Ex Vivo Models to Decipher the Molecular Mechanisms of Genetic Notch Cardiovascular Disorders

Impact statementIn this review, a comprehensive overview of the limitations of current in vivo models of genetic Notch cardiovascular diseases is provided, followed by a discussion over the potential of microphysiological systems and computational models in overcoming these limitations and in potentiating drug testing and modeling of these pathologies.Notch is an evolutionary, conserved, cell-cell signaling pathway that is central to several biological processes, from tissue morphogenesis to homeostasis. It is therefore not surprising that several genetic mutations of Notch components cause inherited human diseases, especially cardiovascular disorders. Despite numerous efforts, current in vivo models are still insufficient to unravel the underlying mechanisms of these pathologies, hindering the development of utmost needed medical therapies. In this perspective review, we discuss the limitations of current murine models and outline how the combination of microphysiological systems (MPSs) and targeted computational models can lead to breakthroughs in this field. In particular, while MPSs enable the experimentation on human cells in controlled and physiological environments, in silico models can provide a versatile tool to translate the in vitro findings to the more complex in vivo setting. As a showcase example, we focus on Notch-related cardiovascular diseases, such as Alagille syndrome, Adams-Oliver syndrome, and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)

From structural resilience to cell specification - Intermediate filaments as regulators of cell fate

During the last decades intermediate filaments (IFs) have emerged as important regulators of cellular signaling events, ascribing IFs with functions beyond the structural support they provide. The organ and developmental stage-specific expression of IFs regulate cell differentiation within developing or remodeling tissues. Lack of IFs causes perturbed stem cell differentiation in vasculature, intestine, nervous system, and mammary gland, in transgenic mouse models. The aberrant cell fate decisions are caused by deregulation of different stem cell signaling pathways, such as Notch, Wnt, YAP/TAZ, and TGF beta. Mutations in genes coding for IFs cause an array of different diseases, many related to stem cell dysfunction, but the molecular mechanisms remain unresolved. Here, we provide a comprehensive overview of how IFs interact with and regulate the activity, localization and function of different signaling proteins in stem cells, and how the assembly state and PTM profile of IFs may affect these processes. Identifying when, where and how IFs and cell signaling congregate, will expand our understanding of IF-linked stem cell dysfunction during development and disease

In Situ Coupled Electrochemical-Goniometry as a Tool to Reveal Conformational Changes of Charged Peptides

The opportunity to manipulate cell functions by regulating bioactive surfaces is a potentially promising approach for organic bioelectronics. Here, the tuning of the orientation of charged peptides by means of an electrical input observed via optical tensiometry is reported. A stimuli-responsive self-assembled monolayer (SAM) with specially designed charged peptides is used as a model system to switch between two separate hydrophilic states. The underwater contact angle (UCA) technique is used to measure changes in the wetting property of a dichloromethane droplet under electrical stimuli. The observed changes in the UCA of the bio-interface can be understood in terms of a change in the surface energy between the ON and OFF states. Molecular dynamics simulations in an electric field have been performed to verify the hypothesis of the orientational change of the charged peptides upon electrical stimulation. In addition, X-ray photoelectron spectroscopy (XPS) is performed to clarify the stability of the functionalized electrodes. Finally, the possibility of using such a novel switching system as a tool to characterize bioactive surfaces is discussed

Mouse Model of Alagille Syndrome and Mechanisms of Jagged1 Missense Mutations.

