Molecular and Cellular Characterization of Primary Endothelial Cells from a Familial Cavernomatosis Patient.

Cerebral cavernous malformation (CCM) or familial cavernomatosis is a rare, autosomal dominant, inherited disease characterized by the presence of vascular malformations consisting of blood vessels with an abnormal structure in the form of clusters. Based on the altered gene (CCM1/Krit1, CCM2, CCM3) and its origin (spontaneous or familial), different types of this disease can be found. In this work we have isolated and cultivated primary endothelial cells (ECs) from peripheral blood of a type 1 CCM patient. Differential functional and gene expression profiles of these cells were analyzed and compared to primary ECs from a healthy donor. The mutation of the familial index case consisted of a heterozygous point mutation in the position +1 splicing consensus between exons 15 and 16, causing failure in RNA processing and in the final protein. Furthermore, gene expression analysis by quantitative PCR revealed a decreased expression of genes involved in intercellular junction formation, angiogenesis, and vascular homeostasis. Cell biology analysis showed that CCM1 ECs were impaired in angiogenesis and cell migration. Taken together, the results obtained suggest that the alterations found in CCM1 ECs are already present in the heterozygous condition, suffering from vascular impairment and somewhat predisposed to vascular damage.

CCM signaling complex (CSC) couples both classic and non-classic Progesterone receptor signaling.

BACKGROUND: Breast cancer, the most diagnosed cancer, remains the second leading cause of cancer death in the United States, and excessive Progesterone (PRG) or Mifepristone (MIF) exposure may be at an increased risk for developing breast cancer. PRG exerts its cellular responses through signaling cascades involving classic, non-classic, or combined responses by binding to either classic nuclear PRG receptors (nPRs) or non-classic membrane PRG receptors (mPRs). Currently, the intricate balance and switch mechanisms between these two signaling cascades remain elusive. Three genes, CCM1-3, form the CCM signaling complex (CSC) which mediates multiple signaling cascades. METHODS: Utilizing molecular, cellular, Omics, and systems biology approaches, we analyzed the relationship among the CSC, PRG, and nPRs/mPRs during breast cancer tumorigenesis. RESULTS: We discovered that the CSC plays an essential role in coupling both classic and non-classic PRG signaling pathways by mediating crosstalk between them, forming the CmPn (CSC-mPRs-PRG-nPRs) signaling network. We found that mPR-specific PRG actions (PRG + MIF) play an essential role in this CmPn network during breast cancer tumorigenesis. Additionally, we have identified 4 categories of candidate biomarkers (9 intrinsic, 2 PRG-inducible, 1 PRG-repressive, 1 mPR-specific PRG-repressive, and 2 mPR-responsive) for Luminal-A breast cancers during tumorigenesis and have confirmed the prognostic application of RPL13 and RPL38 as intrinsic biomarkers using a dual validation method. CONCLUSIONS: We have discovered that the CSC plays an essential role in the CmPn signaling network for Luminal-A breast cancers with identification of two intrinsic biomarkers. Video Abstract.

Distant Recurrence of a Cerebral Cavernous Malformation in the Vicinity of a Developmental Venous Anomaly: Case Report of Local Oxy-Inflammatory Events.

BACKGROUND: Cerebral cavernous malformations (CCMs) are a major type of cerebrovascular lesions of proven genetic origin that occur in either sporadic (sCCM) or familial (fCCM) forms, the latter being inherited as an autosomal dominant condition linked to loss-of-function mutations in three known CCM genes. In contrast to fCCMs, sCCMs are rarely linked to mutations in CCM genes and are instead commonly and peculiarly associated with developmental venous anomalies (DVAs), suggesting distinct origins and common pathogenic mechanisms. CASE REPORT: A hemorrhagic sCCM in the right frontal lobe of the brain was surgically excised from a symptomatic 3 year old patient, preserving intact and pervious the associated DVA. MRI follow-up examination performed periodically up to 15 years after neurosurgery intervention demonstrated complete removal of the CCM lesion and no residual or relapse signs. However, 18 years after surgery, the patient experienced acute episodes of paresthesia due to a distant recurrence of a new hemorrhagic CCM lesion located within the same area as the previous one. A new surgical intervention was, therefore, necessary, which was again limited to the CCM without affecting the pre-existing DVA. Subsequent follow-up examination by contrast-enhanced MRI evidenced a persistent pattern of signal-intensity abnormalities in the bed of the DVA, including hyperintense gliotic areas, suggesting chronic inflammatory conditions. CONCLUSIONS: This case report highlights the possibility of long-term distant recurrence of hemorrhagic sCCMs associated with a DVA, suggesting that such recurrence is secondary to focal sterile inflammatory conditions generated by the DVA.

