Contribution of protein-protein interactions to the endothelial-barrier-stabilizing function of KRIT1.

Krev-interaction trapped protein 1 (KRIT1) is an endothelial scaffold protein that promotes adherens junction (AJ) stability. The precise mechanism by which KRIT1 promotes barrier stabilization is unclear. We tested the ability of a panel of KRIT1 constructs containing mutations that inhibit Rap1 binding, ICAP1α binding, disrupt KRIT1's phosphotyrosine-binding (PTB) domain, or direct KRIT1 to the plasma membrane, either alone or in combination, to restore barrier function in KRIT1-deficient endothelial cells. We found that ablating the 192NPAY195 motif or disrupting the PTB domain was sufficient to restore AJ protein localization and barrier function to control levels, irrespective of the junctional localization of KRIT1 or Rap1 binding. The ability of our KRIT1 constructs to rescue AJ and barrier function in KRIT1-depleted endothelial cells correlated with decreased ß1 integrin activity and maintenance of cortical actin fibers. Taken together, our findings indicate that Rap1 binding, ICAP1α binding and junctional localization are not required for the ability of KRIT1 to stabilize endothelial contacts, and suggest that the ability of KRIT1 to limit integrin activity could be involved in barrier stabilization.

Somatic PIK3CA Mutations in Sporadic Cerebral Cavernous Malformations.

BACKGROUND: Cerebral cavernous malformations (CCMs) are common sporadic and inherited vascular malformations of the central nervous system. Although familial CCMs are linked to loss-of-function mutations in KRIT1 (CCM1), CCM2, or PDCD10 (CCM3), the genetic cause of sporadic CCMs, representing 80% of cases, remains incompletely understood. METHODS: We developed two mouse models harboring mutations identified in human meningiomas with the use of the prostaglandin D2 synthase (PGDS) promoter. We performed targeted DNA sequencing of surgically resected CCMs from patients and confirmed our findings by droplet digital polymerase-chain-reaction analysis. RESULTS: We found that in mice expressing one of two common genetic drivers of meningioma - Pik3ca H1047R or AKT1 E17K - in PGDS-positive cells, a spectrum of typical CCMs develops (in 22% and 11% of the mice, respectively) instead of meningiomas, which prompted us to analyze tissue samples from sporadic CCMs from 88 patients. We detected somatic activating PIK3CA and AKT1 mutations in 39% and 1%, respectively, of lesion tissue from the patients. Only 10% of lesions harbored mutations in the CCM genes. We analyzed lesions induced by the activating mutations Pik3ca H1074R and AKT1 E17K in mice and identified the PGDS-expressing pericyte as the probable cell of origin. CONCLUSIONS: In tissue samples from sporadic CCMs, mutations in PIK3CA were represented to a greater extent than mutations in any other gene. The contribution of somatic mutations in the genes that cause familial CCMs was comparatively small. (Funded by the Fondation ARC pour la Recherche contre le Cancer and others.).

Plasma water T2 detects age-stratified differences in cardiometabolic health among familial CCM patients with Hispanic CCM1 mutation.

Cerebral cavernous malformations (CCMs) are abnormal clusters of capillaries in the nervous system. This pilot study analyzed the cardiometabolic health status of individuals with familial CCMs caused by a rare mutation in the CCM1 gene (fCCM1). The aim was to compare plasma water T2 values from individuals with fCCM1 with values from metabolically unhealthy and healthy individuals with no known CCM mutations. This observational, cross-sectional study included 75 participants: 11 fCCM1 patients, 24 metabolically unhealthy and 40 metabolically healthy individuals. Plasma water T2, an early, global and practical marker of cardiometabolic health, was measured in the time domain using benchtop magnetic resonance relaxometry. The results were stratified by age (equal to or less than 45 vs. older than 45 years). Group means were compared using Welch's one-way ANOVA and post hoc Tukey-Kramer tests. Multivariable linear regression, with T2 as the outcome variable, was used to explore associations with age, gender, Hispanic ethnicity and fCCM1 status. In the younger age stratum, the fCCM1 group had a mean plasma water T2 value comparable to the metabolically healthy group (p = 0.6388), but higher than the unhealthy group (p < 0.0001). By contrast, in the older stratum, the mean plasma water T2 value for the fCCM1 group was comparable to the metabolically unhealthy group (p = 0.7819) and lower than the healthy group (p = 0.0005). Multivariable linear regression revealed that age and the interaction between age and fCCM1 status were significant predictors of T2, even after adjusting for gender and Hispanic ethnicity. Plasma water T2 shows potential as a biomarker for assessing the health status of individuals with fCCM1. Further research is needed to validate these preliminary observations and elucidate the association between CCMs and cardiometabolic health.