BACKGROUND & AIMS: Alagille syndrome is a genetic disorder characterized by cholestasis, ocular abnormalities, characteristic facial features, heart defects, and vertebral malformations. Most cases are associated with mutations in JAGGED1 (JAG1), which encodes a Notch ligand, although it is not clear how these contribute to disease development. We aimed to develop a mouse model of Alagille syndrome to elucidate these mechanisms. METHODS: Mice with a missense mutation (H268Q) in Jag1 (Jag1+/Ndr mice) were outbred to a C3H/C57bl6 background to generate a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice). Liver tissues were collected at different timepoints during development, analyzed by histology, and liver organoids were cultured and analyzed. We performed transcriptome analysis of Jag1Ndr/Ndr livers and livers from patients with Alagille syndrome, cross-referenced to the Human Protein Atlas, to identify commonly dysregulated pathways and biliary markers. We used species-specific transcriptome separation and ligand-receptor interaction assays to measure Notch signaling and the ability of JAG1Ndr to bind or activate Notch receptors. We studied signaling of JAG1 and JAG1Ndr via NOTCH 1, NOTCH2, and NOTCH3 and resulting gene expression patterns in parental and NOTCH1-expressing C2C12 cell lines. RESULTS: Jag1Ndr/Ndr mice had many features of Alagille syndrome, including eye, heart, and liver defects. Bile duct differentiation, morphogenesis, and function were dysregulated in newborn Jag1Ndr/Ndr mice, with aberrations in cholangiocyte polarity, but these defects improved in adult mice. Jag1Ndr/Ndr liver organoids collapsed in culture, indicating structural instability. Whole-transcriptome sequence analyses of liver tissues from mice and patients with Alagille syndrome identified dysregulated genes encoding proteins enriched at the apical side of cholangiocytes, including CFTR and SLC5A1, as well as reduced expression of IGF1. Exposure of Notch-expressing cells to JAG1Ndr, compared with JAG1, led to hypomorphic Notch signaling, based on transcriptome analysis. JAG1-expressing cells, but not JAG1Ndr-expressing cells, bound soluble Notch1 extracellular domain, quantified by flow cytometry. However, JAG1 and JAG1Ndr cells each bound NOTCH2, and signaling from NOTCH2 signaling was reduced but not completely inhibited, in response to JAG1Ndr compared with JAG1. CONCLUSIONS: In mice, expression of a missense mutant of Jag1 (Jag1Ndr) disrupts bile duct development and recapitulates Alagille syndrome phenotypes in heart, eye, and craniofacial dysmorphology. JAG1Ndr does not bind NOTCH1, but binds NOTCH2, and elicits hypomorphic signaling. This mouse model can be used to study other features of Alagille syndrome and organ development

Vimentin regulates Notch signaling strength and arterial remodeling in response to hemodynamic stress

The intermediate filament (IF) cytoskeleton has been proposed to regulate morphogenic processes by integrating the cell fate signaling machinery with mechanical cues. Signaling between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) through the Notch pathway regulates arterial remodeling in response to changes in blood flow. Here we show that the IF-protein vimentin regulates Notch signaling strength and arterial remodeling in response to hemodynamic forces. Vimentin is important for Notch transactivation by ECs and vimentin knockout mice (VimKO) display disrupted VSMC differentiation and adverse remodeling in aortic explants and in vivo. Shear stress increases Jagged1 levels and Notch activation in a vimentin-dependent manner. Shear stress induces phosphorylation of vimentin at serine 38 and phosphorylated vimentin interacts with Jagged1 and increases Notch activation potential. Reduced Jagged1-Notch transactivation strength disrupts lateral signal induction through the arterial wall leading to adverse remodeling. Taken together we demonstrate that vimentin forms a central part of a mechanochemical transduction pathway that regulates multilayer communication and structural homeostasis of the arterial wall

The Vampire as both a Segregated and an Integrated Other. Discussing humanisation of the vampire in "Twilight" contrasting "Dracula"

This essay discusses how the view of the vampire has changed from Dracula to Twilight where the wish to be integrated into humanity and society is fundamental for both main vampire characters of the novels. By viewing the vampire through narration, the authors and contemporary social ideals it has developed from a segregated solitary animal to an integrated humanised creature. This humanisation has given the vampire a conscious and an individual personality contributing to the reader’s possibility to identify with it through attraction

Estetikens vara eller icke vara i skolan – En textanalytisk undersökning av entreprenöriellt lärande, kreativitet och estetiska läroprocesser

