PIK3CA and CCM mutations fuel cavernomas through a cancer-like mechanism.

Vascular malformations are thought to be monogenic disorders that result in dysregulated growth of blood vessels. In the brain, cerebral cavernous malformations (CCMs) arise owing to inactivation of the endothelial CCM protein complex, which is required to dampen the activity of the kinase MEKK31-4. Environmental factors can explain differences in the natural history of CCMs between individuals5, but why single CCMs often exhibit sudden, rapid growth, culminating in strokes or seizures, is unknown. Here we show that growth of CCMs requires increased signalling through the phosphatidylinositol-3-kinase (PI3K)-mTOR pathway as well as loss of function of the CCM complex. We identify somatic gain-of-function mutations in PIK3CA and loss-of-function mutations in the CCM complex in the same cells in a majority of human CCMs. Using mouse models, we show that growth of CCMs requires both PI3K gain of function and CCM loss of function in endothelial cells, and that both CCM loss of function and increased expression of the transcription factor KLF4 (a downstream effector of MEKK3) augment mTOR signalling in endothelial cells. Consistent with these findings, the mTORC1 inhibitor rapamycin effectively blocks the formation of CCMs in mouse models. We establish a three-hit mechanism analogous to cancer, in which aggressive vascular malformations arise through the loss of vascular 'suppressor genes' that constrain vessel growth and gain of a vascular 'oncogene' that stimulates excess vessel growth. These findings suggest that aggressive CCMs could be treated using clinically approved mTORC1 inhibitors.

Transcriptomic signatures of individual cell types in cerebral cavernous malformation.

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.

Cerebral Hemorrhage: Pathophysiology, Treatment, and Future Directions.

Intracerebral hemorrhage (ICH) is a devastating form of stroke with high morbidity and mortality. This review article focuses on the epidemiology, cause, mechanisms of injury, current treatment strategies, and future research directions of ICH. Incidence of hemorrhagic stroke has increased worldwide over the past 40 years, with shifts in the cause over time as hypertension management has improved and anticoagulant use has increased. Preclinical and clinical trials have elucidated the underlying ICH cause and mechanisms of injury from ICH including the complex interaction between edema, inflammation, iron-induced injury, and oxidative stress. Several trials have investigated optimal medical and surgical management of ICH without clear improvement in survival and functional outcomes. Ongoing research into novel approaches for ICH management provide hope for reducing the devastating effect of this disease in the future. Areas of promise in ICH therapy include prognostic biomarkers and primary prevention based on disease pathobiology, ultra-early hemostatic therapy, minimally invasive surgery, and perihematomal protection against inflammatory brain injury.

Impact of socioeconomics and race on clinical follow-up and trial enrollment and adherence in cerebral cavernous malformation.

OBJECTIVES: Cerebral cavernous malformation (CCM) affects more than a million Americans but advanced care for symptomatic lesions and access to research studies is largely limited to referral academic centers MATERIALS AND METHODS: A cohort of CCM patients screened for research studies at an accredited center of excellence for CCM was analyzed. Demographics, lesion location, history of hemorrhage, insurance type and area of deprivation index (ADI) were collected. Primary outcomes were clinical follow-up within a year from initial evaluation, and enrollment and adherence in clinical trials among eligible subjects RESULTS: A majority (52.8%) of CCM patients evaluated had a high socioeconomic status (SES) (ADI 1-3), and only 11.5% were African American. Patients who had a symptomatic bleed were more likely to follow-up (p=0.01), and those with brainstem lesion were more likely to enroll/adhere in a clinical trial (p=0.02). Rates of clinical follow-up were similar across different ADI groups, insurance coverage and race. Patients who were uninsured/self-paying, and African Americans were more likely to decline/drop from clinical trials (OR 2.4, 95% CI 0.46-10.20 and OR 2.2, 95% CI 0.33-10.75, respectively), but differences were not statistically significant CONCLUSIONS: Access of disadvantaged patients to center of excellence care and research remains limited despite geographic proximity to their community. Patients with lower SES and African Americans are as likely to follow-up clinically, but there were trends of differences in enrollment/adherence in clinical trials. Mitigation efforts should target systemic causes of low access to specialized care among uninsured and African American patients.