Comprehensive analysis of Novel mutations in CCM1/KRIT1 and CCM2/MGC4607 and their clinical implications in Cerebral Cavernous malformations.

BACKGROUND: Cerebral Cavernous Malformations (CCM) is a genetic disease characterized by vascular abnormalities in the brain and spinal cord, affecting 0.4-0.5 % of the population. We identified two novel pathogenic mutations, CCM1/KRIT1 c.811delT (p.Trp271GlyfsTer5) and CCM2/MGC4607 c.613_614insGG p.Glu205GlyfsTer31), which disrupt crucial protein domains and potentially alter disease progression. OBJECTIVE: The study aims to comprehensively analyze a Brazilian cohort of CCM patients, integrating genetic, clinical, and structural aspects. Specifically, we sought to identify novel mutations within the CCM complex, and explore their potential impact on disease progression. METHODS: We conducted a detailed examination of neuroradiological and clinical features in both symptomatic and asymptomatic CCM patients, performing genetic analyses through sequencing of the CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10 genes In silico structural predictions were carried out using PolyPhen-2, SIFT, and Human Genomics Community tools. Protein-protein interactions and docking analyses were explored using the STRING database. RESULTS: Genetic analysis identifies 6 pathogenic mutations, 4 likely pathogenic, 1 variants of uncertain significance, and 7 unclassified mutations, including the novel mutations in CCM1 c.811delT and CCM2 c.613_614insGG. In silico structural analysis revealed significant alterations in protein structure, supporting their pathogenicity. Protein-protein interaction analysis indicated nuanced impacts on cellular processes. Clinically, we observed a broad spectrum of symptoms, including seizures and focal neurological deficits. However, no statistically significant differences were found in lesion burden, age of first symptom onset, or sex between the identified CCM1/KRIT1 and CCM2/MGC4607 mutations among all patients studied. CONCLUSION: This study enhances the understanding of CCM by linking clinical variability, genetic mutations, and structural effects. The identification of these novel mutations opens new avenues for research and potential therapeutic strategies.

A novel KRIT1/CCM1 mutation accompanied by a NOTCH3 mutation in a Chinese family with multiple cerebral cavernous malformations.

Family cerebral cavernous malformations (FCCMs) are mainly inherited through the mutation of classical CCM genes, including CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10. FCCMs can cause severe clinical symptoms, including epileptic seizures, intracranial hemorrhage (ICH), or functional neurological deficits (FNDs). In this study, we reported a novel mutation in KRIT1 accompanied by a NOTCH3 mutation in a Chinese family. This family consists of 8 members, 4 of whom had been diagnosed with CCMs using cerebral MRI (T1WI, T2WI, SWI). The proband (II-2) and her daughter (III-4) had intracerebral hemorrhage and refractory epilepsy, respectively. Based on whole-exome sequencing (WES) data and bioinformatics analysis from 4 patients with multiple CCMs and 2 normal first-degree relatives, a novel KRIT1 mutation, NG_012964.1 (NM_194456.1): c.1255-1G > T (splice-3), in intron 13 was considered a pathogenic gene in this family. Furthermore, based on 2 severe and 2 mild CCM patients, we found an SNV missense mutation, NG_009819.1 (NM_000435.2): c.1630C > T (p.R544C), in NOTCH3. Finally, the KRIT1 and NOTCH3 mutations were validated in 8 members using Sanger sequencing. This study revealed a novel KRIT1 mutation, NG_012964.1 (NM_194456.1): c.1255-1G > T (splice-3), in a Chinese CCM family, which had not been reported previously. Moreover, the NOTCH3 mutation NG_009819.1 (NM_000435.2): c.1630C > T (p.R544C) might be a second hit and associated with the progression of CCM lesions and severe clinical symptoms.