De nya styrdokumenten för den svenska gymnasieskolan har medfört att de estetiska ämnena nedprioriterats och borttagits som obligatorium. Istället har fokus förflyttats till ett entreprenöriellt lärande. Uppsatsens syfte är att undersöka vilka belägg som kan finnas i litteratur, forskning och historik för att påvisa betydelsen av estetik och estetiska läroprocesser i skolan. Detta görs i förhållande till styrdokumenten och det entreprenöriella lärandet som eftersträvas i den nya gymnasiereformen, Gy11. Uppsatsen utgår från frågeställningen: Måste det entreprenöriella lärandet innebära att estetik och estetiska läroprocesser inte längre får plats i undervisningen? Vi har valt att genomgående utföra en textanalytisk undersökning av tidigare forskning av entreprenöriellt lärande, kreativitet och estetik. På så sätt hoppas vi belysa valet av att förminska det estetiska ur styrdokumenten för gymnasieskolan. Estetiska läroprocesser kan i hög grad öka de kompetenser som tillskrivs det entreprenöriella lärandet såsom bland andra självförtroende, förmåga att reflektera, behov och lust, handlingskraft och lösningsorienterat tänkande. Estetiska upplevelsers betydelse för mänsklig utveckling stöds av filosofer, forskare och teoretiker genom tiderna, från Platon via John Dewey till Anne Bamford. Det entreprenöriella lärandet arbetar med kreativitetsfrämjande metoder för ett målinriktat syfte. Den kreativa förmågan ska gå via individen för att utveckla samhället och därmed leda till något konkret och produktivt. Estetiska läroprocesser syftar istället mot att utveckla individen, dess individuella förmågor och lusten att lära. Genom att stärka och utveckla individen stärker man i förlängningen också samhället. Estetiska läroprocesser är ett individinriktat lärande medan det entreprenöriella är produktionsinriktat men båda eftersträvar samma individuella förmågor. I så väl det entreprenöriella lärandet som i de estetiska läroprocesserna framhålls betydelsen av ett kreativt skapande och förhållningssätt. De estetiska läroprocesserna är ett medel för att nå ett entreprenöriellt lärande genom att nyttja kreativiteten i motivationsberikande undervisning och som redskap eller metod för en varierad undervisningsform

Networking Notch : regulation of Notch traffic and signaling crosstalk

Utvecklingen av flercelliga organismer är en mångfacetterad process som kräver kommunikation celler emellan. Under utvecklingen av en organism måste cellerna göra vissa val, vilket bestämmer riktningen för deras fortsatta utveckling. Utgående från dessa val erhåller cellerna egenskaper som är karaktäristiska för olika celltyper. Notch-signalräckan är en viktig reglerare av valet mellan olika cellöden. Notch-signalräckan aktiveras när Notch-receptorer som uttrycks på ytan av en cell binder till Notch-ligander som uttrycks på ytan av en annan närliggande cell. Denna avhandling belyser mekanismerna som reglerar omsättningen av såväl Notch-receptorer som -ligander till och från cellmembranen, samt ökar förståelsen för hur dessa mekanismer påverkar Notch-medierade cellöden i stamceller. Internalisering av Notch receptorer anses nödvändigt för fullständig aktivering av Notch-signalvägen. De bakomliggande molekylära mekanismerna är dock fortfarande oklara. Vi har upptäckt att atypiskt protein kinas Cζ (aPKCζ) reglerar internaliseringen av Notch-receptorer. aPKCζ fosforylerar Notch, vilket leder till receptorns internalisering, men effekten är beroende av receptorns signaleringsstatus. Vi visar att aPKCζ reglerar Notch-signaleringen och styr både neuroners och muskelcellers differentiering. Ytterligare har vi analyserat samspelet mellan cellskelettet och Notch-signalvägen. Våra resultat visar att intermediärfilamenten, en del av cellskelettet, är viktiga reglerare av Notch-signaleringen både under neuronal och vaskulär utveckling. Intermediärfilamenten vimentin och GFAP reglerar uttrycket av Notch-ligander vid cellmembranen i hjärnans stödceller, astrocyterna, och påverkar därmed neuronala stamcellers cellödesbeslut. Vimentin är även viktigt reglerare av Notch-signalräckan vid angiogenesen. Celler som saknar vimentin uppvisar avvikande Notch-signalering emedan möss som saknar vimentin påvisar en fördröjd utveckling av vaskulaturen under embryonalstadiet. ------------------------------------------------- Monisoluisten organismien kehittyminen on monimutkainen prosessi, joka vaatii viestintää solujen välillä. Kehittymisen aikana solut joutuvat tiettyjen valintojen eteen, mitkä tulevat määrittämään niiden erilaistumisen suunnan. Solut omaksuvat solutyypille ominaisia ominaisuuksia näihin valintoihin perustuen Notch-signalointireitti säätelee solujen erilaistumista eri suuntiin. Notch-signalointireitti aktivoituu, kun Notch-reseptori yhden solun pinnalla sitoo Notch-ligandin toisen, viereisen solun solukalvolla. Tutkimukseni lisää tuntemusta Notch-reseptoreiden ja ligandien solun sisäisestä liikennöinnistä ja sitä säätelevistä mekanismeista, sekä tämän säätelyn vaikutuksista kantasulojen erilaistumiseen. Notch-signalointireitin aktivoituminen vaatii reseptoreiden ja ligandien sisäistämisen solukalvolta, mutta taustalla olevat ja sisäistymistä säätelevät mekanismit ovat vielä epäselviä. Tutkimukseni osoittaa, että atyyppinen proteiinikinaasi Cζ (aPKCζ) säätelee Notch-reseptoreiden endosytoosia. Endosytoosin lopputulos riippuu siitä onko reseptori aktivoitunut ligandin välityksellä vai ei. Tuloksemme osoittavat aPKCζ säätelevän Notch-signalointia ja kontrolloivan sekä hermosolujen, että lihassolujen erilaistumista. Analysoimme myös Notch-signaloinnin ja solun tukirangan vuorovaikutuksia. Välikokoiset filamentit, jotka ovat osa tukirankaa, säätelevät Notch-signalointia neuronaalisen erilaistumisen sekä verisuonten uudismuodostumisen aikana. Vimentiini ja GFAP ovat välikokoisia säikeitä, jotka säätelevät Notch-ligandien ekspressiota astrosyyttien, eli aivojen hermotukisolujen solukalvolla. Vimentiini säätelee myös Notch-signalointireittiä angiogeneesin aikana. Vimentiiniä vailla olevilla soluilla ilmenee heikentynyttä Notch-signalointia, joka voidaan liittää hiirillä ilmenevään vimenttiinin puutteesta johtuvaan viivästyneeseen verisuonien kehitykseen.

