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Regulation of cardiovascular development and integrity by the heart of glass–cerebral cavernous malformation protein pathway

Nature Medicine volume 15, pages 169–176 (2009)Cite this article

A Corrigendum to this article was published on 01 May 2009

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Abstract

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: KRIT1, CCM2 and PDCD10. Here we show that the heart of glass (HEG1) receptor, which in zebrafish has been linked to ccm gene function, is selectively expressed in endothelial cells. Heg1−/− mice showed defective integrity of the heart, blood vessels and lymphatic vessels. Heg1−/−; Ccm2lacZ/+ and Ccm2lacZ/lacZ mice had more severe cardiovascular defects and died early in development owing to a failure of nascent endothelial cells to associate into patent vessels. This endothelial cell phenotype was shared by zebrafish embryos deficient in heg, krit1 or ccm2 and reproduced in CCM2-deficient human endothelial cells in vitro. Defects in the hearts of zebrafish lacking heg or ccm2, in the aortas of early mouse embryos lacking CCM2 and in the lymphatic vessels of neonatal mice lacking HEG1 were associated with abnormal endothelial cell junctions like those observed in human CCMs. Biochemical and cellular imaging analyses identified a cell-autonomous pathway in which the HEG1 receptor couples to KRIT1 at these cell junctions. This study identifies HEG1-CCM protein signaling as a crucial regulator of heart and vessel formation and integrity.

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Figure 1: Heart and blood vessel integrity defects in HEG1-deficient mouse embryos and neonates.
Figure 2: Lymphatic vessel dilatation and leakage in Heg1−/− neonatal mice.
Figure 3: Heg1−/−; Ccm2lacZ/+ mouse embryos do not establish a patent blood vascular network.
Figure 4: Endothelial cells of zebrafish lacking heg or ccm2 form vessels that are normally patterned but not patent.
Figure 5: HEG1 is required to form normal endothelial junctions in vivo.
Figure 6: HEG1 receptor intracellular tails associate with CCM2 through KRIT1.

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Change history

  • 27 January 2009

    In the version of the supplementary information for this article originally posted online, the descriptions of Supplementary Videos 1–6 were incorrect. The errors have been corrected as of 27 January 2009.

  • 12 February 2009

    In this version of the article initially published, Shawn M. Sweeney was not included in the list of authors. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank C. Bertozzi, C. Chen, A. Granger, J. Lee, D. Li, P. Mericko, A. Schmaier, E. Sebzda, N. Shanbhag and K. Whitehead for valuable insights; M. Pack and J. He for assistance with zebrafish studies; R. Meade for preparation of the electron microscopy samples; and T. Branson for animal husbandry. This work was supported by the Swiss National Science Foundation, a Network of Excellence grant from the European Community (M.A.) and the US National Institutes of Health grants T32 HL07439 (to B.K.), T32 HL07971 (to X.Z.), HL078784 and AR27214 (to M.G.), HL62454 (to J.K.) and HL075380 and HL095326 (to M.L.K.).

Author information

Authors and Affiliations

  1. Department of Medicine and Cardiovascular Institute, University of Pennsylvania, 421 Curie Blvd., Philadelphia, 19104, Pennsylvania, USA

    Benjamin Kleaveland, Xiangjian Zheng, Zhiying Zou, Shawn M Sweeney, Mei Chen, Lili Guo, Min-min Lu, Diane Zhou & Mark L Kahn

  2. Department of Medicine, University of California, San Diego, 9500 Gilman Drive, MC 0726, La Jolla, 92093-0726, California, USA

    Jian J Liu & Mark H Ginsberg

  3. Biozentrum, University of Basel, Klingelbergstrasse 50/70, Basel, CH-4056, Switzerland

    Yannick Blum & Markus Affolter

  4. Department of Pathology and Department of Obstetrics and Gynecology, Institute of Cancer Genetics and Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, 10032, New York, USA

