Circulating biomarkers in familial cerebral cavernous malformation.

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.

Kinases in cerebral cavernous malformations: Pathogenesis and therapeutic targets.

Cerebral cavernous malformations (CCMs) are low-flow, hemorrhagic vascular lesions of the central nervous system of genetic origin, which can cause stroke-like symptoms and seizures. From the identification of CCM1, CCM2 and CCM3 as genes related to disease progression, molecular and cellular mechanisms for CCM pathogenesis have been established and the search for potential drugs to target CCM has begun. Broadly speaking, kinases are the major group signaling in CCM pathogenesis. These include the MEKK3/MEK5/ERK5 cascade, Rho/Rock signaling, CCM3/GCKIII signaling, PI3K/mTOR signaling, and others. Since the discovery of Rho/Rock in CCM pathogenesis, inhibitors for Rho signaling and subsequently other components in CCM signaling were discovered and applied in preclinical and clinical trials to ameliorate CCM progression. This review discusses the general aspects of CCM disease, kinase-mediated signaling in CCM pathogenesis and the current state of potential treatment options for CCM. It is suggested that kinase target drug development in the context of CCM might facilitate and meet the unmet requirement - a non-surgical option for CCM disease.

Cerebral cavernous malformations: Typical and atypical imaging characteristics.

Cavernous malformations (CMs) are benign vascular malformations that maybe seen anywhere in the central nervous system. They are dynamic lesions, growing or shrinking over time and only rarely remaining stable. Size varies from a few millimeters to a few centimeters. CMs can be sporadic or familial, and while most of them are congenital, de novo and acquired lesions may also be seen. Etiology is still unknown. A genetic molecular mechanism has been proposed since a cerebral cavernous malformation gene loss of function was found in both familial and sporadic lesions. Additionally, recent studies suggest that formation of CMs in humans may be associated with a distinctive bacterial gut composition (microbioma). Imaging is fairly typical but may vary according to age, location, and etiology. Follow-up is not well established because CMs patients have a highly unpredictable clinical course. Angiogenic and inflammatory mechanisms have been implicated in disease activity, as well as lesional hyperpermeability and iron deposition. Imaging and serum biomarkers of these mechanisms are under current investigation. Treatment options, including surgery or radiosurgery, are not well defined and are dependent upon multiple factors, including clinical presentation, lesion location, number of hemorrhagic events, and medical comorbidities. Our purpose is to review the imaging features of CMs based on their size, location, and etiology, as well as their differential diagnosis and best imaging approach. New insights in etiology will be briefly considered. Follow-up strategies, including serum and imaging biomarkers, and treatment options will also be discussed.

Radiation-induced cerebral cavernous malformation: is it just a childhood disease?

Cerebral cavernous malformations (CCM) are clusters of dilated sinusoidal channels, lined by a single layer of endothelium. Acquired form of these lesions was related to previous radiation-therapy, and might be related to its pathophysiology, because the vascular endothelial growth factor has higher expression in the immature brain of the child. Consequently, although it occurs mainly in the paediatric population, it can happen in adults. We report a case of radiation-induced CCM in an adult patient presenting with aggressive behaviour and cerebral haemorrhage.

Radiosurgery effects and adverse effects in symptomatic eloquent brain-located Cavernomas.

In this study, the dose schedule efficacy, safety and late adverse effects of stereotactic radiosurgery (SRS) were evaluated for patients with symptomatic cavernomas who were not eligible for surgery and treated with SRS. Between January 2013 and December 2018, 53 patients with cavernomas were treated using SRS with the CyberKnife® system. Patients' diseases were deeply located or were in subcortical functional brain regions. In addition to bleeding, 23 (43.4%) patients had epilepsy, 12 (22.6%) had neurologic symptoms and 16 patients (30.2%) had severe headaches. The median volume was 741 (range, 421-1351) mm3, and the median dose was 15 (range, 14-16) Gy in one fraction. After treatment, six (50%) of 12 patients with neurologic deficits still had deficits. Rebleeding after treatment developed in only two (3.8%) patients. The drug was completely stopped in 14 (60.9%) out of 23 patients who received epilepsy treatment, and the dose of levetiracetam decreased from 2000 mg to 1000 mg in four (17.3%) of nine patients. Radiologically, complete response (CR) was observed in 13 (24.5%) patients, and partial responses (PR) were observed in 32 (60.2%) patients. Clinical response of CR was observed in 30 (56.6%) patients, PR was observed in 16 (30.2%), stable disease (SD) was observed in three (5.7%) and four (7.5%) patients progressed. In conclusion, SRS applied in the appropriate dose schedule may be an effective and reliable method in terms of symptom control and prevention of rebleeding, especially in patients with inoperable cavernomas.