KRIT1 Deficiency Promotes Aortic Endothelial Dysfunction.

Loss-of-function mutations of the gene encoding Krev interaction trapped protein 1 (KRIT1) are associated with the pathogenesis of Cerebral Cavernous Malformation (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries and affecting 0.5% of the human population. However, growing evidence demonstrates that KRIT1 is implicated in the modulation of major redox-sensitive signaling pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, suggesting that its loss-of-function mutations may have pathological effects not limited to CCM disease. The aim of this study was to address whether KRIT1 loss-of-function predisposes to the development of pathological conditions associated with enhanced endothelial cell susceptibility to oxidative stress and inflammation, such as arterial endothelial dysfunction (ED) and atherosclerosis. Silencing of KRIT1 in human aortic endothelial cells (HAECs), coronary artery endothelial cells (HCAECs), and umbilical vein endothelial cells (HUVECs) resulted in increased expression of endothelial proinflammatory adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) and in enhanced susceptibility to tumor necrosis factor alpha (TNF-α)-induced apoptosis. These effects were associated with a downregulation of Notch1 activation that could be rescued by antioxidant treatment, suggesting that they are consequent to altered intracellular redox homeostasis induced by KRIT1 loss-of-function. Furthermore, analysis of the aorta of heterozygous KRIT1+/- mice fed a high-fructose diet to induce systemic oxidative stress and inflammation demonstrated a 1.6-fold increased expression of VCAM-1 and an approximately 2-fold enhanced fat accumulation (7.5% vs 3.6%) in atherosclerosis-prone regions, including the aortic arch and aortic root, as compared to corresponding wild-type littermates. In conclusion, we found that KRIT1 deficiency promotes ED, suggesting that, besides CCM, KRIT1 may be implicated in genetic susceptibility to the development of atherosclerotic lesions.

Abdominal Pain Mimicking a Neurological Disorder: A Case Report of Spinal Cavernous Malformation in a Pediatric Patient.

We present a case of a 13-year-old boy with abdominal pain initially misdiagnosed as gastrointestinal in origin. Despite initial outpatient management, his symptoms rapidly deteriorated, revealing a central-medullary cavernous malformation causing spinal cord compression. This case underscores the importance of a comprehensive pediatric examination and highlights new treatment approaches for spinal cavernous malformations.

Imaging Features and Misdiagnosis of Giant Cerebral Cavernous Malformations.

