Pathogenic effects of TIE2-mutations causing venous malformation

Venous malformations (VM) are localized defects of angiogenesis, which typically involve the skin, mucosa and subcutis. They are characterized by enlarged venous channels with a relative lack of surrounding smooth muscle cells (SMCs). While mostly sporadic (>98%), 1-2% occur as an autosomal dominantly inherited trait, named cutaneomucosal venous malformation (VMCM). Both forms can be caused by gain-of-function mutations in TEK, encoding the endothelial cell (EC) -specific tyrosine kinase receptor TIE2. Eight different inherited TIE2 changes have been implicated in VMCM, all of which induce a ligand-independent receptor hyperphosphorylation in vitro, from 2- to 30-fold that of the wild-type. The most common of these (10/17 families; 58.8%) is the mildly activating R849W (Publication I). A somatic second-hit was identified in tissue-derived cDNA from a patient carrying the R849W-mutation in the germline. It was located on the wild-type allele and partially deleted the ligand-binding domain of TIE2, resulting in a loss-of-function of the receptor (Publication II). At least 50% of common sporadic VMs are also caused by somatic activating mutations in TIE2. The mutations include a frequent L914F change (85.7%), and a series of double-mutations on the same allele (in cis). Like the inherited changes, all somatic TIE2 mutations cause hyperphosphorylation in vitro. L914F is phosphorylated 12-13 fold the level of wild-type TIE2, 4 times as strongly as R849W. Double-mutants cause higher (roughly additive) levels of chronic phosphorylation as compared to their constituent single-mutant forms. Interestingly, the most common TIE2 mutants (germline R849W and somatic L914F) have distinct but overlapping effects on receptor compartmentalization and translocation in response to angiopoietin-1 (ANGPT1) ligand (Publication II). Global gene expression profiling of human umbilical vein endothelial cells (HUVECs) overexpressing wild-type TIE2, the most frequent “weak” inherited mutant R849W, and the most frequent somatic mutant L914F revealed that L914F strongly dysregulates pathways involved in vascular development and cell migration. By contrast, R849W, in the absence of ligand-stimulation, has extremely weak effects, making it indistinguishable from wild-type cells in global analyses. In addition, we inferred that the transcription factor FOXO1 is inhibited in L914F mutant cells as compared to wild-type, based on differential expression of its target genes. Amongst these is the platelet derived growth factor beta (PDGFB), a known recruiter of SMCs, which shows a significant drop at the level of secreted protein from cultured cells, as well as ex vivo, around malformed veins as compared to normal veins or arteries. AKT, which has been shown to phosphorylate and therefore inhibit FOXO1, is highly activated by the TIE2 mutant forms. Inhibition of AKT increases PDGFB secretion in mutant cells, demonstrating an AKT/FOXO1-dependent role for TIE2 in the regulation of PDGFB production. This allowed us to hypothesize that TIE2 dysregulation causes smooth muscle cell paucity in VMs at least partially due to lack of PDGFB (Publication III, in preparation). In conclusion, the insights we have gained about the effects of TIE2 mutants allow us to explain some of the key features of the disease phenotype they mediate: lack of SMCs around lesions, lack of whose support may contribute to vessel dilation. In concert with in vivo mouse models of VMs currently being generated, these data will be of great value in deciphering why VMs occur, and which pathways are potentially attractive therapeutic targets. Les malformations veineuses (VM) sont des défauts localisés survenant lors de l'angiogenèse qui impliquent généralement la peau, les muqueuses et l'hypoderme. Elles sont caractérisées par des canaux veineux élargis avec un manque relatif de cellules musculaires lisses (SMC) les entourant. Alors que la plupart des malformations veineuses sont sporadiques (>98%), elles peuvent également se transmettre selon le mode autosomique dominant (1-2%). On parle alors de malformations veineuses mucocutanées (VMCM). Les deux formes sont causées par des mutations du gène TEK, codant un récepteur tyrosine kinase spécifique des cellules endothéliales (EC), TIE2. Toutes les mutations identifiées induisent un gain de fonction du récepteur. Huit changements héréditaires différents dans le gène TEK sont impliqués dans les VMCM. Ils induisent, in vitro, une hyperphosphorylation indépendante du ligand de 2 à 30 fois supérieure à celle du récepteur sauvage. Le changement le plus commun (10/17 familles; 58,8%) est la mutation légèrement autophosphorylante R849W (publication I). Un second-hit somatique a été identifié dans l’ADNc dérivé du tissu d'un patient porteur de la mutation germinale R849W. Il s’agit d’une délétion partielle de l'allèle sauvage entraînant la suppression d’une partie du domaine de liaison au ligand de TIE2. Ceci résulte en une perte de fonction du récepteur (publication II). Au moins 50% des malformations veineuses sporadiques sont également causées par des mutations somatiques activatrices dans TIE2. Les mutations comprennent le changement fréquent L914F (85,7%), et une série de doubles mutations situées sur le même allèle (en cis). Comme les changements héréditaires, toutes les mutations somatiques identifiées dans le gène TEK provoquent une hyperphosphorylation in vitro. Le niveau d’autophosphorylation de L914F est de 12 à 13 fois supérieur à celui du récepteur TIE2 sauvage et donc 4 fois plus élevé que celui de R849W. Les doubles mutations causent un niveau supérieur de phosphorylation (approximativement additif) par rapport à leurs constituants exprimés indépendamment. Fait intéressant, les mutations les plus communes de TIE2 (R849W germinale et L914F somatique) ont des effets distincts mais aussi communs sur la localisation intracellulaire et la translocation du récepteur en réponse au ligand Angpt1 (publication II). Des profils d'expressions géniques ont été réalisés sur 3 séries de cellules endothéliales provenant de la veine ombilicale humaine (HUVEC). Ces cellules étaient non infectées ou surexprimaient soit le récepteur TIE2 sauvage, le mutant le plus fréquemment identifié dans les cas familiaux R849W, ou le mutant le plus fréquemment identifié dans les cas somatiques L914F. Ces profils d’expressions géniques ont révélé que L914F modifie fortement les voies impliquées dans le développement vasculaire et la migration cellulaire, alors que R849W, en l'absence d’une stimulation par le ligand, a des effets extrêmement faibles. De plus, nous avons déduit que le facteur de transcription FOXO1 est inhibé dans les cellules mutantes L914F par rapport aux cellules exprimant le récepteur sauvage. Ce résultat est basé sur l'expression différentielle de ses gènes cibles. Parmi ces gènes cibles, le « Platelet Derived Growth Factor Beta » (PDGFB), un recruteur connu des cellules musculaires lisses, montre une baisse significative au niveau de la protéine sécrétée par les cellules en culture, ainsi que ex vivo, dans les veines malformés par rapport aux veines ou artères normales. AKT, qui a été montré comme phosphorylant et donc inhibant FOXO1, est fortement activé par les formes mutantes de TIE2. L'inhibition d'AKT augmente la sécrétion de PDGFB par les cellules mutantes, ce qui démontre le rôle de TIE2, dépendant de AKT/FOXO1, dans la régulation de la production de PDGFB. Ces résultats nous ont permis de montrer que le dérèglement de TIE2 entraîne une pénurie de cellules musculaires lisses dans les VM à cause du manque de PDGFB (Publication III, en préparation). En conclusion, les données que nous avons acquises sur les effets physiopathologiques des mutants TIE2 nous permettent d'expliquer une des caractéristiques principales de la maladie qu'ils entraînent: le manque de SMC autour des lésions contribuerait à la dilatation des vaisseaux. De concert avec des modèles de souris in vivo en cours de génération, ces données seront d'une grande valeur pour comprendre pourquoi les VM se produisent, et quelles sont les voies qui pourraient se révéler être des cibles thérapeutiques intéressantes.(SBIM 3) -- UCL, 201