Endothelial Differentiation of CCM1 Knockout iPSCs Triggers the Establishment of a Specific Gene Expression Signature.

Cerebral cavernous malformation (CCM) is a neurovascular disease that can lead to seizures and stroke-like symptoms. The familial form is caused by a heterozygous germline mutation in either the CCM1, CCM2, or CCM3 gene. While the importance of a second-hit mechanism in CCM development is well established, it is still unclear whether it immediately triggers CCM development or whether additional external factors are required. We here used RNA sequencing to study differential gene expression in CCM1 knockout induced pluripotent stem cells (CCM1-/- iPSCs), early mesoderm progenitor cells (eMPCs), and endothelial-like cells (ECs). Notably, CRISPR/Cas9-mediated inactivation of CCM1 led to hardly any gene expression differences in iPSCs and eMPCs. However, after differentiation into ECs, we found the significant deregulation of signaling pathways well known to be involved in CCM pathogenesis. These data suggest that a microenvironment of proangiogenic cytokines and growth factors can trigger the establishment of a characteristic gene expression signature upon CCM1 inactivation. Consequently, CCM1-/- precursor cells may exist that remain silent until entering the endothelial lineage. Collectively, not only downstream consequences of CCM1 ablation but also supporting factors must be addressed in CCM therapy development.

Mesenteric cysts, lymphatic leak, and cerebral cavernous malformation in a proband with KRIT1-related disease.

Cerebral cavernous malformations (CCMs) of the central nervous system arise sporadically or secondary to genomic variation. Established genetic etiologies include deleterious variants in KRIT1 (CCM1), malcavernin (CCM2), and PDCD10 (CCM3). KRIT1-related disease has not been described in conjunction with lymphatic defects, although lymphatic defects with abnormal endothelial cell junctions have been observed in mice deficient in HEG1-KRIT1 signaling. We report a proband with CCMs, multiple chylous mesenteric cysts, and chylous ascites with leaky lymphatic vasculature. Clinical short-read exome sequencing detected a disease-associated KRIT1 variant (NM_194456.1:c.[1927C>T];[=], p.(Gln643*)). We postulate an expansion of KRIT1-related disease to include lymphatic malformations and lymphatic endothelial dysfunction.

Identification of a novel LATS1 variant associated with familial cerebral cavernous malformations in a Chinese family.

BACKGROUND: Cerebral cavernous malformations (CCMs) are common sporadic or hereditary vascular malformations in the central nervous system. CCM1-3 variants have been identified that are associated with the majority of familial cerebral cavernous malformations (FCCMs). However, there are still a few CCM1-3 wild-type FCCMs. The aim of the present study was to identify an additional pathogenic variant of FCCMs. METHODS: In this study, a large five-generation Chinese Han family affected by CCMs was recruited. Magnetic resonance imaging (MRI) was done for the detection of CCMs. Whole-exome sequencing (WES) was performed, and the identified variants were co-segregation analyzed by Sanger sequencing. The function of candidate variants was predicted in silico and experimental validated by angiogenesis assay in human umbilical vein endothelial cells (HUVECs) in vitro. RESULTS: Twenty-four family members and one healthy spouse were enrolled. We found that CCMs were exhibited on MRI in nine family members. Overall, twenty-seven candidate variants were identified using WES, and no CCM1-3 variants were detected. The missense variant in LATS1 (c.821C > T, p.Thr274Ile) was verified to be associated with the clinical and pathological phenotype of FCCMs. CONCLUSION: Our findings indicated that the LATS1 variant could be a potential pathogenic factor for FCCMs in this Chinese family.

Improving clinical interpretation of five KRIT1 and PDCD10 intronic variants.