BACKGROUND: While cerebral cavernous malformations (CCMs) have been extensively described, few reports have described the imaging appearance of giant CCMs (GCCMs). OBJECTIVE: To describe the imaging characteristics of GCCMs and study the reasons for preoperative misdiagnosis. METHODS: We retrospectively analyzed the data of 12 patients (5 men, 7 women; mean age, 35.23 ± 12.64 years) with histopathologically confirmed GCCMs. Two radiologists analyzed the CT (n = 12) and MRI (n = 10) features: location, number, size, shape, boundary, signal intensity, and enhancement. RESULTS: The sellar region, cerebral hemisphere, skull bone, and ventricle were involved in 5, 4, 2, and 1 patients, respectively. Three tumors were irregularly shaped, while nine were oval. Eleven lesions showed slightly high- and/or high-density on CT; 1 lesion appeared as a low-density cyst. Calcifications were found in 11 lesions. Four tumors showed uniform hypointensity on T1-weighted imaging (T1WI) and hyperintense signals on T2-weighted imaging (T2WI). Six tumors showed mixed low-, equal-, and high-intensity signals on T1WI and T2WI. Noticeable contrast enhancement and gradual strengthening were noted on T1WI. Ten lesions showed hemorrhage and hemosiderin deposition. The GCCMs were wrongly diagnosed as cartilage-derived tumors/ meningioma (3 patients); tumor and hematoma (2 patients each); and pituitary tumor/ meningioma, chondroma, chordoma, ependymoma, and macroadenoma (1 patient each). CONCLUSIONS: GCCMs present as an oval mass with slightly high- and/or high-density calcifications on CT and show hemorrhage and hemosiderin accumulation on MRI. Therefore, slightly high- and/or high-density calcification and hemosiderin accumulation are critical clinical characteristics of GCCMs.

The First Potentially Causal Genetic Variant Documented in a Polish Woman with Multiple Cavernous Malformations of the Brain.

Cerebral cavernous malformations (CCMs) are relatively common in the central nervous system. They occur in two forms, sporadic and familial (FCCMs). Three genes are recognized to be associated with FCCM, including CCM1, CCM2, and CCM3, the latter also called PDCD10. In this article, we describe a single-nucleotide variant in the PDCD10 gene in a 23-year-old Polish female with CCM. The NM_007217.4 (PDCD10): c.395+1G>A variant destroys the canonical splice donor site following exon 6. This is the first reported genetically characterized case of CCM (FCCM) in Poland.

Multidrug-Loaded Lipid Nanoemulsions for the Combinatorial Treatment of Cerebral Cavernous Malformation Disease.

Cerebral cavernous malformation (CCM) or cavernoma is a major vascular disease of genetic origin, whose main phenotypes occur in the central nervous system, and is currently devoid of pharmacological therapeutic strategies. Cavernomas can remain asymptomatic during a lifetime or manifest with a wide range of symptoms, including recurrent headaches, seizures, strokes, and intracerebral hemorrhages. Loss-of-function mutations in KRIT1/CCM1 are responsible for more than 50% of all familial cases, and have been clearly shown to affect cellular junctions, redox homeostasis, inflammatory responses, and angiogenesis. In this study, we investigated the therapeutic effects of multidrug-loaded lipid nanoemulsions in rescuing the pathological phenotype of CCM disease. The pro-autophagic rapamycin, antioxidant avenanthramide, and antiangiogenic bevacizumab were loaded into nanoemulsions, with the aim of reducing the major molecular dysfunctions associated with cavernomas. Through Western blot analysis of biomarkers in an in vitro CCM model, we demonstrated that drug-loaded lipid nanoemulsions rescue antioxidant responses, reactivate autophagy, and reduce the effect of pro-angiogenic factors better than the free drugs. Our results show the importance of developing a combinatorial preventive and therapeutic approach to reduce the risk of lesion formation and inhibit or completely revert the multiple hallmarks that characterize the pathogenesis and progression of cavernomas.

KRIT1: A Traffic Warden at the Busy Crossroads Between Redox Signaling and the Pathogenesis of Cerebral Cavernous Malformation Disease.

