96. Activating MAP2K1 Mutation in Zebrafish Endothelial Cells Causes Arteriovenous Shunts

Abstract

Purpose: Arteriovenous malformation (AVM) is a sporadic vascular malformation defined by a nidus of irregular blood vessels connecting arteries to veins instead of a normal capillary bed. Somatic activating mutations in MAP2K1 cause extracranial AVM. The purpose of this study was to create an AVM animal model using zebrafish. Methods: Single-cell stage casper;Tg(gata1:dsred) zebrafish embryos were injected with 1 nL of transgenesis mixture: 100 ng/μL of either (1) Control (pTol2-Fli:GFP) or (2) Mutant (pTol2-Fli:GFP-kdrl:MAP2K1K57N) plasmid DNA + 40 ng/μL Tol2 transposase mRNA. Erythrocytes in these fish express the fluorescent protein dsRed. Embryos were anesthetized with 0.4 mg/mL Tricaine, embedded in 1% low-melt agarose and then imaged using a Zeiss LSM 800 Confocal Microscope or a Nikon SMZ18 Fluorescence Microscope. Confocal images were obtained using Zen Blue version 2.5. Embryos were imaged 72 hours post-fertilization. Blood flow was visualized using dsRed fluorescence. Results: Injection of MAP2K1K57N plasmid resulted in abnormal arteriovenous shunts (58/96 transgenic embryos, 60%). The phenotype consisted of either (1) a proximal shunt with blood flowing through a direct connection between proximal aortic vessels to the common cardinal vein and immediately back into the heart (39/58 embryos, 67%) or (2) a mid-trunk shunt between the dorsal artery and posterior cardinal vein (19/58 embryos, 33%). Shunts were not present in control zebrafish (n=65). Endothelial cells at the site of the shunt expressed high levels of the marker transgene confirming shunts contained mutant endothelial cells. Conclusion: Zebrafish endothelial cells expressing mutant MAP2K1 form abnormal arteriovenous shunts. The phenotype recapitulates extracranial human AVM. Mutant MAP2K1 zebrafish are a promising animal model for testing pharmacotherapy to treat AVM.