Cerebral cavernous malformation (CCM) is a vascular malformation of the central nervous system which may occur sporadically or segregate within families due to heterozygous variants in KRIT1/CCM1, MGC4607/CCM2 or PDCD10/CCM3. Intronic variants are not uncommon in familial CCM, but their clinical interpretation is often hampered by insufficient data supporting in silico predictions. Here, the mRNA analysis for two intronic unpublished variants (KRIT1 c.1147-7 T > G and PDCD10 c.395 + 2 T > G) and three previously published variants in KRIT1 but without data supporting their effects was carried out. This study demonstrated that all variants can induce a frameshift with the lack of residues located in the C-terminal regions and involved in protein-protein complex formation, which is essential for vascular homeostasis. These results support the introduction of mRNA analysis in the diagnostic pathway of familial CCM and expand the knowledge of abnormal splicing patterning in this disorder.

Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3.

Loss-of-function variants in CCM1/KRIT1, CCM2, and CCM3/PDCD10 are associated with autosomal dominant cerebral cavernous malformations (CCMs). CRISPR/Cas9-mediated CCM3 inactivation in human endothelial cells (ECs) has been shown to induce profound defects in cell-cell interaction as well as actin cytoskeleton organization. We here show that CCM3 inactivation impairs fibronectin expression and consequently leads to reduced fibers in the extracellular matrix. Despite the complexity and high molecular weight of fibronectin fibrils, our in vitro model allowed us to reveal that fibronectin supplementation restored aberrant spheroid formation as well as altered EC morphology, and suppressed actin stress fiber formation. Yet, fibronectin replacement neither enhanced the stability of tube-like structures nor inhibited the survival advantage of CCM3-/- ECs. Importantly, CRISPR/Cas9-mediated introduction of biallelic loss-of-function variants into either CCM1 or CCM2 demonstrated that the impaired production of a functional fibronectin matrix is a common feature of CCM1-, CCM2-, and CCM3-deficient ECs.

CDC42 Deletion Elicits Cerebral Vascular Malformations via Increased MEKK3-Dependent KLF4 Expression.

RATIONALE: Aberrant formation of blood vessels precedes a broad spectrum of vascular complications; however, the cellular and molecular events governing vascular malformations are not yet fully understood. OBJECTIVE: Here, we investigated the role of CDC42 (cell division cycle 42) during vascular morphogenesis and its relative importance for the development of cerebrovascular malformations. METHODS AND RESULTS: To avoid secondary systemic effects often associated with embryonic gene deletion, we generated an endothelial-specific and inducible knockout approach to study postnatal vascularization of the mouse brain. Postnatal endothelial-specific deletion of Cdc42 elicits cerebrovascular malformations reminiscent of cerebral cavernous malformations (CCMs). At the cellular level, loss of CDC42 function in brain endothelial cells (ECs) impairs their sprouting, branching morphogenesis, axial polarity, and normal dispersion within the brain tissue. Disruption of CDC42 does not alter EC proliferation, but malformations occur where EC proliferation is the most pronounced during brain development-the postnatal cerebellum-indicating that a high, naturally occurring EC proliferation provides a permissive state for the appearance of these malformations. Mechanistically, CDC42 depletion in ECs elicited increased MEKK3 (mitogen-activated protein kinase kinase kinase 3)-MEK5 (mitogen-activated protein kinase kinase 5)-ERK5 (extracellular signal-regulated kinase 5) signaling and consequent detrimental overexpression of KLF (Kruppel-like factor) 2 and KLF4, recapitulating the hallmark mechanism for CCM pathogenesis. Through genetic approaches, we demonstrate that the coinactivation of Klf4 reduces the severity of vascular malformations in Cdc42 mutant mice. Moreover, we show that CDC42 interacts with CCMs and that CCM3 promotes CDC42 activity in ECs. CONCLUSIONS: We show that endothelial-specific deletion of Cdc42 elicits CCM-like cerebrovascular malformations and that CDC42 is engaged in the CCM signaling network to restrain the MEKK3-MEK5-ERK5-KLF2/4 pathway.

Postzygotic mosaicism in cerebral cavernous malformation.

