RESUMO
KRIT1 is a 75 kDa scaffolding protein which regulates endothelial cell phenotype by limiting the response to inflammatory stimuli and maintaining a quiescent and stable endothelial barrier. Loss-of-function mutations in KRIT1 lead to the development of cerebral cavernous malformations (CCM), a disease marked by the formation of abnormal blood vessels which exhibit a loss of barrier function, increased endothelial proliferation, and altered gene expression. While many advances have been made in our understanding of how KRIT1, and the functionally related proteins CCM2 and PDCD10, contribute to the regulation of blood vessels and the vascular barrier, some important open questions remain. In addition, KRIT1 is widely expressed and KRIT1 and the other CCM proteins have been shown to play important roles in non-endothelial cell types and tissues, which may or may not be related to their role as pathogenic originators of CCM. In this review, we discuss some of the unsettled questions regarding the role of KRIT1 in vascular physiology and discuss recent advances that suggest this ubiquitously expressed protein may have a role beyond the endothelial cell.
Assuntos
Células Endoteliais , Hemangioma Cavernoso do Sistema Nervoso Central , Proteína KRIT1 , Humanos , Proteína KRIT1/metabolismo , Proteína KRIT1/genética , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Animais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , MutaçãoRESUMO
BACKGROUND: Cerebral vascular malformations (CCMs) are primarily found within the brain, where they result in increased risk for stroke, seizures, and focal neurological deficits. The unique feature of the brain vasculature is the blood-brain barrier formed by the brain neurovascular unit. Recent studies suggest that loss of CCM genes causes disruptions of blood-brain barrier integrity as the inciting events for CCM development. CCM lesions are proposed to be initially derived from a single clonal expansion of a subset of angiogenic venous capillary endothelial cells (ECs) and respective resident endothelial progenitor cells (EPCs). However, the critical signaling events in the subclass of brain ECs/EPCs for CCM lesion initiation and progression are unclear. METHODS: Brain EC-specific CCM3-deficient (Pdcd10BECKO) mice were generated by crossing Pdcd10fl/fl mice with Mfsd2a-CreERT2 mice. Single-cell RNA-sequencing analyses were performed by the chromium single-cell platform (10× genomics). Cell clusters were annotated into EC subtypes based on visual inspection and GO analyses. Cerebral vessels were visualized by 2-photon in vivo imaging and tissue immunofluorescence analyses. Regulation of mTOR (mechanistic target of rapamycin) signaling by CCM3 and Cav1 (caveolin-1) was performed by cell biology and biochemical approaches. RESULTS: Single-cell RNA-sequencing analyses from P10 Pdcd10BECKO mice harboring visible CCM lesions identified upregulated CCM lesion signature and mitotic EC clusters but decreased blood-brain barrier-associated EC clusters. However, a unique EPC cluster with high expression levels of stem cell markers enriched with mTOR signaling was identified from early stages of the P6 Pdcd10BECKO brain. Indeed, mTOR signaling was upregulated in both mouse and human CCM lesions. Genetic deficiency of Raptor (regulatory-associated protein of mTOR), but not of Rictor (rapamycin-insensitive companion of mTOR), prevented CCM lesion formation in the Pdcd10BECKO model. Importantly, the mTORC1 (mTOR complex 1) pharmacological inhibitor rapamycin suppressed EPC proliferation and ameliorated CCM pathogenesis in Pdcd10BECKO mice. Mechanistic studies suggested that Cav1/caveolae increased in CCM3-depleted EPC-mediated intracellular trafficking and complex formation of the mTORC1 signaling proteins. CONCLUSIONS: CCM3 is critical for maintaining blood-brain barrier integrity and CCM3 loss-induced mTORC1 signaling in brain EPCs initiates and facilitates CCM pathogenesis.
