Neuronal expression of the Ccm2 gene in a new mouse model of cerebral cavernous malformations

Cerebral cavernous malformations are vascular defects of the central nervous system consisting of clusters of dilated vessels that are subject to frequent hemorrhaging. The genes mutated in three forms of autosomal dominant cerebral cavernous malformations have been cloned, but it remains unclear which cell type is ultimately responsible for the lesion. In this article we describe mice with a gene trap insertion in the Ccm2 gene. Consistent with the human phenotype, heterozygous animals develop cerebral vascular malformations, although penetrance is low. β-galactosidase activity in heterozygous brain and in situ hybridization in wild-type brain revealed Ccm2 expression in neurons and choroid plexus but not in vascular endothelium of small vessels in the brain. The expression pattern of Ccm2 is similar to that of the Ccm1 gene and its interacting protein ICAP1 (Itgb1bp1). These data suggest that cerebral cavernous malformations arise as a result of defects in the neural parenchyma surrounding the vascular endothelial cells in the brain. Nicholas W. Plummer, Teresa L. Squire and Sudha Srinivasan contributed equally to this work.     

Ccm3 functions in a manner distinct from Ccm1 and Ccm2 in a zebrafish model of CCM vascular disease

Cerebral cavernous malformations (CCMs) are vascular anomalies of the central nervous system that arise due to mutations in genes encoding three unrelated proteins: CCM1 (KRIT1); CCM2 (Malcavernin/OSM) and CCM3 (PDCD10). Both biochemical and mutant studies suggest that CCM1 and CCM2 act as part of a physical complex to regulate vascular morphogenesis and integrity. In contrast, mouse Ccm3 mutant and in vitro cell culture data suggests an independent role for Ccm3. In this study, we sought to use the zebrafish model system to examine for the first time the role of ccm3 in cranial vessel development. We report that inhibition of zebrafish ccm3a/b causes heart and circulation defects distinct from those seen in ccm1 (santa) and ccm2 (valentine) mutants, and leads to a striking dilation and mispatterning of cranial vessels reminiscent of the human disease pathology. ccm3, but not ccm2, defects can be rescued upon overexpression of stk25b, a GCKIII kinase previously shown to interact with CCM3. Morpholino knockdown of the GCKIII gene stk25b results in heart and vasculature defects similar to those seen in ccm3 morphants. Finally, additional loss of ccm3 in ccm2 mutants leads to a synergistic increase in cranial vessel dilation. These results support a model in which CCM3 plays a role distinct from CCM1/2 in CCM pathogenesis, and acts via GCKIII activity to regulate cranial vasculature integrity and development. CCM3/GCKIII activity provides a novel therapeutic target for CCMs, as well as for the modulation of vascular permeability.     

PDCD10 Gene Mutations in Multiple Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) are vascular abnormalities that may cause seizures, intracerebral haemorrhages, and focal neurological deficits. Familial form shows an autosomal dominant pattern of inheritance with incomplete penetrance and variable clinical expression. Three genes have been identified causing familial CCM: KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3. Aim of this study is to report additional PDCD10/CCM3 families poorly described so far which account for 10-15% of hereditary cerebral cavernous malformations. Our group investigated 87 consecutive Italian affected individuals (i.e. positive Magnetic Resonance Imaging) with multiple/familial CCM through direct sequencing and Multiplex Ligation-Dependent Probe Amplification (MLPA) analysis. We identified mutations in over 97.7% of cases, and PDCD10/CCM3 accounts for 13.1%. PDCD10/CCM3 molecular screening revealed four already known mutations and four novel ones. The mutated patients show an earlier onset of clinical manifestations as compared to CCM1/CCM2 mutated patients. The study of further families carrying mutations in PDCD10/CCM3 may help define a possible correlation between genotype and phenotype; an accurate clinical follow up of the subjects would help define more precisely whether mutations in PDCD10/CCM3 lead to a characteristic phenotype.     

Mutations in a gene encoding a novel protein containing a phosphotyrosine-binding domain cause type 2 cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are congenital vascular anomalies of the central nervous system that can result in hemorrhagic stroke, seizures, recurrent headaches, and focal neurologic deficits. Mutations in the gene KRIT1 are responsible for type 1 CCM (CCM1). We report that a novel gene, MGC4607, exhibits eight different mutations in nine families with type 2 CCM (CCM2). MGC4607, similar to the KRIT1 binding partner ICAP1alpha, encodes a protein with a phosphotyrosine-binding domain. This protein may be part of the complex pathway of integrin signaling that, when perturbed, causes abnormal vascular morphogenesis in the brain, leading to CCM formation.     