BACKGROUND: Cerebral cavernous malformations (CCMs) can cause severe neurological morbidity but our understanding of the mechanisms that drive CCM formation and growth is still incomplete. Recent experimental data suggest that dysfunctional CCM3-deficient endothelial cell clones form cavernous lesions in conjunction with normal endothelial cells. OBJECTIVE: In this study, we addressed the question whether endothelial cell mosaicism can be found in human cavernous tissue of CCM1 germline mutation carriers. METHODS AND RESULTS: Bringing together single-molecule molecular inversion probes in an ultra-sensitive sequencing approach with immunostaining to visualise the lack of CCM1 protein at single cell resolution, we identified a novel late postzygotic CCM1 loss-of-function variant in the cavernous tissue of a de novo CCM1 germline mutation carrier. The extended unilateral CCM had been located in the right central sulcus causing progressive proximal paresis of the left arm at the age of 15 years. Immunohistochemical analyses revealed that individual caverns are lined by both heterozygous (CCM1+/- ) and compound heterozygous (CCM1-/- ) endothelial cells. CONCLUSION: We here demonstrate endothelial cell mosaicism within single caverns of human CCM tissue. In line with recent in vitro data on CCM1-deficient endothelial cells, our results provide further evidence for clonal evolution in human CCM1 pathogenesis.

Novel CCM2 missense variants abrogating the CCM1-CCM2 interaction cause cerebral cavernous malformations.

BACKGROUND: Cerebral cavernous malformations (CCMs) are vascular malformations mostly located within the central nervous system. Most deleterious variants are loss of function mutations in one of the three CCM genes. These genes code for proteins that form a ternary cytosolic complex with CCM2 as a hub. Very few CCM2 missense variants have been shown to be deleterious by modifying the ternary CCM complex stability. OBJECTIVES: To investigate the causality of novel missense CCM2 variants detected in patients with CCM. METHODS: The three CCM genes were screened in 984 patients referred for CCM molecular screening. Interaction between CCM1 and CCM2 proteins was tested using co-immunoprecipitation experiments for the CCM2 missense variants located in the phosphotyrosine binding (PTB) domain. RESULTS: 11 distinct CCM2 rare missense variants were found. Six variants predicted to be damaging were located in the PTB domain, four of them were novel. When co-transfected with CCM1 in HEK293T cells, a loss of interaction between CCM1 and CCM2 was observed for all six variants. CONCLUSION: We showed, using co-immunoprecipitation experiments, that CCM2 missense variants located in the PTB domain were actually damaging by preventing the normal interaction between CCM1 and CCM2. These data are important for diagnosis and genetic counselling, which are challenging in patients harbouring such variants.

Description of Two Families with New Mutations in Familial Cerebral Cavernous Malformations Genes.

Cerebral cavernous malformations (CCMs) are dilated aberrant leaky capillaries located in the Central Nervous System. Familial CCM is an autosomal dominant inherited disorder related to mutations in KRIT1, Malcavernin or PDCD10. We show two unrelated families presenting familial CCM due to two new mutations in KRIT1 and PDCD10, producing truncated proteins. Clinical phenotype was highly variable among patients from asymptomatic individuals to diplopia, seizures or severe intracranial hemorrhage. PDCD10 patients usually show a more aggressive course and they frequently showed multiple meningiomas. This work provides evidence for the pathogenicity of two new mutations in CCM genes and supports previous findings regarding familial CCM and multiple meningiomas.

The CCM1-CCM2 complex controls complementary functions of ROCK1 and ROCK2 that are required for endothelial integrity.