Assuntos
Células Progenitoras Endoteliais , Hemangioma Cavernoso do Sistema Nervoso Central , Alvo Mecanístico do Complexo 1 de Rapamicina , Transdução de Sinais , Animais , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Camundongos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/irrigação sanguínea , Camundongos Knockout , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Camundongos Endogâmicos C57BL , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genéticaRESUMO
We previously identified talin rod domain-containing protein 1 (TLNRD1) as a potent actin-bundling protein in vitro. Here, we report that TLNRD1 is expressed in the vasculature in vivo. Its depletion leads to vascular abnormalities in vivo and modulation of endothelial cell monolayer integrity in vitro. We demonstrate that TLNRD1 is a component of the cerebral cavernous malformations (CCM) complex through its direct interaction with CCM2, which is mediated by a hydrophobic C-terminal helix in CCM2 that attaches to a hydrophobic groove on the four-helix domain of TLNRD1. Disruption of this binding interface leads to CCM2 and TLNRD1 accumulation in the nucleus and actin fibers. Our findings indicate that CCM2 controls TLNRD1 localization to the cytoplasm and inhibits its actin-bundling activity and that the CCM2-TLNRD1 interaction impacts endothelial actin stress fiber and focal adhesion formation. Based on these results, we propose a new pathway by which the CCM complex modulates the actin cytoskeleton and vascular integrity.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Células Endoteliais da Veia Umbilical Humana , Humanos , Animais , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais/metabolismo , Adesões Focais/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Fibras de Estresse/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Ligação Proteica , Camundongos , Núcleo Celular/metabolismo , TalinaRESUMO
Loss-of-function variants in CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10 genes are identified in the vast majority of familial cases with multiple cerebral cavernous malformations. However, genomic DNA sequencing combined with large rearrangement screening fails to detect a pathogenic variant in 5% of the patients. We report a family with two affected members harboring multiple CCM lesions, one with severe hemorrhages and one asymptomatic. No causative variant was detected using DNA sequencing of the three CCM genes, CNV detection analysis, and RNA sequencing. However, a loss of heterozygosity in CCM2 was observed on cDNA sequences in one of the two affected members, which strongly suggested that this locus might be involved. Whole genome sequencing (WGS) identified a balanced structural variant on chromosome 7 with a breakpoint interrupting the CCM2 gene, preventing normal mRNA synthesis. These data underline the importance of WGS in undiagnosed patients with typical multiple CCM.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Perda de Heterozigosidade , Linhagem , Humanos , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/diagnóstico , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Feminino , Masculino , Adulto , Proteínas de Transporte/genética , Cromossomos Humanos Par 7/genética , DNA Complementar/genética , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Hispânico ou Latino , Proteína KRIT1 , Mutação , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Estudos Transversais , Proteína KRIT1/genética , Hispânico ou Latino/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Projetos Piloto , Água , Fatores Etários , Adulto Jovem , Idoso , Imageamento por Ressonância MagnéticaRESUMO
Cerebral cavernous malformations (CCMs) are abnormally packed blood vessels lined with endothelial cells, that do not exhibit intervening tight junctions, lack muscular and elastic layers and are usually surrounded by hemosiderin and gliosis. CCMs may be sporadic or familial autosomal dominant (FCCMs) caused by loss of function mutations in CCM1 (KRIT1), CCM2 (MGC4607), and CCM3 (PDCD10) genes. In the FCCMs, patients have multiple CCMs, different family members are affected, and developmental venous anomalies are absent. CCMs may be asymptomatic or may manifest with focal neurological deficits with or without associated hemorrhage andseizures. Recent studies identify a digenic "triple-hit" mechanism involving the aquisition of three distinct genetic mutations that culminate in phosphatidylinositol-3-kinase (PIK3CA) gain of function, as the basis for rapidly growing and clinically symptomatic CCMs. The pathophysiology of CCMs involves signaling aberrations in the neurovascular unit, including proliferative dysangiogenesis, blood-brain barrier hyperpermeability, inflammation and immune mediated processes, anticoagulant vascular domain, and gut microbiome-driven mechanisms. Clinical trials are investigating potential therapies, magnetic resonance imaging and plasma biomarkers for hemorrhage and CCMs-related epilepsy, as well as different techniques of neuronavigation and neurosonology to guide surgery in order to minimize post-operatory morbidity and mortality. This review addresses the recent data about the natural history, genetics, neuroimaging and therapeutic approaches for CCMs.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Humanos , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/terapia , MutaçãoRESUMO
