Arteriovenous malformation Map2k1 mutation affects vasculogenesis

Abstract

Somatic activating MAP2K1 mutations in endothelial cells (ECs) cause extracranial arteriovenous malformation (AVM). We previously reported the generation of a mouse line allowing inducible expression of constitutively active MAP2K1 (p.K57N) from the Rosa locus (R26^{GT-Map2k1-GFP/+}) and showed, using Tg-Cdh5CreER, that EC expression of mutant MAP2K1 is sufficient for the development of vascular malformations in the brain, ear, and intestines. To gain further insight into the mechanism by which mutant MAP2K1 drives AVM development, we induced MAP2K1 (p.K57N) expression in ECs of postnatal-day-1 pups (P1) and investigated the changes in gene expression in P9 brain ECs by RNA-seq. We found that over-expression of MAP2K1 altered the transcript abundance of > 1600 genes. Several genes had > 20-fold changes between MAP2K1 expressing and wild-type ECs; the highest were Col15a1 (39-fold) and Itgb3 (24-fold). Increased expression of COL15A1 in R26^{GT-Map2k1-GFP/+}; Tg-Cdh5CreER^+/- brain ECs was validated by immunostaining. Ontology showed that differentially expressed genes were involved in processes important for vasculogenesis (e.g., cell migration, adhesion, extracellular matrix organization, tube formation, angiogenesis). Understanding how these genes and pathways contribute to AVM formation will help identify targets for therapeutic intervention.

Figure 1

Expression of MAP2K1 (p.K57N) causes brain vascular malformations in a R26^{GT-Map2k1-GFP/+}; Tg:Cdh5-CreER^+/− mouse line. (A) Pups at P9 after subcutaneous tamoxifen injection at P1. Notice the growth retardation in the R26^{GT-Map2k1-GFP/+}; Tg:Cdh5-CreER^+/− pup. (B,C) Brains from the pups shown in (A). R26^{GT-Map2k1-GFP/+}; Tg:Cdh5-CreER^+/− brains contain multifocal vascular malformations. (c) cerebellum, (ic) inferior colliculus, (sc) superior colliculus, (cc) cerebral cortex, and (ob) olfactory bulb. (C,D) Hematoxylin and eosin stained sections through the cerebral cortex demonstrate enlarged vessels (asterisks) and hemorrhage (arrows and insert) in the R26^{GT-Map2k1-GFP/+}; Tg:Cdh5-CreER^+/− brain. Transverse sinus (ts).

Figure 2

Principal component analysis shows R26^{GT-Map2k1-GFP/+}; Tg:Cdh5-CreER^+/− ECs have a distinct molecular signature.

Figure 3

(A) Volcano plot for the 1,677 significantly differentially expressed genes. Adjusted p-value (y-axis) is plotted against Log(2) fold change (x-axis). The most significantly affected gene is Col15a1. Note that Map2k1 transcripts are increased (arrow). (B) Comparison of Map2k1 (p.K57N) expression levels to wild type Map2k1 in brain ECs with a recombined R26^{GT-Map2k1-GFP/+} allele. Expression from the activated R26^{GT-Map2k1-GFP} allele is on average 69 × greater than expression from an endogenous Map2k1 allele.

Figure 4

Ontology using the top 200 differentially expressed genes ranked by adjusted p-value. The most impacted pathways were cell migration, cell adhesion, and matrix organization associated with tube morphogenesis, angiogenesis, and blood vessel development.

Figure 5

Endothelial cell Map2k1 mutation causes increased COL15A1 protein production. Top panels (A–C) Immunostaining of P9 R26^{GT-Map2k1-GFP/+} brain sections. Bottom panels (D–F) Immunostaining of P9 R26^{GT-Map2k1-GFP/+}; Tg:Cdh5-CreER^+/− brain sections. (A,D) CD-31; (B,E) COL15A1; (C,F) CD-31 + COL15A1 + DAPI. Note abnormal vasculature and COL15A1 expression in the R26^{GT-Map2k1-GFP/+}; Tg:Cdh5-CreER^+/− brain.