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OBJECTIVE: Hemorrhoids are a common anorectal disease that causes pain, itching, and burning. The prevalence of hemorrhoids is estimated to be as high as 36% in the general population, with approximately 50% of individuals experiencing symptomatic hemorrhoids at least once in their life. Middle age, obesity, and pregnancy are risk factors. The combination of tribenoside and lidocaine (Procto-Glyvenol©, Recordati) has been used for decades to treat low-grade hemorrhoids, and its efficacy and safety are well supported by clinical experience. Tribenoside has been shown to have an anti-inflammatory effect, ameliorate the local microcirculation and vascular tone, and promote the healing of basement membrane. However, the molecular mechanism behind its wound-healing properties is still unclear. MATERIALS AND METHODS: Human dermal fibroblasts were used to test the effect of tribenoside on cell proliferation, cell migration, and production of reactive oxygen species in vitro. Full-thickness excisional wound model in rats was used to test the wound-healing properties of Procto-Glyvenol© in vivo. RESULTS: Tribenoside has been found to increase the migration rate of fibroblasts in vitro and to improve the wound healing process by promoting re-epithelialization in rats. Furthermore, novel antioxidant activity of tribenoside has been reported, which may represent a further mechanism of action in wound healing. CONCLUSIONS: Procto-Glyvenol© improves the natural healing process of wounds by stimulating cell migration and protecting against the toxic effects of reactive oxygen species. Therefore, it may represent a first-line treatment for hemorrhoids, which are a significant medical and socioeconomic problem that can deteriorate the quality of life. [ABSTRACT FROM AUTHOR]

We describe here an innovative, non-transgenic animal model of Alzheimer's disease. This model mimics early stages of sporadic disease, which represents the vast majority of cases. The model was obtained by interfering with the complex between a disintegrin and metalloproteinase domain containing protein 10 (ADAM10), the main α-secretase candidate, and its partner, synapse-associated protein 97, a protein of the postsynaptic density-membrane associated guanylate kinase family. Association of ADAM10 with synapse-associated protein 97 governs enzyme trafficking and activity at synapses. Interfering with the ADAM10/synapse-associated protein 97 complex for 2 weeks by means of a cell-permeable peptide strategy is sufficient to shift the metabolism of the amyloid precursor protein towards amyloidogenesis and allows the reproduction of initial phases of sporadic Alzheimer's disease. After 2 weeks of treatment, we detected progressive Alzheimer's disease-like neuropathology, with an increase of β-amyloid aggregate production and of tau hyperphosphorylation, and a selective alteration of N-methyl-d-aspartic acid receptor subunit composition in the postsynaptic compartment of mouse brain. Behavioural and electrophysiological deficits were also induced by peptide treatment. [ABSTRACT FROM AUTHOR]

Cerebral cavernous malformations (CCMs) are anomalies of the cerebral vasculature. Loss of the CCM proteins CCM1/KRIT1, CCM2, or CCM3/PDCD10 trigger a MAPK-Krüppel-like factor 2 (KLF2) signaling cascade, which induces a pathophysiological pattern of gene expression. The downstream target genes that are activated by KLF2 are mostly unknown. Here we show that Chromobox Protein Homolog 7 (CBX7), component of the Polycomb Repressive Complex 1, contributes to pathophysiological KLF2 signaling during zebrafish cardiovascular development. CBX7/cbx7a mRNA is strongly upregulated in lesions of CCM patients, and in human, mouse, and zebrafish CCM-deficient endothelial cells. The silencing or pharmacological inhibition of CBX7/Cbx7a suppresses pathological CCM phenotypes in ccm2 zebrafish, CCM2-deficient HUVECs, and in a pre-clinical murine CCM3 disease model. Whole-transcriptome datasets from zebrafish cardiovascular tissues and human endothelial cells reveal a role of CBX7/Cbx7a in the activation of KLF2 target genes including TEK, ANGPT1, WNT9, and endoMT-associated genes. Our findings uncover an intricate interplay in the regulation of Klf2-dependent biomechanical signaling by CBX7 in CCM. This work also provides insights for therapeutic strategies in the pathogenesis of CCM., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests B.B is inventor of patents on PTX3 and obtains royalties on PTX3-related reagents. N.B. serves on Advisory Board and Speakers Bureau for Celgene and Janssen, and on Speakers Bureau for Takeda, Amgen., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

