Mutations in 2 distinct genetic pathways result in cerebral cavernous malformations in mice

Cerebral cavernous malformations (CCMs) are a common type of vascular malformation in the brain that are a major cause of hemorrhagic stroke. This condition has been independently linked to 3 separate genes: Krev1 interaction trapped (KRIT1), Cerebral cavernous malformation 2 (CCM2), and Programmed cell death 10 (PDCD10). Despite the commonality in disease pathology caused by mutations in these 3 genes, we found that the loss of Pdcd10 results in significantly different developmental, cell biological, and signaling phenotypes from those seen in the absence of Ccm2 and Krit1. PDCD10 bound to germinal center kinase III (GCKIII) family members, a subset of serine-threonine kinases, and facilitated lumen formation by endothelial cells both in vivo and in vitro. These findings suggest that CCM may be a common tissue manifestation of distinct mechanistic pathways. Nevertheless, loss of heterozygosity (LOH) for either Pdcd10 or Ccm2 resulted in CCMs in mice. The murine phenotype induced by loss of either protein reproduced all of the key clinical features observed in human patients with CCM, as determined by direct comparison with genotype-specific human surgical specimens. These results suggest that CCM may be more effectively treated by directing therapies based on the underlying genetic mutation rather than treating the condition as a single clinical entity.

Cerebral cavernous malformations (CCMs) are common vascular malformations with a prevalence of 1 in 200 to 250 individuals in unselected populations (1, 2). CCMs can lead to focal neurological deficits, seizures, and hemorrhagic stroke, but no pharmacologic therapy currently exists (3). CCMs predominantly occur in the central nervous system and are characterized by subclinical bleeding and consequential hemosiderin deposits that are detected by MRI (4). MRI is the primary clinical modality for detection, diagnosis, and management of CCMs. Hemosiderin deposits give CCMs an MRI appearance of a central mass with a dark perilesional halo, whose appearance is nearly diagnostic (pathognomonic) of cavernous malformation (5). Cavernous malformations are characterized by a complex of vascular channels of varying sizes lined by a single layer of endothelial cells without any abnormally large arteries, arterialized veins, or large venous outflow vessels. Although dense fibrillary neuroglial tissue may penetrate the mass, vascular channels are generally arranged in a back-to-back pattern with little or no intervening brain parenchyma. There is often a peripheral margin of gliotic tissue containing hemosiderin-laden macrophages (6).

CCMs can occur sporadically or be inherited in an autosomal dominant pattern. Familial CCM has been linked to heterozygosity for any of 3 genes: Krev1 interaction trapped (KRIT1), Cerebral cavernous malformation 2 (CCM2), and Programmed cell death 10 (PDCD10) (7). All 3 proteins bind each other in coimmunoprecipitation experiments on cells overexpressing these proteins, leading to the hypothesis that they function as a complex to affect a common signaling mechanism (8). Both Krit1 and Ccm2 are required for proper connection of the developing heart with the aorta to establish circulation in the mouse embryo (9, 10). Mice lacking either Krit1 or Ccm2 fail to form a lumenized first branchial arch artery to link the heart and aorta; as a result, mice lacking either gene die at the same age. The requirement for Ccm2 is endothelial autonomous (9, 11). In the endothelium, both KRIT1 and CCM2 suppress the activity of the small GTPase RhoA (9, 12). Loss of either gene leads to RhoA activation and signaling through Rho kinase (ROCK) resulting in increased actin stress fibers, impaired cell-cell interactions, and increased vascular permeability (9, 12, 13). These defects can be reversed in cell culture and in mice with inhibitors of RhoA including HMG-CoA reductase inhibitors (statins) (9, 12).

A recent report suggested a similar mechanism of Rho activation for PDCD10 (14). However, other studies have suggested a different cell-signaling role for PDCD10. While KRIT1, CCM2, and PDCD10 all occupy the cytoplasmic compartment of the cell, unique subcellular localization for each has been described, including nuclear localization of KRIT1 (15) and Golgi localization of PDCD10 (16). Each protein has also been found to have unique binding partners (17–19). PDCD10’s binding partners include members of the germinal center kinase III (GCKIII) subfamily of serine-threonine kinases with homology to yeast sterile-20 (STE20) kinase (16, 20–23). Furthermore, the clinical features of CCM in KRIT1, CCM2, and PDCD10 families have important differences (24, 25), with PDCD10 resulting in the most severe disease (24, 26). It is possible that CCM disease is the common result of multiple unique mechanisms and may require unique therapeutic strategies to target the underlying disturbed cellular and signaling pathways.

Though the signaling and cellular mechanisms associated with the 3 known CCM proteins may be different, a common genetic mechanism of loss of heterozygosity (LOH) has been suggested for familial CCM disease (27–29). Familial CCM is more aggressive than sporadic disease, with an earlier age of onset, increased risk of hemorrhage and seizure, and an increased number of lesions (3, 30–32). These observations have led to the hypothesis that CCM disease occurs by the Knudson 2-hit mechanism, similar to retinoblastoma (33). There is limited evidence from human pathologic specimens to support an association of LOH with CCM lesions. In a series of challenging experiments, a number of investigators have identified biallelic mutations, 1 somatic and 1 germline, in the endothelium of a subset of patient samples (27, 28, 34). While LOH could not be confirmed for a number of samples, a total of 4 cases have been described for KRIT1 and 1 case each for CCM2 and PDCD10. Mice heterozygous for Krit1 or Ccm2 do not develop cavernous malformations, and no examples of secondary somatic mutations have been reported (35, 36).

To determine whether endothelial LOH is not simply associated with, but is causative for, CCM pathology, requires an animal model with a controlled genetic mutation that can be directed and detected in a tissue-specific manner. In this work, we use drug-inducible, tissue-specific strains of Cre recombinase to target conditional null alleles of the CCM genes to test directly the 2-hit hypothesis for cavernous malformations. We demonstrate that Pdcd10 differs substantially from Ccm2 in development, cell biology, and signaling, yet LOH is the common genetic mechanism to cause CCMs in both genotypes. These findings suggest that PDCD10 influences different endothelial signaling pathways from KRIT1/CCM2 to lead to a common histopathology and imply that medical treatment to stabilize familial CCM may need to be developed and evaluated in a genotype-specific manner.

