Introduction
Arteriovenous malformation (AVM) is a sporadic congenital vascular anomaly that is present at birth and progresses over time. Lesions contain a nidus of abnormal blood vessels connecting arteries directly to veins without a normal capillary bed. Patients suffer deformity, ulceration, bleeding, pain, and occasionally congestive heart failure. Treatment includes embolization or resection, but recurrence is common and patients rarely are cured. Our group showed that extracranial AVMs contain an activating somatic mutation in MAP2K1 isolated to endothelial cells.1 The goals of this study were to: (1) determine if the MAP2K1 mutation was sufficient to cause arteriovenous shunts in vivo, (2) establish a MAP2K1-mutant animal model of AVM to further investigate the etiopathogenesis of the lesion, and (3) develop an in vivo assay to test novel pharmacotherapies for AVM.
Methods
Creation of MAP2K1 zebrafish
Experiments were performed in accordance with Boston Children’s Hospital Institutional Animal Care and Use Committee. Zebrafish were maintained under standard conditions.2 A description of the husbandry and environmental conditions is available at https://www.protocols.io/ (dx.doi.org/10.17504/protocols.io.mrjc54n). Control (pTol2-fli1ep:egfp) and mutant (pTol2-fli1ep:egfp-kdrl:MAP2K1K57N) plasmids were synthesized. Plasmid JDW 770 (pTol2-kdrl:EGFP-hsKRAS4B-G12V-ac/Y) was obtained from Addgene.3 The KRAS mutation was replaced with the MAP2K1 K57N mutation under control of the endothelial-specific kdrl promoter.4 A fli1ep:egfp reporter5 was substituted using in-fusion cloning. Because the MAP2K1 mutation in human AVM is isolated to the endothelial cell, the kdrl promoter was chosen to express MAP2K1K57N in zebrafish endothelial cells. Plasmids were co-injected with Tol2 transposase mRNA at the single-cell stage.
All experiments were performed in transgenic Tg(gata1a:DsRed)sd2 fish (Leonard Zon ZDB-ALT-051223-6) in the casper background.6Tg(gata1a:DsRed)sd2 fish were selected because red blood cells fluoresce red allowing for visualization of blood flow. Embryos were dechorinated using forceps for imaging before hatching and housed at 28°C in E3 medium. Single-cell stage embryos were injected with 1 nL of transgenesis mixture (100 ng/μL of either control (pTol2-fli1ep:egfp) or mutant (pTol2-fli1ep:egfp-kdrl:MAP2K1K57N plasmid DNA) + 20 ng/μL Tol2 transposase mRNA. To establish the phenotype of injected fish 161 embryos were examined (group 1).
Treatment with MEK inhibition
To determine the effects of MEK inhibition on shunt formation, a second cohort of 126 fish was used (group 2). Trametinib, a MAP2K1 inhibitor7 (Cat. No. HY-10999; MedChemExpress, Monmouth Junction, NJ) was dissolved in DMSO and heated to 42°C. The solution then was cooled to room temperature and added to E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, 5% methylene blue). Three groups were studied: (1) DMSO control (1 µM), (2) 0.2 μM trametinib, and (3) 0.4 μM trametinib. The solutions were added to E3 media 28 hours post-fertilization. Trametinib-treated E3 media was replaced at 24 hours.
Phenotype analysis
Embryos were anesthetized with 0.4 mg/mL tricaine, embedded in 1% low-melt agarose (Biobasic, Markham ON, Canada), and examined using a Zeiss LSM 800 confocal microscope with Zen Blue (version 2.5). Blood flow was assessed using a Nikon SMZ18 fluorescence microscope. Non-living fish, those with failed transgenesis, or embryos with gross morphological changes excluding the vasculature were not analyzed. Fish with evidence of transgenesis were assessed for changes in blood flow 72 hours post-fertilization by visualizing fluorescence in red blood cells and/or blood flow using light microscopy. Tg(fli1ep:egfp)-injected control fish also were examined if evidence of transgenesis was present. The investigator was blinded to treatment group during phenotype quantification. Statistical significance was determined using 2-sample t test for phenotype quantification and 1-way analysis of variance with Tukey multiple comparisons for drug treatment.