In the study of cerebral cavernous malformations (CCMs), the quantification of lesion burden is the main parameter for evaluation of disease severity and efficacy of drugs. We describe a reliable and cost-effective protocol to evaluate the number and the size of vascular malformations in the murine brain. This approach is based on histology and confocal imaging and can be performed with standard laboratory equipment. We detail the preparation of brain sections followed by image acquisition and analysis. For complete details on the use and execution of this protocol, please refer to Maderna et al. (2022)., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Cerebral cavernous malformations (CCM) are low-flow vascular lesions prone to cause severe hemorrhage-associated neurological complications. Pathogenic germline variants in CCM1, CCM2, or CCM3 can be identified in nearly 100% of CCM patients with a positive family history. In line with the concept that tumor-like mechanisms are involved in CCM formation and growth, we here demonstrate an abnormally increased proliferation rate of CCM3-deficient endothelial cells in co-culture with wild-type cells and in mosaic human iPSC-derived vascular organoids. The observation that NSC59984, an anticancer drug, blocked the abnormal proliferation of mutant endothelial cells further supports this intriguing concept. Fluorescence-activated cell sorting and RNA sequencing revealed that co-culture induces upregulation of proangiogenic chemokine genes in wild-type endothelial cells. Furthermore, genes known to be significantly downregulated in CCM3 -/- endothelial cell mono-cultures were upregulated back to normal levels in co-culture with wild-type cells. These results support the hypothesis that wild-type ECs facilitate the formation of a niche that promotes abnormal proliferation of mutant ECs. Thus, targeting the cancer-like features of CCMs is a promising new direction for drug development., (© 2022. The Author(s).)

Cerebral cavernous malformations (CCM) are capillary malformations affecting the central nervous system and commonly present with headaches, epilepsy and stroke. Treatment of CCM is symptomatic, and its prevention is limited. CCM are often sporadic but sometimes may be multifocal and/or affect multiple family members. Heterozygous pathogenic variants in PDCD10 cause the rarest and apparently most severe genetic variant of familial CCM. We carried out an RNA-Seq and a Q-PCR validation analysis in Pdcd10-silenced and wild-type mouse endothelial cells in order to better elucidate CCM molecular pathogenesis. Ninety-four differentially expressed genes presented an FDR-corrected p-value < 0.05. A functionally clustered dendrogram showed that differentially expressed genes cluster in cell proliferation, oxidative stress, vascular processes and immune response gene-ontology functions. Among differentially expressed genes, the major cluster fell in signaling related to inflammation and pathogen recognition, including HIF1α and Nos2 signaling and immune regulation. Validation analysis performed on wild-type, Pdcd10-null and Pdcd10-null reconstituted cell lines was consistent with RNA-Seq data. This work confirmed previous mouse transcriptomic data in endothelial cells, which are recognized as a critical tissue for CCM formation and expands the potential molecular signatures of PDCD10-related familial CCM to alterations in inflammation and pathogen recognition pathways.

Cerebral Cavernous Malformation (CCM) is a brain vascular disease with various neurological symptoms. In this study, we describe the inflammatory profile in CCM and show for the first time the formation of neutrophil extracellular traps (NETs) in rodents and humans with CCM. Through RNA-seq analysis of cerebellum endothelial cells from wild-type mice and mice with an endothelial cell-specific ablation of the Ccm3 gene (Ccm3 iECKO ), we show that endothelial cells from Ccm3 iECKO mice have an increased expression of inflammation-related genes. These genes encode proinflammatory cytokines and chemokines, as well as adhesion molecules, which promote recruitment of inflammatory and immune cells. Similarly, immunoassays showed elevated levels of these cytokines and chemokines in the cerebellum of the Ccm3 iECKO mice. Consistently, both flow cytometry and immunofluorescence analysis showed infiltration of different subsets of leukocytes into the CCM lesions. Neutrophils, which are known to fight against infection through different strategies, including the formation of NETs, represented the leukocyte subset within the most pronounced increase in CCM. Here, we detected elevated levels of NETs in the blood and the deposition of NETs in the cerebral cavernomas of Ccm3 iECKO mice. Degradation of NETs by DNase I treatment improved the vascular barrier. The deposition of NETs in the cavernomas  of patients with CCM confirms the clinical relevance of NETs in CCM., (© 2022. The Author(s).)