Cross-talk between signaling and metabolism in the vasculature.

The link between signaling and metabolism was first recognized with insulin signal transduction. Efficient glucose uptake by the endothelium requires insulin receptor activation to deliver GLUT receptors to the cell surface. More recently however, additional evidence has emerged for a broader crosstalk as signaling events have been shown to regulate a large number of metabolic enzymes. Changes in the metabolic status of endothelial and smooth muscle cells are observed at times of increased proliferative activity and these coincide with activation of cell surface receptors. Intriguingly, a rise in glycolysis appears to be associated with remodeling of the actin cytoskeleton during migration and angiogenesis. Overall, understanding how do signaling and metabolic pathways intersect and cross-regulate each other has become an important question and an emerging cornerstone in vascular biology

Interleukins, growth factors, and transcription factors are key targets for gene therapy in osteoarthritis: A scoping review.

peer reviewed[en] OBJECTIVE: Osteoarthritis (OA) is the most common degenerative joint disease, characterized by a progressive loss of cartilage associated with synovitis and subchondral bone remodeling. There is however no treatment to cure or delay the progression of OA. The objective of this manuscript was to provide a scoping review of the preclinical and clinical studies reporting the effect of gene therapies for OA. METHOD: This review followed the JBI methodology and was reported in accordance with the PRISMA-ScR checklist. All research studies that explore in vitro, in vivo, or ex vivo gene therapies that follow a viral or non-viral gene therapy approach were considered. Only studies published in English were included in this review. There were no limitations to their date of publication, country of origin, or setting. Relevant publications were searched in Medline ALL (Ovid), Embase (Elsevier), and Scopus (Elsevier) in March 2023. Study selection and data charting were performed by two independent reviewers. RESULTS: We found a total of 29 different targets for OA gene therapy, including studies examining interleukins, growth factors and receptors, transcription factors and other key targets. Most articles were on preclinical in vitro studies (32 articles) or in vivo animal models (39 articles), while four articles were on clinical trials related to the development of TissueGene-C (TG-C). CONCLUSION: In the absence of any DMOAD, gene therapy could be a highly promising treatment for OA, even though further development is required to bring more targets to the clinical stage