Results
Control zebrafish exhibited normal circulatory patterns with clearly separated arteries and veins in the trunk and head with no evidence of arteriovenous shunts (n = 65; Figures 1A, 2A, B). In contrast, embryos expressing endothelial MAP2K1K57N exhibited 2 major shunt phenotypes (group 1). The most common phenotype was a proximal shunt with a terminal connection between a branch of the lateral dorsal aorta and the adjacent common cardinal vein with minimal or no distal blood flow (39/96 embryos; 40%; Figures 1B, 2C, 3). The second major phenotype was a direct abnormal connection between the major artery and vein within the trunk or tail (19/96 embryos, 20%). These shunts had either: (1) a direct fistula with intact distal blood flow (Figures 1C, 2D, E) or (2) a terminal connection with minimal or no distal blood flow (Figures 1D, 2F). Several endothelial cells lining the shunt expressed high levels of the marker transgene confirming shunts contained MAP2K1K57N-expressing endothelial cells (Figure 2E). Endothelial cells expressing the transgene also were identified in areas of phenotypically normal vasculature. MEK inhibition led to a statistically significant dose-dependent reduction of shunt formation in embryos expressing endothelial MAP2K1K57N from 84% to 55% (0.2 μM) and 25% (0.4 μM) (P = 0.006; Figure 4).
;)
Figure 1.: Schematic of observed phenotypes. A, Normal zebrafish vasculature, (B) proximal shunt phenotype observed in embryos expressing endothelial MAP2K1K57N. C, Distal fistula phenotype observed in MAP2K1K57N-expressing embryos. D, Terminal connection with minimal or no distal flow observed in some MAP2K1K57N embryos. Boxes indicate area of embryo represented by confocal images in Figure 2.
Figure 2.: Expression of activated MAP2K1 in zebrafish endothelial cells causes arteriovenous shunts. A and B, Confocal images of normal rostral (A) and tail (B) vasculature in zebrafish injected with control GFP plasmid. C, Confocal image of MAP2K1K57N embryo showing an abnormal proximal shunt between the VA and CCV. D, Confocal image of MAP2K1K57N embryo with fistula between the CA and CV at 10×. E, Confocal image at 20× demonstrates the fistula contains mutant endothelial cells expressing high levels of the marker transgene. F, Confocal image of MAP2K1K57N embryo with terminal connection occurring between the DA and PCV. Arteriovenous shunts are identified by white circles. Arrowheads indicate transgene-expressing endothelial cells. CA, CCV, CV, DA, H, PCV, VA. CA indicates caudal artery; CCV, common cardinal vein; CV, caudal vein; DA, dorsal artery; H, heart; PCV, posterior cardinal vein; VA, ventral aorta.
Figure 3.: Group 1 MAP2K1 mutant zebrafish phenotype quantification (control n = 65, MAP2K1 mutant n = 96). Statistical significance was determined by 2-sample t test. *P < .05.
Figure 4.: Group 2 trametinib treatment of MAP2K1 mutant zebrafish reduced the frequency of shunt formation (dimethyl sulfoxide n = 44, trametinib 0.2 µM n = 51, trametinib 0.4 µM n = 31). Statistical significance was determined by 1-way analysis of variance with Tukey multiple comparisons. *P < .05; NS indicates not significant.
Discussion
Expression of mutant MAP2K1 (K57N) in zebrafish endothelial cells resulted in abnormal arteriovenous shunts, supporting the causality of this variant in human AVMs. Our model shared some similarities and differences with other investigators who have created zebrafish with RAS/MAPK signaling pathway variants. Al-Olabi et al8 demonstrated postzygotic mosaic expression of mutant BRAF (V600E) and MAP2K1 (Q58del) caused disordered formation of the caudal vein plexus and impeded blood flow. A modest improvement in blood flow was seen after treatment with a RAF inhibitor. Bell et al9 created a MAP2K1 (K57N) fish as a comparison group while evaluating mutations in TEK. These fish were made using the Gal4-UAS system and formed abnormal arteriovenous connections at 30–48 hours postfertilization in the distal tail. Fish et al3 most closely mirrored our phenotype injecting zebrafish with kdrl:EGFP-KRAS (G12V); 48% had dorsal artery and cardinal vein shunts reduced with MEK inhibition using SL327.