Molecular characterization of vascular anomalies has revealed that affected endothelial cells (ECs) harbor gain-of-function (GOF) mutations in the gene encoding the catalytic α subunit of PI3Kα (PIK3CA). These PIK3CA mutations are known to cause solid cancers when occurring in other tissues. PIK3CA-related vascular anomalies, or "PIKopathies," range from simple, i.e., restricted to a particular form of malformation, to complex, i.e., presenting with a range of hyperplasia phenotypes, including the PIK3CA-related overgrowth spectrum. Interestingly, development of PIKopathies is affected by fluid shear stress (FSS), a physiological stimulus caused by blood or lymph flow. These findings implicate PI3K in mediating physiological EC responses to FSS conditions characteristic of lymphatic and capillary vessel beds. Consistent with this hypothesis, increased PI3K signaling also contributes to cerebral cavernous malformations, a vascular disorder that affects low-perfused brain venous capillaries. Because the GOF activity of PI3K and its signaling partners are excellent drug targets, understanding PIK3CA's role in the development of vascular anomalies may inform therapeutic strategies to normalize EC responses in the diseased state. This Review focuses on PIK3CA's role in mediating EC responses to FSS and discusses current understanding of PIK3CA dysregulation in a range of vascular anomalies that particularly affect low-perfused regions of the vasculature. We also discuss recent surprising findings linking increased PI3K signaling to fast-flow arteriovenous malformations in hereditary hemorrhagic telangiectasias.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases , Malformações Vasculares , Humanos , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Animais , Malformações Vasculares/genética , Malformações Vasculares/patologia , Malformações Vasculares/fisiopatologia , Malformações Vasculares/metabolismo , Malformações Vasculares/enzimologia , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Células Endoteliais/metabolismo , Estresse Mecânico , Mutação com Ganho de Função , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/fisiopatologia , Hemangioma Cavernoso do Sistema Nervoso Central/patologiaRESUMO
BACKGROUND: Cerebral cavernous malformation (CCM) is a low-flow, bleeding-prone vascular disease that can cause cerebral hemorrhage, seizure and neurological deficits. Its inheritance mode includes sporadic or autosomal dominant inheritance with incomplete penetrance, namely sporadic CCM (SCCM) and familial CCM. SCCM is featured by single lesion and single affection in a family. Among CCM patients especially SCCM, the pathogenesis of the corresponding phenotypes and pathological features or candidate genes have not been fully elucidated yet. METHODS: Here, we performed in-depth single-cell RNA sequencing (scRNA-Seq) and bulk assay for transposase-accessible chromatin sequencing (ATAC-Seq) in SCCM and control patients. Further validation was conducted for the gene of interest using qPCR and RNA in situ hybridization (RNA FISH) techniques to provide further atlas and evidence for SCCM generative process. RESULTS: We identified six cell types in the SCCM and control vessels and found that the expression of NEK1, RNPC3, FBRSL1, IQGAP2, MCUB, AP3B1, ESCO1, MYO9B and PVT1 were up-regulated in SCCM tissues. Among the six cell types, we found that compared with control conditions, PVT1 showed a rising peak which followed the pseudo-time axis in endothelial cell clusters of SCCM samples, while showed an increasing trend in smooth muscle cell clusters of SCCM samples. Further experiments indicated that, compared with the control vessels, PVT1 exhibited significantly elevated expression in SCCM samples. CONCLUSION: In SCCM conditions, We found that in the process of development from control to lesion conditions, PVT1 showed a rising peak in endothelial cells and showed an increasing trend in smooth muscle cells at the same time. Overall, there was a significantly elevated expression of NEK1, RNPC3, FBRSL1, IQGAP2, MCUB, AP3B1, ESCO1, MYO9B and PVT1 in SCCM specimens compared to control samples.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Análise de Célula Única , Humanos , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Análise de Célula Única/métodos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Células Endoteliais/metabolismo , Células Endoteliais/patologiaRESUMO
