In the present paper, we used an inducible, EC-specific deletion of the Ccm2
gene, to bypass the embryonic lethality encountered after constitutive or endothelial-specific Ccm2
ablation (Boulday et al., 2009
; Whitehead et al., 2009
). Early post-natal deletion of Ccm2
resulted in vascular lesions strikingly recapitulating human CCM lesions in the brain as well as in the retina, in 100% of Ccm2
-deleted animals. In this robust and relevant mouse model for the human CCM disease, we showed that CCM lesions affect only the venous bed and that CCM development is restricted to key time periods temporally related to intense angiogenesis.
In the past few years, different groups working in the field of CCM in vivo models concluded to a vascular, EC-specific, autonomous function of the 3 Ccm
genes (Hogan et al., 2008
; Boulday et al., 2009
; Whitehead et al., 2009
; He et al., 2010
). Recently, a paper challenged those results showing CCM-like lesions after neuroglial-specific loss of Ccm3
. In contrast to what was previously published, Louvi et al. (2011)
demonstrated a cell autonomous effect of Ccm3
in astrocytes, resulting in increased cell proliferation and cell survival, as well as a cell nonautonomous effect, resulting in a vascular phenotype. This data suggest that Ccm
genes play a role in vascular and non vascular cells within the CNS, pointing out the importance of the communication between cells composing the neurovascular unit, which may partly explain the CNS restriction of the CCM lesions.
Another approach to obtain a mouse model for human CCM has been developed using genetic sensitizers, attempting to increase the rate of somatic mutation of CCM genes and obtain vascular lesions in heterozygous Ccm1+/−
mice. On a tumor repressor Trp53-null background, 30% of the Ccm1+/−
mice developed lesions (Plummer et al., 2004
), but the frequency of early onset malignancies was a limitation of this model. With a similar approach, 50% of the Ccm1+/−
mice with a mismatch repair complex null background (Msh2−/−
) developed lesions (McDonald et al., 2011
). No lesion was obtained in the Msh2−/−
This sensitized mouse model, as well as the model from Louvi et al. (2011)
, has the advantage of progressing slowly, with a relative normal lifetime for the animal. In addition, according to what is thought to happen in human, lesion development in the heterozygous McDonald’s model occurs as a stochastic event throughout the brain (McDonald et al., 2011
). In contrast, in the model described herein, a very efficient loss of both Ccm2
alleles is obtained in ECs, providing a more aggressive model. iCCM2 animals after P1-induction show a rapid onset of the disease, with lesion development restricted to some specific locations of the CNS, followed by an early death caused at least in part by severe hemorrhages within the CNS. However, our results using later postnatal Ccm2
deletion (Fig. S6) suggested that a milder mouse model, useful for long term studies, could be obtained that might allow lesion development within the brain hemispheres. This will be the subject for further analysis. Finally, it would be unrealistic to expect one animal model to fully recapitulate a human phenotype and we believe that the different CCM mouse models available so far will be complementary for mechanistic exploration.
In our study, AJ and TJ organization was strongly affected in CCM2-deficient ECs in vitro and in the CCM2 lesions in vivo,
consistent with the impaired TJs described in human CCM lesions. CCM2 deletion caused complex changes in AJ and TJ organization, which included down-regulation of junctional components and alteration of their distribution at intercellular contacts. Surprisingly, endothelial cell–cell TJs were maintained in the Msh2−/−
mouse model as assessed by electronic microscopy (McDonald et al., 2011
). The apparent discordance between this model and our data may come from the different approaches used by the two groups. In our model, we cannot exclude that, even though we observed a down-regulation and alteration of some AJ and TJ components using immunostaining approaches, the TJ ultrastructure could be preserved. Previous studies (Whitehead et al., 2009
; Stockton et al., 2010
) showed that in CCM2-deficient ECs, cortical actin cytoskeleton was severely affected. This effect required the association of CCM2 with CCM1/Krit. Data presented here add to these observations and show that deletion of CCM2 causes complex changes in AJ and TJ organization, which include down-regulation of junctional components and alteration of their distribution at intercellular contacts. In a previous paper, we showed (Lampugnani et al., 2010
) that similar junctions’ alterations could also be observed in CCM1-depleted ECs, further supporting the idea of a physical and functional interaction between CCM1 and CCM2. One of the most striking effects of CCM2 depletion observed in our study was the strong reduction of claudin-5 expression, which is likely the cause of altered TJ organization. This effect may explain, to a good extent, the defect in permeability control of CCM2 KO ECs in vitro and in vivo (Fig. S4; Stockton et al., 2010
). Furthermore, as already mentioned, TJ are severely affected in the vascular lesions of CCM patients. Genetic deletion of claudin-5
is known to be associated to defects in blood–brain barrier (Morita et al., 1999
), which leads to death immediately after birth.