Loss of Pdcd10 results in embryonic lethal phenotypes distinct from loss of Krit1 or Ccm2. To determine the role of Pdcd10 in development and disease, we developed a conditional null allele, Pdcd10^flox, in which exons 4–8 are flanked with LoxP sites (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI44393DS1). This strategy also allowed us to generate a constitutive null allele, Pdcd10^–, by crossing mice carrying Pdcd10^flox with a mouse strain expressing Cre recombinase in the germline (37). To characterize the role of Pdcd10 in development, we intercrossed Pdcd10^+/– mice and harvested embryos at varying stages of development. Surprisingly, we found that mice lacking Pdcd10 died at a much earlier age than those deficient for either Krit1 or Ccm2. Whereas Krit1 and Ccm2 mice show growth arrest at E9.0 and die at E11 (9–11), the loss of Pdcd10 leads to growth arrest at E8.0, after gastrulation, but prior to the onset of circulation or the requirement for cardiovascular function (Table 1 and Supplemental Figure 1).

Table 1

Early growth arrest and death in embryos lacking Pdcd10

An earlier requirement for Pdcd10 than Krit1 or Ccm2 in development does not preclude a shared role in the vascular system. Mice with endothelial loss of Ccm2 phenocopy the constitutive null mutant (9, 11), so we sought to determine whether mice with endothelial loss of Pdcd10 would also recapitulate the Ccm2 phenotype. Using the endothelial Tie2-Cre driver, the same Cre driver that we used to ablate Ccm2 (9), we found that although mice with endothelial loss of Pdcd10 (Pdcd10^flox/–;Tie2-Cre) did not survive to birth, they had patent branchial arch arteries, a developed circulatory system, and were indistinguishable from control littermates at E9.5 (Supplemental Figure 2). Instead, we found that loss of Pdcd10 in the endothelium leads to progressive enlargement of the cardinal vein and other veins of the rostral embryo at E11.5 (Figure 1). Venous enlargement was not due to abnormal cardiac structure or function (Figure 1 and Supplemental Figure 2), nor were defects observed in arteries of the embryo (Figure 1 and Supplemental Figure 2). Death occurred due to hemorrhage from venous rupture by E13.5 (Table 2). Additionally, mice with loss of Pdcd10 in neural and glial tissues induced by Nestin-Cre showed no vascular or any other obvious defects and were born alive (Supplemental Figure 2 and Supplemental Table 1). These observations suggest that Pdcd10 is required in the endothelium for control of venous size and integrity, yet Pdcd10 differs from Ccm2 in that it is not required for the establishment of circulation.

Figure 1

Pdcd10 is required in the endothelium for venous integrity. (A–H) H&E staining of developmental time course of Pdcd10 endothelial knockout. Pdcd10^flox/+;Tie2-Cre is shown in A–D. Pdcd10^flox/–;Tie2-Cre is shown in E–H. Close-up images of the cardinal vein at E12.5 are shown in D and H. Asterisks denote the cardinal veins. Circles denote the external jugular veins. (I and J) Echocardiography of hearts from Pdcd10^flox/– (I) and Pdcd10^flox/–;Tie2-Cre (J) mice at E11.5. Top panels show M-mode images of hearts contracting over time. Bottom panels show waveforms corresponding to blood flow across the atrioventricular valves. s, systole; d, diastole; e, early filling; a, atrial contraction; R, valvular regurgitation. Scale bars: 1 mm (A–C and E–G); 500 μm (D and H). n ≥ 6 embryos at each age.

Table 2

Loss of Pdcd10 in the endothelium leads to embryonic death after E12.5

Loss of PDCD10 does not affect RhoA signaling but results in lumen formation defects. To explore the role of PDCD10 in endothelial cells, we depleted PDCD10 in human dermal microvascular endothelial cells (HMVEC) with siRNA (Supplemental Figure 3). Whereas the loss of CCM2 leads to an increase in actin stress fibers as a result of RhoA activation (9, 12, 13) and phosphorylation of myosin light chain-2 by Rho kinase (Figure 2), we observed none of these indicators of RhoA activation with the loss of PDCD10 (Figure 2). Thus, the role of PDCD10 in endothelial cell biology and signaling differs from that of CCM2.

Figure 2

PDCD10 differs from CCM2 in downstream signaling. (A) Phalloidin staining of human microvascular endothelial cells treated with siRNA directed against CCM2, PDCD10, or nonsense control. (B) Quantification of stress fiber response of HMVEC cells. Stress fiber content is determined by adding the total length of stress fibers divided by the total number of cells. Results indicate mean ± SEM and are representative of at least 3 independent experiments. *P < 0.001 versus control or PDCD10; **P = NS versus control. (C) Immunoblot for phospho-myosin light chain-2 (with α-actinin immunoblot as a loading control). Results are representative of 3 independent experiments. Scale bars: 100 μm.

Having found that PDCD10’s function in development differs from that of CCM2, we sought to identify defects associated with PDCD10 depletion in assays relevant to vascular development. PDCD10-depleted HUVECs showed defective function in a 3D angiogenesis assay. Endothelial cells plated in a collagen matrix spontaneously organize into complex multicellular capillary-like networks with lumens (38), but cells depleted of PDCD10 failed to organize themselves into a lumenized network (Supplemental Figure 4, A and B). We explored potential downstream signaling pathways using this assay. A growing body of evidence suggests that PDCD10 interacts with the GCKIII subfamily of serine-threonine kinases (16, 21–23); we used siRNA to deplete cells of each of the subfamily members (Supplemental Figure 3). We observed no effect on lumen formation with the knockdown of STK25, STK24, or MST4 in HUVECs (Supplemental Figure 4C).