BACKGROUND: Heterogeneity in the severity of cerebral cavernous malformations (CCMs) disease, including brain bleedings and thrombosis that cause neurological disabilities in patients, suggests that environmental, genetic, or biological factors act as disease modifiers. Still, the underlying mechanisms are not entirely understood. Here, we report that mild hypoxia accelerates CCM disease by promoting angiogenesis, neuroinflammation, and vascular thrombosis in the brains of CCM mouse models. METHODS: We used genetic studies, RNA sequencing, spatial transcriptome, micro-computed tomography, fluorescence-activated cell sorting, multiplex immunofluorescence, coculture studies, and imaging techniques to reveal that sustained mild hypoxia via the CX3CR1-CX3CL1 (CX3C motif chemokine receptor 1/chemokine [CX3C motif] ligand 1) signaling pathway influences cell-specific neuroinflammatory interactions, contributing to heterogeneity in CCM severity. RESULTS: Histological and expression profiles of CCM neurovascular lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) in male and female mice found that sustained mild hypoxia (12% O2, 7 days) accelerates CCM disease. Our findings indicate that a small reduction in oxygen levels can significantly increase angiogenesis, neuroinflammation, and thrombosis in CCM disease by enhancing the interactions between endothelium, astrocytes, and immune cells. Our study indicates that the interactions between CX3CR1 and CX3CL1 are crucial in the maturation of CCM lesions and propensity to CCM immunothrombosis. In particular, this pathway regulates the recruitment and activation of microglia and other immune cells in CCM lesions, which leads to lesion growth and thrombosis. We found that human CX3CR1 variants are linked to lower lesion burden in familial CCMs, proving it is a genetic modifier in human disease and a potential marker for aggressiveness. Moreover, monoclonal blocking antibody against CX3CL1 or reducing 1 copy of the Cx3cr1 gene significantly reduces hypoxia-induced CCM immunothrombosis. CONCLUSIONS: Our study reveals that interactions between CX3CR1 and CX3CL1 can modify CCM neuropathology when lesions are accelerated by environmental hypoxia. Moreover, a hypoxic environment or hypoxia signaling caused by CCM disease influences the balance between neuroinflammation and neuroprotection mediated by CX3CR1-CX3CL1 signaling. These results establish CX3CR1 as a genetic marker for patient stratification and a potential predictor of CCM aggressiveness.
Assuntos
Receptor 1 de Quimiocina CX3C , Quimiocina CX3CL1 , Modelos Animais de Doenças , Hemangioma Cavernoso do Sistema Nervoso Central , Transdução de Sinais , Animais , Feminino , Humanos , Masculino , Camundongos , Quimiocina CX3CL1/metabolismo , Quimiocina CX3CL1/genética , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Hipóxia/metabolismo , Hipóxia/complicações , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/metabolismo , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Doenças Neuroinflamatórias/genéticaRESUMO
Linking variants from genome-wide association studies (GWAS) to underlying mechanisms of disease remains a challenge1-3. For some diseases, a successful strategy has been to look for cases in which multiple GWAS loci contain genes that act in the same biological pathway1-6. However, our knowledge of which genes act in which pathways is incomplete, particularly for cell-type-specific pathways or understudied genes. Here we introduce a method to connect GWAS variants to functions. This method links variants to genes using epigenomics data, links genes to pathways de novo using Perturb-seq and integrates these data to identify convergence of GWAS loci onto pathways. We apply this approach to study the role of endothelial cells in genetic risk for coronary artery disease (CAD), and discover 43 CAD GWAS signals that converge on the cerebral cavernous malformation (CCM) signalling pathway. Two regulators of this pathway, CCM2 and TLNRD1, are each linked to a CAD risk variant, regulate other CAD risk genes and affect atheroprotective processes in endothelial cells. These results suggest a model whereby CAD risk is driven in part by the convergence of causal genes onto a particular transcriptional pathway in endothelial cells. They highlight shared genes between common and rare vascular diseases (CAD and CCM), and identify TLNRD1 as a new, previously uncharacterized member of the CCM signalling pathway. This approach will be widely useful for linking variants to functions for other common polygenic diseases.
