

    Article  CAS  PubMed  Google Scholar 

  56. Carmeliet, P. et al. Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis. Cell 98, 147–157 (1999).

    Article  CAS  PubMed  Google Scholar 

  57. Gory-Fauré, S. et al. Role of vascular endothelial-cadherin in vascular morphogenesis. Development 126, 2093–2102 (1999).

    PubMed  Google Scholar 

  58. Sigurbjörnsdóttir, S., Mathew, R. & Leptin, M. Molecular mechanisms of de novo lumen formation. Nat. Rev. Mol. Cell Biol. 15, 665–676 (2014).

    Article  CAS  PubMed  Google Scholar 

  59. Song, Y., Eng, M. & Ghabrial, A.S. Focal defects in single-celled tubes mutant for cerebral cavernous malformation 3, GCKIII, or NSF2. Dev. Cell 25, 507–519 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Li, S. et al. Retro-orbital injection of FITC-dextran is an effective and economical method for observing mouse retinal vessels. Mol. Vis. 17, 3566–3573 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Unc13B-deficient mice were a gift from N. Brose (Max Planck Institute of Experimental Medicine, Germany). We thank J. Pober, R. Liu and T. Manes for reagents and discussion. This work was partly supported by US National Institutes of Health (NIH) grants R01 HL109420 (W.M.), HL115148 (W.M.), GM109487 (D.W.), National Natural Science Foundation of China (no. 91539110) (W.M.), CT Stem Cell Innovation (Established Investigator Grant) award no. 14-SCB-YALE-17 (W.M.) and 2016YFC1300600 (W.M.), Scientific Grants of Guangdong (no. 2015B020225002 and 2015A050502018) (W.M.), and American Heart Association grants 13SDG17110045 (H.Z.) and 14SDG20490020 (W.T.).

Author information

Authors and Affiliations

Authors

Contributions

H.J.Z., L.Q., H.Z. and W.M. conceived the study, designed experiments and wrote the manuscript; H.J.Z., L.Q., H.Z., W.T., W.J., Y.H., Z.W., Q.Y. and M.S.K. performed experiments; W.J., G.F. and M.Y. generated the anti-angiopoietin-2 antibodies; X.L. interpreted retinal data; A.V. provided the human CCM blocks; and H.J.Z., L.Q., H.Z., D.T., D.W. and W.M. interpreted data. M.S.K. edited the manuscript.

Corresponding authors

Correspondence to Dianqing Wu or Wang Min.

Ethics declarations

Competing interests

G.F. and M.Y. are employees of Genentech. W.J. is an employee of Guangzhou Darron Medscience.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 4939 kb)

Supplementary Source Data (XLSX 43 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhou, H., Qin, L., Zhang, H. et al. Endothelial exocytosis of angiopoietin-2 resulting from CCM3 deficiency contributes to cerebral cavernous malformation. Nat Med 22, 1033–1042 (2016). https://doi.org/10.1038/nm.4169

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.4169

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research