Significance: KRIT1 (Krev interaction trapped 1) is a scaffolding protein that plays a critical role in vascular morphogenesis and homeostasis. Its loss-of-function has been unequivocally associated with the pathogenesis of Cerebral Cavernous Malformation (CCM), a major cerebrovascular disease of genetic origin characterized by defective endothelial cell-cell adhesion and ensuing structural alterations and hyperpermeability in brain capillaries. KRIT1 contributes to the maintenance of endothelial barrier function by stabilizing the integrity of adherens junctions and inhibiting the formation of actin stress fibers. Recent Advances: Among the multiple regulatory mechanisms proposed so far, significant evidence accumulated over the past decade has clearly shown that the role of KRIT1 in the stability of endothelial barriers, including the blood-brain barrier, is largely based on its involvement in the complex machinery governing cellular redox homeostasis and responses to oxidative stress and inflammation. KRIT1 loss-of-function has, indeed, been demonstrated to cause an impairment of major redox-sensitive mechanisms involved in spatiotemporal regulation of cell adhesion and signaling, which ultimately leads to decreased cell-cell junction stability and enhanced sensitivity to oxidative stress and inflammation. Critical Issues: This review explores the redox mechanisms that influence endothelial cell adhesion and barrier function, focusing on the role of KRIT1 in such mechanisms. We propose that this supports a novel model wherein redox signaling forms the common link between the various pathogenetic mechanisms and therapeutic approaches hitherto associated with CCM disease. Future Directions: A comprehensive characterization of the role of KRIT1 in redox control of endothelial barrier physiology and defense against oxy-inflammatory insults will provide valuable insights into the development of precision medicine strategies. Antioxid. Redox Signal. 38, 496-528.

Contralateral Anterior Interhemispheric-Transcallosal-Transrostral Approach for the Resection of a Subcallosal Cavernous Malformation: A Case Report and an Operative Video.

This case report demonstrates the surgical resection of a cerebral cavernous malformation located in the subcallosal region. The authors present a detailed operative video explaining the steps to successfully remove the lesion through a contralateral interhemispheric-transcallosal-transrostral approach with the patient in lateral decubitus. The surgical procedure was uneventful, and the patient had no postoperative deficits and no residual lesions in a three-month follow-up.

Next-Generation Sequencing Advances the Genetic Diagnosis of Cerebral Cavernous Malformation (CCM).

Cerebral Cavernous Malformation (CCM) is a cerebrovascular disease of genetic origin that predisposes to seizures, focal neurological deficits and fatal intracerebral hemorrhage. It may occur sporadically or in familial forms, segregating as an autosomal dominant condition with incomplete penetrance and highly variable expressivity. Its pathogenesis has been associated with loss-of-function mutations in three genes, namely KRIT1 (CCM1), CCM2 and PDCD10 (CCM3), which are implicated in defense mechanisms against oxidative stress and inflammation. Herein, we screened 21 Italian CCM cases using clinical exome sequencing and found six cases (~29%) with pathogenic variants in CCM genes, including a large 145−256 kb genomic deletion spanning the KRIT1 gene and flanking regions, and the KRIT1 c.1664C>T variant, which we demonstrated to activate a donor splice site in exon 16. The segregation of this cryptic splicing mutation was studied in a large Italian family (five affected and seven unaffected cases), and showed a largely heterogeneous clinical presentation, suggesting the implication of genetic modifiers. Moreover, by analyzing ad hoc gene panels, including a virtual panel of 23 cerebrovascular disease-related genes (Cerebro panel), we found two variants in NOTCH3 and PTEN genes, which could contribute to the abnormal oxidative stress and inflammatory responses to date implicated in CCM disease pathogenesis.

Towards precision nanomedicine for cerebrovascular diseases with emphasis on Cerebral Cavernous Malformation (CCM).

Introduction: Cerebrovascular diseases encompass various disorders of the brain vasculature, such as ischemic/hemorrhagic strokes, aneurysms, and vascular malformations, also affecting the central nervous system leading to a large variety of transient or permanent neurological disorders. They represent major causes of mortality and long-term disability worldwide, and some of them can be inherited, including Cerebral Cavernous Malformation (CCM), an autosomal dominant cerebrovascular disease linked to mutations in CCM1/KRIT1, CCM2, or CCM3/PDCD10 genes.Areas covered: Besides marked clinical and etiological heterogeneity, some commonalities are emerging among distinct cerebrovascular diseases, including key pathogenetic roles of oxidative stress and inflammation, which are increasingly recognized as major disease hallmarks and therapeutic targets. This review provides a comprehensive overview of the different clinical features and common pathogenetic determinants of cerebrovascular diseases, highlighting major challenges, including the pressing need for new diagnostic and therapeutic strategies, and focusing on emerging innovative features and promising benefits of nanomedicine strategies for early detection and targeted treatment of such diseases.Expert opinion: Specifically, we describe and discuss the multiple physico-chemical features and unique biological advantages of nanosystems, including nanodiagnostics, nanotherapeutics, and nanotheranostics, that may help improving diagnosis and treatment of cerebrovascular diseases and neurological comorbidities, with an emphasis on CCM disease.