Assuntos
Doença da Artéria Coronariana , Células Endoteliais , Estudo de Associação Genômica Ampla , Hemangioma Cavernoso do Sistema Nervoso Central , Humanos , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Predisposição Genética para Doença/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Polimorfismo de Nucleotídeo Único , Epigenômica , Transdução de Sinais/genética , Herança MultifatorialRESUMO
BACKGROUND: Cerebral Cavernous Malformations (CCMs) are brain vascular abnormalities associated with an increased risk of hemorrhagic strokes. Familial CCMs result from autosomal dominant inheritance involving three genes: KRIT1 (CCM1), MGC4607 (CCM2), and PDCD10 (CCM3). CCM1 and CCM3 form the CCM Signal Complex (CSC) by binding to CCM2. Both CCM1 and CCM2 exhibit cellular heterogeneity through multiple alternative spliced isoforms, where exons from the same gene combine in diverse ways, leading to varied mRNA transcripts. Additionally, both demonstrate nucleocytoplasmic shuttling between the nucleus and cytoplasm, suggesting their potential role in gene expression regulation as transcription factors (TFs). Due to the accumulated data indicating the cellular localization of CSC proteins in the nucleus and their interaction with progesterone receptors, which serve dual roles as both cellular signaling components and TFs, a question has arisen regarding whether CCMs could also function in both capacities like progesterone receptors. METHODS: To investigate this potential, we employed our proprietary deep-learning (DL)-based algorithm, specifically utilizing a biased-Support Vector Machine (SVM) model, to explore the plausible cellular function of any of the CSC proteins, particularly focusing on CCM gene isoforms with nucleocytoplasmic shuttling, acting as TFs in gene expression regulation. RESULTS: Through a comparative DL-based predictive analysis, we have effectively discerned a collective of 11 isoforms across all CCM proteins (CCM1-3). Additionally, we have substantiated the TF functionality of 8 isoforms derived from CCM1 and CCM2 proteins, marking the inaugural identification of CCM isoforms in the role of TFs. CONCLUSIONS: This groundbreaking discovery directly challenges the prevailing paradigm, which predominantly emphasizes the involvement of CSC solely in endothelial cellular functions amid various potential cellular signal cascades during angiogenesis.
Assuntos
Aprendizado Profundo , Hemangioma Cavernoso do Sistema Nervoso Central , Humanos , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Receptores de Progesterona/metabolismo , Proteínas de Transporte/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Cerebral cavernous malformation (CCM) is a hemorrhagic neurovascular disease with no currently available therapeutics. Prior evidence suggests that different cell types may play a role in CCM pathogenesis. The contribution of each cell type to the dysfunctional cellular crosstalk remains unclear. Herein, RNA-seq was performed on fluorescence-activated cell sorted endothelial cells (ECs), pericytes, and neuroglia from CCM lesions and non-lesional brain tissue controls. Differentially Expressed Gene (DEG), pathway and Ligand-Receptor (LR) analyses were performed to characterize the dysfunctional genes of respective cell types within CCMs. Common DEGs among all three cell types were related to inflammation and endothelial-to-mesenchymal transition (EndMT). DEG and pathway analyses supported a role of lesional ECs in dysregulated angiogenesis and increased permeability. VEGFA was particularly upregulated in pericytes. Further pathway and LR analyses identified vascular endothelial growth factor A/ vascular endothelial growth factor receptor 2 signaling in lesional ECs and pericytes that would result in increased angiogenesis. Moreover, lesional pericytes and neuroglia predominantly showed DEGs and pathways mediating the immune response. Further analyses of cell specific gene alterations in CCM endorsed potential contribution to EndMT, coagulation, and a hypoxic microenvironment. Taken together, these findings motivate mechanistic hypotheses regarding non-endothelial contributions to lesion pathobiology and may lead to novel therapeutic targets. Video Abstract.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Fator A de Crescimento do Endotélio Vascular , Humanos , Fator A de Crescimento do Endotélio Vascular/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Células Endoteliais , Perfilação da Expressão Gênica , Transcriptoma , Microambiente TumoralRESUMO
