Loss-of-function mutations of Ccm
genes in humans can lead to lesions in brain capillaries that result in hemorrhages that in turn cause apparent neurologic defects and even death in patients. However, the roles of CCM proteins in brain vasculature and their significance in the disease context have remained poorly understood. In the present study, we first provide genetic evidence that endothelial and/or endocardial Ccm2
expression in mice is required for proper angiogenesis and heart development during embryogenesis, in agreement with previous studies (8
). We have extended these observations to show that expression of Ccm2
in ECs is required for formation of smooth and stable vessel lumens, implying a role for CCM2 in endothelial cell–cell interactions. Importantly, we now demonstrate that conditional Ccm2
gene inactivation in adult mice induces brain hemorrhages highly reminiscent of those observed in human CCM patients. This provides the first CCM2 mouse model for CCMs. The CCM-like brain lesions induced in mice display disrupted vascular lumens and enlarged capillary cavities, lacking neuro-vascular associations; in addition, these lesions are surrounded by accumulations of activated microglia. This CCM animal model should prove to be very valuable to explore the signaling pathways and functions of CCM2 and to understand how the absence of CCM2 can lead to severe CCMs in brain capillaries. Furthermore, this animal model may prove useful in testing therapeutic intervention strategies for the human disease.
Our data demonstrate that CCM2 is required in endothelium for the angiogenic remodeling of the vasculature in the yolk sac, the head and intersomitic vessels, but is not required for the initial primary capillary formation. We also find that expression of arterial markers and recruitment of vascular SMCs are severely impaired in Ccm2
mutants. Previous studies by others and us have indicated that VEGF-A induces arterial differentiation by acting through the Notch pathway (27
). Furthermore, VEGF-A has also recently been shown to stabilize VEGR2 via CCM3, thereby facilitating signaling by this receptor (11
). Given the ability of CCM2 to associate with both CCM1 and CCM3, CCM2 could conceivably have a role in VEGFR2 signaling as well.
Complete lumen formation in the dorsal aorta is essential for a proper circulatory system. Our extensive confocal imaging analyses, which have made use of several markers for luminal membranes, have revealed that deletion of endothelial Ccm2
resulted in a disrupted lumen in dorsal aorta. This observation suggests that CCM2 may regulate cytoskeletal architecture in ECs. Recent biochemical studies using human microvascular endothelial cells indicate that CCM2 can directly associate with and inhibit RhoA activity, decreasing stress fiber formation; therefore, loss of Ccm2
could lead to disruption of cell–cell junctions and thus lumen formation as a consequence of cytoskeletal changes (9
During heart development, essential and reciprocal interactions occur between endocardial cells and between endocardial and myocardial cells. Notch has been shown to mediate the endocardium–myocardium interaction that is critical for trabeculation and ventricular chamber morphogenesis (30
). Interestingly, endothelial/endocardial deletion of Notch1
results in defective ventricular patterning and trabeculation that is almost identical to the cardiac phenotype seen in the Ccm2
mutants. A recent report also demonstrates that CCM1 may activate Delta–Notch signaling, which leads to AKT phosphorylation in ECs in vitro
The induced loss of Ccm2 in a limited number of blood-accessible cells and ECs in adult mice generated specific, localized lesions in brain capillaries, resulting over time in neurologic defects, hemorrhages and ultimately the death of animals. The lesions observed in the Ccm2 mutants appear to be very similar to those seen in human CCM patients. Mouse brain lesions were characterized by grossly dilated malformed vessels that were prone to rupture, releasing erythrocytes. Furthermore, the normal EC–astrocyte interaction was severely disrupted. These findings indicate defects in EC association of the lesional vessels that are somewhat reminiscent of those seen during angiogenesis in mice lacking CCM2 in ECs during embryonic development. Similar to human lesions, those in brains of adult mice were associated with an immune response as well, as documented by the recruitment of microglia.
How might loss of CCM2 cause lesions in adult brain? Since only a small subset of brain capillaries develops malformations and hemorrhages, we hypothesize that such lesions may develop in newly forming vessels, a process that may be initiated in response to local inflammation or some other event (32
). If so, one may imagine a scenario, wherein resident Ccm2
-deleted ECs and/or EC precursors are recruited into newly sprouting vessels, resulting in a partially defective/disrupted lumen on account of the impaired functions of the Ccm2
-deficient ECs. In an alternative and possibly additional scenario, one may imagine that mutant resident ECs and/or their precursors may be prone to initiate new vessel formation and/or preferentially contribute after the process has been initiated, thus increasing the representation of mutant cells in the new vessel wall. In support of this hypothesis, recent reports have suggested that loss of CCM proteins might in fact promote angiogenic sprouting, migration and proliferation of ECs in culture (31
). Conceivably, CCM proteins could have a positive role in transmitting angiogenic signals, while at the same time, setting thresholds to limit spontaneous growth and sprouting.
The brain lesions induced by pIpC-treatment of Ccm2flox/flox;MX1-Cre
mice over time appear to faithfully recapitulate the pathogenesis of the lesions in human CCM patients. Therefore, this represents the first CCM2 mouse model for the CCM disease. After completion of our work, another mouse model for CCM was recently reported (37
). In this model, heterozygous Ccm1+/−
mice were crossed onto a background of Msh2
deficiency, a gene critical for mismatch repair. In this model, complete loss of Ccm1
was suggested to occur in some cells as a result of the increased somatic mutational load, resulting in brain lesions resembling those seen in human patients. In contrast, backcrossing of Ccm2+/−
mice onto an Msh2
deficient background failed to generate lesions; the reasons for this are unknown. Unlike our model, wherein we induce the loss of Ccm2
in adult mice, the timing of the deletion of Ccm1
in the Msh2
-deficient model cannot be controlled and loss of Ccm1
and the generation of lesions could occur at any time starting during embryonic development; furthermore, penetrance in this model was reported at 50% only. Finally, and importantly, Msh2
deficiency likely predisposes towards mutations in other genes, which potentially compromises the usefulness of the model. Nevertheless, the two models are also complementary as they provide a means to compare and contrast the pathogenesis of lesions induced by loss of two different CCM proteins; CCM proteins may have distinct activities in the context of CCMs (36
The pIpC-induced, MX1-Cre-mediated deletion of Ccm2 in adult mice described here represents a temporally controllable and fully penetrant model for generating brain vascular lesions that resemble those observed in human CCM patients. This mouse disease model should greatly facilitate further investigations into the cellular and molecular mechanisms underlying CCM vascular disease and may ultimately allow for testing of possible therapeutic interventions to slow or stop the progression of this disease.