Cerebral Cavernous Malformation: Immune and Inflammatory Perspectives.

Cerebral cavernous malformation (CCM) is a type of vascular anomaly that arises due to the dyshomeostasis of brain capillary networks. In the past two decades, many advances have been made in this research field. Notably, as a more reasonable current view, the CCM lesions should be attributed to the results of a great number of additional events related to the homeostasis disorder of the endothelial cell. Indeed, one of the most fascinating concerns in the research field is the inflammatory perturbation in the immune microenvironment, which would affect the disease progression as well as the patients' outcomes. In this work, we focused on this topic, and underlined the immune-related factors' contribution to the CCM pathologic progression.

Laser Interstitial Thermal Therapy for Cerebral Cavernous Malformations: A Systematic Review of Indications, Safety, and Outcomes.

BACKGROUND: Cerebral cavernous malformations (CCM) in deep eloquent areas present a surgical challenge. Laser interstitial thermal therapy (LITT) may present itself as a safe minimally invasive treatment option. OBJECTIVE: To systematically review the indications, safety, and outcomes of LITT for CCM. METHODS: Electronic databases were searched from inception to October 7, 2021 for articles with CCM and LITT keywords. Studies describing CCMs treated with LITT were included. RESULTS: A total of 32 patients with CCMs in lobar (79%), basal ganglia (12%), and brainstem (9%) locations were treated with LITT. Indications for LITT included drug-resistant seizures (75%), unacceptable surgical risk (22%), recurrent hemorrhage (16%), and early intervention to discontinue antiepileptic drugs (3%). No death or CCM-associated intracranial hemorrhage occurred intraoperatively or postoperatively, and most patients experienced no adverse effects or transient effects that resolved at follow-up (84%). Of those treated for CCM-associated epilepsy, 83% experienced Engel class I seizure freedom and most were class IA (61%). Most patients experienced symptomatic improvement (93%), and a decrease in antiepileptic drugs was reported in more than half of patients (56%), with 28% able to discontinue all antiepilepsy medications after LITT. CONCLUSIONS: LITT seems to be a safe treatment for CCMs located in deep eloquent areas and in lesions presenting with medically refractory seizures or recurrent hemorrhages. Randomized studies are needed to further elucidate its efficacy in treating CCM.

The Impact of Volume Factor on the Long-Term Outcome of Gamma Knife Radiosurgery for Sporadic Cerebral Cavernous Malformations.

OBJECTIVE: We aimed to evaluate the long-term outcome of gamma knife radiosurgery (GKRS) for the treatment of sporadic cerebral cavernous malformation (CCM), especially the influence of lesion volume on annual hemorrhage rate (AHR) of patients with CCM after GKRS. METHODS: Fifty-one single-lesion patients with a history of hemorrhage who underwent radiosurgery at our institution were included and divided into 2 groups (A and B), based on their lesion volume. Group A included 25 patients with lesion volumes >1 cm3, whereas group B included 26 patients with lesion volumes ≤1 cm3. The clinical data of the patients were retrospectively analyzed. RESULTS: All patients were followed up for more than 4 years after GKRS. The calculated AHR before GKRS was 18.49% in group A and 10.16% in group B. The calculated AHR after GKRS was 5.43% and 0.99% for groups A and B, respectively. Significant differences in AHR after GKRS were identified between group A and group B (P = 0.011). Thirty-seven patients with sporadic CCM (14 in group A, 23 in group B) experienced symptom improvement, and significant differences in symptom improvement were observed between group A and group B (P = 0.009). CONCLUSIONS: GKRS decreased the risk of hemorrhage and was beneficial for symptomatic improvement in patients with sporadic CCM with a history of hemorrhage. The long-term clinical outcomes for patients with sporadic CCM with small lesion volumes (≤1 cm3) were better than those of patients with sporadic CCM with large lesion volumes (>1 cm3).

Assessing the association of common genetic variants in EPHB4 and RASA1 with phenotype severity in familial cerebral cavernous malformation.

