Biology subjects

Lendahl, U.

Publications and source records attributed to Lendahl, U..

10 recordsLinked to original sources

bioRxiv2025

Disrupting Notch signalling by a small molecule inhibiting dihydroorotate dehydrogenase activity

The Notch signalling pathway is highly evolutionarily conserved and regulates differentiation and homeostasis in most organs. Given the critical role of Notch signalling for normal development, dysregulated Notch signalling is frequently linked to pathogenesis of disease and cancer. Hence, developing Notch-targeting therapeutics is warranted but has been challenging and Notch inhibitors have not yet reached broad clinical use. In this report, we identify potential Notch inhibitors, using a novel cell-based Notch reporter system for unbiased screening of compounds reducing Notch signalling. A library of 37.966 small organic compounds was screened for inhibitor candidates, followed by a counter screen to eliminate {gamma}-secretase inhibitor-like compounds and an orthogonal screen based on the role of Notch signalling in myogenic differentiation. This triage led to the identification of five Notch inhibitor candidate hits with different chemical backbones and unrelated to previous Notch antagonists. One candidate hit showed structural similarities to dihydroorotate dehydrogenase (DHODH) inhibitors, and we provide evidence that inhibition of DHODH activity reduces Notch signalling. In conclusion, our data support the notion that DHODH inhibition may be an interesting avenue to explore for the development of novel Notch inhibitors.

cancer biology↗

bioRxiv2025

A comprehensive molecular atlas of the cell types in the mouse liver

The liver plays a crucial role in essential physiological processes, and its impaired function due to liver fibrosis from various causes is an increasingly significant health issue. The livers functionality relies on the precise arrangement of its cellular structures, yet the molecular architecture of these units remains only partially understood. We created a comprehensive molecular atlas detailing all the major cell types present in the adult mouse liver through deep single-cell RNA sequencing. Our analysis offers new insights into hepatic endothelial and mesenchymal cells, specifically highlighting the differences between the cells of the periportal microvasculature, the sinusoids, and the portal vein, the latter exhibiting a mixed arterio-venous phenotype. We identified distinct subpopulations of hepatic stellate cells, fibroblasts, and vascular mural cells located in different anatomical regions. Comparisons with transcriptomic data from disease models indicate that a previously unrecognized capsular population of hepatic stellate cells expands in response to fibrotic disease. Our findings reveal that various fibroblast subpopulations respond differently to pathological insults. This data resource will be invaluable for advancing therapeutic interventions targeting hepatic diseases.

cell biology↗

bioRxiv2024

Design of a Soluble Multivalent Notch Agonist

Designed protein agonists can enhance the efficiency of endogenous signaling pathways, and provide a powerful means to control cellular functions and develop disease therapeutics. Designing a soluble cytokine-like agonist for Notch signaling, an evolutionarily conserved pathway that regulates cell fate in embryonic and adult development, is especially challenging because Notch receptor activation requires a mechanical force that is typically mediated by cell-associated transmembrane ligands at sites of cell-cell contact. Moreover, free soluble Notch ligand is signal inhibitory. Here, we exploit computationally designed protein oligomers with precise geometries and valencies to generate cytokine-like, protein only, multivalent soluble Notch agonists. These tools promote cell-cell contact, cluster Notch proteins in synapses at the cell surface, and activate Notch signaling in reporter cell lines and cells expressing endogenous receptors. We demonstrate the utility of these soluble Notch agonists in T cell differentiation from cord blood (CB) and human induced pluripotent stem cells (iPSCs), and in bioreactor production of T cells in liquid suspension. Soluble multivalent Notch agonists can be applied broadly to in vitro cellular differentiation methods to generate clinical cell products and to develop immunotherapies.