Pdcd10 functionally associates with GCKIII in lumen formation. Because the GCKIII family members may be functionally redundant, we sought to validate the importance of the PDCD10-GCKIII interaction in vivo and in a simpler genetic model. We chose the fruit fly, Drosophila melanogaster, in which the GCKIII family is represented by a single protein, GCKIII. This model avoids the complexity of human or mouse, which has 3 GCKIII kinases, or zebrafish, which has 2 orthologs for Pdcd10, 2 orthologs for STK25, and single orthologs for STK24 and MST4 (23). Furthermore, the Drosophila genome contains a single ortholog of PDCD10, but no orthologs for KRIT1 or CCM2. Thus, Drosophila represents a simple model organism for studying Pdcd10-specific biology. To ensure that the biochemical interaction between PDCD10 and GCKIII was conserved in Drosophila, we performed immunoprecipitation assays with the Drosophila proteins. Drosophila Pdcd10 binds to GCKIII (Supplemental Figure 4D) in a manner analogous to that of human, mouse, or zebrafish protein (21, 23). A human disease PDCD10 mutation exists, which deletes 18 amino acids crucial for PDCD10-GCKIII binding (21). Removal of the analogous 18 amino acids from the Drosophila Pdcd10 protein abrogated binding to GCKIII (Supplemental Figure 4D).

Whereas Drosophila do not develop a vascular system, they do form a branched, lumenized network of tubes in the tracheal (respiratory) system. This epithelial network requires coordinated cell-cell interactions and specialized cell-cell junctions, analogous to the mammalian vascular system (39, 40). To determine the necessity of Pdcd10 in developing fly tracheal tubes, we expressed a dsRNA directed against Pdcd10 (41) under the control of the tracheal-specific Breathless promoter (42) using the GAL4-UAS system (43). We found that tracheal tubes lacking Pdcd10 grow and branch normally, but fail to lumenize and fill with air (Supplemental Figure 5), indicating that Pdcd10 is required for normal lumen formation in fly tracheal tubes as it is in human endothelial tubes. This effect is highly penetrant; nearly all dsRNA-expressing flies show lack of lumenization in multiple tracheal tubes (Supplemental Figure 4E). The specificity of this effect is confirmed, as it could be rescued by coexpression of Drosophila Pdcd10 in RNAi-expressing cells (Supplemental Figure 4E). Notably, coexpression of the 18–amino acid deletion form of Drosophila Pdcd10, which does not bind to GCKIII, fails to rescue this phenotype (Supplemental Figure 4E). We used the same RNAi strategy to inactivate GCKIII (44) in the developing tracheal system. Tracheal tubes lacking GCKIII exhibit failure of lumenization (Supplemental Figure 5), and the phenotype appears very similar to that of Pdcd10-deficient tubes. Thus, Pdcd10 is essential for normal lumen formation in the absence of Krit1 and Ccm2 and in a manner that requires interaction with GCKIII kinases.

Our experiments in Drosophila suggested that GCKIII kinases are required for lumen formation but that functional redundancy in mammalian cells may have accounted for the lack of a lumen formation defect when any single kinase was lost. We therefore performed combinatorial knockdown experiments for pairs of the GCKIII kinases. Consistent with our hypothesis, we found that the loss of STK25, if coupled with the loss of either STK24 or MST4, was sufficient to reproduce the lumen formation defects seen with PDCD10 depletion (Supplemental Figure 4). Thus, PDCD10 interaction with GCKIII kinases is critical in lumen formation.

LOH of either Pdcd10 or Ccm2 causes murine cavernous malformations that phenocopy human CCMs. Pdcd10 and Ccm2’s functions clearly differ in embryonic development, in endothelial cell culture, and in signaling, and Pdcd10 is necessary for lumen formation in a system that lacks Ccm2 or Krit1. The 3 genes, however, are linked to the same human disease. Limited human genetics suggested an association between LOH and CCM disease, so we investigated whether Pdcd10 and Ccm2 share this genetic mechanism for causing disease. To control the timing of the endothelial “second-hit,” we used a drug-inducible Cre strain under the control of the PDGF-B promoter. This PDGFb-iCreER^T2 strain expresses a Cre recombinase activated only after administration of the drug tamoxifen. Consistent with the original report describing this recombinase (37), we found that the administration of tamoxifen on the first postnatal day (P1) led to efficient, endothelial-specific recombination throughout the entire brain vasculature (Supplemental Figure 6). The induction of endothelial LOH of either Pdcd10 or Ccm2 by this Cre at birth resulted in CCMs in mice as early as 1 month of age (Figure 3). Induction of endothelial LOH of either Pdcd10 or Ccm2 resulted in a spectrum of vascular malformations, from capillary telangiectasias, to isolated caverns, to multiple back-to-back caverns with thrombosis, hemorrhage, and formation of secondary channels (Supplemental Figure 7). Loss of protein product via LOH was confirmed by antibody staining (Supplemental Figure 8). The retinal vasculature is another location for human CCMs, and murine cavernous malformations were also observed in mouse retinal vasculature (Supplemental Figure 9). To formally prove LOH in the endothelium of these CCMs, we performed laser capture microdissection to obtain tissue-specific DNA as previously done in human studies of CCM (28). We found that DNA from lesion endothelium had lost the conditional allele for either Pdcd10 or Ccm2, confirming LOH, whereas this allele could still be detected in adjacent neuronal tissue (Figure 3). In contrast, loss of Ccm2 in neural tissues using Nestin-Cre did not result in the development of vascular malformations (Supplemental Figure 10).

Figure 3

Cavernous malformations result from LOH of either Ccm2 or Pdcd10. (A) Cavernous malformation (arrow) shown in an H&E-stained section of brain cerebrum from a mouse with induced endothelial knockout of Ccm2. (B) Confirmation of LOH of Ccm2 in 2 mice with loss of Ccm2^flox allele by PCR, compared with Pdcd10 wild-type allele as a control. (C) Cavernous malformations (arrows) and a less complex telangiectasia (arrowhead) shown in an H&E-stained section of brain cerebrum from a mouse with induced endothelial knockout of Pdcd10. (D) Confirmation of LOH of Pdcd10 in 2 mice by PCR with loss of Pdcd10^flox allele compared with Ccm2 wild-type allele as a control. Samples in B and D were obtained via laser capture microdissection of sectioned mouse brains. Scale bars: 1 mm.