Endothelial integrity relies on a mechanical crosstalk between intercellular and cell-matrix interactions. This crosstalk is compromised in hemorrhagic vascular lesions of patients carrying loss-of-function mutations in cerebral cavernous malformation (CCM) genes. RhoA/ROCK-dependent cytoskeletal remodeling is central to the disease, as it causes unbalanced cell adhesion towards increased cell-extracellular matrix adhesions and destabilized cell-cell junctions. This study reveals that CCM proteins directly orchestrate ROCK1 and ROCK2 complementary roles on the mechanics of the endothelium. CCM proteins act as a scaffold, promoting ROCK2 interactions with VE-cadherin and limiting ROCK1 kinase activity. Loss of CCM1 (also known as KRIT1) produces excessive ROCK1-dependent actin stress fibers and destabilizes intercellular junctions. Silencing of ROCK1 but not ROCK2 restores the adhesive and mechanical homeostasis of CCM1 and CCM2-depleted endothelial monolayers, and rescues the cardiovascular defects of ccm1 mutant zebrafish embryos. Conversely, knocking down Rock2 but not Rock1 in wild-type zebrafish embryos generates defects reminiscent of the ccm1 mutant phenotypes. Our study uncovers the role of the CCM1-CCM2 complex in controlling ROCK1 and ROCK2 to preserve endothelial integrity and drive heart morphogenesis. Moreover, it solely identifies the ROCK1 isoform as a potential therapeutic target for the CCM disease.

KRIT1 as a possible new player in melanoma aggressiveness.

Krev interaction trapped protein 1 (KRIT1) is a scaffold protein known to form functional complexes with distinct proteins, including Malcavernin, PDCD10, Rap1 and others. It appears involved in several cellular signaling pathways and exerts a protective role against inflammation and oxidative stress. KRIT1 has been studied as a regulator of endothelial cell functions and represents a determinant in the pathogenesis of Cerebral Cavernous Malformation (CCM), a cerebrovascular disease characterized by the formation of clusters of abnormally dilated and leaky blood capillaries, which predispose to seizures, neurological deficits and intracerebral hemorrhage. Although KRIT1 is ubiquitously expressed, few studies have described its involvement in pathologies other than CCM including cancer. Cutaneous melanoma represents the most fatal skin cancer due to its high metastatic propensity. Despite the numerous efforts made to define the signaling pathways activated during melanoma progression, the molecular mechanisms at the basis of melanoma growth, phenotype plasticity and resistance to therapies are still under investigation.

The cerebral cavernous malformation disease causing gene KRIT1 participates in intestinal epithelial barrier maintenance and regulation.

Epithelial barrier maintenance and regulation requires an intact perijunctional actomyosin ring underneath the cell-cell junctions. By searching for known factors affecting the actin cytoskeleton, we identified Krev interaction trapped protein 1 (KRIT1) as a major regulator for epithelial barrier function through multiple mechanisms. KRIT1 is expressed in both small intestinal and colonic epithelium, and KRIT1 knockdown in differentiated Caco-2 intestinal epithelium decreases epithelial barrier function and increases cation selectivity. KRIT1 knockdown abolished Rho-associated protein kinase-induced and myosin II motor inhibitor-induced barrier loss by limiting both small and large molecule permeability but did not affect myosin light chain kinase-induced increases in epithelial barrier function. These data suggest that KRIT1 participates in Rho-associated protein kinase- and myosin II motor-dependent (but not myosin light chain kinase-dependent) epithelial barrier regulation. KRIT1 knockdown exacerbated low-dose TNF-induced barrier loss, along with increased cleaved caspase-3 production. Both events are blocked by pan-caspase inhibition, indicating that KRIT1 regulates TNF-induced barrier loss through limiting epithelial apoptosis. These data indicate that KRIT1 controls epithelial barrier maintenance and regulation through multiple pathways, suggesting that KRIT1 mutation in cerebral cavernous malformation disease may alter epithelial function and affect human health.-Wang, Y., Li, Y., Zou, J., Polster, S. P., Lightle, R., Moore, T., Dimaano, M., He, T.-C., Weber, C. R., Awad, I. A., Shen, L. The cerebral cavernous malformation disease causing gene KRIT1 participates in intestinal epithelial barrier maintenance and regulation.

Cutaneous findings of familial cerebral cavernous malformation syndrome due to the common Hispanic mutation.