Three genes are associated with cerebral cavernous malformations (CCMs): CCM1, CCM2 and CCM3. These genes participate in microvascular angiogenesis, cell-to-cell junctions, migration and apoptosis. We evaluated the expression in vivo of CCM genes in primary tumors and metastastases in a murine model of metastatic breast carcinoma. We used cell lines obtained from metastasis of 4T1, 4TLM and 4THM breast cancer to liver and heart. These cells were injected into the mammary ridge of Balb/C female mice. After 27 days, the primary tumors, liver and lung were removed and CCM proteins were assessed using immunohistochemistry and western blot analysis. CCM proteins were expressed in primary tumor tissues of all tumor-injected animals; however, no CCM protein was expressed in metastatic tumor cells that migrated into other tissues. CCM proteins still were observed in the lung and liver tissue cells. Our findings suggest that CCM proteins are present during primary tumor formation, but when these cells develop metastatic potential, they lose CCM protein expression. CCM protein expression was lost or reduced in metastatic tissues compared to the primary tumor, which indicates that CCM proteins might participate in tumorigenesis and metastasis.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Neoplasias , Feminino , Animais , Camundongos , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Membrana/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Cerebral cavernous malformation (CCM) is a vascular disease that affects the central nervous system, which familial form is due to autosomal dominant mutations in the genes KRIT1(CCM1), MGC4607(CCM2), and PDCD10(CCM3). Patients affected by the PDCD10 mutations usually have the onset of symptoms at an early age and a more aggressive phenotype. The aim of this study is to investigate the molecular mechanism involved with CCM3 disease pathogenesis. Herein, we report two typical cases of CCM3 phenotype and compare the clinical and neuroradiological findings with five patients with a familial form of KRIT1 or CCM2 mutations and six patients with a sporadic form. In addition, we evaluated the PDCD10 gene expression by qPCR and developed a bioinformatic pipeline to understand the structural changes of mutations. The two CCM3 patients had an early onset of symptoms and a high lesion burden. Furthermore, the sequencing showed that Patient 1 had a frameshift mutation in c.222delT; p.(Asn75Thrfs*14) that leads to lacking the last 124 C-terminal amino acids on its primary structure and Patient 2 had a variant on the splicing site region c.475-2A > G. The mRNA expression was fourfold lower in both patients with PDCD10 mutation. Using in silico analysis, we identify that the frameshift mutation transcript lacks the C-terminal FAT-homology domain compared to the wild-type PDCD10 and preserves the N-terminal dimerization domain. The two patients studied here allow estimating the potential impact of mutations in clinical interpretation as well as support to better understand the mechanism and pathogenesis of CCM3.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Humanos , Sistema Nervoso Central , Hemangioma Cavernoso do Sistema Nervoso Central/diagnóstico por imagem , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Mutação/genética , Fenótipo , Proteínas Proto-Oncogênicas/genéticaRESUMO
Cavernous-type malformations are venous lesions that occur in multiple locations throughout the body, and when present in the CNS, they have canonically been referred to as cavernomas, cavernous angiomas, and cerebral cavernous malformations. Herein all these lesions are referred to as "cavernous venous malformations" (CavVMs), which is congruent with the current International Society for the Study of Vascular Anomalies classification system. Even though histologically similar, depending on their location relative to the dura mater, these malformations can have different features. In Part 1 of this review, the authors discuss and review pertinent clinical knowledge with regard to CavVMs as influenced by anatomical location, starting with the dural and extradural malformations. They particularly emphasize dural CavVMs (including those in the cavernous sinus), orbital CavVMs, and spinal CavVMs. The genetic and histopathological features of CavVMs in these locations are reviewed, and commonalities in their presumed mechanisms of pathogenesis support the authors' conceptualization of a spectrum of a single disease entity. Illustrative cases for each subtype are presented, and the pathophysiological and genetic features linking dural and extradural to intradural CavVMs are examined. A new classification is proposed to segregate CavVMs based on the location from which they arise, which guides their natural history and treatment.