We characterized the histopathologic features of cavernous malformations in mice with induced endothelial loss of either Pdcd10 (Figure 4) or Ccm2 (Figure 5). We compared mouse CCMs with surgical specimens from CCM patients with germline mutations of either PDCD10 (Figure 4) or CCM2 (Figure 5). Cavernous malformations in these induced mouse models share all of the key histologic features of CCM lesions with human specimens. Furthermore, both Pdcd10 and Ccm2 resulted in identical pathologic findings for all of these defining characteristics in both human and mouse (Table 3). Mouse specimens showed numerous vascular channels of variable diameter (Figure 4, A and B, and Figure 5, A and B), some of them with organized thrombi. Immunohistochemical stains for endothelium highlighted endothelial cells in the channels, with focal attenuation or loss of endothelial cells in larger channels (Figure 4, G and H, and Figure 5, G and H). Iron stain highlighted the presence of hemosiderin-laden macrophages and hemosiderin in the wall of the channels as well as in the periphery and brain tissue (Figure 4, C and D, and Figure 5, C and D). Additionally, we also examined gliosis, trichrome staining, elastin, laminin, and collagen type IV, and these features were all identical between mouse and human lesions (Figure 4, E and N, Figure 5, E and N, and Table 3).

Figure 4

Pathologic analysis of mouse and human PDCD10-associated CCM. Paired analysis of histologic sections with human tissue on the left and mouse on the right. (A and B) H&E staining revealing back-to-back vascular channels (arrows) and hemosiderin pigment (arrowheads) in surrounding tissues. (C and D) Iron (blue) detected by Prussian blue stain highlights hemosiderin deposits in macrophages and surrounding brain tissue. (E and F) Fibrous matrix deposits (blue) identified by Masson’s trichrome staining with fibrous tissue surrounding vascular channels (arrows) and in surrounding gliotic brain. (G and H) Endothelial staining for CD34 (G) or CD31 (H) is positive in the cells lining the channels. (I and J) Elastin staining shows that vascular channels lack elastic laminae (arrows) unlike normal vessels of similar caliber (arrowhead, inset in I). The fibrous matrix surrounding channels includes laminin (K and L) and collagen IV (M and N). Scale bars: 200 μm.

Figure 5

Pathologic analysis of mouse and human CCM2-associated CCM. Paired analysis of histologic sections with human tissue on the left and mouse on the right. (A and B) H&E staining revealing back-to-back vascular channels (arrows) and hemosiderin pigment (arrowhead) in surrounding tissues. (C and D) Iron (blue) detected by Prussian blue stain highlights hemosiderin deposits in macrophages and surrounding brain tissue. (E and F) Fibrous matrix deposits (blue) identified by Masson’s trichrome staining with fibrous tissue surrounding vascular channels (arrows) and in surrounding gliotic brain. (G and H) Endothelial staining for CD34 (G) or CD31 (H) is positive in the cells lining the channels. (I and J) Elastin staining shows that vascular channels lack elastic laminae (arrows). The fibrous matrix surrounding channels includes laminin (K and L) and collagen IV (M and N). Scale bars: 200 μm.

Table 3

Pathologic findings in human and murine CCMs

We also examined mouse lesions at the ultrastructural level using transmission electron microscopy. Murine cavernous malformations showed endothelial cells lining vascular channels with associated basal lamina (Figure 6A). Some of the larger channels showed segmental multilayering (lamellated appearance) of the basal lamina (Figure 6B). The most dilated channels showed focally marked attenuation of the endothelial cells (Figure 6C); however, tight junctions were identified between cells, and definitive gaps were not seen. Connective tissue composed mainly of collagen fibers separated the vascular channels (Figure 6D). Pericytes or astrocytic foot processes were missing, as seen in human lesions. Foci of mononuclear inflammatory cells were also seen (Figure 6E), including hemosiderin-laden macrophages (Figure 6F). Ultrastructural analysis of murine CCM lesions was similar to that described in human CCMs (45), further solidifying the fidelity of the mouse model to the human disease.

Figure 6

Ultrastructural findings in murine cavernous malformations. (A) Dilated vascular channels are lined by endothelial cells (arrowheads) with associated basal lamina (arrows). (B) Occasional channels have segments with a multilayered appearance (arrows indicate lamellae of endothelium with basal laminae). Tight junctions appear intact (arrowhead). (C) Focal areas of endothelial attenuation are observed (arrow) without apparent gaps or disruption of tight junctions (arrowhead indicates a junctional complex). (D) Channels are separated by loose connective tissue composed mostly of collagen (arrows). (E) Foci of mononuclear inflammatory cells are present (arrows). (F) Hemosiderin-laden macrophages (arrow) are among the inflammatory cells observed. Images are representative of 5 lesions from 3 Pdcd10 mice. Scale bars: 4 μm.

Murine cavernous malformations can be detected and followed noninvasively by MRI. MRI is used to document the natural history of human cavernous malformations and will be necessary for prospective therapeutic trials in both mice and humans. We thus employed monthly live MRI studies to follow the onset and progression of disease in mice until at least 6 months of age (Figure 7). As in humans (24), mice lacking Pdcd10 had an earlier onset of disease, with a more severe phenotype than Ccm2. All Pdcd10 mice studied at 1 month of age had lesions, whereas no Ccm2 mice had yet developed lesions by 2 months of age (Figure 7K). Mice with Pdcd10 mutations had a greater disease burden when assessed by total lesion burden (Figure 7L) or by number of complex lesions (increased signal intensity within the lesion; Figure 7M). Cavernous malformations were fully penetrant in mice with Ccm2 LOH at 6 months of age and most had lesions at 4 months, at which time the disparity between the genotypes began to narrow. This parity reflected not only the increased burden of disease in Ccm2, but also the onset of death in the most severely affected Pdcd10 mice. Mice of both genotypes began to die of hemorrhage, but mortality was greater in mice with LOH for Pdcd10 (Figure 7N), a finding that reflects the reported experience in humans (24).