A Roadmap for Developing Plasma Diagnostic and Prognostic Biomarkers of Cerebral Cavernous Angioma With Symptomatic Hemorrhage (CASH).

BACKGROUND: Cerebral cavernous angioma (CA) is a capillary microangiopathy predisposing more than a million Americans to premature risk of brain hemorrhage. CA with recent symptomatic hemorrhage (SH), most likely to re-bleed with serious clinical sequelae, is the primary focus of therapeutic development. Signaling aberrations in CA include proliferative dysangiogenesis, blood-brain barrier hyperpermeability, inflammatory/immune processes, and anticoagulant vascular domain. Plasma levels of molecules reflecting these mechanisms and measures of vascular permeability and iron deposition on magnetic resonance imaging are biomarkers that have been correlated with CA hemorrhage. OBJECTIVE: To optimize these biomarkers to accurately diagnose cavernous angioma with symptomatic hemorrhage (CASH), prognosticate the risk of future SH, and monitor cases after a bleed and in response to therapy. METHODS: Additional candidate biomarkers, emerging from ongoing mechanistic and differential transcriptome studies, would further enhance the sensitivity and specificity of diagnosis and prediction of CASH. Integrative combinations of levels of plasma proteins and characteristic micro-ribonucleic acids may further strengthen biomarker associations. We will deploy advanced statistical and machine learning approaches for the integration of novel candidate biomarkers, rejecting noncorrelated candidates, and determining the best clustering and weighing of combined biomarker contributions. EXPECTED OUTCOMES: With the expertise of leading CA researchers, this project anticipates the development of future blood tests for the diagnosis and prediction of CASH to clinically advance towards precision medicine. DISCUSSION: The project tests a novel integrational approach of biomarker development in a mechanistically defined cerebrovascular disease with a relevant context of use, with an approach applicable to other neurological diseases with similar pathobiologic features.

Polymorphisms in genes related to oxidative stress and inflammation: Emerging links with the pathogenesis and severity of Cerebral Cavernous Malformation disease.

Cerebral Cavernous Malformation (CCM) is a cerebrovascular disease of genetic origin affecting 0.5% of the population and characterized by abnormally enlarged and leaky capillaries that predispose to seizures, neurological deficits, and intracerebral hemorrhage (ICH). CCM occurs sporadically or is inherited as dominant condition with incomplete penetrance and highly variable expressivity. Three disease genes have been identified: KRIT1 (CCM1), CCM2 and CCM3. Previous results demonstrated that loss-of-function mutations of CCM genes cause pleiotropic effects, including defective autophagy, altered reactive oxygen species (ROS) homeostasis, and enhanced sensitivity to oxidative stress and inflammatory events, suggesting a novel unifying pathogenetic mechanism, and raising the possibility that CCM disease onset and severity are influenced by the presence of susceptibility and modifier genes. Consistently, genome-wide association studies (GWAS) in large and homogeneous cohorts of patients sharing the familial form of CCM disease and identical mutations in CCM genes have led to the discovery of distinct genetic modifiers of major disease severity phenotypes, such as development of numerous and large CCM lesions, and susceptibility to ICH. This review deals with the identification of genetic modifiers with a significant impact on inter-individual variability in CCM disease onset and severity, including highly polymorphic genes involved in oxidative stress, inflammatory and immune responses, such as cytochrome P450 monooxygenases (CYP), matrix metalloproteinases (MMP), and Toll-like receptors (TLR), pointing to their emerging prognostic value, and opening up new perspectives for risk stratification and personalized medicine strategies.

Comparative omics of CCM signaling complex (CSC).