Assuntos
Malformações Vasculares do Sistema Nervoso Central , Hemangioma Cavernoso do Sistema Nervoso Central , Hemangioma Cavernoso , Humanos , Sistema Nervoso Central/patologia , Hemangioma Cavernoso do Sistema Nervoso Central/diagnóstico por imagem , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/cirurgia , Malformações Vasculares do Sistema Nervoso Central/diagnóstico por imagem , Malformações Vasculares do Sistema Nervoso Central/cirurgia , Malformações Vasculares do Sistema Nervoso Central/patologia , Veias/patologiaRESUMO
BACKGROUND: Cerebral Cavernous Malformation (CCM) is a rare cerebrovascular disease, characterized by the presence of multiple vascular malformations that may result in intracerebral hemorrhages (ICHs), seizure(s), or focal neurological deficits (FND). Familial CCM (fCCM) is due to loss of function mutations in one of the three independent genes KRIT1 (CCM1), Malcavernin (CCM2), or Programmed Cell death 10 (PDCD10/CCM3). The aim of this study was to identify plasma protein biomarkers of fCCM to assess the severity of the disease and predict its progression. METHODS: Here, we have investigated plasma samples derived from n = 71 symptomatic fCCM patients (40 female/31 male) and n = 17 healthy donors (HD) (9 female/8 male) of the Phase 1/2 Treat_CCM trial, using multiplexed protein profiling approaches. FINDINGS: Biomarkers as sCD14 (p = 0.00409), LBP (p = 0.02911), CXCL4 (p = 0.038), ICAM-1 (p = 0.02013), ANG2 (p = 0.026), CCL5 (p = 0.00403), THBS1 (p = 0.0043), CRP (p = 0.0092), and HDL (p = 0.027), were significantly different in fCCM compared to HDs. Of note, sENG (p = 0.011), THBS1 (p = 0.011) and CXCL4 (p = 0.011), were correlated to CCM genotype. sROBO4 (p = 0.014), TM (p = 0.026) and CRP (p = 0.040) were able to predict incident adverse clinical events, such as ICH, FND or seizure. GDF-15, FLT3L, CXCL9, FGF-21 and CDCP1, were identified as predictors of the formation of new MRI-detectable lesions over 2-year follow-up. Furthermore, the functional relevance of ang2, thbs1, robo4 and cdcp1 markers was validated by zebrafish pre-clinical model of fCCM. INTERPRETATION: Overall, our study identifies a set of biochemical parameters to predict CCM progression, suggesting biological interpretations and potential therapeutic approaches to CCM disease. FUNDING: Italian Medicines Agency, Associazione Italiana per la Ricerca sul Cancro (AIRC), ERC, Leducq Transatlantic Network of Excellence, Swedish Research Council.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Animais , Humanos , Masculino , Feminino , Hemangioma Cavernoso do Sistema Nervoso Central/etiologia , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Associadas aos Microtúbulos/genética , Peixe-Zebra/metabolismo , Biomarcadores , Convulsões , Antígenos de Neoplasias , Moléculas de Adesão CelularRESUMO
Intracranial vascular malformations manifest on a continuum ranging from predominantly arterial to predominantly venous in pathology. Cerebral cavernous malformations (CCMs) are capillary malformations that exist at the midpoint of this continuum. The axon guidance factor Ephrin B2 and its receptor EphB4 are critical regulators of vasculogenesis in the developing central nervous system. Ephrin B2/EphB4 dysregulation has been implicated in the pathogenesis of arterial-derived arteriovenous malformations and vein-based vein of Galen malformations. Increasing evidence supports the hypothesis that aberrant Ephrin B2/EphB4 signaling may contribute to developing vascular malformations, but their role in CCMs remains largely uncharacterized. Evidence of Ephrin dysregulation in CCMs would be important to establish a common link in the pathogenic spectrum of EphrinB2/Ephb4 dysregulation. By studying patient-derived primary CCM endothelial cells (CCMECs), we established that CCMECs are functionally distinct from healthy endothelial cell controls; CCMECs demonstrated altered patterns of migration, motility, and impaired tube formation. In addition to the altered phenotype, the CCMECs also displayed an increased ratio of EphrinB2/EphB4 compared to the healthy endothelial control cells. Furthermore, whole exome sequencing identified mutations in both EphrinB2 and EphB4 in the CCMECs. These findings identify functional alterations in the EphrinB2/EphB4 ratio as a feature linking pathophysiology across the spectrum of arterial, capillary, and venous structural malformations in the central nervous system while revealing a putative therapeutic target.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Receptor EphB2 , Receptor EphB4 , Humanos , Receptor EphB4/genética , Receptor EphB2/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Células Endoteliais/patologia , Cultura Primária de Células , Sequenciamento do Exoma , Masculino , Feminino , Pré-Escolar , Criança , AdolescenteRESUMO
