A novel mouse model of cerebral cavernous malformations based on the two-hit mutation hypothesis recapitulates the human disease

Abstract

Cerebral cavernous malformations (CCMs) are vascular lesions of the central nervous system appearing as multicavernous, blood-filled capillaries, leading to headache, seizure and hemorrhagic stroke. CCM occurs either sporadically or as an autosomal dominant disorder caused by germline mutation of one of the three genes: CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10. Surgically resected human CCM lesions have provided molecular and immunohistochemical evidence for a two-hit (germline plus somatic) mutation mechanism. In contrast to the equivalent human genotype, mice heterozygous for a Ccm1- or Ccm2-null allele do not develop CCM lesions. Based on the two-hit hypothesis, we attempted to improve the penetrance of the model by crossing Ccm1 and Ccm2 heterozygotes into a mismatch repair-deficient Msh2(-/-) background. Ccm1(+/-)Msh2(-/-) mice exhibit CCM lesions with high penetrance as shown by magnetic resonance imaging and histology. Significantly, the CCM lesions range in size from early-stage, isolated caverns to large, multicavernous lesions. A subset of endothelial cells within the CCM lesions revealed somatic loss of CCM protein staining, supporting the two-hit mutation mechanism. The late-stage CCM lesions displayed many of the characteristics of human CCM lesions, including hemosiderin deposits, immune cell infiltration, increased endothelial cell proliferation and increased Rho-kinase activity. Some of these characteristics were also seen, but to a lesser extent, in early-stage lesions. Tight junctions were maintained between CCM lesion endothelial cells, but gaps were evident between endothelial cells and basement membrane was defective. In contrast, the Ccm2(+/-)Msh2(-/-) mice lacked cerebrovascular lesions. The CCM1 mouse model provides an in vivo tool to investigate CCM pathogenesis and new therapies.

Figure 1.

Breeding scheme to create Ccm1^+/−Msh2^−/− mice. A two-generation cross was used to produce the Msh2 knockout allele (box). The first cross generated the Msh2 knockout allele using CRE-lox technology and the second cross aimed at breeding out the CRE transgene by back-crossing mice to C57BL/6J. From that point, a three-generation cross produces Ccm1^+/−Msh2^−/− mice. First, Msh2 heterozygotes without the CRE transgene were crossed with mice heterozygous for Ccm1. Double heterozygotes from this cross were mated with each other in the fourth generation to produce Ccm1^+/−Msh2^−/− mice as well as littermate controls. A similar breeding scheme was used to generate Ccm2^+/−Msh2^−/− mice.

Figure 2.

Characterization of lesions in Ccm1^+/–Msh2^–/– mice. Both stage 1 (isolated dilated vessels) and stage 2 (clusters of dilated vessels) are found in the brains of the mice. Images are shown of coronal sections of brains from gradient recalled echo magnetic resonance (left) and H&E staining. The boxes in the middle panels denote the area represented under higher magnification in the right panels. In the middle row, black arrows denote representative stage 1 lesions and white arrows denote a stage 2 lesion. Scale bars are 1 mm (left), 0.5 mm (center) and 0.1 mm (right).

Figure 3.

Phenotypic maturation in stage 1 versus stage 2 lesions. Stage 2 lesions (right panels) in brains from Ccm1^+/−Msh2^−/− mice harbor B cells (brown B220), proliferating endothelium (brown Ki67) and iron (blue Perl stain). These are not visible in stage 1 lesions (left panels). Images are shown at ×3 higher magnification in the respective insets. Circles indicate blue iron particles in stage 2 multicavernous lesions (bottom right panel). Scale bar is 100 µm.

Figure 4.

ROCK activation in mouse CCM lesions. ROCK activation was assessed by pMLC immunohistochemistry (dark brown staining, left panels). Normal capillaries (arrowheads) from an Msh2^−/− control mouse (top row) do not show evidence of ROCK activation. By contrast, in Ccm1^+/−Msh2^−/− mice, endothelial cells (ECs) lining stage 1 lesions (arrows) stain weakly for pMLC (middle row) and ECs lining three caverns (asterisks) of a stage 2 lesion show stronger pMLC staining. Corresponding serial sections show no staining with an isotype control (right panels). All tissue sections were counterstained blue with hematoxylin. Scale bar is 100 μm.

Figure 5.

ROCK activation increases as CCM lesions grow. (A) The relative intensities of pMLC staining were compared between normal capillaries, stage 1 CCM lesions and stage 2 lesions. The intensity of pMLC staining increases with the size and complexity of the lesions in Ccm1^+/−Msh2^−/− mouse brains with CCMs (regression coefficient = 0.7655, P < 0.0001; common odds ratio = 2.150). (B) The pMLC staining intensities of normal capillaries were compared between the genotypes and MRI phenotypes of the mice. Normal capillaries in brains from Ccm1^+/−Msh2^−/− mice with CCM lesions show stronger pMLC staining than those from mice with the same genotype without CCM lesions, which in turn show stronger pMLC staining than those from Msh2^−/− control mice (P < 0.0001, exact Mantel–Haenszel test). Post hoc Bonferroni-adjusted P-values were <0.001 between two groups in all three comparisons.

Figure 6.

Reduced KRIT1 expression in the endothelium of mouse CCM lesions. Capillaries in Msh2^−/− control brains (first row) showed prominent brown staining for both KRIT1 (left) and CCM2 (right). Consistent with the genotype of the mice, KRIT1 staining was reduced, while CCM2 staining remained at control levels in normal capillaries (second row) of the Ccm1^+/−Msh2^−/− mouse brain. Endothelial cells lining two stage 1 CCM lesions (third row) and two caverns of a stage 2 CCM lesion (fourth row) showed normal staining for CCM2 (right), but either reduced (white arrow) or no staining (black arrow) for KRIT1 (left). Scale bar is 50 µm.

Figure 7.

Electron microscopy reveals abnormal ultrastructure of CCM lesion endothelium. (A) Three CCM lesions (arrows) in a Ccm1^+/−Msh2^−/− mouse brain shown by Toluidine Blue staining (×40, scale bar 25 µm). (B) Intact tight junctions (arrows) and basal lamina (arrowheads) are present within a normal capillary in a control C57BL/6 mouse (×4600, scale bar 1 µm). Intact tight junctions (arrows) and basal lamina (arrowheads) are also present within a normal capillary (C, ×35000, scale bar 200 nm) and a CCM lesion (D, ×35000, scale bar 200 nm) in a Ccm1^+/−Msh2^−/− mouse brain. (E) In the same lesion, filopodia (arrows) are present (×4060, scale bar 2 µm). (F) A tight junction and the basal lamina (arrowhead) are both broken in this lesion and erythrocyte extravasation is visible (×8260, scale bar 1 µm). RBC, red blood cell; NU, endothelial nucleus; BL, basal lamina.