Figure 7

Natural history of murine CCM by MRI — Pdcd10 onsets earlier and is more severe than Ccm2. (A–D) Live MRI scans of the same Pdcd10^flox/+;PDGFb-iCreER^T2 mouse at 2 months and 3 months (A and B) and its Pdcd10^flox/–;PDGFb-iCreER^T2 littermate (C and D). Both mice were given tamoxifen at birth. (E–J) Live MRI scans of the same Ccm2^flox/+;PDGFb-iCreER^T2 mouse (E–G) and its Ccm2^flox/–;PDGFb-iCreER^T2 littermate (H–J) at 4, 6, and 7 months. Both mice were given tamoxifen at birth. Arrows indicate CCM lesions. (K) Disease penetrance (proportion affected) by age in Ccm2 and Pdcd10 induced knockout mice as assessed by live MRI. (L) Lesion burden assessed as total number of lesions observed on each tomographic view (slice) of the MRI per mouse. (M) Number of complex lesions (lesions with bright cores) per mouse. (N) Kaplan-Meier survival curve of Ccm2 and Pdcd10 induced knockout mice. For K–N, n = 11 Ccm2, n = 13 Pdcd10. Data in L and M represent mean ± SEM. *P < 0.01; **P < 0.05; ***P < 0.001. Scale bars: 1 mm.

Although CCM has been associated with mutations in 3 distinct loci, it has been clinically treated as a single pathophysiologic entity. In this manuscript, we describe for what we believe is the first time the pathologic features of CCM in a genotype-specific manner. We find that mutations of both PDCD10 and CCM2 result in a common pathologic expression of disease in both humans and mice. Surprisingly, this common disease endpoint does not constitute proof of a common disease mechanism. Whereas our previous work and the work of others found that Ccm2 and Krit1 play similar roles in embryonic development, in vitro cell biology, and cell signaling (9, 10, 12, 13), these roles differ from those we observe with Pdcd10. Unlike Krit1 and Ccm2, Pdcd10 is not required for development of the branchial arch arteries that connect the heart to the aorta. Rather, Pdcd10 has an essential, nonendothelial role in development not shared with Krit1 or Ccm2 as well as an essential function in venous maturation. We further observe differences in cell biology and signaling between PDCD10 and KRIT1 or CCM2; whereas the loss of KRIT1 or CCM2 leads to RhoA activation, increased Rho kinase activity, myosin light chain phosphorylation, and actin stress fiber formation (9, 12, 13), we do not observe activation of this signaling pathway in cells depleted of PDCD10. Instead, we found that PDCD10 signals primarily through the GCKIII family of kinases.

The similarity in human CCM disease caused by mutations in KRIT1, CCM2, and PDCD10 have led to an assumption that the proteins encoded by these genes function in a common signaling pathway. This assumption has been supported by experimental evidence showing binding between ectopically expressed, epi­tope-tagged proteins (7, 8). However, the complexities of signaling pathways and pathophysiology allow for multiple mechanisms to converge on a common disease phenotype (Figure 8A). An example is hypertrophic cardiomyopathy, which was considered 1 disease until molecular genetics revealed that 2 different mechanisms (sarcomere function or metabolism), each affected by distinct genes, both result in pathologic hypertrophy (Figure 8B and ref. 46). We propose that a similar scenario is involved in the pathogenesis of CCM (Figure 8C).

Figure 8

Convergence of different mechanistic pathways in common pathology. (A) Proposed schema for different genes acting on separate mechanistic pathways, yet ultimately resulting in a common expression of disease. (B) Genetic studies of hypertrophic cardiomyopathy highlight genes that can be grouped broadly into 2 separate mechanistic pathways: sarcomeric proteins such as β-myosin heavy chain (MYH7), and metabolic genes including adenosine monophosphate-activated protein kinase (PRKAG2). (C) Studies of mouse development, cell biology, and signaling suggest that KRIT1 and CCM2 signal through RhoA GTPase, while PDCD10 signals through GCKIII kinases to lead to cavernous malformations.

There is controversy concerning the signaling pathways affected by PDCD10. Several reports suggest an essential role for binding GCKIII family serine-threonine kinases (16, 21–23). However, a recent characterization of mice carrying a different conditional allele of Pdcd10 showed that the loss of Pdcd10 in endothelial cells substantially blocks VEGFR2 signaling and inhibits the earliest stages of developmental angiogenesis (47). The implication of VEGFR2 signaling through MAP kinases agrees with a previous report linking GCKIII kinases and ERK signaling (20). Our data contrast with these reports; we observe that the absence of Pdcd10 in vivo leads to a localized vascular defect at a much later developmental stage, inconsistent with a panendothelial block of VEGF signaling. In cell culture, we have not found any interaction between PDCD10 and VEGF or ERK signaling (Supplemental Figure 11). We suspect that this disparity may be due to a difference in knockout strategy; however, our confidence in our allele is bolstered by our ability to induce CCM disease. Our data support the model that PDCD10 signals through the GCKIII family kinases, as we have observed in human endothelial cells and in Drosophila that PDCD10 binds to GCKIII family kinases and both are required for lumen formation. Previous reports have further suggested that GCKIII signals through RhoA to converge with KRIT1/CCM2 signaling (23). By direct comparison of Ccm2 and Pdcd10 in mouse and cell biology, however, our data suggest that PDCD10 signaling is distinct from the CCM2-RhoA axis.

