| 1. |
- Claesson-Welsh, Lena, et al.
(author)
-
Permeability of the Endothelial Barrier : Identifying and Reconciling Controversies
- 2021
-
In: Trends in Molecular Medicine. - : Elsevier. - 1471-4914 .- 1471-499X. ; 27:4, s. 314-331
-
Research review (peer-reviewed)abstract
- Leakage from blood vessels into tissues is governed by mechanisms that control endothelial barrier function to maintain homeostasis. Dysregulated endothelial permeability contributes to many conditions and can influence disease morbidity and treatment. Diverse approaches used to study endothelial permeability have yielded a wealth of valuable insights. Yet, ongoing questions, technical challenges, and unresolved controversies relating to the mechanisms and relative contributions of barrier regulation, transendothelial sieving, and transport of fluid, solutes, and particulates complicate interpretations in the context of vascular physiology and pathophysiology. Here, we describe recent in vivo findings and other advances in understanding endothelial barrier function with the goal of identifying and reconciling controversies over cellular and molecular processes that regulate the vascular barrier in health and disease.
|
|
| 2. |
- Cottarelli, Azzurra, et al.
(author)
-
Fgfbp1 promotes blood-brain barrier development by regulating collagen IV deposition and maintaining Wnt/beta-catenin signaling
- 2020
-
In: Development. - : COMPANY BIOLOGISTS LTD. - 0950-1991 .- 1477-9129. ; 147:16
-
Journal article (peer-reviewed)abstract
- Central nervous system (CNS) blood vessels contain a functional blood-brain barrier (BBB) that is necessary for neuronal survival and activity. Although Wnt/beta-catenin signaling is essential for BBB development, its downstream targets within the neurovasculature remain poorly understood. To identify targets of Wnt/beta-catenin signaling underlying BBB maturation, we performed a microarray analysis that identified Fgfbp1 as a novel Wnt/beta-catenin-regulated gene in mouse brain endothelial cells (mBECs). Fgfbp1 is expressed in the CNS endothelium and secreted into the vascular basement membrane during BBB formation. Endothelial genetic ablation of Fgfbp1 results in transient hypervascularization but delays BBB maturation in specific CNSregions, as evidenced by both upregulation of Plvap and increased tracer leakage across the neurovasculature due to reduced Wnt/beta-catenin activity. In addition, collagen IV deposition in the vascular basement membrane is reduced in mutant mice, leading to defective endothelial cell-pericyte interactions. Fgfbp1 is required cell-autonomously in mBECs to concentrate Wnt ligands near cell junctions and promote maturation of their barrier properties in vitro. Thus, Fgfbp1 is a crucial extracellular matrix protein during BBB maturation that regulates cell-cell interactions and Wnt/beta-catenin activity.
|
|
| 3. |
- Globisch, Maria A., et al.
(author)
-
Immunothrombosis and vascular heterogeneity in cerebral cavernous malformation
- 2022
-
In: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 140:20, s. 2154-2169
-
Journal article (peer-reviewed)abstract
- Cerebral cavernous malformation (CCM) is a neurovascular disease that results in various neurological symptoms. Thrombi have been reported in surgically resected CCM patient biopsies, but the molecular signatures of these thrombi remain elusive. Here, we investigated the kinetics of thrombi formation in CCM and how thrombi affect the vasculature and contribute to cerebral hypoxia. We used RNA sequencing to investigate the transcriptome of mouse brain endothelial cells with an inducible endothelial-specific Ccm3 knock-out (Ccm3-iECKO). We found that Ccm3-deficient brain endothelial cells had a higher expression of genes related to the coagulation cascade and hypoxia when compared with wild-type brain endothelial cells. Immunofluorescent assays identified key molecular signatures of thrombi such as fibrin, von Willebrand factor, and activated platelets in Ccm3-iECKO mice and human CCM biopsies. Notably, we identified polyhedrocytes in Ccm3-iECKO mice and human CCM biopsies and report it for the first time. We also found that the parenchyma surrounding CCM lesions is hypoxic and that more thrombi correlate with higher levels of hypoxia. We created an in vitro model to study CCM pathology and found that human brain endothelial cells deficient for CCM3 expressed elevated levels of plasminogen activator inhibitor-1 and had a redistribution of von Willebrand factor. With transcriptomics, comprehensive imaging, and an in vitro CCM preclinical model, this study provides experimental evidence that genes and proteins related to the coagulation cascade affect the brain vasculature and promote neurological side effects such as hypoxia in CCMs. This study supports the concept that antithrombotic therapy may be beneficial for patients with CCM.