BACKGROUND: Cerebral cavernous malformations (CCMs), a major neurosurgical condition, characterized by abnormally dilated intracranial capillaries, result in increased susceptibility to stroke. KRIT1 (CCM1), MGC4607 (CCM2), and PDCD10 (CCM3) have been identified as causes of CCMs in which at least one of them is disrupted in most familial cases. Our goal is to identify potential biomarkers and genetic modifiers of CCMs, using a global comparative omics approach across several in vitro studies and multiple in vivo animal models. We hypothesize that through analysis of the CSC utilizing various omics, we can identify potential biomarkers and genetic modifiers, by systemically evaluating effectors and binding partners of the CSC as well as second layer interactors. METHODS: We utilize a comparative omics approach analyzing multiple CCMs deficient animal models across nine independent studies at the genomic, transcriptomic, and proteomic levels to dissect alterations in various signaling cascades. RESULTS: Our analysis revealed a large set of genes that were validated across multiple independent studies, suggesting an important role for these identified genes in CCM pathogenesis. CONCLUSION: This is currently one of the largest comparative omics analysis of CCM deficiencies across multiple models, allowing us to investigate global alterations among multiple signaling cascades involved in both angiogenic and non-angiogenic events and to also identify potential biomarker candidates of CCMs, which can be used for new therapeutic strategies.

Vitamin D Deficiency and the Risk of Cerebrovascular Disease.

Vitamin D deficiency has been clearly linked to major chronic diseases associated with oxidative stress, inflammation, and aging, including cardiovascular and neurodegenerative diseases, diabetes, and cancer. In particular, the cardiovascular system appears to be highly sensitive to vitamin D deficiency, as this may result in endothelial dysfunction and vascular defects via multiple mechanisms. Accordingly, recent research developments have led to the proposal that pharmacological interventions targeting either vitamin D deficiency or its key downstream effects, including defective autophagy and abnormal pro-oxidant and pro-inflammatory responses, may be able to limit the onset and severity of major cerebrovascular diseases, such as stroke and cerebrovascular malformations. Here we review the available evidence supporting the role of vitamin D in preventing or limiting the development of these cerebrovascular diseases, which are leading causes of disability and death all over the world.

From Genes and Mechanisms to Molecular-Targeted Therapies: The Long Climb to the Cure of Cerebral Cavernous Malformation (CCM) Disease.

Cerebral cavernous malformation (CCM) is a rare cerebrovascular disorder of genetic origin consisting of closely clustered, abnormally dilated and leaky capillaries (CCM lesions), which occur predominantly in the central nervous system. CCM lesions can be single or multiple and may result in severe clinical symptoms, including focal neurological deficits, seizures, and intracerebral hemorrhage. Early human genetic studies demonstrated that CCM disease is linked to three chromosomal loci and can be inherited as autosomal dominant condition with incomplete penetrance and highly variable expressivity, eventually leading to the identification of three disease genes, CCM1/KRIT1, CCM2, and CCM3/PDCD10, which encode for structurally unrelated intracellular proteins that lack catalytic domains. Biochemical, molecular, and cellular studies then showed that these proteins are involved in endothelial cell-cell junction and blood-brain barrier stability maintenance through the regulation of major cellular structures and mechanisms, including endothelial cell-cell and cell-matrix adhesion, actin cytoskeleton dynamics, autophagy, and endothelial-to-mesenchymal transition, suggesting that they act as pleiotropic regulators of cellular homeostasis, and opening novel therapeutic perspectives. Indeed, accumulated evidence in cellular and animal models has eventually revealed that the emerged pleiotropic functions of CCM proteins are mainly due to their ability to modulate redox-sensitive pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, thus contributing to the preservation of cellular homeostasis and stress defenses.In this introductory review, we present a general overview of 20 years of amazing progress in the identification of genetic culprits and molecular mechanisms underlying CCM disease pathogenesis, and the development of targeted therapeutic strategies.

Next Generation Sequencing (NGS) Strategies for Genetic Testing of Cerebral Cavernous Malformation (CCM) Disease.