Familial cerebral cavernous malformations due to the common Hispanic mutation (FCCM1-CHM) is an endemic condition among the Hispanic population of the Southwestern United States associated with significant morbidity and mortality. Cutaneous vascular malformations (CVMs) can be found in individuals with FCCM1-CHM, but their morphology, prevalence, and association with cerebral cavernous malformations (CCMs) has not been well characterized. A cross-sectional study of 140 individuals with confirmed FCCM1-CHM was performed with statistical analyses of CVM, CCM, and patient characteristics. We then compared these findings to other cohorts with Familial cerebral cavernous malformations (FCCM) due to other mutations. We observed a higher overall prevalence and a different predominant morphological subtype of CVM compared to previous FCCM cohorts. While the number of CVMs was not a reliable indicator of the number of CCMs present, each person with one or more CVMs had evidence of central nervous system (CNS) disease. Awareness of the morphology of these cutaneous lesions can aid in the diagnosis of individuals with FCCM-CHM in Hispanic patients or those with family history of CCM.

Rho Kinase Inhibition Blunts Lesion Development and Hemorrhage in Murine Models of Aggressive Pdcd10/Ccm3 Disease.

Background and Purpose- Previously, murine models Krit1 +/- Msh2 -/- and Ccm2 +/- Trp53 -/- showed a reduction or no effect on cerebral cavernous malformation (CCM) burden and favorable effects on lesional hemorrhage by the robust Rock (Rho-associated protein kinase) inhibitor fasudil and by simvastatin (a weak pleiotropic inhibitor of Rock). Herein, we concurrently investigated treatment of the more aggressive Pdcd10/Ccm3 model with fasudil, simvastatin, and higher dose atorvastatin to determined effectiveness of Rock inhibition. Methods- The murine models, Pdcd10 +/- Trp53 -/- and Pdcd10 +/- Msh2 -/-, were contemporaneously treated from weaning to 5 months of age with fasudil (100 mg/kg per day in drinking water, n=9), simvastatin (40 mg/kg per day in chow, n=11), atorvastatin (80 mg/kg per day in chow, n=10), or with placebo (n=16). We assessed CCM volume in mouse brains by microcomputed tomography. Lesion burden was calculated as lesion volume normalized to total brain volume. We analyzed chronic hemorrhage in CCM lesions by quantitative intensity of Perls staining in brain sections. Results- The Pdcd10 +/- Trp53 -/- /Msh2 -/- models showed a mean CCM lesion burden per mouse reduction from 0.0091 in placebos to 0.0042 ( P=0.027) by fasudil, and to 0.0047 ( P=0.025) by atorvastatin treatment, but was not changed significantly by simvastatin. Hemorrhage intensity per brain was commensurately decreased by Rock inhibition. Conclusions- These results support the exploration of proof of concept effect of high-dose atorvastatin on human CCM disease for potential therapeutic testing.

Phenotypic characterization of murine models of cerebral cavernous malformations.

Cerebral cavernous malformations (CCMs) are clusters of dilated capillaries that affect around 0.5% of the population. CCMs exist in two forms, sporadic and familial. Mutations in three documented genes, KRIT1(CCM1), CCM2, and PDCD10(CCM3), cause the autosomal dominant form of the disease, and somatic mutations in these same genes underlie lesion development in the brain. Murine models with constitutive or induced loss of respective genes have been applied to study disease pathobiology and therapeutic manipulations. We aimed to analyze the phenotypic characteristic of two main groups of models, the chronic heterozygous models with sensitizers promoting genetic instability, and the acute neonatal induced homozygous knockout model. Acute model mice harbored a higher lesion burden than chronic models, more localized in the hindbrain, and largely lacking iron deposition and inflammatory cell infiltrate. The chronic model mice showed a lower lesion burden localized throughout the brain, with significantly greater perilesional iron deposition, immune B- and T-cell infiltration, and less frequent junctional protein immunopositive endothelial cells. Lesional endothelial cells in both models expressed similar phosphorylated myosin light chain immunopositivity indicating Rho-associated protein kinase activity. These data suggest that acute models are better suited to study the initial formation of the lesion, while the chronic models better reflect lesion maturation, hemorrhage, and inflammatory response, relevant pathobiologic features of the human disease.