|
|
| 4. |
- Globisch, Maria Ascención
(author)
-
Inflammation and immunothrombosis in cerebral cavernous malformation : Novel molecular targets for the treatment of an incurable disease
- 2023
-
Doctoral thesis (other academic/artistic)abstract
- Cerebral cavernous malformation (CCM) is a vascular disease that causes mulberry-like lesions (cavernomas) in the central nervous system (CNS). Cavernomas are fragile, leaky and prone to rupture which may cause symptoms such as epileptic seizures, focal neurological deficits and hemorrhagic strokes. CCM lesions can appear sporadically in 0.5% of the general population. Alternatively, CCM lesions appear as a consequence of a loss-of-function germline mutation in either CCM1, CCM2, or CCM3 in endothelial cells of the CNS. Inherited CCM is termed familial, and it affects approximately one in ten thousand individuals in an autosomal dominant manner. The aim of this thesis project is to identify novel cell and molecular mechanisms that contribute to the development and progression of cavernous malformations. Additionally, this thesis project aims to identify and validate inhibitors that may reduce lesions and alleviate the side effects in CCM. In this thesis project, two inducible endothelial specific Ccm3 deficient mouse models (acute and chronic) were evaluated with methods such as RNA-sequencing, immunofluorescence, ELISA, scanning and transmission electron microscopy, flow cytometry, and in situ hybridization. Moreover, in vitro cell cultures were used with methods such as immunofluorescence, qPCR, and western blot. Importantly, sporadic and familial human CCM samples were used to show the clinical relevance of our studies. In paper I we focused on the role and kinetics of inflammation in CCM. We analyzed the transcriptome of healthy and Ccm3 deficient mouse brain endothelial cells and found that genes related to inflammation were upregulated in CCM pathology. We identified various inflammatory cytokines in vivo and also identified neutrophils as the most prominent immune cell subtype in CCM. Moreover, we found that neutrophils in CCM produce neutrophil extracellular traps (NETs), that can be inhibited with DNase I. The inhibition of NETs stabilized cavernoma vasculature by reducing fibrinogen and IgG leakage. In paper II we re-used the acute RNA-seq database from paper 1 and focused on endothelial hemostasis and hypoxia. We found that genes related to procoagulation, anticoagulation, and hypoxia were highly upregulated in Ccm3 deficient mice. We validated the findings in vivo and found that the hemostatic system in CCM is dysregulated and that it causes, bleeding, thrombosis, and cerebral hypoxia. In paper III we evaluated the effect of propranolol in CCM. We treated chronic CCM mice with the beta-blocker propranolol and found that propranolol was able to reduce lesions in the brains and retinas of CCM mice as well as reduce cadaverine leakage. Importantly, we identified endothelial plasmalemmal pits and a thick basal membrane between endothelial cells and pericytes, pathological features which reduced upon propranolol treatment. Altogether this thesis significantly contributes to the CCM field as it identified pathological features of cavernomas such as neutrophils with NETs, endothelial plasmalemmal pits, and polyhedrocytes. This thesis work also evaluated pharmacological inhibitors (DNase I and propranolol) in mouse models of CCM and supports the use of anticoagulant therapies in patients with CCM.
|
|
| 5. |
- Huang, Hua, 1986-, et al.
(author)
-
ELTD1-deletion reduces vascular abnormality and improves T-cell recruitment after PD-1 blockade in glioma.
- 2021
-
In: Neuro-Oncology. - : Oxford University Press. - 1522-8517 .- 1523-5866. ; 24:3, s. 398-411
-
Journal article (peer-reviewed)abstract
- BACKGROUND: Tumor vessels in glioma are molecularly and functionally abnormal, contributing to treatment resistance. Proteins differentially expressed in glioma vessels can change vessel phenotype and be targeted for therapy. ELTD1 (Adgrl4) is an orphan member of the adhesion G-protein-coupled receptor family upregulated in glioma vessels, and has been suggested as a potential therapeutic target. However, the role of ELTD1 in regulating vessel function in glioblastoma is poorly understood.METHODS: ELTD1 expression in human gliomas and its association with patient survival was determined using tissue microarrays and public databases. The role of ELTD1 in regulating tumor vessel phenotype was analyzed using orthotopic glioma models and ELTD1 -/- mice. Endothelial cells isolated from murine gliomas were transcriptionally profiled to determine differentially expressed genes and pathways. The consequence of ELTD1-deletion on glioma immunity was determined by treating tumor bearing mice with PD-1-blocking antibodies.RESULTS: ELTD1 levels were upregulated in human glioma vessels, increased with tumor malignancy, and were associated with poor patient survival. Progression of orthotopic gliomas was not affected by ELTD1-deletion, however, tumor vascular function was improved in ELTD1 -/- mice. Bioinformatic analysis of differentially expressed genes indicated increased inflammatory response and decreased proliferation in tumor endothelium in ELTD1 -/- mice. Consistent with an enhanced inflammatory response, ELTD1-deletion improved T-cell infiltration in GL261-bearing mice after PD-1 checkpoint blockade.CONCLUSION: Our data demonstrate that ELTD1 participates in inducing vascular dysfunction in glioma, and suggests that targeting of ELTD1 may normalize the vessels and improve the response to immunotherapy.