Another functional consequence of alterations in AJ or TJ architecture is defective cell polarity. We previously reported that Ccm1
silencing altered VE-cadherin and AJ organization and inhibited the localization of the polarity complex at cell–cell junctions (Lampugnani et al., 2010
). As a consequence, the polarized expression of apical (podocalyxin) and basal (collagen IV) proteins was affected. In this study, although junctions are altered in vivo in CCM2 lesions, apical and basal proteins seem to be correctly distributed (Fig. S2 H). It is reasonable that because CCM1, but not CCM2, also directly interacts with integrins and modulates their functions, this additional property may be required for cell polarity (Zovein et al., 2010
Our results clearly showed that despite pan-endothelial Ccm2 ablation, CCM lesions did not affect all vascular beds. CCM lesions developed only in the cerebellum and the retina after P1 ablation. At the time of analysis, other highly vascularized organs, such as the heart and lungs, did not show evidence of CCM lesions upon dissection, even though Cre-mediated recombination was confirmed in those organs (unpublished data). Thus, our data clearly demonstrate that loss of Ccm2 is not sufficient to induce CCM lesions. In addition to the complete endothelial absence of CCM2, additional factors, possibly specific for the neurovascular microenvironment, might be necessary to cause the CCM disease.
Within the brain and the retina, CCM lesions affect only the venous bed. This is consistent with what is observed in human retinal CCM lesion when using retinal angiography. The bubblelike vascular structures composing the CCM retinal lesion are the last vessels to be filled up by the fluorescent dye, suggesting that lesions are composed by pocket-like capillaries connected to the venous system. In zebrafish, Ccm1
knockdown using morpholinos did not affect dorsal aorta and intersomitic vessel development, but resulted in abnormal morphogenesis with major dilation of the posterior cardinal vein and the caudal vein (Hogan et al., 2008
). In our study, mechanisms explaining the venous restriction of CCM2 lesions remain to be elucidated. The venous-specific effect of CCM2 ablation cannot be explained by a difference in the timing of excision that would affect a vascular bed with a later development, because veins and arteries of the superficial vascular plexus develop concomitantly in the retina (Dorrell and Friedlander, 2006
; Fruttiger, 2007
). We then excluded a difference in recombination efficiency between veins and arteries. As assessed by XGal staining on retinas or cerebral hemispheres, P1-tamoxifen–induced recombination was clearly affecting arteries and veins to the same extent (Fig. S1, A and B). Another trivial explanation would be a venous restriction of Ccm2
expression during late embryogenesis and the postnatal period. In a previous work, we detected a moderate labeling for all three Ccm
transcripts in the heart, arterial, and venous large vessels by E14.5, decreasing at late embryogenic stages (Petit et al., 2006
). To further address this issue, we compared CCM2 mRNA and protein expression in mesenteric arteries and veins. No significant difference in Ccm2
gene expression level was observed in these two vascular beds at the perinatal period (unpublished data). CCM2 protein expression was also confirmed in both types of vessels (unpublished data). The venous specificity of CCM lesions could also reflect a different level of TJ component expression (i.e., claudin-5 expression) in veins versus arteries. However, claudin-5 expression, evaluated by quantitative RT-PCR was similar in mesenteric arteries and veins (unpublished data). Thus, additional work is needed to clarify what differs between veins and arteries that could explain the specific response of venous EC to Ccm2
The main characteristic feature of affected veins at early stages of lesion development in retinas of the iCCM2 mice was an increase in the size of the veins. To understand the mechanisms of this venous dilation, we analyzed EC proliferation before lesion formation. We did not find any enhancement in EC proliferation, suggesting that proliferation is not the primary event leading to lesions. This is consistent with what was found by other groups in the mouse as well as in the zebrafish (Hogan et al., 2008
; McDonald et al., 2011
). In iCCM2 cerebellum, the endothelium lining the already formed CCM lesions (single or multicavernous) did not show any increase in cell proliferation compared with endothelium from controls, as assessed by stainings for the proliferation-associated nuclear protein Ki67 at P8 and P14 ( and not depicted). Our results contrast with other data, showing an increase in proliferation of EC lining multiple mature caverns as compared with single, early cavernous lesions (McDonald et al., 2011
). It is possible that the relatively short median survival in our mouse model may be a limit for analyzing EC proliferation in mature, multicavernous CCM lesions. Interestingly, loss of Ccm1
in zebrafish resulted in impaired EC morphology rather than increase in EC proliferation, with a progressive spreading and thinning of the ECs forming the dilated vessel (Hogan et al., 2008
). We do hypothesize that such a mechanism could explain the phenotype described in our CCM2 mouse model.