Ex Vivo Models to Decipher the Molecular Mechanisms of Genetic Notch Cardiovascular Disorders

Notch is an evolutionary, conserved, cell-cell signaling pathway that is central to several biological processes, from tissue morphogenesis to homeostasis. It is therefore not surprising that several genetic mutations of Notch components cause inherited human diseases, especially cardiovascular disorders. Despite numerous efforts, current in vivo models are still insufficient to unravel the underlying mechanisms of these pathologies, hindering the development of utmost needed medical therapies. In this perspective review, we discuss the limitations of current murine models and outline how the combination of microphysiological systems (MPSs) and targeted computational models can lead to breakthroughs in this field. In particular, while MPSs enable the experimentation on human cells in controlled and physiological environments, in silico models can provide a versatile tool to translate the in vitro findings to the more complex in vivo setting. As a showcase example, we focus on Notch-related cardiovascular diseases, such as Alagille syndrome, Adams-Oliver syndrome, and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). In this review, a comprehensive overview of the limitations of current in vivo models of genetic Notch cardiovascular diseases is provided, followed by a discussion over the potential of microphysiological systems and computational models in overcoming these limitations and in potentiating drug testing and modeling of these pathologies

PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner

\u3cp\u3eActivation of Notch signaling requires intracellular routing of the receptor, but the mechanisms controlling the distinct steps in the routing process is poorly understood. We identify PKCζ as a key regulator of Notch receptor intracellular routing. When PKCζ was inhibited in the developing chick central nervous system and in cultured myoblasts, Notch-stimulated cells were allowed to undergo differentiation. PKCζ phosphorylates membrane-tethered forms of Notch and regulates two distinct routing steps, depending on the Notch activation state. When Notch is activated, PKCζ promotes re-localization of Notch from late endosomes to the nucleus and enhances production of the Notch intracellular domain, which leads to increased Notch activity. In the non-activated state, PKCζ instead facilitates Notch receptor internalization, accompanied with increased ubiquitylation and interaction with the endosomal sorting protein Hrs. Collectively, these data identify PKCζ as a key regulator of Notch trafficking and demonstrate that distinct steps in intracellular routing are differentially modulated depending on Notch signaling status.\u3c/p\u3