Notch1 activation in mice causes arteriovenous malformations phenocopied by ephrinB2 and EphB4 mutants

Notch signaling is essential for embryonic vascular development in mammals and other vertebrates. Here we show that mouse embryos with conditional activation of the Notch1 gene in endothelial cells (Notch1 gain of function embryos) exhibit defects in vascular remodeling increased diameter of the dorsal aortae, and form arteriovenous malformations. Conversely, embryos with either constitutive or endothelial cell‐specific Notch1 gene deletion also have vascular defects, but exhibit decreased diameter of the dorsal aortae and form arteriovenous malformations distinctly different from the Notch1 gain of function mutants. Surprisingly, embryos homozygous for mutations of the ephrinB/EphB pathway genes Efnb2 and Ephb4 exhibit vascular defects and arteriovenous malformations that phenocopy the Notch1 gain of function mutants. These results suggest that formation of arteriovenous malformations in Notch1 gain of function mutants and ephrinB/EphB pathway loss of function mutant embryos occurs by different mechanisms. genesis 48:146–150, 2010. © 2010 Wiley‐Liss, Inc.     

CCM1–ICAP-1 complex controls β1 integrin–dependent endothelial contractility and fibronectin remodeling

The endothelial CCM complex regulates blood vessel stability and permeability. Loss-of-function mutations in CCM genes are responsible for human cerebral cavernous malformations (CCMs), which are characterized by clusters of hemorrhagic dilated capillaries composed of endothelium lacking mural cells and altered sub-endothelial extracellular matrix (ECM). Association of the CCM1/2 complex with ICAP-1, an inhibitor of β1 integrin, prompted us to investigate whether the CCM complex interferes with integrin signaling. We demonstrate that CCM1/2 loss resulted in ICAP-1 destabilization, which increased β1 integrin activation and led to increased RhoA-dependent contractility. The resulting abnormal distribution of forces led to aberrant ECM remodeling around lesions of CCM1- and CCM2-deficient mice. ICAP-1–deficient vessels displayed similar defects. We demonstrate that a positive feedback loop between the aberrant ECM and internal cellular tension led to decreased endothelial barrier function. Our data support that up-regulation of β1 integrin activation participates in the progression of CCM lesions by destabilizing intercellular junctions through increased cell contractility and aberrant ECM remodeling.     

Mutations within the programmed cell death 10 gene cause cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are hamartomatous vascular malformations characterized by abnormally enlarged capillary cavities without intervening brain parenchyma. They cause seizures and cerebral hemorrhages, which can result in focal neurological deficits. Three CCM loci have been mapped, and loss-of-function mutations were identified in the KRIT1 (CCM1) and MGC4607 (CCM2) genes. We report herein the identification of PDCD10 (programmed cell death 10) as the CCM3 gene. The CCM3 locus has been previously mapped to 3q26-27 within a 22-cM interval that is bracketed by D3S1763 and D3S1262. We hypothesized that genomic deletions might occur at the CCM3 locus, as reported previously to occur at the CCM2 locus. Through high-density microsatellite genotyping of 20 families, we identified, in one family, null alleles that resulted from a deletion within a 4-Mb interval flanked by markers D3S3668 and D3S1614. This de novo deletion encompassed D3S1763, which strongly suggests that the CCM3 gene lies within a 970-kb region bracketed by D3S1763 and D3S1614. Six additional distinct deleterious mutations within PDCD10, one of the five known genes mapped within this interval, were identified in seven families. Three of these mutations were nonsense mutations, and two led to an aberrant splicing of exon 9, with a frameshift and a longer open reading frame within exon 10. The last of the six mutations led to an aberrant splicing of exon 5, without frameshift. Three of these mutations occurred de novo. All of them cosegregated with the disease in the families and were not observed in 200 control chromosomes. PDCD10, also called "TFAR15," had been initially identified through a screening for genes differentially expressed during the induction of apoptosis in the TF-1 premyeloid cell line. It is highly conserved in both vertebrates and invertebrates. Its implication in cerebral cavernous malformations strongly suggests that it is a new player in vascular morphogenesis and/or remodeling.     

