Developmental timing of CCM2 loss influences cerebral cavernous malformations in mice

Abstract

Cerebral cavernous malformations (CCM) are vascular malformations of the central nervous system (CNS) that lead to cerebral hemorrhages. Familial CCM occurs as an autosomal dominant condition caused by loss-of-function mutations in one of the three CCM genes. Constitutive or tissue-specific ablation of any of the Ccm genes in mice previously established the crucial role of Ccm gene expression in endothelial cells for proper angiogenesis. However, embryonic lethality precluded the development of relevant CCM mouse models. Here, we show that endothelial-specific Ccm2 deletion at postnatal day 1 (P1) in mice results in vascular lesions mimicking human CCM lesions. Consistent with CCM1/3 involvement in the same human disease, deletion of Ccm1/3 at P1 in mice results in similar CCM lesions. The lesions are located in the cerebellum and the retina, two organs undergoing intense postnatal angiogenesis. Despite a pan-endothelial Ccm2 deletion, CCM lesions are restricted to the venous bed. Notably, the consequences of Ccm2 loss depend on the developmental timing of Ccm2 ablation. This work provides a highly penetrant and relevant CCM mouse model.

Figure 1.

Endothelial Ccm2 deletion at P1 results in CCM malformations mimicking the human CCM lesions in the cerebellum and in the retina. All animals were injected at P1 with 10 µl tamoxifen (equivalent to 20 µg) and dissected at the indicated time. Genotypes of inducible CCM2 KO (iCCM2) and control animals were respectively Cdh5(PAC)-CreERT2; Ccm2^Del/fl and Cdh5(PAC)-CreERT2; Ccm2^+/fl. (A) Kaplan-Meier survival curve from the control group (blue line, n = 110) and the iCCM2 group (dotted red line, n = 56). Circles represent censored animals, which were sacrificed for analysis. (B) Control and iCCM2 mouse brains upon dissection (top) and after H&E staining (bottom; n = 6 in each group from 4 different litters, analyzed between P11 and P19). CCM malformations, located in the cerebellum of iCCM2 animals, are composed by single or multiple caverns (asterisks) with extensive hemorrhage (black arrows) around the juxtaposed vascular cavities. Note the dilation of meningeal vessels in the iCCM2 (yellow arrow). (C) Control and iCCM2 mouse retinas at P13 upon dissection (left and middle) and after isolectin-B4 staining (right, n = 7 in each group from 4 different litters, analyzed between P11 and P15). (D) Mouse lesions phenocopy human CCM lesions. (left) Histology of the cerebral lesions in mouse and human. (right) Mouse retina upon dissection and human retinal angiography Bars: 2 mm (B, top); 500 µm (C and D, mouse retina); 100 µm (B [bottom] and D [mouse cerebellum]).

Figure 2.

Ccm2 deletion alters AJ and TJ organization in CCM lesions. Analysis of cell–cell junctions in CCM2 malformations on frozen sections of iCCM2 brain. For all immunofluorescence experiments, cell nuclei are visualized with DAPI (blue). Data are representative of 3 independent observations (n = 5 in each group, from 2 different litters). (A) H&E staining (left) and confocal microscopy analysis showing vessels stained using anti-PECAM1 (red, right). (B) Co-staining of the vessels using PECAM1 staining (red) and the TJ components (green) using claudin-5 (top) and ZO.1 (bottom). Claudin-5 and ZO.1 are normally expressed in peri-lesion vessels (arrowheads), whereas they are strongly down-regulated in abnormally dilated and hemorrhagic vessels of the lesion (dotted area). (C) VE-cadherin staining (red) of the endothelium lining lesion and peri-lesion vessels. (right) Magnification of the boxed area. Pink arrows indicate VE-cadherin expressed outside of the junctions. Bars: 200 µm (A); 100 µm (B); 60 µm (C, top); 4 µm (C, bottom and right).

Figure 3.