BACKGROUND: To investigate whether common variants in EPHB4 and RASA1 are associated with cerebral cavernous malformation (CCM) disease severity phenotypes, including intracranial hemorrhage (ICH), total and large lesion counts. METHODS: Familial CCM cases enrolled in the Brain Vascular Malformation Consortium were included (n = 338). Total lesions and large lesions (≥5 mm) were counted on MRI; clinical history of ICH at enrollment was assessed by medical records. Samples were genotyped on the Affymetrix Axiom Genome-Wide LAT1 Human Array. We tested the association of seven common variants (three in EPHB4 and four in RASA1) using multivariable logistic regression for ICH (odds ratio, OR) and multivariable linear regression for total and large lesion counts (proportional increase, PI), adjusting for age, sex, and three principal components. Significance was based on Bonferroni adjustment for multiple comparisons (0.05/7 variants = 0.007). RESULTS: EPHB4 variants were not significantly associated with CCM severity phenotypes. One RASA1 intronic variant (rs72783711 A>C) was significantly associated with ICH (OR = 1.82, 95% CI = 1.21-2.37, p = 0.004) and nominally associated with large lesion count (PI = 1.17, 95% CI = 1.03-1.32, p = 0.02). CONCLUSION: A common RASA1 variant may be associated with ICH and large lesion count in familial CCM. EPHB4 variants were not associated with any of the three CCM severity phenotypes.

Are radiation-induced cavernomas clinically relevant findings? Results from long-term follow-up with brain magnetic resonance imaging of childhood cancer survivors.

INTRODUCTION: Radiation-induced cavernomas (RIC) after cranial radiotherapy have an unknown risk of hemorrhage. Zabramski magnetic resonance imaging (MRI) classification is touted as being able to indicate non-radiation-induced cavernomas hemorrhage risk. The aim of our study was to assess the hemorrhage risk of RIC during long-term follow-up of childhood cancer survivors based on brain MRI examinations. PATIENTS AND METHODS: We analyzed retrospectively long-term follow-up data of 36 childhood cancer survivors after initial diagnosis with acute leukemia (n = 18) or brain tumor (n = 18), all treated with cranial radiotherapy. Detected RIC in long-term follow-up brain MRI (1.5 or 3 Tesla) were classified following the Zabramski MRI classification and were categorized into "high" (Zabramski type I, II or V) or "low" (type III or IV) risk of hemorrhage. RESULTS: 18 patients (50%) showed RIC with a significant relation to the original tumor entity (p = 0.023) and the cumulative radiation dose to the brain (p = 0.016): all 9 childhood cancer survivors diagnosed with medulloblastoma developed RIC. We classified RIC in only 3/36 childhood cancer survivors (8%) (1 patient with acute lymphoblastic leukemia [Zabramski type II] and 2 patients with medulloblastoma [type I and type II]) as high risk for hemorrhage, the remaining RIC were classified as Zabramski type IV with low risk for hemorrhage. None of the childhood cancer survivors with RIC showed symptomatic hemorrhages. CONCLUSIONS: RIC are common late effects in childhood cancer survivors treated with cranial radiotherapy affecting half of these patients. However, only a few RIC (occurring in 8% of all reviewed childhood cancer survivors) were classified as high risk for hemorrhage and none of the childhood cancer survivors with RIC developed symptomatic hemorrhages. Thus, we conclude that RIC are low-risk findings in brain MRI and the course is mainly benign.

Astrocytes propel neurovascular dysfunction during cerebral cavernous malformation lesion formation.

Cerebral cavernous malformations (CCMs) are common neurovascular lesions caused by loss-of-function mutations in 1 of 3 genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3), and generally regarded as an endothelial cell-autonomous disease. Here we reported that proliferative astrocytes played a critical role in CCM pathogenesis by serving as a major source of VEGF during CCM lesion formation. An increase in astrocyte VEGF synthesis is driven by endothelial nitric oxide (NO) generated as a consequence of KLF2- and KLF4-dependent elevation of eNOS in CCM endothelium. The increased brain endothelial production of NO stabilized HIF-1α in astrocytes, resulting in increased VEGF production and expression of a "hypoxic" program under normoxic conditions. We showed that the upregulation of cyclooxygenase-2 (COX-2), a direct HIF-1α target gene and a known component of the hypoxic program, contributed to the development of CCM lesions because the administration of a COX-2 inhibitor significantly prevented the progression of CCM lesions. Thus, non-cell-autonomous crosstalk between CCM endothelium and astrocytes propels vascular lesion development, and components of the hypoxic program represent potential therapeutic targets for CCMs.