Perfusion and Permeability MRI Predicts Future Cavernous Angioma Hemorrhage and Growth.

BACKGROUND: Cerebral cavernous angioma (CA) is a capillary vasculopathy affecting more than a million Americans with a small fraction of cases demonstrating lesional bleed or growth with major clinical sequelae. Perfusion and permeability are fundamental features of CA pathophysiology, but their role as prognostic biomarkers is unclear. PURPOSE: To investigate whether perfusion or permeability lesional descriptors derived from dynamic contrast-enhanced quantitative perfusion (DCEQP) magnetic resonance imaging (MRI) can predict subsequent lesional bleed/growth in the year following imaging. STUDY TYPE: Single-site case-controlled study. SUBJECTS: Two hundred and five consecutively enrolled patients (63.4% female). FIELD STRENGTH/SEQUENCE: Three-Tesla/T1 -mapping with contrast-enhanced dynamic two-dimensional (2D) spoiled gradient recalled acquisition (SPGR) sequences. ASSESSMENT: Prognostic associations with bleed/growth (present or absent) in the following year were assessed in 745 CA lesions evaluated by DCEQP in the 205 patients in relation to lesional descriptors calculated from permeability and perfusion maps. A subgroup of 30 cases also underwent peripheral blood collection at the time of DCEQP scans and assays of plasma levels of soluble CD14, IL-1ß, VEGF, and soluble ROBO4 proteins, whose weighted combination had been previously reported in association with future CA bleeding. STATISTICAL TESTS: Mann-Whitney U-test for univariate analyses. Logistic regression models minimizing the Bayesian information criterion (BIC), testing sensitivity and specificity (receiver operating characteristic curves) of weighted combinations of parameters. RESULTS: The best prognostic biomarker for lesional bleed or growth included brainstem lesion location, mean lesional permeability, and low-value perfusion cluster mean (BIC = 201.5, sensitivity = 77%, specificity = 72%, P < 0.05). Adding a previously published prognostic plasma protein biomarker improved the performance of the imaging model (sensitivity = 100%, specificity = 88%, P < 0.05). DATA CONCLUSION: A combination of MRI-based descriptors reflecting higher lesional permeability and lower perfusion cluster may potentially predict future bleed/growth in CAs. The sensitivity and specificity of the prognostic imaging biomarker can be enhanced when combined with brainstem lesion location and a plasma protein biomarker of CA hemorrhage. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: Stage 5.

Antibodies in cerebral cavernous malformations react with cytoskeleton autoantigens in the lesional milieu.

Previous studies have reported robust inflammatory cell infiltration, synthesis of IgG, B-cell clonal expansion, deposition of immune complexes and complement within cerebral cavernous malformation (CCM) lesions. B-cell depletion has also been shown to reduce the maturation of CCM in murine models. We hypothesize that antigen(s) within the lesional milieu perpetuate the pathogenetic immune responses in CCMs. This study aims to identify those putative antigen(s) using monoclonal antibodies (mAbs) derived from plasma cells found in surgically removed human CCM lesions. We produced human mAbs from laser capture micro-dissected plasma cells from four CCM patients, and also germline-reverted versions. CCM mAbs were assayed using immunofluorescence on central nervous system (CNS) tissues and immunocytochemistry on human primary cell lines. Antigen characterization was performed using a combination of confocal microscopy, immunoprecipitation and mass spectrometry. Affinity was determined by enzyme-linked immunosorbent assay, and specificity by multi-color confocal microscopy and quantitative co-localization. CCM mAbs bound CNS tissue, especially endothelial cells and astrocytes. Non-muscle myosin heavy chain IIA (NMMHCIIA), vimentin and tubulin are three cytoskeleton proteins that were commonly targeted. Selection of cytoskeleton proteins by plasma cells was supported by a high frequency of immunoglobulin variable region somatic hypermutations, high affinity and selectivity of mAbs in their affinity matured forms, and profoundly reduced affinity and selectivity in the germline reverted forms. Antibodies produced by plasma cells in CCM lesions commonly target cytoplasmic and cytoskeletal autoantigens including NMMHCIIA, vimentin and tubulin that are abundant in endothelial cells and astrocytes. Binding to, and selection on autoantigen(s) in the lesional milieu likely perpetuates the pathogenetic immune response in CCMs. Blocking this in situ autoimmune response may yield a novel treatment for CCM.