The nature and severity of disease in familial forms of CCM in comparison with sporadic CCM suggested a genetic mechanism consistent with Knudson’s 2-hit hypothesis (33): LOH for a CCM gene induces lesions. Limited evidence to support this theory has come from a few human surgical samples amid multiple cases in which the second genetic hit could not be found (27, 28). Further supportive evidence comes from mice with heterozygous mutations for Krit1 or Ccm2 that have been mated into strains with high rates of spontaneous mutations. Mice heterozygous for Krit1 develop CCM lesions on either a p53- or Msh2-null background, whereas mice heterozygous for Ccm2 develop lesions only on the p53-, but not the Msh2-null background (35, 36). Whereas these models provide suggestive evidence of LOH and employ stochastic events to induce CCM formation, LOH was not demonstrated at either locus, nor can the LOH hypothesis be supported for all CCM genes, as Ccm2^+/–;Msh2^–/– mice do not develop CCMs. These models also do not rule out a role for mutations in other, non-CCM genes and do not control tissue specificity of mutation. The penetrance of CCMs in these models is incomplete, complicating the use of these models in prospective trials to study therapeutics or natural history of CCM disease. Concurrent development of neoplasms in both the p53- and the Msh2-null backgrounds also adds confounding physiological stressors and increases the mortality of the animals. In contrast, we employ a strategy that allows direct testing of the LOH mechanism in CCM disease. Using an inducible Cre-recombinase, we have targeted gene-specific LOH for both Pdcd10 and Ccm2 to the endothelium of mice. In the case of both genes, we have found that LOH is sufficient to cause a fully penetrant CCM phenotype that recapitulates every key pathologic and radiologic hallmark of human disease.

Much work remains to translate the observations and insights regarding disease signaling mechanisms into viable therapeutic strategies in patients. This work underscores the importance of carefully considering disease mechanisms in a genotype-specific manner. The availability of faithful, genotype-specific, and highly penetrant mouse models of CCM disease unlocks the tremendous opportunities to study the natural history of lesion genesis and progression as well as opportunities for preclinical testing of therapeutic interventions. In order for mouse models of CCM disease to be useful in informing human studies, the same tools used to follow patients with CCM need to be developed for serial observation of affected mice. We have demonstrated that noninvasive MRI of live mice, the same modality used to follow CCM in humans, can detect and follow murine CCMs. Although our mouse models share a measurable mortality, as with humans, they are compatible with prolonged survival and serial, noninvasive observation. The ability to follow these mice noninvasively over time is a crucial prerequisite for judging the effectiveness of any preclinical therapeutic strategy in the future and for testing the timing and intensity of LOH required for lesion formation. Our mouse models of CCM phenocopy human disease closely, supplanting previously available surrogate phenotypes, and are a powerful new tool in the armamentarium to decipher and combat CCM disease.

View Supplemental data

We thank N. London, S. Navankasattusas, L. Shi, Y. Xiong, C. Jensen, J. Zhu, D. Zurcher, A. Fang, T. Mleynek, and D. Lim for technical assistance; O. Abdullah and E. Hsu and the University of Utah Small Animal Imaging Facility; C. Rodesch and the University of Utah Cell Imaging/Fluorescence Facility; N. Chandler and the University of Utah Electron Microscopy Facility; S. Tripp and the Immunohistochemistry Research and Development Lab at ARUP Laboratories; J. Hansen at Central Labs at Intermountain Medical Center; K. Thomas and S. Odelberg for critical comments and helpful scientific discussions; and S. Chin for helpful scientific discussions. This work was funded by the US NIH (to G.E. Davis, M.M. Metzstein, K.J. Whitehead, and D.Y. Li), including training grant T32-GM007464 (to A.C. Chan and O.E. Ruiz), the Hellenic Cardiological Society (to N.A. Diakos), the American Heart Association (to K.J. Whitehead and D.Y. Li), the H.A. and Edna Benning Foundation, the Juvenile Diabetes Research Foundation, and the Burroughs Wellcome Fund (to D.Y. Li).

Address correspondence to: Kevin J. Whitehead, Room 4A100, 30 N 1900 East, Salt Lake City, Utah 84132, USA. Phone: 801.581.7715; Fax: 801.581.7735; E-mail: kevin.whitehead@u2m2.utah.edu. Or to: Dean Y. Li, Building 533 Room 4220, 15 N 2030 East, Salt Lake City, Utah 84112, USA. Phone: 801.585.5505; Fax: 801.585.0701; E-mail: dean.li@u2m2.utah.edu.

Conflict of interest: The University of Utah seeks to commercialize this technology and has filed patent applications related to this manuscript.

Reference information: J Clin Invest. 2011;121(5):1871–1881. doi:10.1172/JCI44393.