The application of next generation sequencing (NGS) technique has a great impact on complex disease studies. Indeed, genetic heterogeneity, phenotypic variability, and disease rarity are all factors that make the traditional diagnostic approach to genetic disorders, whereby a specific gene is selected for sequencing based on the clinical phenotype, very challenging and obsolete.Exome sequencing, which sequences the protein-coding region of the genome, has been rapidly applied to variant discovery in research settings. Recent coverage and accuracy improvements have accelerated the development of clinical exome sequencing (CES) platforms targeting disease-related genes and enabling variant identification in patients with suspected genetic diseases. Nowadays, CES is rapidly becoming the diagnostic test of choice in patients with suspected Mendelian diseases, especially for those with heterogeneous etiology and clinical presentation. Reporting large CES series can improve guidelines on best practices for test utilization, and a better variant interpretation through clinically oriented data sharing.Herein, we suggest a feasible CES procedure for the genetic testing of Cerebral Cavernous Malformation (CCM) disease, including proband identification, library preparation, data analysis, and variant interpretation.

Production of KRIT1-knockout and KRIT1-knockin Mouse Embryonic Fibroblasts as Cellular Models of CCM Disease.

The development of distinct cellular and animal models has allowed the identification and characterization of molecular mechanisms underlying the pathogenesis of cerebral cavernous malformation (CCM) disease. This is a major cerebrovascular disorder of proven genetic origin, affecting 0.5% of the population. Three disease genes have been identified: CCM1/KRIT1, CCM2, and CCM3. These genes encode for proteins implicated in the regulation of major cellular structures and mechanisms, such as cell-cell and cell-matrix adhesion, actin cytoskeleton dynamics, and endothelial-to-mesenchymal transition, suggesting that they may act as pleiotropic regulators of cellular homeostasis. Indeed, accumulated evidence in cellular and animal models demonstrates that emerged pleiotropic functions of CCM proteins are mainly due to their ability to modulate redox-sensitive pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, thus contributing to the preservation of cellular homeostasis and stress defenses. In particular, we demonstrated that KRIT1 loss-of-function affects master regulators of cellular redox homeostasis and responses to oxidative stress, including major redox-sensitive transcriptional factors and antioxidant proteins, and autophagy, suggesting that altered redox signaling and oxidative stress contribute to CCM pathogenesis, and opening novel preventive and therapeutic perspectives.In this chapter, we describe materials and methods for isolation of mouse embryonic fibroblast (MEF) cells from homozygous KRIT1-knockout mouse embryos, and their transduction with a lentiviral vector encoding KRIT1 to generate cellular models of CCM disease that contributed significantly to the identification of pathogenetic mechanisms.

Fluorescence Analysis of Reactive Oxygen Species (ROS) in Cellular Models of Cerebral Cavernous Malformation Disease.

Cerebral cavernous malformation (CCM) is a vascular disease of proven genetic origin, which may arise sporadically or can be inherited as an autosomal dominant condition with incomplete penetrance and highly variable expressivity. CCM disease exhibits a range of different phenotypes, including wide interindividual differences in lesion number, size, and susceptibility to intracerebral hemorrhage (ICH). Mutations of the KRIT1 gene account for over 50% of familial cases. Previously, we demonstrated that KRIT1 loss-of-function is associated with altered homeostasis of intracellular reactive oxygen species (ROS) and abnormal activation of redox-sensitive transcription factors, which collectively result in pro-oxidative, pro-inflammatory, and pro-angiogenic effects, suggesting a novel pathogenic mechanism for CCM disease. Consistently, these original discoveries have been confirmed and extended by subsequent findings showing mechanistic relationships between pleiotropic redox-dependent effects of KRIT1 loss-of-function and enhanced cell sensitivity to oxidative stress, which may eventually lead to cellular dysfunctions and CCM disease pathogenesis. In this chapter, we describe few basic methods used for qualitative and quantitative analysis of intracellular ROS in cellular models of CCM disease.