Circulating Plasma miRNA Homologs in Mice and Humans Reflect Familial Cerebral Cavernous Malformation Disease.

Patients with familial cerebral cavernous malformation (CCM) inherit germline loss of function mutations and are susceptible to progressive development of brain lesions and neurological sequelae during their lifetime. To date, no homologous circulating molecules have been identified that can reflect the presence of germ line pathogenetic CCM mutations, either in animal models or patients. We hypothesize that homologous differentially expressed (DE) plasma miRNAs can reflect the CCM germline mutation in preclinical murine models and patients. Herein, homologous DE plasma miRNAs with mechanistic putative gene targets within the transcriptome of preclinical and human CCM lesions were identified. Several of these gene targets were additionally found to be associated with CCM-enriched pathways identified using the Kyoto Encyclopedia of Genes and Genomes. DE miRNAs were also identified in familial-CCM patients who developed new brain lesions within the year following blood sample collection. The miRNome results were then validated in an independent cohort of human subjects with real-time-qPCR quantification, a technique facilitating plasma assays. Finally, a Bayesian-informed machine learning approach showed that a combination of plasma levels of miRNAs and circulating proteins improves the association with familial-CCM disease in human subjects to 95% accuracy. These findings act as an important proof of concept for the future development of translatable circulating biomarkers to be tested in preclinical studies and human trials aimed at monitoring and restoring gene function in CCM and other diseases.

Plasma metabolites with mechanistic and clinical links to the neurovascular disease cavernous angioma.

BACKGROUND: Cavernous angiomas (CAs) affect 0.5% of the population, predisposing to serious neurologic sequelae from brain bleeding. A leaky gut epithelium associated with a permissive gut microbiome, was identified in patients who develop CAs, favoring lipid polysaccharide producing bacterial species. Micro-ribonucleic acids along with plasma levels of proteins reflecting angiogenesis and inflammation were also previously correlated with CA and CA with symptomatic hemorrhage. METHODS: The plasma metabolome of CA patients and CA patients with symptomatic hemorrhage was assessed using liquid-chromatography mass spectrometry. Differential metabolites were identified using partial least squares-discriminant analysis (p < 0.05, FDR corrected). Interactions between these metabolites and the previously established CA transcriptome, microbiome, and differential proteins were queried for mechanistic relevance. Differential metabolites in CA patients with symptomatic hemorrhage were then validated in an independent, propensity matched cohort. A machine learning-implemented, Bayesian approach was used to integrate proteins, micro-RNAs and metabolites to develop a diagnostic model for CA patients with symptomatic hemorrhage. RESULTS: Here we identify plasma metabolites, including cholic acid and hypoxanthine distinguishing CA patients, while arachidonic and linoleic acids distinguish those with symptomatic hemorrhage. Plasma metabolites are linked to the permissive microbiome genes, and to previously implicated disease mechanisms. The metabolites distinguishing CA with symptomatic hemorrhage are validated in an independent propensity-matched cohort, and their integration, along with levels of circulating miRNAs, enhance the performance of plasma protein biomarkers (up to 85% sensitivity and 80% specificity). CONCLUSIONS: Plasma metabolites reflect CAs and their hemorrhagic activity. A model of their multiomic integration is applicable to other pathologies.

Cavernous angiomas (CAs) are clusters of abnormal blood vessels found in the brain or spinal cord. A blood test that could identify people with CAs that have recently bled would help determine who need surgery or closer medical monitoring. We looked at the blood of people with CAs to compare the levels of metabolites, a type of small molecule produced within the body, in those who had recently bled and those who had not. We found that some metabolites may contribute to CA and have an impact on CA symptoms. Monitoring the levels of these metabolites can determine whether there had been a recent bleed. In the future, drugs or other therapies could be developed that would block or change the levels of these molecules and possibly be used to treat CA disease.

Rapamycin in Cerebral Cavernous Malformations: What Doses to Test in Mice and Humans.