Cerebral Cavernous Malformations (CCMs) are vascular malformations of the central nervous system which can lead to moderate to severe neurological phenotypes in patients. A majority of CCM lesions are driven by a cancer-like three-hit mutational mechanism, including a somatic, activating mutation in the oncogene PIK3CA, as well as biallelic loss-of-function mutations in a CCM gene. However, standard sequencing approaches often fail to yield a full complement of pathogenic mutations in many CCMs. We suggest this reality reflects the limited sensitivity to identify low-frequency variants and the presence of mutations undetectable with bulk short-read sequencing. Here we report a single-nucleus DNA-sequencing approach that leverages the underlying biology of CCMs to identify lesions with somatic loss-of-heterozygosity, a class of such hidden mutations. We identify an alternative genetic mechanism for CCM pathogenesis and establish a method that can be repurposed to investigate the genetic underpinning of other disorders with multiple somatic mutations.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Humanos , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Proteína KRIT1/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Reguladoras de Apoptose/genética , Mutação , Análise de Sequência de DNARESUMO
BACKGROUND: Cerebral cavernous malformations (CCMs) are vascular malformations that frequently cause stroke. CCMs arise due to loss of function in one of the genes that encode the CCM complex, a negative regulator of MEKK3-KLF2/4 signaling in vascular endothelial cells. Gain-of-function mutations in PIK3CA (encoding the enzymatic subunit of the PI3K (phosphoinositide 3-kinase) pathway associated with cell growth) synergize with CCM gene loss-of-function to generate rapidly growing lesions. METHODS: We recently developed a model of CCM formation that closely reproduces key events in human CCM formation through inducible CCM loss-of-function and PIK3CA gain-of-function in mature mice. In the present study, we use this model to test the ability of rapamycin, a clinically approved inhibitor of the PI3K effector mTORC1, to treat rapidly growing CCMs. RESULTS: We show that both intraperitoneal and oral administration of rapamycin arrests CCM growth, reduces perilesional iron deposition, and improves vascular perfusion within CCMs. CONCLUSIONS: Our findings further establish this adult CCM model as a valuable preclinical model and support clinical testing of rapamycin to treat rapidly growing human CCMs.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Animais , Humanos , Adulto , Camundongos , Hemangioma Cavernoso do Sistema Nervoso Central/tratamento farmacológico , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Células Endoteliais/metabolismo , Sirolimo/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/metabolismoRESUMO
OBJECTIVE: Cerebral cavernous malformation (CCM) is a subtype of the vascular malformations found within the cerebral cortex. Although rare and usually discovered incidentally, these vascular abnormalities can predispose patients to spontaneous cerebral hemorrhage and subsequently lead to a myriad of neurological symptoms at presentation such as seizures and other focal neurological deficits. Although the symptoms and presentations of CCM have been adequately described in the adult population, disease characteristics and outcomes have not been extensively described in the pediatric population. Furthermore, the etiology of CCM-e.g., familial versus sporadic disease, as well as the risk factors for hemorrhage and neurological deficits and predictors of clinical and surgical outcomes-has not been adequately explored in the pediatric population. The current study attempts to classify and characterize differences in the clinical presentation, characteristics, and outcomes of CCMs between familial and sporadic cases within the pediatric population. METHODS: A retrospective review identified 131 pediatric patients with radiographically confirmed diagnosis of CCM. All pertinent demographic and clinical variables were collected. CCM lesions were characterized using T2-weighted and susceptibility-weighted angiography (SWAN) MRI. Statistical analysis was conducted using the t-test for continuous variables, whereas categorical variables were analyzed with the Fisher exact test or chi-square test. Multivariate analysis was performed using a Cox proportional hazards model with R version 4.2.0. RESULTS: This retrospective study identified 131 pediatric CCM patients with a mean age of 8.4 years, and 54% (n = 71) were male. Twenty-seven percent (n = 35) were identified as cases with familial CCM, with the remainder classified as sporadic. The most common symptoms at presentation included generalized symptoms (headaches, nausea, and vomiting) or seizures, with a large proportion of patients also presenting as asymptomatic. No significant differences were observed in severity of symptoms between patients harboring different forms of the disease. Patients with familial CCM were noted to have a larger lesion size on average (5.26 cm3 vs 1.6 cm3, p = 0.047). These patients also had a shorter progression-free follow-up interval, with 50% of patients showing progression by 888 days, compared with only 15% of sporadic CCM patients during the same period (p = 0.0019). Familial etiology of the disease and larger average lesion volume were independent, significant predictors of disease progression (p = 0.001, HR 3.29, 95% CI 1.65-6.54) and future hemorrhage (p = 0.023, HR 1.1, 95% CI 1.01-1.10), respectively. CONCLUSIONS: Familial and sporadic CCMs tend to present with similar characteristics within the pediatric population. Patients with the familial form of the disease have an increased risk of progressive disease in terms of further hemorrhagic events.