In this paper, we showed that the timing of Ccm2
deletion (E14.5, P1, and 3 wk of age) defines the cerebral (or retinal) EC response to CCM2 loss. We first excluded differences in tamoxifen-induced recombination efficiency that could explain the disparate temporal and spatial responses to Ccm2
deletion. XGal staining confirmed a high recombination efficiency at E14.5, P1, and 3 wk of age, in all the cerebral and retinal vessels (Fig. S1, A–F). Mice induced at 3 wk, after vessel development, did not develop CCM lesion in the CNS. In contrast, mice induced at P1 showed CCM lesions in the cerebellum and the retina, whereas late in utero Ccm2
deletion elicits vascular malformations in the cerebral hemispheres. In those two situations, the location of CCM lesions in the CNS corresponds to specific places undergoing intense angiogenesis at the time of deletion (Plate, 1999
; Acker et al., 2001
; Dorrell and Friedlander, 2006
). Thus, our results comparing the different timing of Ccm2
deletion strongly suggest that angiogenesis might be the extra trigger leading to CCM lesion development. Interestingly, in human CCM patients, the number of lesions increases significantly with age, particularly after 50 yr old (Denier et al., 2006
; Labauge et al., 2007
). In addition, it has been shown that angiogenesis can occur in human adult brain in response to cerebral ischemia (Beck and Plate, 2009
). We speculate that an increase in CCM lesion number over 50 yr old may be related to proangiogenic stimuli that may be caused by hypoxic events that occur with aging.
The mechanisms of this restricted temporal CCM competence are thus far unknown. In some aspects, it is reminiscent of the previously reported restricted temporal cystogenic competence of renal epithelial cells. The autosomal dominant polycystic kidney disease is characterized by a progressive increase in renal tubular diameter followed by multiple cysts formation. A key postnatal developmental switch has been involved in this cystogenic process and has been related to abnormal planar cell polarity signaling (PCP; Fischer et al., 2006
; Piontek et al., 2007
; Karner et al., 2009
; Verdeguer et al., 2010
). PCP controls, through the coupling of cell division and morphogenesis, the growth and the size of the normal renal tube. Interestingly, in the normal developing retinal vasculature, orientation of mitosis along the vessel axis was also reported (Zeng et al., 2007
), suggesting that vessel growth is determined by PCP.
While this manuscript was in revision, two other studies were published (Chan et al., 2011
; Cunningham et al., 2011
) that independently validated the use of inducible Ccm
KO approaches to obtain mouse models for the CCM disease. All these very recent complementary in vivo studies show similarities but also differences, most likely linked to distinct methodological approaches, which will be useful to decipher the mechanisms of CCM development.
In this study, we describe a relevant and robust mouse model for CCM disease with a complete penetrance in the CNS. We believe this model to be of importance in better deciphering molecular mechanisms involved in the CCM pathogenesis. Moreover, the rapid onset of the disease in the iCCM2 mouse model makes it particularly suitable for therapeutic preclinical evaluation, especially for a fast first screening of novel agents targeting lesion genesis. Indeed, prevention of lesion development/progression/bleeding, or induction of lesion regression are now the real challenge to pursue for the CCM disease (Yadla et al., 2010
Herein, analysis of the iCCM2 model suggests that the loss of Ccm2 is required and sufficient for the development of CCM lesions but only in a restricted spatial and temporal manner. We propose that, within an appropriate time window, a pro-angiogenic stimulus in the neurovascular unit microenvironment provides a permissive signal for venous EC from the CNS to eventually form CCM lesions.