Cerebral cavernous malformations (CCMs) are hemorrhagic neurovascular lesions that affect more than 1 million people in the United States. Rapamycin inhibits CCM development and bleeding in murine models. The appropriate dosage to modify disease phenotype remains unknown. Current approved indications by the U.S. Food and Drug Administration and clinicaltrials.gov were queried for rapamycin human dosing for various indications. A systematic literature search was conducted on PubMed to investigate mouse dosimetry of rapamycin. In humans, low daily doses of <2 mg/day or trough level targets <15 ng/mL were typically used for benign indications akin to CCM disease, with relatively low complication rates. Higher oral doses in humans, used for organ rejection, result in higher complication rates. Oral dosing in mice, between 2 and 4 mg/kg/day, achieved blood trough levels in the 5-15 ng/mL range, a concentration likely to be targeted in human studies to treat CCM. Preclinical studies are needed utilizing dosing strategies which achieve blood levels corresponding to likely human dosimetry.

Perfusion and permeability as diagnostic biomarkers of cavernous angioma with symptomatic hemorrhage.

Cavernous angiomas with symptomatic hemorrhage (CASH) have a high risk of rebleeding, and hence an accurate diagnosis is needed. With blood flow and vascular leak as established mechanisms, we analyzed perfusion and permeability derivations of dynamic contrast-enhanced quantitative perfusion (DCEQP) MRI in 745 lesions of 205 consecutive patients. Thirteen respective derivations of lesional perfusion and permeability were compared between lesions that bled within a year prior to imaging (N = 86), versus non-CASH (N = 659) using machine learning and univariate analyses. Based on logistic regression and minimizing the Bayesian information criterion (BIC), the best diagnostic biomarker of CASH within the prior year included brainstem lesion location, sporadic genotype, perfusion skewness, and high-perfusion cluster area (BIC = 414.9, sensitivity = 74%, specificity = 87%). Adding a diagnostic plasma protein biomarker enhanced sensitivity to 100% and specificity to 85%. A slightly modified derivation achieved similar accuracy (BIC = 321.6, sensitivity = 80%, specificity = 82%) in the cohort where CASH occurred 3-12 months prior to imaging after signs of hemorrhage would have disappeared on conventional MRI sequences. Adding the same plasma biomarker enhanced sensitivity to 100% and specificity to 87%. Lesional blood flow on DCEQP may distinguish CASH after hemorrhagic signs on conventional MRI have disappeared and are enhanced in combination with a plasma biomarker.

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.

Subclinical imaging changes in cerebral cavernous angiomas during prospective surveillance.

OBJECTIVE: The purpose of this study was to systematically assess asymptomatic changes (ACs), including subclinical hemorrhage, growth, or new lesion formation (NLF) during longitudinal follow-up of cerebral cavernous angiomas (CAs), and to correlate these with symptomatic hemorrhage (SH) during the same period and with clinical features of the disease. METHODS: One hundred ninety-two patients were included in this study, among 327 consecutive patients with CA, prospectively identified between September 2009 and February 2019. Included patients had undergone clinical and MRI follow-up, in conjunction with institutional review board-approved biomarker studies, and harbored ≥ 1 CA with a maximum diameter of ≥ 5 mm on T2-weighted MRI. Rates of AC and SH per lesion-year and patient-year were assessed using prospectively articulated criteria. In multifocal/familial cases, rates of NLF were also assessed. RESULTS: There were no differences in demographic or disease features among cases included or excluded in the study cohort, except for a higher proportion of included patients with CCM3 mutation. Follow-up was 411 patient-years (2503 lesion-years). The rate of AC was higher than the rate of SH (12.9% vs 7.5% per patient-year, and 2.1% vs 1.2% per lesion-year, both p = 0.02). Patients presenting with a prior history of SH had a higher rate of AC than those with other forms of presentation (19.7% and 8.2% per patient-year, respectively; p = 0.003). A higher rate of NLF on T2-weighted MRI (p = 0.03) was observed in patients with prior SH. Three of 6 solitary/sporadic and 2 of 28 multifocal/familial patients underwent resection of the lesion after AC. CONCLUSIONS: Rates of AC are greater than SH during prospective follow-up of CAs, and greater in cases with prior SH. AC may be a more sensitive biomarker of lesional activity, and a more efficient surrogate outcome in clinical trials than SH. Patients experiencing an AC are more likely to undergo a surgical intervention when CAs are solitary/sporadic than when they are multifocal/familial.