Cavernomas are multi-lumen and blood-filled vascular malformations which form in the brain and the spinal cord. They lead to hemorrhage, epileptic seizures, neurological deficits, and paresthesia. An effective medical treatment is still lacking, and the available murine models for cavernomas have several limitations for preclinical studies. These include disease phenotypes that differ from human diseases, such as restriction of the lesions to the cerebellum, and absence of acute hemorrhage. Additional limitations of current murine models include rapid development of lesions, which are lethal before the first month of age. Here, we have characterized a murine model that recapitulates features of the human disease: lesions develop after weaning throughout the entire CNS, including the spinal cord, and undergo acute hemorrhage. This provides a preclinical model to develop new drugs for treatment of acute hemorrhage in the brain and spinal cord, as an unmet medical emergency for patients with cavernomas., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Endothelial-to-mesenchymal transition (EndMT) is the biological process through which endothelial cells transdifferentiate into mesenchymal cells. During embryo development, EndMT regulates endocardial cushion formation via TGFβ/BMP signaling. In adults, EndMT is mainly activated during pathological conditions. Hence, it is necessary to characterize molecular regulators cooperating with TGFβ signaling in driving EndMT, to identify potential novel therapeutic targets to treat these pathologies. Here, we studied YAP, a transcriptional co-regulator involved in several biological processes, including epithelial-to-mesenchymal transition (EMT). As EndMT is the endothelial-specific form of EMT, and YAP (herein referring to YAP1) and TGFβ signaling cross-talk in other contexts, we hypothesized that YAP contributes to EndMT by modulating TGFβ signaling. We demonstrate that YAP is required to trigger TGFβ-induced EndMT response, specifically contributing to SMAD3-driven EndMT early gene transcription. We provide novel evidence that YAP acts as SMAD3 transcriptional co-factor and prevents GSK3β-mediated SMAD3 phosphorylation, thus protecting SMAD3 from degradation. YAP is therefore emerging as a possible candidate target to inhibit pathological TGFβ-induced EndMT at early stages., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

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Aims of the Study: The European Society of Medical Oncology (ESMO) recommends that countries should have reference centres to provide adequate diagnosis and treatment of gestational trophoblastic disease. A trophoblastic disease centre in the French-speaking part of Switzerland was inaugurated in 2009. The objectives of this study were to report the activity of the centre during the last 10 years and analyse gestational trophoblastic disease outcomes., Methods: This was a retrospective study with data collected from all cases of gestational trophoblastic disease referred to the centre from 2009 to 2018. All histological specimens as well as data for treatment and follow-up of gestational trophoblastic disease and neoplasia were reviewed. Clinical features, including age, prognostic score and International Federation of Gynecology and Obstetrics (FIGO) stages (in the case of gestational trophoblastic neoplasia), human chorionic gonadotropin (hCG) follow-up, treatment and outcome were reported., Results: The centre registered 354 patients, and these patients presented 156 cases of partial hydatidiform moles, 163 cases of complete hydatidiform moles and 14 cases of gestational trophoblastic neoplasia. During follow-up, 35 gestational trophoblastic neoplasms were diagnosed after hCG persistence. After pathology review, the overall agreement rates between our centre and a participating provider hospital was 82%. Methotrexate was the first line of single-agent chemotherapy for most patients, with resistance rates of 23%. Multi-agent chemotherapy was used as first-line treatment for five patients. None of the patients followed up by the centre died from gestational trophoblastic disease., Conclusions: This study reflects the activity of the Swiss trophoblastic disease centre from the French-speaking part of Switzerland created in 2009, and its role as local and national reference centre, in terms of global health, for women with gestational trophoblastic disease.

The programmed cell death 10 ( PDCD10 ) gene was originally identified as an apoptosis-related gene, although it is now usually known as CCM3 , as the third causative gene of cerebral cavernous malformation (CCM). CCM is a neurovascular disease that is characterized by vascular malformations and is associated with headaches, seizures, focal neurological deficits, and cerebral hemorrhage. The PDCD10/CCM3 protein has multiple subcellular localizations and interacts with several multi-protein complexes and signaling pathways. Thus PDCD10/CCM3 governs many cellular functions, which include cell-to-cell junctions and cytoskeleton organization, cell proliferation and apoptosis, and exocytosis and angiogenesis. Given its central role in the maintenance of homeostasis of the cell, dysregulation of PDCD10/CCM3 can result in a wide range of altered cell functions. This can lead to severe diseases, including CCM, cognitive disability, and several types of cancers. Here, we review the multifaceted roles of PDCD10/CCM3 in physiology and pathology, with a focus on its functions beyond CCM., (© 2021 Chongqing Medical University. Production and hosting by Elsevier B.V.)