1. Otten P, Pizzolato GP, Rilliet B, Berney J. A propos de 131 cas d’angiomes caverneux (cavernomes) du S.N.C. repérés par l’analyse rétrospective de 24 535 autopsies. Neurochirurgie. 1989;35(2):82–83.
   View this article via: PubMed Google Scholar
2. Vernooij MW, et al. Incidental findings on brain MRI in the general population. N Engl J Med. 2007;357(18):1821–1828.
   View this article via: PubMed CrossRef Google Scholar
3. Hasegawa T, McInerney J, Kondziolka D, Lee JY, Flickinger JC, Lunsford LD. Long–term results after stereotactic radiosurgery for patients with cavernous malformations. Neurosurgery. 2002;50(6):1190–1197.
   View this article via: PubMed CrossRef Google Scholar
4. Chappell PM, Steinberg GK, Marks MP. Clinically documented hemorrhage in cerebral arteriovenous malformations: MR characteristics. Radiology. 1992;183(3):719–724.
   View this article via: PubMed Google Scholar
5. Burger PC, Scheithauer BW. Tumors of the Central Nervous System . Washington, DC, USA: American Registry of Pathology; 2007.
6. Wang H, Gujrati M. Pathology of cerebral cavernous malformations. In: Lanzino G, Spetzler RF, eds. Cavernous Malformations of the Brain and Spinal Cord . New York, New York, USA: Thieme Medical Publishers, Inc; 2008:22–25.
7. Faurobert E, Albiges-Rizo C. Recent insights into cerebral cavernous malformations: a complex jigsaw puzzle under construction. FEBS J. 2010;277(5):1084–1096.
   View this article via: PubMed CrossRef Google Scholar
8. Hilder TL, et al. Proteomic identification of the cerebral cavernous malformation signaling complex. J Proteome Res. 2007;6(11):4343–4355.
   View this article via: PubMed CrossRef Google Scholar
9. Whitehead KJ, et al. The cerebral cavernous malformation signaling pathway promotes vascular integrity via Rho GTPases. Nat Med. 2009;15(2):177–184.
   View this article via: PubMed CrossRef Google Scholar
10. Whitehead KJ, Plummer NW, Adams JA, Marchuk DA, Li DY. Ccm1 is required for arterial morphogenesis: implications for the etiology of human cavernous malformations. Development. 2004;131(6):1437–1448.
    View this article via: PubMed CrossRef Google Scholar
11. Boulday G, et al. Tissue-specific conditional CCM2 knockout mice establish the essential role of endothelial CCM2 in angiogenesis: implications for human cerebral cavernous malformations. Dis Model Mech. 2009;2(3–4):168–177.
    View this article via: PubMed CrossRef Google Scholar
12. Stockton RA, Shenkar R, Awad IA, Ginsberg MH. Cerebral cavernous malformations proteins inhibit Rho kinase to stabilize vascular integrity. J Exp Med. 2010;207(4):881–896.
    View this article via: PubMed CrossRef Google Scholar
13. Glading A, Han J, Stockton RA, Ginsberg MH. KRIT-1/CCM1 is a Rap1 effector that regulates endothelial cell cell junctions. J Cell Biol. 2007;179(2):247–254.
    View this article via: PubMed CrossRef Google Scholar
14. Borikova AL, et al. Rho kinase inhibition rescues the endothelial cell cerebral cavernous malformation phenotype. J Biol Chem. 2010;285(16):11760–11764.
    View this article via: PubMed CrossRef Google Scholar
15. Zawistowski JS, et al. CCM1 and CCM2 protein interactions in cell signaling: implications for cerebral cavernous malformations pathogenesis. Hum Mol Genet. 2005;14(17):2521–2531.
    View this article via: PubMed CrossRef Google Scholar
16. Fidalgo M, Fraile M, Pires A, Force T, Pombo C, Zalvide J. CCM3/PDCD10 stabilizes GCKIII proteins to promote Golgi assembly and cell orientation. J Cell Sci. 2010;123(pt 8):1274–1284.
    View this article via: PubMed CrossRef Google Scholar
17. Zhang J, Clatterbuck RE, Rigamonti D, Chang DD, Dietz HC. Interaction between krit1 and icap1alpha infers perturbation of integrin beta1-mediated angiogenesis in the pathogenesis of cerebral cavernous malformation. Hum Mol Genet. 2001;10(25):2953–2960.
    View this article via: PubMed CrossRef Google Scholar
18. Zawistowski JS, Serebriiskii IG, Lee MF, Golemis EA, Marchuk DA. KRIT1 association with the integrin-binding protein ICAP-1: a new direction in the elucidation of cerebral cavernous malformations (CCM1) pathogenesis. Hum Mol Genet. 2002;11(4):389–396.
    View this article via: PubMed CrossRef Google Scholar
19. Uhlik MT, et al. Rac-MEKK3-MKK3 scaffolding for p38 MAPK activation during hyperosmotic shock. Nat Cell Biol. 2003;5(12):1104–1110.
    View this article via: PubMed CrossRef Google Scholar
20. Ma X, et al. PDCD10 interacts with Ste20-related kinase MST4 to promote cell growth and transformation via modulation of the ERK pathway. Mol Biol Cell. 2007;18(6):1965–1978.
    View this article via: PubMed CrossRef Google Scholar
21. Voss K, et al. Functional analyses of human and zebrafish 18-amino acid in-frame deletion pave the way for domain mapping of the cerebral cavernous malformation 3 protein. Hum Mutat. 2009;30(6):1003–1011.
    View this article via: PubMed CrossRef Google Scholar
22. Goudreault M, et al. A PP2A phosphatase high-density interaction network identifies a novel striatin-interacting phosphatase and kinase complex linked to the cerebral cavernous malformation 3 (CCM3) protein. Mol Cell Proteomics. 2008;8(1):157–171.
    View this article via: PubMed Google Scholar
23. Zheng X, et al. CCM3 signaling through sterile 20-like kinases plays an essential role during zebrafish cardiovascular development and cerebral cavernous malformations. J Clin Invest. 2010;120(8):2795–2804.
    View this article via: JCI PubMed CrossRef Google Scholar
24. Denier C, et al. Genotype-phenotype correlations in cerebral cavernous malformations patients. Ann Neurol. 2006;60(5):550–556.
    View this article via: PubMed CrossRef Google Scholar
25. Sirvente J, Enjolras O, Wassef M, Tournier-Lasserve E, Labauge P. Frequency and phenotypes of cutaneous vascular malformations in a consecutive series of 417 patients with familial cerebral cavernous malformations. J Eur Acad Dermatol Venereol. 2009;23(9):1066–1072.
    View this article via: PubMed CrossRef Google Scholar
26. Labauge P, et al. Multiple dural lesions mimicking meningiomas in patients with CCM3/PDCD10 mutations. Neurology. 2009;72(23):2044–2046.
