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Bone Marrow-Derived Alk1 Mutant Endothelial Cells and Clonally Expanded Somatic Alk1 Mutant Endothelial Cells Contribute to the Development of Brain Arteriovenous Malformations in Mice

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Abstract

We have previously demonstrated that deletion of activin receptor-like kinase 1 (Alk1) or endoglin in a fraction of endothelial cells (ECs) induces brain arteriovenous malformations (bAVMs) in adult mice upon angiogenic stimulation. Here, we addressed three related questions: (1) could Alk1− mutant bone marrow (BM)-derived ECs (BMDECs) cause bAVMs? (2) is Alk1− ECs clonally expended during bAVM development? and (3) is the number of mutant ECs correlates to bAVM severity? For the first question, we transplanted BM from PdgfbiCreER;Alk12f/2f mice (EC-specific tamoxifen-inducible Cre with Alk1-floxed alleles) into wild-type mice, and then induced bAVMs by intra-brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor and intra-peritoneal injection of tamoxifen. For the second question, clonal expansion was analyzed using PdgfbiCreER;Alk12f/2f;confetti+/− mice. For the third question, we titrated tamoxifen to limit Alk1 deletion and compared the severity of bAVM in mice treated with low and high tamoxifen doses. We found that wild-type mice with PdgfbiCreER;Alk12f/2f BM developed bAVMs upon VEGF stimulation and Alk1 gene deletion in BMDECs. We also observed clusters of ECs expressing the same confetti color within bAVMs and significant proliferation of Alk1− ECs at early stage of bAVM development, suggesting that Alk1− ECs clonally expanded by local proliferation. Tamoxifen dose titration revealed a direct correlation between the number of Alk1− ECs and the burden of dysplastic vessels in bAVMs. These results provide novel insights for the understanding of the mechanism by which a small fraction of Alk1 or endoglin mutant ECs contribute to development of bAVMs.

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The authors declare that all data supporting the findings of this study are available in the paper and its Supplementary Information.

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Acknowledgements

We thank Dr. Ludmila Pawlikowska for assistance with manuscript preparation.

Funding

This study was supported by the National Institutes of Health (R01 HL122774, NS027713 and NS112819) and from the Michael Ryan Zodda Foundation to H.S.; AHA fellowship (20POST35120371) to N.S.; and the US Department of Defense (PR161205), the Leducq Foundation (ATTRACT), and Barrow Neurological Foundation to S.P.O.

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Authors and Affiliations

  1. Center for Cerebrovascular Research, University of California, San Francisco, CA, USA

    Sonali S. Shaligram, Rui Zhang, Wan Zhu, Li Ma, Man Luo, Rich Liang, Leandro do Prado, Chaoliang Tang, Felix Pan, Peipei Pan & Hua Su

  2. Department of Anesthesia and Perioperative Care, University of California, San Francisco, CA, USA

    Sonali S. Shaligram, Rui Zhang, Wan Zhu, Li Ma, Man Luo, Rich Liang, Leandro do Prado, Chaoliang Tang, Felix Pan, Peipei Pan & Hua Su

  3. Department of Neurosurgery, University of California, San Francisco, CA, USA

    Qiang Li & Miriam Weiss

  4. Department of Pediatrics, University of California, San Francisco, CA, USA

    Thomas Arnold & Nicolas Santander

  5. Barrow Aneurysm & AVM Research Center, Department of Neurobiology, Barrow Neurological Institute, Phoenix, AZ, USA

    S. Paul Oh

Authors
  1. Sonali S. Shaligram
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  2. Rui Zhang
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  3. Wan Zhu
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  4. Li Ma
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  5. Man Luo
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  6. Qiang Li
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  7. Miriam Weiss
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  8. Thomas Arnold
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  9. Nicolas Santander
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  10. Rich Liang
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  11. Leandro do Prado
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  13. Felix Pan
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  14. S. Paul Oh
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  15. Peipei Pan
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  16. Hua Su
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Contributions

HS, SS, and TA designed the experiment. SS, ML, QL, RZ, LM, MW, SNG, RL, LP, CT, and FP collected and analyzed the data. SS and HS drafted the manuscript. TA, PP, SPO, and HS critically read the manuscript.

Corresponding author

Correspondence to Hua Su.

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Ethics Approval

The protocol and experimental procedures for using laboratory animals were approved by the Institution of Animal Care and Use Committee (IACUC) at the University of California, San Francisco.

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The authors declare no competing interests.

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Shaligram, S.S., Zhang, R., Zhu, W. et al. Bone Marrow-Derived Alk1 Mutant Endothelial Cells and Clonally Expanded Somatic Alk1 Mutant Endothelial Cells Contribute to the Development of Brain Arteriovenous Malformations in Mice. Transl. Stroke Res. 13, 494–504 (2022). https://doi.org/10.1007/s12975-021-00955-9

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  • DOI: https://doi.org/10.1007/s12975-021-00955-9

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