Although MEK inhibition caused a dose-dependent reduction in total and proximal shunt formation, it did not significantly lower the frequency of distal shunts. This may have occurred because distal shunt formation was less common and thus underpowered to observe an effect. Clinically, MEK inhibition may slow AVM growth by preventing progressive shunt formation. MEK inhibition also may reduce recurrent shunt formation following excision or embolization of AVMs. The ability of MEK inhibition to treat established shunts remains unclear.
Shunts in our fish contained a combination of mutant and wild-type endothelial cells. Mutant endothelial cells were present in areas of phenotypically normal vessel development, indicating mutant endothelial cells are necessary but not sufficient to form shunts. Shunts may develop from a cell nonautonomous mechanism which occurs with human AVM growth.10 This could be tested using time-lapse confocal imaging of mutant fish tracking differences in which mutant endothelial cells form shunts. This technique also could be used to examine if shunt formation originates from an arterial or venous endothelial cell.
Improved treatment options for patients with AVM are desperately needed and MEK inhibition appears to be a promising strategy in humans.11,12 Clinical trials with MEK inhibitors are ongoing in Europe and the United States. Our finding that MEK inhibition reduced shunt formation in zebrafish embryos with endothelial-expressed MAP2K1K57N supports the use of MEK inhibitors for patients with AVM. This zebrafish model allows for further testing of novel pharmacotherapeutic options.
References
1. Couto JA, Huang AY, Konczyk DJ, et al. Somatic MAP2K1 mutations are associated with extracranial arteriovenous malformation. Am J Hum Genet. 2017;100:546–554.
2. Westerfield M. The
Zebrafish Book. A Guide for the Laboratory Use of
Zebrafish (Danio Rerio). 4th ed. Eugene: University of Oregon Press; 2000.
3. Fish JE, Wythe JD, Perfecto C, et al. Somatic gain of KRAS function in the endothelium is sufficient to cause vascular malformations that require MEK but not PI3K signaling. Circ Res. 2020;127:727–743.
4. Covassin LD, Siekmann AF, Kacergis MC, et al. A genetic screen for vascular mutants in
zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development. Dev Biol. 2009;329:212–226.
5. Villefranc JA, Amigo J, Lawson ND. Gateway compatible vectors for analysis of gene function in the
zebrafish. Dev Dyn. 2007;236:3077–3087.
6. White RM, Sessa A, Burke C, et al. Transparent adult
Zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell. 2008;2:183–189.
7. Smits PJ, Konczyk DJ, Sudduth CL, Goss JA, Greene AK. Endothelial MAP2K1 mutations in arteriovenous malformation activate the RAS/MAPK pathway. Biochem Biophys Res Commun. 2020;529:450–454.
8. Al-Olabi L, Polubothu S, Dowsett K, et al. Mosaic RAS/MAPK variants cause sporadic vascular malformations which respond to targeted therapy. J Clin Invest. 2018;128:1496–1508.
9. Bell LM, Holm A, Matysiak U, et al. Functional assessment of two variants of unknown significance in TEK by endothelium-specific expression in
Zebrafish embryos. Hum Mol Genet. 2021;31:10–17.
10. Konczyk DJ, Goss JA, Smits PJ, Sudduth CL, Al-Ibraheemi A, Greene AK. Arteriovenous malformation MAP2K1 mutation causes local cartilage overgrowth by a cell-non autonomous mechanism. Sci Rep. 2020;10:4428.
11. Lekwuttikarn R, Lim YH, Admani S, Choate KA, Teng JMC. Genotype-guided medical treatment of an arteriovenous malformation in a child. JAMA Dermatol. 2019;155:256–257.
12. Edwards EA, Phelps AS, Cooke D, et al. Monitoring arteriovenous malformation response to genotype-targeted therapy. Pediatrics. 2020;146:e20193206.