The development of numerous radiation-induced cavernous malformations in a germ cell tumor patient: A case report.

Radiation-induced cavernous malformations (RICMs) are most commonly reported in young patients who have previously received radiotherapy. Here, we report a case of a patient with a germ cell tumor who was treated with whole brain radiotherapy (WBRT) and then incidentally found to have numerous RICMs. A 31-year-old male visited the hospital for a testicular mass. On examination, he was diagnosed with a mixed germ cell tumor with lung/brain metastases. The patient underwent a left orchiectomy and received 4 cycles of chemotherapy. He was then treated with WBRT for residual lesions in the brain and a wedge resection for the lung metastasis. Four years later, approximately 250-300 RICMs were incidentally observed in a follow-up brain image. Because the patient had not noticed any symptoms and the RICMs were small in size, he was not treated. To our knowledge, this is the first reported case of numerous (approximately 250-300) RICMs in a germ cell tumor patient after WBRT. Herein, we report details of this case and discuss the typical clinical features of RICM.

Radiation-induced cavernous malformation after stereotactic radiosurgery for cavernous sinus meningioma: a case report.

BACKGROUND: Radiation-induced cavernous malformation (RICM) is a rare sequela of stereotactic radiosurgery (SRS) treatment of intracranial tumors. To date, no study reported on RICM after SRS for meningiomas originating from the skull base. The relationship between locus of initial meningioma and RICM has not been studied. CASE PRESENTATION: A 57-year-old woman presented with persistent headaches and blepharoptosis at initial episode. MRI disclosed a right parasellar lesion, diagnosed as a cavernous sinus meningioma (CSM). After receiving a single-fractionated SRS, headache relieved, but blepharoptosis did not significantly improve. Three years and three months later, she returned with headaches and dizziness. MRI showed an enlarged CSM. Moreover, a new mass-like lesion, suspected hemangioma, appeared in the nearby right temporal lobe. After surgical removal of the new lesion and the CSM, the patient's neurological symptoms significantly improved. Pathology confirmed CSM and temporal RICM. CONCLUSIONS: We report the first rare case of RICM occurring after SRS for CSM. The RICM may be in the same region as the initial tumor. Surgical intervention was preferred for symptomatic RICM and initial meningioma. We recommend long-term regular followup MRIs for patients with meningioma after SRS treatment.

Signalling through cerebral cavernous malformation protein networks.

Cerebral cavernous malformations (CCMs) are neurovascular abnormalities characterized by thin, leaky blood vessels resulting in lesions that predispose to haemorrhages, stroke, epilepsy and focal neurological deficits. CCMs arise due to loss-of-function mutations in genes encoding one of three CCM complex proteins, KRIT1, CCM2 or CCM3. These widely expressed, multi-functional adaptor proteins can assemble into a CCM protein complex and (either alone or in complex) modulate signalling pathways that influence cell adhesion, cell contractility, cytoskeletal reorganization and gene expression. Recent advances, including analysis of the structures and interactions of CCM proteins, have allowed substantial progress towards understanding the molecular bases for CCM protein function and how their disruption leads to disease. Here, we review current knowledge of CCM protein signalling with a focus on three pathways which have generated the most interest-the RhoA-ROCK, MEKK3-MEK5-ERK5-KLF2/4 and cell junctional signalling pathways-but also consider ICAP1-ß1 integrin and cdc42 signalling. We discuss emerging links between these pathways and the processes that drive disease pathology and highlight important open questions-key among them is the role of subcellular localization in the control of CCM protein activity.

Cerebral cavernous malformations are driven by ADAMTS5 proteolysis of versican.

Cerebral cavernous malformations (CCMs) form following loss of the CCM protein complex in brain endothelial cells due to increased endothelial MEKK3 signaling and KLF2/4 transcription factor expression, but the downstream events that drive lesion formation remain undefined. Recent studies have revealed that CCM lesions expand by incorporating neighboring wild-type endothelial cells, indicative of a cell nonautonomous mechanism. Here we find that endothelial loss of ADAMTS5 reduced CCM formation in the neonatal mouse model. Conversely, endothelial gain of ADAMTS5 conferred early lesion genesis in the absence of increased KLF2/4 expression and synergized with KRIT1 loss of function to create large malformations. Lowering versican expression reduced CCM burden, indicating that versican is the relevant ADAMTS5 substrate and that lesion formation requires proteolysis but not loss of this extracellular matrix protein. These findings identify endothelial secretion of ADAMTS5 and cleavage of versican as downstream mechanisms of CCM pathogenesis and provide a basis for the participation of wild-type endothelial cells in lesion formation.

