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Arteriovenous malformation MAP2K1-mutant endothelial cells exhibit dysregulated vasculogenesis in-vitro and in-vivo
Matthew P. Vivero*1, Cheng Yu Sheng1, Patrick Smits1, Joyce Bischoff2, Matthew Warman3, Arin Greene1
1Plastic Surgery, Boston Children's Hospital, MA, MA; 2Surgery, Boston Children's Hospital, Boston, MA; 3Orthopedic Surgery, Boston Children's Hospital, Boston, MA


Objective: Arteriovenous malformation (AVM) is a fast-flow vascular anomaly characterized by abnormal arterial to venous circulatory shunts causing ischemia, ulceration, and bleeding. Extracranial AVMs contain a somatic mutation in the MAP2K1 gene. The purposes of this study was to: (1) create a stable MAP2K1 mutant endothelial cell (EC) line, (2) determine how the MAP2K1 mutation affects EC behavior, and (3) create in-vitro and in-vivo models to test pharmacotherapy.

Design: Utilizing CRISPR-Cas9 genome editing, the MAP2K1 p.K57N mutation was introduced into immortalized arterial endothelial cells. Expression of mutant transcript and elevated phosphorylated ERK (P-ERK) was tested using digital droplet PCR (ddPCR) and Western Blot. Bulk-RNAseq and differential gene expression analysis was conducted comparing mutant and pseudo-wildtype (pWT) populations. Gene-edited lines were injected into immunodeficient mice and implant phenotype was characterized. In-vitro and in-vivo models were treated with a MEK inhibitor and assessed.

Setting: All experiments were conducted in the Plastic Surgery Research Laboratory and the ARCH Animal Care Facility at Boston Children"ôs Hospital.

Participants: TeloHAECs, an immortalized arterial EC cell commercially available, were used for the generation of our AVM cellular module. Immunodeficient mice, Nu/Nu genotype publicly available from Jackson Laboratory, were utilized for our murine model.

Interventions: Trametinib, an FDA-approved MEK inhibitor, was utilized to treat both cells and mice.

Main Outcome Measures: Transcriptomic and phenotypic differences secondary to both AVM-causing MAP2K1 mutation and Trametinib administration

Results: Six mutant (both heterozygous and hemizygous) and three pWT cell lines were engineered; genotype and GNAQ mutant or pWT transcript expression was confirmed. Protein analysis confirmed equal levels of MEK1/2 expression but increased P-ERK in mutant cells. Bulk-RNAseq identified differentially expressed angiogenic, cellular adhesion, and inflammatory genes between mutant and pWT cell lines. Nude mouse implants of mutant ECs displayed malformed, dilated, and leaky vasculature. Significant transcriptomic and histologic changes were prevented with MEK inhibition.

Conclusions: The p.K57N MAP2K1 mutation in ECs activates RAS/MEK signaling, stimulates vasculogenic gene expression, and forms abnormal, vascularized lesions in mice. These in-vitro and in-vivo models provide insight into the pathophysiology of human AVM and enable preclinical testing of novel drug therapies.


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