    View this article via: PubMed CrossRef Google Scholar
27. Akers AL, Johnson E, Steinberg GK, Zabramski JM, Marchuk DA. Biallelic somatic and germline mutations in cerebral cavernous malformations (CCMs): evidence for a two-hit mechanism of CCM pathogenesis. Hum Mol Genet. 2009;18(5):919–930.
    View this article via: PubMed Google Scholar
28. Gault J, et al. Cerebral cavernous malformations: somatic mutations in vascular endothelial cells. Neurosurgery. 2009;65(1):138–144.
    View this article via: PubMed CrossRef Google Scholar
29. Pagenstecher A, Stahl S, Sure U, Felbor U. A two-hit mechanism causes cerebral cavernous malformations: complete inactivation of CCM1, CCM2 or CCM3 in affected endothelial cells. Hum Mol Genet. 2009;18(5):911–918.
    View this article via: PubMed Google Scholar
30. Rigamonti D, et al. Cerebral cavernous malformations. Incidence and familial occurrence. N Engl J Med. 1988;319(6):343–347.
    View this article via: PubMed CrossRef Google Scholar
31. Labauge P, Brunereau L, Levy C, Laberge S, Houtteville JP. The natural history of familial cerebral cavernomas: a retrospective MRI study of 40 patients. Neuroradiology. 2000;42(5):327–332.
    View this article via: PubMed CrossRef Google Scholar
32. Del Curling O Jr, Kelly DL Jr, Elster AD, Craven TE. An analysis of the natural history of cavernous angiomas. J Neurosurg. 1991;75(5):702–708.
    View this article via: PubMed Google Scholar
33. Knudson AG Jr. Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci U S A. 1971;68(4):820–823.
    View this article via: PubMed CrossRef Google Scholar
34. Gault J, Shenkar R, Recksiek P, Awad IA. Biallelic somatic and germ line CCM1 truncating mutations in a cerebral cavernous malformation lesion. Stroke. 2005;36(4):872–874.
    View this article via: PubMed CrossRef Google Scholar
35. Shenkar R, et al. Advanced magnetic resonance imaging of cerebral cavernous malformations: part II. Imaging of lesions in murine models. Neurosurgery. 2008;63(4):790–797.
    View this article via: PubMed CrossRef Google Scholar
36. McDonald DA, et al. A novel mouse model of cerebral cavernous malformations based on the two-hit mutation hypothesis recapitulates the human disease. Hum Mol Genet. 2011;20(2):211–222.
    View this article via: PubMed CrossRef Google Scholar
37. Claxton S, Kostourou V, Jadeja S, Chambon P, Hodivala-Dilke K, Fruttiger M. Efficient, inducible Cre-recombinase activation in vascular endothelium. Genesis. 2008;46(2):74–80.
    View this article via: PubMed CrossRef Google Scholar
38. Davis GE, Camarillo CW. An alpha 2 beta 1 integrin-dependent pinocytic mechanism involving intracellular vacuole formation and coalescence regulates capillary lumen and tube formation in three-dimensional collagen matrix. Exp Cell Res. 1996;224(1):39–51.
    View this article via: PubMed CrossRef Google Scholar
39. Ghabrial A, Luschnig S, Metzstein MM, Krasnow MA. Branching morphogenesis of the Drosophila tracheal system. Annu Rev Cell Dev Biol. 2003;19:623–647.
    View this article via: PubMed CrossRef Google Scholar
40. Manning G, Krasnow MA. Development of the Drosophila tracheal system. In The Development of Drosophila melanogaster . Bate M, Martinez Arias A, eds. Plainview, New York, USA: Cold Spring Harbor Laboratory Press; 1993:609–685.
41. Mummery-Widmer JL, et al. Genome-wide analysis of Notch signalling in Drosophila by transgenic RNAi. Nature. 2009;458(7241):987–992.
    View this article via: PubMed CrossRef Google Scholar
42. Shiga Y, Tanaka-Matakatsu M, Hayashi S. A nuclear GFP/beta-galactosidase fusion protein as a marker for morphogenesis in living Drosophila. Dev Growth Differ. 1996;38(1):99–106.
    View this article via: CrossRef Google Scholar
43. Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993;118(2):401–415.
    View this article via: PubMed Google Scholar
44. Dietzl G, et al. A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature. 2007;448(7150):151–156.
    View this article via: PubMed CrossRef Google Scholar
45. Clatterbuck RE, Eberhart CG, Crain BJ, Rigamonti D. Ultrastructural and immunocytochemical evidence that an incompetent blood-brain barrier is related to the pathophysiology of cavernous malformations. J Neurol Neurosurg Psychiatry. 2001;71(2):188–192.
    View this article via: PubMed CrossRef Google Scholar
46. Wang L, Seidman JG, Seidman CE. Narrative review: harnessing molecular genetics for the diagnosis and management of hypertrophic cardiomyopathy. Ann Intern Med. 2010;152(8):513–520.
    View this article via: PubMed CrossRef Google Scholar
47. He Y, et al. Stabilization of VEGFR2 signaling by cerebral cavernous malformation 3 is critical for vascular development. Sci Signal. 2010;3(116):ra26.
    View this article via: PubMed CrossRef Google Scholar
48. Navankasattusas S, et al. The netrin receptor UNC5B promotes angiogenesis in specific vascular beds. Development. 2008;135(4):659–667.
    View this article via: PubMed CrossRef Google Scholar
49. di Tomaso E, et al. PDGF-C induces maturation of blood vessels in a model of glioblastoma and attenuates the response to anti-VEGF treatment. PLoS ONE. 2009;4(4):e5123.
    View this article via: PubMed CrossRef Google Scholar
50. Jones CA, et al. Robo4 stabilizes the vascular network by inhibiting pathologic angiogenesis and endothelial hyperpermeability. Nat Med. 2008;14(4):448–453.
    View this article via: PubMed CrossRef Google Scholar
51. Saunders WB, Bayless KJ, Davis GE. MMP-1 activation by serine proteases and MMP-10 induces human capillary tubular network collapse and regression in 3D collagen matrices. J Cell Sci. 2005;118(pt 10):2325–2340.
    View this article via: PubMed CrossRef Google Scholar
52. Groth AC, Fish M, Nusse R, Calos MP. Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics. 2004;166(4):1775–1782.
    View this article via: PubMed CrossRef Google Scholar
53. Venken KJ, He Y, Hoskins RA, Bellen HJ. P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science. 2006;314(5806):1747–1751.
    View this article via: PubMed CrossRef Google Scholar