Common transcriptome, plasma molecules, and imaging signatures in the aging brain and a Mendelian neurovascular disease, cerebral cavernous malformation.

Brain senescence is associated with impaired endothelial barrier function, angiogenic and inflammatory activity, and propensity to brain hemorrhage. The same pathological changes occur in cerebral cavernous malformations (CCM), a genetic neurovascular anomaly. We hypothesized common transcriptomic and plasma cytokine signatures in the aging brain and CCM. We identified 320 genes [fold change ≥1.5; p < 0.05; false discovery rate (FDR) corrected] commonly dysregulated in the aging brain and CCM. Ontology and pathway analyses of the common differentially expressed genes were related to inflammation and extracellular matrix organization. Plasma levels of C-reactive protein and angiopoietin-2 were significantly greater in older compared to younger healthy non-CCM subjects and were also greater in CCM (Sporadic and Familial) subjects regardless of age (all: p < 0.05; FDR corrected). Plasma levels of vascular endothelial growth factor were significantly greater in older compared to younger subjects, in both healthy non-CCM and Sporadic-CCM groups (all: padj < 0.05). Plasma levels of vascular endothelial growth factor were also significantly greater in Familial-CCM cases with germ line mutations regardless of age (all: padj < 0.05) compared to both healthy non-CCM and Sporadic-CCM subjects. Brain white matter vascular permeability assessed by MRI followed the same pattern as vascular endothelial growth factor across all groups. In addition, quantitative susceptibility mapping of brain white matter, a measure of iron deposition, was increased in older compared to younger healthy non-CCM subjects. Genetic aberrations, plasma molecules, and imaging biomarkers in a well characterized Mendelian neurovascular disease may also be applicable in the aging brain. Graphical abstract.

Permissive microbiome characterizes human subjects with a neurovascular disease cavernous angioma.

Cavernous angiomas (CA) are common vascular anomalies causing brain hemorrhage. Based on mouse studies, roles of gram-negative bacteria and altered intestinal homeostasis have been implicated in CA pathogenesis, and pilot study had suggested potential microbiome differences between non-CA and CA individuals based on 16S rRNA gene sequencing. We here assess microbiome differences in a larger cohort of human subjects with and without CA, and among subjects with different clinical features, and conduct more definitive microbial analyses using metagenomic shotgun sequencing. Relative abundance of distinct bacterial species in CA patients is shown, consistent with postulated permissive microbiome driving CA lesion genesis via lipopolysaccharide signaling, in humans as in mice. Other microbiome differences are related to CA clinical behavior. Weighted combinations of microbiome signatures and plasma inflammatory biomarkers enhance associations with disease severity and hemorrhage. This is the first demonstration of a sensitive and specific diagnostic microbiome in a human neurovascular disease.

Biomarkers of cavernous angioma with symptomatic hemorrhage.

BACKGROUNDCerebral cavernous angiomas (CAs) with a symptomatic hemorrhage (CASH) have a high risk of recurrent hemorrhage and serious morbidity.METHODSEighteen plasma molecules with mechanistic roles in CA pathobiology were investigated in 114 patients and 12 healthy subjects. The diagnostic biomarker of a CASH in the prior year was derived as that minimizing the Akaike information criterion and validated using machine learning, and was compared with the prognostic CASH biomarker predicting bleeding in the subsequent year. Biomarkers were longitudinally followed in a subset of cases. The biomarkers were queried in the lesional neurovascular unit (NVU) transcriptome and in plasma miRNAs from CASH and non-CASH patients.RESULTSThe diagnostic CASH biomarker included a weighted combination of soluble CD14 (sCD14), VEGF, C-reactive protein (CRP), and IL-10 distinguishing CASH patients with 76% sensitivity and 80% specificity (P = 0.0003). The prognostic CASH biomarker (sCD14, VEGF, IL-1ß, and sROBO-4) was confirmed to predict a bleed in the subsequent year with 83% sensitivity and 93% specificity (P = 0.001). Genes associated with diagnostic and prognostic CASH biomarkers were differentially expressed in CASH lesional NVUs. Thirteen plasma miRNAs were differentially expressed between CASH and non-CASH patients.CONCLUSIONShared and unique biomarkers of recent symptomatic hemorrhage and of future bleeding in CA are mechanistically linked to lesional transcriptome and miRNA. The biomarkers may be applied for risk stratification in clinical trials and developed as a tool in clinical practice.FUNDINGNIH, William and Judith Davis Fund in Neurovascular Surgery Research, Be Brave for Life Foundation, Safadi Translational Fellowship, Pritzker School of Medicine, and Sigrid Jusélius Foundation.