NASAFYTOL® supplementation in adults hospitalized with COVID-19 infection: results from an exploratory open-label randomized controlled trial

peer reviewedObjectivesThe effect and safety of Nasafytol®, a food supplement combining curcumin, quercetin, and Vitamin D, on hospitalized COVID-19-positive patients as support to standard of care were to be assessed.MethodsThis exploratory, open-label, randomized, controlled trial was carried out among hospitalized adults with COVID-19 infection. Participants were randomly assigned to receive Nasafytol® or Fultium® control. The improvement of the clinical condition and occurrence of (serious) adverse events were evaluated. The study was registered on clincaltrials.gov with the identifier NCT04844658.ResultsTwenty-five patients received Nasafytol®, and 24 received Fultium®. Demographic characteristics were well balanced between the groups. On day 14 (or at hospital leave if < 14 days), no difference was observed between groups regarding their clinical condition, fever, or the need of oxygen therapy. At day 7, however, 19 participants had been discharged from the hospital in the Nasafytol® arm compared to 10 participants in the Fultium® arm. No participants were transferred to the ICU or died in the Nasafytol® arm, vs. 4 transfers and 1 death in the Fultium® arm. The clinical condition of participants in the Nasafytol® arm had improved, as evidenced by a decrease in the COVID-19 WHO score. Interestingly, five SAEs occurred with Fultium®, while no SAE was observed with Nasafytol®.ConclusionSupplementation with Nasafytol®, in addition to standard-of-care treatment, led to a faster discharge from the hospital, improved clinical conditions of participants, and a reduced risk of serious outcomes, including transfer to the intensive care unit or death, in patients hospitalized with COVID-19

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.status: publishe

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue.

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue.

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue

Oral supplementation with fish cartilage hydrolysate in an adult population suffering from knee pain and function discomfort: results from an innovative approach combining an exploratory clinical study and an ex vivo clinical investigation

Abstract Background Aging is frequently associated with impairments of the musculoskeletal system and many elderly people experience joint discomfort or pain which might reduce their ability to move and consequently alter their quality of life. A beneficial effect of fish cartilage hydrolysate (FCH) on pain and joint function has recently been shown in an ACLT/pMMx osteoarthritis rat model. Methods We therefore performed an exploratory, non-comparative, multi-centric clinical trial including 33 subjects with moderate knee joint discomfort and loss of functionality to investigate the efficacy of FCH on their algo-functional status. We further determined the potential health benefit of FCH in an original clinical ex vivo study investigating the role of FCH human metabolites on primary human chondrocytes. Results FCH significantly improved knee pain and function, as assessed by the Knee injury and Osteoarthritis Outcome Score (KOOS). Moreover, FCH significantly reduced pain at rest and while walking, and patient global assessment (PGA), as assessed by the Visual Analogue Scale (VAS), and improved patients’ quality of life (SF-36). FCH metabolites decreased the synthesis of catabolic factors (MMP-13) and pro-inflammatory mediators (NO, PGE2) and limited the inhibitory effect of IL-1β on the synthesis of cartilage matrix components (GAG and collagen). Conclusions Thus, these data provide insights on the mode of action of FCH in humans and contribute to explain how FCH may relieve pain and improve joint function in subjects with knee discomfort. Although these preliminary data need to be confirmed in a randomized controlled trial, they strongly support the potential health benefit of such an active ingredient. Trial registration: The study was registered on clinicaltrials.gov with the identifier NCT04420091 (09/06/2020)

Osteoarthritis endotype discovery via clustering of biochemical marker data.

[en] OBJECTIVES: Osteoarthritis (OA) patient stratification is an important challenge to design tailored treatments and drive drug development. Biochemical markers reflecting joint tissue turnover were measured in the IMI-APPROACH cohort at baseline and analysed using a machine learning approach in order to study OA-dominant phenotypes driven by the endotype-related clusters and discover the driving features and their disease-context meaning. METHOD: Data quality assessment was performed to design appropriate data preprocessing techniques. The k-means clustering algorithm was used to find dominant subgroups of patients based on the biochemical markers data. Classification models were trained to predict cluster membership, and Explainable AI techniques were used to interpret these to reveal the driving factors behind each cluster and identify phenotypes. Statistical analysis was performed to compare differences between clusters with respect to other markers in the IMI-APPROACH cohort and the longitudinal disease progression. RESULTS: Three dominant endotypes were found, associated with three phenotypes: C1) low tissue turnover (low repair and articular cartilage/subchondral bone turnover), C2) structural damage (high bone formation/resorption, cartilage degradation) and C3) systemic inflammation (joint tissue degradation, inflammation, cartilage degradation). The method achieved consistent results in the FNIH/OAI cohort. C1 had the highest proportion of non-progressors. C2 was mostly linked to longitudinal structural progression, and C3 was linked to sustained or progressive pain. CONCLUSIONS: This work supports the existence of differential phenotypes in OA. The biomarker approach could potentially drive stratification for OA clinical trials and contribute to precision medicine strategies for OA progression in the future. TRIAL REGISTRATION NUMBER: NCT03883568