Cerebral cavernous malformation (CCM) is a rare neurovascular disease that is characterized by enlarged and irregular blood vessels that often lead to cerebral hemorrhage. Loss-of-function mutations to any of three genes results in CCM lesion formation; namely, KRIT1 , CCM2 , and PDCD10 (CCM3) . Here, we report for the first time in-depth single-cell RNA sequencing, combined with spatial transcriptomics and immunohistochemistry, to comprehensively characterize subclasses of brain endothelial cells (ECs) under both normal conditions and after deletion of Pdcd10 ( Ccm3) in a mouse model of CCM. Integrated single-cell analysis identifies arterial ECs as refractory to CCM transformation. Conversely, a subset of angiogenic venous capillary ECs and respective resident endothelial progenitors appear to be at the origin of CCM lesions. These data are relevant for the understanding of the plasticity of the brain vascular system and provide novel insights into the molecular basis of CCM disease at the single cell level., Competing Interests: FO, LC, SJ, MC, FL, MM, VS, SC, JB, MG, CM, ML, PM No competing interests declared, ED Reviewing editor, eLife, (© 2020, Orsenigo et al.)

Rationale: Intercellular tight junctions are crucial for correct regulation of the endothelial barrier. Their composition and integrity are affected in pathological contexts, such as inflammation and tumor growth. JAM-A (junctional adhesion molecule A) is a transmembrane component of tight junctions with a role in maintenance of endothelial barrier function, although how this is accomplished remains elusive., Objective: We aimed to understand the molecular mechanisms through which JAM-A expression regulates tight junction organization to control endothelial permeability, with potential implications under pathological conditions., Methods and Results: Genetic deletion of JAM-A in mice significantly increased vascular permeability. This was associated with significantly decreased expression of claudin-5 in the vasculature of various tissues, including brain and lung. We observed that C/EBP-α (CCAAT/enhancer-binding protein-α) can act as a transcription factor to trigger the expression of claudin-5 downstream of JAM-A, to thus enhance vascular barrier function. Accordingly, gain-of-function for C/EBP-α increased claudin-5 expression and decreased endothelial permeability, as measured by the passage of fluorescein isothiocyanate (FITC)-dextran through endothelial monolayers. Conversely, C/EBP-α loss-of-function showed the opposite effects of decreased claudin-5 levels and increased endothelial permeability. Mechanistically, JAM-A promoted C/EBP-α expression through suppression of β-catenin transcriptional activity, and also through activation of EPAC (exchange protein directly activated by cAMP). C/EBP-α then directly binds the promoter of claudin-5 to thereby promote its transcription. Finally, JAM-A-C/EBP-α-mediated regulation of claudin-5 was lost in blood vessels from tissue biopsies from patients with glioblastoma and ovarian cancer., Conclusions: We describe here a novel role for the transcription factor C/EBP-α that is positively modulated by JAM-A, a component of tight junctions that acts through EPAC to up-regulate the expression of claudin-5, to thus decrease endothelial permeability. Overall, these data unravel a regulatory molecular pathway through which tight junctions limit vascular permeability. This will help in the identification of further therapeutic targets for diseases associated with endothelial barrier dysfunction. Graphic Abstract: An graphic abstract is available for this article.

Cerebral cavernous malformation (CCM) is a neurovascular familial or sporadic disease that is characterised by capillary-venous cavernomas, and is due to loss-of-function mutations to any one of three CCM genes. Familial CCM follows a two-hit mechanism similar to that of tumour suppressor genes, while in sporadic cavernomas only a small fraction of endothelial cells shows mutated CCM genes. We reported that in mouse models and in human patients, endothelial cells lining the lesions have different features from the surrounding endothelium, as they express mesenchymal/stem-cell markers. Here we show that cavernomas originate from clonal expansion of few Ccm3-null endothelial cells that express mesenchymal/stem-cell markers. These cells then attract surrounding wild-type endothelial cells, inducing them to express mesenchymal/stem-cell markers and to contribute to cavernoma growth. These characteristics of Ccm3-null cells are reminiscent of the tumour-initiating cells that are responsible for tumour growth. Our data support the concept that CCM has benign tumour characteristics.