Comprehensive transcriptome analysis of cerebral cavernous malformation across multiple species and genotypes.

The purpose of this study was to determine important genes, functions, and networks contributing to the pathobiology of cerebral cavernous malformation (CCM) from transcriptomic analyses across 3 species and 2 disease genotypes. Sequencing of RNA from laser microdissected neurovascular units of 5 human surgically resected CCM lesions, mouse brain microvascular endothelial cells, Caenorhabditis elegans with induced Ccm gene loss, and their respective controls provided differentially expressed genes (DEGs). DEGs from mouse and C. elegans were annotated into human homologous genes. Cross-comparisons of DEGs between species and genotypes, as well as network and gene ontology (GO) enrichment analyses, were performed. Among hundreds of DEGs identified in each model, common genes and 1 GO term (GO:0051656, establishment of organelle localization) were commonly identified across the different species and genotypes. In addition, 24 GO functions were present in 4 of 5 models and were related to cell-to-cell adhesion, neutrophil-mediated immunity, ion transmembrane transporter activity, and responses to oxidative stress. We have provided a comprehensive transcriptome library of CCM disease across species and for the first time to our knowledge in Ccm1/Krit1 versus Ccm3/Pdcd10 genotypes. We have provided examples of how results can be used in hypothesis generation or mechanistic confirmatory studies.

Transcriptome clarifies mechanisms of lesion genesis versus progression in models of Ccm3 cerebral cavernous malformations.

Cerebral cavernous malformations (CCMs) are dilated capillaries causing epilepsy and stroke. Inheritance of a heterozygous mutation in CCM3/PDCD10 is responsible for the most aggressive familial form of the disease. Here we studied the differences and commonalities between the transcriptomes of microdissected lesional neurovascular units (NVUs) from acute and chronic in vivo Ccm3/Pdcd10ECKO mice, and cultured brain microvascular endothelial cells (BMECs) Ccm3/Pdcd10ECKO.We identified 2409 differentially expressed genes (DEGs) in acute and 2962 in chronic in vivo NVUs compared to microdissected brain capillaries, as well as 121 in in vitro BMECs with and without Ccm3/Pdcd10 loss (fold change ≥ |2.0|; p < 0.05, false discovery rate corrected). A functional clustered dendrogram generated using the Euclidean distance showed that the DEGs identified only in acute in vivo NVUs were clustered in cellular proliferation gene ontology functions. The DEGs only identified in chronic in vivo NVUs were clustered in inflammation and immune response, permeability, and adhesion functions. In addition, 1225 DEGs were only identified in the in vivo NVUs but not in vitro BMECs, and these clustered within neuronal and glial functions. One miRNA mmu-miR-3472a was differentially expressed (FC = - 5.98; p = 0.07, FDR corrected) in the serum of Ccm3/Pdcd10+/- when compared to wild type mice, and this was functionally related as a putative target to Cand2 (cullin associated and neddylation dissociated 2), a DEG in acute and chronic lesional NVUs and in vitro BMECs. Our results suggest that the acute model is characterized by cell proliferation, while the chronic model showed inflammatory, adhesion and permeability processes. In addition, we highlight the importance of extra-endothelial structures in CCM disease, and potential role of circulating miRNAs as biomarkers of disease, interacting with DEGs. The extensive DEGs library of each model will serve as a validation tool for potential mechanistic, biomarker, and therapeutic targets.