Generation of CCM Phenotype by a Human Microvascular Endothelial Model.

Cerebral cavernous malformations (CCMs) is a disorder of endothelial cells predominantly localized in the brain. Although a complete inactivation of each CCM protein has been found in the affected endothelium of diseased patients and a necessary and additional role of microenvironment has been demonstrated to determine in vivo the occurrence of vascular lesions, a microvascular endothelial model based on knockdown of a CCM gene represents today a convenient method to study some of critical signaling events regulating pathogenesis of CCM. For these reasons, in our laboratory we developed a microvascular cerebral endothelial model of Krit1 deficiency performing silencing experiments of CCM1 gene (Krit1) with siRNA procedure.

Isolation and Purification of Mouse Brain Endothelial Cells to Study Cerebral Cavernous Malformation Disease.

We describe a method to purify primary brain microvascular endothelial cells (BMEC) from mice bearing floxed alleles of Krit1 (Krit1fl/fl) or Pdcd10 (Pdcd10fl/fl) and an endothelial-specific tamoxifen-regulated Cre recombinase (Pdgfb-iCreERT2), and used these to delete Krit1 or Pdcd10 genes in a time-controlled manner. These BMEC culture models contain a high degree of purity and have been used to identify the major molecular processes involved in loss of Krit1/Pdcd10-induced altered brain endothelial phenotype and function. In addition, these in vitro models of cerebral cavernous malformations (CCMs) enable molecular, biochemical, and pharmacological studies that have contributed significantly to understand the pathogenesis of CCMs. The findings using this in vitro CCMs model have been validated in mouse CCM models and observed in human CCMs. In this chapter, we summarize procedures for isolation and purification of BMEC from transgenic mice, as well as our experience to genetically inactivate CCM genes in the brain endothelium.

Generation and Analysis of CCM Phenotypes in C. elegans.

This chapter presents methods for exploiting the powerful tools available in the nematode worm Caenorhabditis elegans to understand the in vivo functions of cerebral cavernous malformation (CCM) genes and the organization of their associated signaling pathways. Included are methods for assessing phenotypes caused by loss-of-function mutations in the worm CCM genes kri-1 and ccm-3, CRISPR-based gene editing techniques, and protocols for conducting high-throughput forward genetic and small molecule screens.

Vertebrate Models to Investigate CCM Pathogenesis: The Zebrafish and Mouse Model.

The use of vertebrate models allows researchers to investigate mechanisms of CCM pathogenesis in vivo, to investigate discrepancies between observations seen in the lab with in vitro experiments and how they translate into animal models; these in vivo models are more relevant in terms of CCM pathogenesis seen in humans than the in vitro counterparts. The use of CCM-deficient Zebrafish model offers advantages given their optical clarity during embryogenesis, short generation time, and high fecundity. When looking at the in vivo mouse model, gene conservation among CCM1, CCM2, and CCM3 is much higher among mammals (>92%), offering higher relevance in terms of similarities between what is seen in a mouse compared to human CCM pathogenesis. With both models, deficiencies in CCM1, CCM2, and CCM3 demonstrate perturbed cardiovascular development and underlying mechanisms of CCM pathogenesis at multiple stages seen in humans. The optimized methods described in this chapter allow researchers to benefit from both in vivo models, investigating impacts of deficiencies in CCM gene expression and its effect on angiogenesis and other signaling cascades, offering a much wider view of the molecular and cellular mechanisms in CCM progression.

Preparation and Analysis of Protein Extracts to Investigate CCM Pathogenesis.

Molecular techniques allow for the rapid discovery of CCM-associated protein targets, crucial to understanding CCM pathogenesis. Here, we describe optimized protein extraction methods that allow for extraction from whole cell, and/or cellular sub-compartments, including nuclear, mitochondria, cytoplasmic, and membrane-bound proteins, from lysates. This allows for the analysis of in vitro co-immunoprecipitation (Co-IP), label-free measurement of protein-protein interactions, multiplex protein-lipid binding assays, and western blots. Together, all these methods allow for a global analysis of the molecular mechanisms underlying CCM pathogenesis.