Blood vascular abnormalities in Rasa1(R780Q) knockin mice: implications for the pathogenesis of capillary malformation-arteriovenous malformation

Abstract

Capillary malformation-arteriovenous malformation (CM-AVM) is an autosomal dominant blood vascular (BV) disorder characterized by CM and fast flow BV lesions. Inactivating mutations of the RASA1 gene are the cause of CM-AVM in most cases. RASA1 is a GTPase-activating protein that acts as a negative regulator of the Ras small GTP-binding protein. In addition, RASA1 performs Ras-independent functions in intracellular signal transduction. Whether CM-AVM results from loss of an ability of RASA1 to regulate Ras or loss of a Ras-independent function of RASA1 is unknown. To address this, we generated Rasa1 knockin mice with an R780Q point mutation that abrogates RASA1 catalytic activity specifically. Homozygous Rasa1(R780Q/R780Q) mice showed the same severe BV abnormalities as Rasa1-null mice and died midgestation. This finding indicates that BV abnormalities in CM-AVM develop as a result of loss of an ability of RASA1 to control Ras activation and not loss of a Ras-independent function of this molecule. More important, findings indicate that inhibition of Ras signaling is likely to represent an effective means of therapy for this disease.

Figure 1

Generation of Rasa1^R780Q knockin allele in mice. A: Exon-intron organization of the murine Rasa1 gene (top) and RASA1 protein (bottom). The positions of the R780Q and R780Q mutations in exon 18 and the GAP domain, respectively, are indicated. B: An R780Q targeting vector with an R780Q mutation in exon 18 and a FRT-flanked NeoR cassette in intron 18 is shown below the endogenous allele. At middle is shown the R780Q targeted allele before and after excision of the NeoR cassette achieved with an actin promoter-driven Flp transgene (Act-Flp). At bottom is depicted a Rasa1 floxed allele in which exon 18 is flanked by loxP sites. Positions of real-time PCR primer/probe pairs used in the detection of ES cell homologous recombinants and PCR primer pairs used in mouse genotyping are indicated. C2, protein kinase C2 homology; GAP, GTPase-activating protein domain; PH, pleckstrin homology; SH2, Src homology 2; SH3, Src homology 3.

Figure 2

Embryonic lethality of homozygous Rasa1^R780Q mice. A:Rasa1^R780Q/fl mice were intercrossed. Tail genomic DNA from progeny at 3 weeks of age was PCR amplified using genotyping primers indicated in Figure 1. Genotyping results from two Rasa1^R780Q/fl (fl/rq) pups and one Rasa1^fl/fl (fl/fl) pup are depicted. B: Graphs show the total number of pups of the indicated genotypes derived from intercrosses of Rasa1^R780Q/fl mice determined at 3 weeks after birth and at E9.5, E11, and E13.5 of development. At E9.5, genotype frequencies are consistent with mendelian inheritance (χ² test). At E11, E13.5, and 3 weeks of age, genotype frequencies are not consistent with mendelian inheritance (P < 0.025, P < 0.05, and P < 0.005, respectively). C and D: Light microscopic appearance (C) and hematoxylin and eosin (H&E)–stained sections (D) of Rasa1^R780Q/R780Q and Rasa1^R780Q/fl embryos and yolk sacs at E9.5. Features to note include reduced size and distended pericardial sac (arrow) and wrinkled appearance of the yolk sac of Rasa1^R780Q/R780Q embryos. E: MEFs derived from E9.5 Rasa1^R780Q/R780Q and Rasa1^fl/fl embryos were analyzed for RASA1 protein abundance by using Western blot analysis. Blots were reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to show equivalent protein loading. Shown are RASA1 amounts in MEF derived from two different embryos of each genotype. E, embryo proper; YS, yolk sac.

Figure 3

Cardiovascular abnormalities in homozygous Rasa1^R780Q mice. A and B: Whole mount (A) and sections (B) of E9.5 Rasa1^R780Q/R780Q and Rasa1^fl/fl embryos were stained with an anti-CD31 antibody to reveal blood vasculature. A: Note the organized BV network in the Rasa1^fl/fl yolk sac (YS) and honeycombed appearance of blood vasculature in the Rasa1^R780Q/R780Q yolk sac (asterisk). Note also irregular blood vasculature in the Rasa1^R780Q/R780Q embryo proper (E). B: Note the disorganized dorsal aorta in the Rasa1^R780Q/R780Q embryo compared with the Rasa1^fl/fl embryo (arrows). DA, dorsal aorta; IS, intersegmental artery.

Figure 4

MAPK activation in homozygous Rasa1^R780Q embryos. Tissue sections of E9.5 Rasa1^R780Q/R780Q and Rasa1^fl/fl embryos were stained with anti-CD31 (red) and anti–phospho-ERK (green) antibodies. Shown are select regions of embryos that include part of the dorsal aorta (arrows). Note the absence of pERK staining in most ECs in Rasa1^fl/fl embryos and the presence of pERK staining in most ECs in Rasa1^R780Q/R780Q embryos.