Alk1 mutant endothelial cells undergo clonal expansion in mouse brain arteriovenous malformations

Shaligram, S.; Zhang, R.; Zhu, W.; Ma, L.; Winkler, E.; Luo, M.; Li, Q.; Arnold, T.; Santander, N.; M. McDougall, C. M.; Wong, J. Y.; Liang, R.; Barbosa Do Prado, L.; Tang, C.; Su, H.

doi:10.1101/2020.05.20.106799

bioRxiv · 10.1101/2020.05.20.106799

Alk1 mutant endothelial cells undergo clonal expansion in mouse brain arteriovenous malformations

Shaligram, S.·Zhang, R.·Zhu, W.·Ma, L.·Winkler, E.·Luo, M.·Li, Q.·Arnold, T.·Santander, N.·M. McDougall, C. M.·Wong, J. Y.·Liang, R.·Barbosa Do Prado, L.·Tang, C.·Su, H.

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Abstract

RationaleMutation in human arteriovenous malformation (AVM) causative genes in a fraction of endothelial cells (ECs) causes AVMs in mice. It is unclear how a small number of mutant ECs can lead to AVM formation. ObjectiveTo understand how a fraction of mutant ECs causes AVM, we tested the following hypotheses: (1) activin receptor-like kinase 1 (Alk1 or Acvlr1) mutant brain ECs undergo clonal expansion upon angiogenic stimulation, (2) Alk1 mutant ECs display growth advantage, (3) the burden of Alk1 mutant ECs correlates with AVM severity, and (4) Alk1 mutant bone marrow (BM) derived ECs alone is sufficient to cause AVM. Methods and ResultsWe used PdgfbiCreER;Alk1f/f;confetti+/- mice which express an EC-specific tamoxifen (TM)-inducible Cre recombinase, a Cre-regulated confetti transgene, and Alk1 floxed alleles. Brain AVMs were induced by direct brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) followed with intra-peritoneal injection of TM two weeks later. Color-predominance of confetti reporter in AVMs compared to control brain ECs suggested that clonal expansion was associated with AVM development. We treated PdgfbiCreER;Alk1f/f with different doses of TM to create a mosaic of wild-type (WT) and mutant ECs and found that equal numbers of Alk1+ and Alk1- ECs were proliferating. Increase of TM dose increased the number of Alk1- ECs, the abnormal vessels in brain AVMs, the number of arteriovenous shunts in the intestines, and mouse mortality. To test if mutation of Alk1 in BM-derived ECs can cause brain AVM, we transplanted WT mice with BM of PdgfbiCreER;Alk1f/f mice. After AAV-VEGF and TM treatment, these mice developed AVMs in their brains and arteriovenous shunts in their intestines. ConclusionClonal expansion of Alk1 mutant ECs could partly explain why a fraction of mutant ECs causes AVM. Mutation of AVM causal genes in BM-derived ECs is sufficient to cause AVM formation.

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Shaligram, S., Zhang, R., Zhu, W., Ma, L., Winkler, E., Luo, M., Li, Q., Arnold, T., Santander, N., M. McDougall, C. M., Wong, J. Y., Liang, R., Barbosa Do Prado, L., Tang, C., Su, H.. 2020-05-21. Alk1 mutant endothelial cells undergo clonal expansion in mouse brain arteriovenous malformations. https://doi.org/10.1101/2020.05.20.106799

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