Ccm2 deletion has no significant effect on Wnt–β-catenin signaling pathway in CCM2 KO ECs in vitro and in iCCM2 lesions. (A) TCF/LEF-β-catenin transcriptional activity in CCM2 WT and null ECs in vitro was determined by transfecting CCM2 WT and KO ECs with the TOP-TK-Luc or the FOP-TK-Luc reporter constructs (containing WT or mutant Tcf/Lef binding sites, respectively, and a basal TK promoter, upstream a luciferase gene). Columns are means ± SD of triplicates from a representative experiment out of three performed. (B-I) Animals were bred with the BAT-Gal reporter mouse to assess β-catenin activation (see Materials and methods for breeding details). All animals were injected with tamoxifen at P1. XGal staining, performed on control and iCCM2 cerebellum, is shown (n = 8 in each group, from 3 different litters). White arrows show the CCM lesions in iCCM2 animals (in C, E, and G). In F–I, H&E staining was performed on cerebellum sections, after XGal staining. The box in G is magnified in I. H shows a CCM lesion composed of multiple juxtaposed caverns. Gcl, granular cell layer; ml, molecular layer; pcl, Purkinje cell layer; wm, white matter. Bars: 1 mm (B and C); 500 µm (D and E); 100 µm (F and G); 50 µm (H and I).

Figure 4.

CCM lesions are capillary-venous and do not affect the arterial compartment. (A) Analysis at P9 or P12 of the retinal vasculature from control or iCCM2 animals using vascular isolectin-B4 staining (left and middle, n = 25 in each group analyzed between P8 and P10), after XGal staining (right, n = 4 in each group, from 2 different litters). Tamoxifen-induced Ccm2 deletion was performed at P1. Arteries (A) are thin and normal in iCCM2 retinas, whereas veins (V) are dilated in iCCM2 animals. Asterisks show CCM lesions developing at the periphery of the retina. (B) Lateral view of cerebral hemispheres of control and iCCM2 embryos dissected at E19.5 (n = 8 in each group, from 4 different litters). Ccm2 deletion was performed at E14.5. Vascular anomalies affect the caudal rhinal vein (crhv) and the capillaries surrounding in the iCCM2 animals. The box in the middle panel is magnified in the image on the right. (C) Analysis of the cerebellar vessels at P10 and P12 after XGal staining on whole brain (left) and after a H&E staining (right). Animals were crossed with either the Rosa26-Stop^fl-LacZ reporter mouse or the artery-specific EphrinB2^tlacZ reporter mouse. Results shown are representative of at least four animals in each group, from two different litters. Bars: 1 mm (A and B, left and middle); 500 µm (B [right] C [left]); 100 µm (A, right); 50 µm (C, right).

Figure 5.

Natural history of the CCM lesions. (A) Retinal CCM lesion development from P7 to P16. The vasculature at the periphery of the retina on controls or iCCM2 animals is shown after isolectin-B4 staining. C, central retina; p, peripheral retina. Data are representative of at least 50 animals in each group, analyzed between P6 and P19. (B) Quantification of the vascular coverage at the venous leading edge of the plexus in the retina at P7. Data are expressed as vascular area ± SEM (isolectin-B4–positive area, relative to total retinal area analyzed; n = 4 in each group, from 2 different litter; 6–8 fields analyzed per retina). (C) Quantification of the EC proliferation in control or iCCM2 retinas at P6, assessed by total number of phospho-histone 3–positive ECs per retina ± SD (n = 6 in each group, from 3 different litters). Bars, 100 µm.

Figure 6.

Timing of ablation determines endothelial response to CCM2 loss. Control and iCCM2 animals were injected with tamoxifen to delete Ccm2 at P1 (left, n = 25 in each group, analyzed between P8 and P10), at 3 wk of age (middle, n = 4 in each group, from 3 different litters) or at E14.5 during gestation (right, n = 8 from 4 different litters). (A) Control and iCCM2 brains upon dissection. (B) Isolectin-B4 staining on control and iCCM2 retinas. Note the CCM lesion in the P1-induced animal (asterisks). V, vein. Bars: 2 mm (A, left and middle); 500 µm (A [right] and B [left]); 100 µm (B, right).