Systems biology and proteomic analysis of cerebral cavernous malformation

Cerebral cavernous malformations (CCM) are vascular anomalies caused by mutations in genes encoding KRIT1, OSM and PDCD10 proteins causing hemorrhagic stroke. We examine proteomic change of loss of CCM gene expression. Using human umbilical vein endothelial cells, label-free differential protein expression analysis with multidimensional liquid chromatography/tandem mass spectrometry was applied to three CCM protein knockdown cell lines and two control cell lines: ProteomeXchange identifier PXD000362. Principle component and cluster analyses were used to examine the differentially expressed proteins associated with CCM. The results from the five cell lines revealed 290 and 192 differentially expressed proteins (p < 0.005 and p < 0.001, respectively). Most commonly affected proteins were cytoskeleton-associated proteins, in particular myosin-9. Canonical genetic pathway analysis suggests that CCM may be a result of defective cell–cell interaction through dysregulation of cytoskeletal associated proteins. Conclusion: The work explores signaling pathways that may elucidate early detection and novel therapy for CCM.     

Notch signaling is required for arterial-venous differentiation during embryonic vascular development

Recent evidence indicates that acquisition of artery or vein identity during vascular development is governed, in part, by genetic mechanisms. The artery-specific expression of a number of Notch signaling genes in mouse and zebrafish suggests that this pathway may play a role in arterial-venous cell fate determination during vascular development. We show that loss of Notch signaling in zebrafish embryos leads to molecular defects in arterial-venous differentiation, including loss of artery-specific markers and ectopic expression of venous markers within the dorsal aorta. Conversely, we find that ectopic activation of Notch signaling leads to repression of venous cell fate. Finally, embryos lacking Notch function exhibit defects in blood vessel formation similar to those associated with improper arterial-venous specification. Our results suggest that Notch signaling is required for the proper development of arterial and venous blood vessels, and that a major role of Notch signaling in blood vessels is to repress venous differentiation within developing arteries. Movies available on-line     

HrT is required for cardiovascular development in zebrafish

The recently identified zebrafish T-box gene hrT is expressed in the developing heart and in the endothelial cells forming the dorsal aorta. Orthologs of hrT are expressed in cardiovascular cells from Drosophila to mouse, suggesting that the function of hrT is evolutionarily conserved. The role of hrT in cardiovascular development, however, has not thus far been determined in any animal model. Using morpholino antisense oligonucleotides, we show that zebrafish embryos lacking hrT function have dysmorphic hearts and an absence of blood circulation. Although the early events in heart formation were normal in hrT morphant embryos, subsequently the hearts failed to undergo looping, and late onset defects in chamber morphology and gene expression were observed. In particular, we found that the loss of hrT function led to a dramatic upregulation of tbx5, a gene required for normal heart morphogenesis. Conversely, we show that overexpression of hrT causes a significant downregulation of tbx5, indicating that one key role of hrT is to regulate the levels of tbx5. Secondly, we found that HrT is required to inhibit the expression of the blood lineage markers gata1 and gata2 in the most posterior lateral plate mesoderm. Finally, we show that HrT is required for vasculogenesis in the trunk, leading to similar vascular defects to those observed in midline mutants such as floating head. hrT expression in the vascular progenitors depends upon midline mesoderm, indicating that this expression is one important component of the response to a midline-derived signal during vascular morphogenesis.     

Structural Basis for the Disruption of the Cerebral Cavernous Malformations 2 (CCM2) Interaction with Krev Interaction Trapped 1 (KRIT1) by Disease-associated Mutations

Familial cerebral cavernous malformations (CCMs) are predominantly neurovascular lesions and are associated with mutations within the KRIT1, CCM2, and PDCD10 genes. The protein products of KRIT1 and CCM2 (Krev interaction trapped 1 (KRIT1) and cerebral cavernous malformations 2 (CCM2), respectively) directly interact with each other. Disease-associated mutations in KRIT1 and CCM2 mostly result in loss of their protein products, although rare missense point mutations can also occur. From gene sequencing of patients known or suspected to have one or more CCMs, we discover a series of missense point mutations in KRIT1 and CCM2 that result in missense mutations in the CCM2 and KRIT1 proteins. To place these mutations in the context of the molecular level interactions of CCM2 and KRIT1, we map the interaction of KRIT1 and CCM2 and find that the CCM2 phosphotyrosine binding (PTB) domain displays a preference toward the third of the three KRIT1 NPX(Y/F) motifs. We determine the 2.75 Å co-crystal structure of the CCM2 PTB domain with a peptide corresponding to KRIT1^NPX(Y/F)3, revealing a Dab-like PTB fold for CCM2 and its interaction with KRIT1^NPX(Y/F)3. We find that several disease-associated missense mutations in CCM2 have the potential to interrupt the KRIT1-CCM2 interaction by destabilizing the CCM2 PTB domain and that a KRIT1 mutation also disrupts this interaction. We therefore provide new insights into the architecture of CCM2 and how the CCM complex is disrupted in CCM disease.     