    Jennifer J Tung & Jan Kitajewski

Authors
  1. Benjamin Kleaveland
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  2. Xiangjian Zheng
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Contributions

B.K. designed and performed all of the studies involving mutant mouse lines, contributed to endothelial cell culture and biochemical studies and wrote the manuscript; X.Z. designed and performed most of the zebrafish and biochemical studies; J.J.L., M.C., Z.Z., S.M.S. and L.G. contributed to the biochemical studies; Y.B. and M.C. contributed to the zebrafish studies; J.J.T. performed the endothelial cell culture studies; M.L. performed the immunohistochemistry studies; D.Z. contributed to the generation of mutant mouse lines; J.K. designed the endothelial cell culture studies and wrote the manuscript; M.A. designed some of the zebrafish studies and wrote the manuscript; M.H.G. designed some of the biochemical studies and wrote the manuscript; M.L.K. contributed to the design of mouse, zebrafish, endothelial and biochemical studies and wrote the manuscript.

Corresponding author

Correspondence to Mark L Kahn.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–12, Supplementary Tables 1 and 2, and Supplementary Methods (PDF 1210 kb)

Supplementary Movie 1

Intersegmental vessels of 24–26-h.p.f. fli1a:EGFP-cdc42 transgenic zebrafish embryos following injection of scrambled morpholino (control). The GFP-cdc42 fusion protein is expressed in endothelial cells and outlines the vacuoles that form and fuse during lumen formation. (AVI 4038 kb)

Supplementary Movie 2

Intersegmental vessels of 24–26-h.p.f. fli1a:EGFP-cdc42 transgenic zebrafish embryos following injection of morpholinos to block expression of heg. The GFP-cdc42 fusion protein is expressed in endothelial cells and outlines the vacuoles that form and fuse during lumen formation. Endothelial vacuole formation in zebrafish embryos lacking heg is similar to that observed in control embryos (Supplementary Movie 1). (AVI 1894 kb)

Supplementary Movie 3

Intersegmental vessels of 24–26-h.p.f. fli1a:EGFP-cdc42 transgenic zebrafish embryos following injection of morpholinos to block expression of ccm2. The GFP-cdc42 fusion protein is expressed in endothelial cells and outlines the vacuoles that form and fuse during lumen formation. Endothelial vacuole formation in zebrafish embryos lacking ccm2 is similar to that observed in control embryos (Supplementary Movie 1). (AVI 5678 kb)

Supplementary Movie 4

Red fluorescent quantum dots were injected into the dorsal aorta of 24–26-h.p.f. fli1a:EGFP-cdc42 transgenic zebrafish embryos following injection of scrambled morpholino (control). Patent intersegmental vessels are indicated by the presence of red fluorescent dots in the vessels. (AVI 2022 kb)

Supplementary Movie 5

Red fluorescent quantum dots were injected into the dorsal aorta of 24–26-h.p.f. fli1a:EGFP-cdc42 transgenic zebrafish embryos following injection of morpholinos to block expression of heg. The presence of red fluorescent dots in the intersegmental vessels indicates vessel patency. The presence of red fluorescent dots in the intersegmental vessels indicates that the intersegmental vessels of zebrafish embryos lacking heg are patent. (AVI 3405 kb)

Supplementary Movie 6

Red fluorescent quantum dots were injected into the dorsal aorta of 24–26-h.p.f. fli1a:EGFP-cdc42 transgenic zebrafish embryos following injection of morpholinos to block expression of ccm2. The presence of red fluorescent dots in the intersegmental vessels indicates vessel patency. The presence of red fluorescent dots in the intersegmental vessels indicates that the intersegmental vessels of zebrafish embryos lacking ccm2 are patent. (AVI 7446 kb)

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Kleaveland, B., Zheng, X., Liu, J. et al. Regulation of cardiovascular development and integrity by the heart of glass–cerebral cavernous malformation protein pathway. Nat Med 15, 169–176 (2009). https://doi.org/10.1038/nm.1918

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