The biological players involved in angiogenesis are only partially defined. Here, we report that endothelial cells (ECs) express a novel isoform of the cell-surface adhesion molecule L1CAM, termed L1-ΔTM. The splicing factor NOVA2, which binds directly to L1CAM pre-mRNA, is necessary and sufficient for the skipping of L1CAM transmembrane domain in ECs, leading to the release of soluble L1-ΔTM. The latter exerts high angiogenic function through both autocrine and paracrine activities. Mechanistically, L1-ΔTM-induced angiogenesis requires fibroblast growth factor receptor-1 signaling, implying a crosstalk between the two molecules. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer, where L1-ΔTM levels correlate with tumor vascularization, supporting the involvement of NOVA2-mediated L1-ΔTM production in tumor angiogenesis. Finally, high NOVA2 expression is associated with poor outcome in ovarian cancer patients. Our results point to L1-ΔTM as a novel, EC-derived angiogenic factor which may represent a target for innovative antiangiogenic therapies., Competing Interests: EB, MG, MC, FF, PP, ML, CB, DP, AD, CG, GJ, CL, GB, SF, MM, MI, CG, RG, DN, DG No competing interests declared, JM, JS Affiliated with Gene Tools, LLC. Funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. ED Reviewing editor, eLife, CG Consultant for Gene-Tools, LLC. Funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript., (© 2019, Angiolini et al.)

We hypothesized that in glioblastoma recurring after radiotherapy, a condition whereby the brain endothelium undergoes radiation-induced senescence, tumor cells with endothelial phenotype may be relevant for tumor neovascularization. Matched glioblastoma samples obtained at primary surgery and at surgery for tumor recurrence after radiotherapy, all expressing epidermal growth factor receptor variant III (EGFRvIII), were assessed by a technique that combines fluorescent in situ hybridization (FISH) for the EGFR/CEP7 chromosomal probe with immunostaining for endothelial cells (CD31) and activated pericytes (α Smooth Muscle Actin). Five EGFRvIII-expressing paired primary/recurrent glioblastoma samples, in which the tumor cells showed EGFR/CEP7 amplification, were then assessed by CD31 and α Smooth Muscle Actin immunofluorescence. In glomeruloid bodies, the ratio between CD31+ cells with amplified EGFR/CEP7 signal and the total CD31+ cells was 0.23 ± 0.09 (mean ± sem) and 0.63 ± 0.07 in primary tumors and in recurrent ones, respectively (p < 0.002, Student-t test). In capillaries, the ratio of CD31+ cells with amplified EGFR/CEP7 over the total CD31+ cells lining the capillary lumen was 0.21 ± 0.06 (mean ± sem) and 0.42 ± 0.07 at primary surgery and at recurrence, respectively (p < 0.005, Student-t test). Expression of α Smooth Muscle Actin by cells with EGFR/CEP7 amplification was not observed. Then, in glioblastoma recurring after radiotherapy, where the brain endothelium suffers from radiation-induced cell senescence, tumor-derived endothelium plays a role in neo-vascularization.

Objective: The assembly of a functional vascular system requires a coordinated and dynamic transition from activation to maturation. High vascular endothelial growth factor activity promotes activation, including junction destabilization and cell motility. Maturation involves junctional stabilization and formation of a functional endothelial barrier. The identity and mechanism of action of prostabilization signals are still mostly unknown. Bone morphogenetic protein receptors and their ligands have important functions during embryonic vessel assembly and maturation. Previous work has suggested a role for growth differentiation factor 6 (GDF6; bone morphogenetic protein 13) in vascular integrity although GDF6's mechanism of action was not clear. Therefore, we sought to further explore the requirement for GDF6 in vascular stabilization., Approach and Results: We investigated the role of GDF6 in promoting endothelial vascular integrity in vivo in zebrafish and in cultured human umbilical vein endothelial cells in vitro. We report that GDF6 promotes vascular integrity by counteracting vascular endothelial growth factor activity. GDF6-deficient endothelium has increased vascular endothelial growth factor signaling, increased vascular endothelial-cadherin Y658 phosphorylation, vascular endothelial-cadherin delocalization from cell-cell interfaces, and weakened endothelial cell adherence junctions that become prone to vascular leak., Conclusions: Our results suggest that GDF6 promotes vascular stabilization by restraining vascular endothelial growth factor signaling. Understanding how GDF6 affects vascular integrity may help to provide insights into hemorrhage and associated vascular pathologies in humans., (© 2017 American Heart Association, Inc.)