ANKS1B Interacts with the Cerebral Cavernous Malformation Protein-1 and Controls Endothelial Permeability but Not Sprouting Angiogenesis

Cerebral cavernous malformations are fragile blood vessel conglomerates in the central nervous system that are caused by mutations in the CCM1/KRIT1, CCM2 or CCM3 genes. The gene products form a protein complex at adherens junctions and loss of either CCM protein disrupts endothelial cell quiescence leading to increased permeability and excessive angiogenesis. We performed a yeast 2-hybrid screen to identify novel proteins directly interacting with KRIT1. The ankyrin repeat and sterile alpha motif domain-containing protein 1B (ANKS1B) was identified as a novel binding partner of KRIT1. Silencing of ANKS1B or the related gene ANKS1A in primary human endothelial cells had no significant effects on cellular proliferation, migration and sprouting angiogenesis. However, silencing of ANKS1B expression disturbed endothelial cell barrier functions leading to increased permeability. Forced ANKS1B expression reduced permeability. This was independent of Rho kinase activity and the presence of KRIT1. Taken together, ANKS1B was identified as a novel KRIT1-interacting protein that selectively controls endothelial permeability but not angiogenesis.     

Patterns of expression of the three cerebral cavernous malformation (CCM) genes during embryonic and postnatal brain development

Cerebral Cavernous Malformation (CCM) is a disease characterized by capillary-venous lesions mostly located in the central nervous system. It occurs both as a sporadic and hereditary autosomal dominant condition. Three CCM genes have been identified and shown to encode the KRIT1 (CCM1), MGC4607 (CCM2) and PDCD10 (CCM3) proteins whose functions are so far unknown. In an attempt to get some insight into the role of the 3 CCM genes, we used in situ hybridization to conduct a comparative analysis of their expression pattern at several time points during murine embryonic, postnatal and adult stages particularly within the central nervous system. A strong expression of the 3 Ccm genes was detected in the various neuronal cell layers of the brain, cerebellum and spinal cord, from embryonic to adult life. By E14.5 a moderate labelling was observed in the heart, arterial and venous large vessels with all 3 Ccm probes. Ccm2 and Ccm3 mRNAs, but not Ccm1, were clearly detected within meningeal and parenchymal cortical vessels at P8. This expression was no more detected by P19 and in adult murine brain, strongly suggesting a role for these 2 proteins in the intensive angiogenesis process occuring within the central nervous system during this period.     

santa and valentine pattern concentric growth of cardiac myocardium in the zebrafish

During embryogenesis, the myocardial layer of the primitive heart tube grows outward from the endocardial-lined lumen, with new cells added to generate concentric thickness to the wall. This is a key evolutionary step, demarcating vertebrates from more primitive chordates, and is essential for normal cardiac function. Zebrafish embryos with the recessive lethal mutations santa (san) and valentine (vtn) do not thicken, but do add the proper number of cells to the myocardium. Consequently, the heart chambers are huge, constituted of a monolayered myocardium lined by endocardium. This phenotype is similar to that of the heart of glass (heg) mutation, which we described previously as a novel endocardial expressed gene. By positional cloning, we here identify san as the zebrafish homolog of human CCM1, and vtn as the homolog of human CCM2. Dominant mutations of either in humans cause vascular anomalies in the brain, known as cerebral cavernous malformations. The synergistic effects of morpholino pairs indicate that san, vtn and heg are in a genetic pathway, and san and vtn contain protein motifs, NPxY and PTB domain, respectively, known to interact. This suggests that concentric growth of the myocardium, crucial for blood pressure generation, is dictated by a heg-san-vtn signaling pathway.     

CCM2–CCM3 interaction stabilizes their protein expression and permits endothelial network formation

Mutations in the essential adaptor proteins CCM2 or CCM3 lead to cerebral cavernous malformations (CCM), vascular lesions that most frequently occur in the brain and are strongly associated with hemorrhagic stroke, seizures, and other neurological disorders. CCM2 binds CCM3, but the molecular basis of this interaction, and its functional significance, have not been elucidated. Here, we used x-ray crystallography and structure-guided mutagenesis to show that an α-helical LD-like motif within CCM2 binds the highly conserved “HP1” pocket of the CCM3 focal adhesion targeting (FAT) homology domain. By knocking down CCM2 or CCM3 and rescuing with binding-deficient mutants, we establish that CCM2–CCM3 interactions protect CCM2 and CCM3 proteins from proteasomal degradation and show that both CCM2 and CCM3 are required for normal endothelial cell network formation. However, CCM3 expression in the absence of CCM2 is sufficient to support normal cell growth, revealing complex-independent roles for CCM3.