|
|
| 6. |
- Kakogiannos, Nikolaos, et al.
(author)
-
JAM-A Acts via C/EBP-alpha to Promote Claudin-5 Expression and Enhance Endothelial Barrier Function
- 2020
-
In: Circulation Research. - : Lippincott Williams & Wilkins. - 0009-7330 .- 1524-4571. ; 127:8, s. 1056-1073
-
Journal article (peer-reviewed)abstract
- 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.
|
|
| 7. |
- Lazzaroni, Francesca, et al.
(author)
-
Circulating biomarkers in familial cerebral cavernous malformation
- 2024
-
In: EBioMedicine. - : Elsevier. - 2352-3964. ; 99
-
Journal article (peer-reviewed)abstract
- 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.
|
|
| 8. |
- Lugano, Roberta, et al.
(author)
-
CD93 maintains endothelial barrier function by limiting the phosphorylation and turnover of VE-cadherin
- 2023
-
In: The FASEB Journal. - : John Wiley & Sons. - 0892-6638 .- 1530-6860. ; 37:4
-
Journal article (peer-reviewed)abstract
- Regulation of vascular permeability to plasma is essential for tissue and organ homeostasis and is mediated by endothelial cell-to-cell junctions that tightly regulate the trafficking of molecules between blood and tissue. The single-pass transmembrane glycoprotein CD93 is upregulated in endothelial cells during angiogenesis and controls cytoskeletal dynamics. However, its role in maintaining homeostasis by regulating endothelial barrier function has not been elucidated yet. Here, we demonstrate that CD93 interacts with vascular endothelial (VE)-cadherin and limits its phosphorylation and turnover. CD93 deficiency in vitro and in vivo induces phosphorylation of VE-cadherin under basal conditions, displacing it from endothelial cell–cell contacts. Consistent with this, endothelial junctions are defective in CD93−/− mice, and the blood–brain barrier permeability is enhanced. Mechanistically, CD93 regulates VE-cadherin phosphorylation and turnover at endothelial junctions through the Rho/Rho kinase-dependent pathway. In conclusion, our results identify CD93 as a key regulator of VE-cadherin stability at endothelial junctions, opening up possibilities for therapeutic strategies directed to control vascular permeability.
|
|
| 9. |
- Maderna, Claudio, et al.
(author)
-
A murine model of cerebral cavernous malformations with acute hemorrhage
- 2022
-
In: iScience. - : Cell Press. - 2589-0042. ; 25:3
-
Journal article (peer-reviewed)abstract
- 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 defi zits, and paresthesia. An effective medical treatment is still lacking, and the available murine models for cavernomas have several limitations for predinical 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.
|
|
| 10. |
- Monzo, Pascale, et al.
(author)
-
Adaptive mechanoproperties mediated by the formin FMN1 characterize glioblastoma fitness for invasion
- 2021
-
In: Developmental Cell. - : Elsevier. - 1534-5807 .- 1878-1551. ; 56:20, s. 2841-2855.e8
-
Journal article (peer-reviewed)abstract
- Glioblastoma are heterogeneous tumors composed of highly invasive and highly proliferative clones, Heterogeneity in invasiveness could emerge from discrete biophysical properties linked to specific molecular expression. We identified clones of patient-derived glioma propagating cells that were either highly proliferative or highly invasive and compared their cellular architecture, migratory, and biophysical properties. We discovered that invasiveness was linked to cellular fitness. The most invasive cells were stiffer, developed higher mechanical forces on the substrate, and moved stochastically. The mechano-chemical-induced expression of the formin FMN1 conferred invasive strength that was confirmed in patient samples. Moreover, FMN1 expression was also linked to motility in other cancer and normal cell lines, and its ectopic expression increased fitness parameters. Mechanistically, FMN1 acts from the microtubule lattice and promotes a robust mechanical cohesion, leading to highly invasive motility.
|
|
