Endothelial cell-cell adhesion is important for proper blood vessel formation, maintaining the integrity of the vasculature, and regulating diffusion of molecules between the luminal and abluminal faces of blood vessels. Weakened endothelial junctions

Endothelial cell-cell adhesion is important for proper blood vessel formation, maintaining the integrity of the vasculature, and regulating diffusion of molecules between the luminal and abluminal faces of blood vessels. Weakened endothelial junctions are implicated in intracranial hemorrhage (ICH), which are severe and debilitating forms of stroke in humans that include the cerebral cavernous malformation (CCM) disorders. CCM occur either sporadically or with an autosomal dominant inheritance pattern. So far, three CCM genes have been identified in the heritable disorders: CCM1/KRIT1 (Laberge et al., 1999), CCM2/malcavernin (Denier et al., 2004) and CCM3/programmed cell death 10 (PDCD10) (Bergametti et al., 2005), accounting for approximately 40%, 20% and 40% of the autosomal dominant disorders, respectively (Craig et al., 1998). The CCM1 locus codes for a protein called KRIT1 that contains four ankyrin repeat domains and a FERM (protein 4.1, ezrin, radixin, moesin) domain that mediates its interaction with Rap1, a small GTPase that facilitates KRIT1 localization to cell-cell junctions. In Drosophila melanogaster, Rap1 is not required for cell proliferation and cell fate specification, but is required for normal tissue morphogenesis and cell shape maintenance (Asha et al., 1999). Rap1 is localized to the adherens junctions and is actively required for their even distribution; loss of Rap1 leads to disrupted epithelial cell behavior (Knox and Brown, 2002). KRIT1/CCM1 also binds CCM2, CCM3, and a variety of additional interacting proteins, and it is thought that these proteins all function together in large multiprotein complexes regulating cell-cell junction formation (Hilder et al., 2007). Despite the identification of the defective genes for the three CCM loci, little is known about the molecular mechanisms underlying CCM lesion formation. Furthermore, not all individuals harboring defective CCM genes develop ICH, reflecting incomplete penetrance of these mutations and/or involvement of additional genetic modifiers predisposing to lesion formation (Lucas et al., 2003). It has been suggested that multiple genetic factors play important roles in predisposition to hemorrhagic stroke and influence the likelihood of ICH events in both familial and sporadic disorders. Recent evidence that CCM genes act together in common intracellular complexes and/or signaling pathways (Dupre et al., 2003; Hilder et al., 2007; Voss et al., 2007; Zawistowski et al., 2005) suggested to us that minor functional perturbations of different genes in this pathway might act together to precipitate ICH. However, demonstrating multigene association is not possible in the small number of available human hemorrhagic stroke pedigrees, nor has functional evidence substantiating this idea been readily available from other model organisms. We sought to explore the possibility that very minor, otherwise silent deficits in genes that function together to regulate endothelial junctional integrity might synergistically initiate hemorrhagic stroke. We turned to the zebrafish, a useful model organism for analysis of human disease genes and their pathophysiology (Lieschke and Currie, 2007). Zebrafish embryos are readily amenable to simultaneous functional knockdown of multiple genes (Pham et al., 2007), making them very useful for functional analysis of multigene families or interacting proteins. In this study, we show that a combined minor reduction in the expression of multiple CCM pathway genes can precipitate hemorrhagic stroke. Our results provide a rationale for understanding the variability in appearance and onset of this disease. RESEARCH REPORT