synthetic biology↗

bioRxiv2022

NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic form of familial small vessel disease and no preventive or curative therapy is available. CADASIL is caused by mutations in the NOTCH3 gene, resulting in a mutated NOTCH3 receptor, with aggregation of the NOTCH3 extracellular domain (ECD) around vascular smooth muscle cells. In this study we have developed a novel active immunization therapy specifically targeting CADASIL-like aggregated NOTCH3 ECD. Immunizing CADASIL TgN3R182C150 mice with aggregates composed of CADASIL-R133C mutated and wild type EGF1-5 repeats for a total of four months resulted in a marked reduction (38-48%) in NOTCH3 deposition around brain capillaries, increased microglia activation and lowered serum levels of NOTCH3 ECD. Active immunization did not impact body weight, general behavior or the number and integrity of vascular smooth muscle cells in the retina, suggesting that the therapy is tolerable. This is the first therapeutic study reporting a successful reduction of CADASIL-like NOTCH3 accumulation in mice supporting further development towards clinical application for the benefit of CADASIL patients.

neuroscience↗

bioRxiv2021

Multimodal characterization of the murine neurovascular niche using a novel microvascular isolation protocol

The blood-brain barrier (BBB) is central to separate blood from the extracellular fluids of the brain. To understand disease-related changes in the BBB is pivotal and such changes can increasingly be studied by single-cell RNA sequencing (scRNAseq), which provides high-resolution insight into gene expression changes related to the pathophysiological response of the vasculature. However, analysis of the vascular cells in the brain is challenging due to the low abundance of these cells relative to neuronal and glial cells, and improved techniques for enrichment of the vascular component is therefore warranted. The present study describes a method whereby panning with CD31-coated magnetic beads allows isolation of brain vasculature without the need for transgenic reporter lines or FACS sorting. The protocol was tested in three modalities: isolation of cells for scRNAseq, western blot (WB) analysis, and primary cell culture. For scRNAseq, a total of 22,515 single-cell transcriptomes were generated from 12-months old mice and separated into 23 clusters corresponding to all known vascular and perivascular cell types. The most abundant cell type was endothelial cells (EC) (Pecam1- and Cdh5-positive), which dispersed into clusters of arterial, capillary, and venous EC according to previously established BBB arterio-venous zonation markers. Furthermore, we identified clusters of microglia (Aif1-positive), one cluster of fenestrated endothelial cells (Plvap-positive; Cldn5-negative), a cluster of pericytes (Kcnj8- and Abcc9-positive) and a cluster of vascular smooth muscle cells (VSMC) (Acta2- and Tagln-positive). WB analysis using established markers for the different cell types (CD31 (EC), SM22 (VSMC), PDGFRB (pericytes), GFAP (astrocytes), and IBA1 (microglia) confirmed their presence in the isolated vascular component and suggests that the protocol is suitable for future proteomic analysis. Finally, we adapted the isolation protocol to accommodate primary culture of brain vascular cells. In conclusion, we have successfully established a simple and fast method for isolating microvasculature from the murine brain independent of cell sorting and alleviating the need to use reporter mouse lines. The protocol is suitable for a multitude of testing modalities, including single-cell analyses, WB and primary cell culture.

cell biology↗

bioRxiv2021

Bleeding in patients and mice with Alagille syndrome: sex differences and risk factors

Spontaneous lethal bleeds are major cause of death in the pediatric liver disease Alagille syndrome (ALGS), yet risk factors and screening methods have not been established. We performed a systematic review and identified significantly more female than male patients with idiopathic intracranial hemorrhage (10:1). We investigated bleeding and vasculature in patients and a mouse model for ALGS (Jag1Ndr/Ndr mice) and asked whether phenotypes identified in mice could be detected in patients non-invasively. Jag1Ndr/Ndr mice bled spontaneously, exhibiting a thin skull and vascular defects including artery-vein crossings, tortuous vessels, capillary breakdown and CADASIL-like sparse vascular smooth muscle cell coverage which was aggravated by hypertension. Retinographs from patients confirmed tortuous blood vessels and artery-vein crossings in ALGS. In conclusion, Jag1Ndr/Ndr mice could be used to develop interventions for vascular defects in ALGS, and retinography could provide a non-invasive method for vascular analysis in these pediatric patients.

developmental biology↗

bioRxiv2020

Lack of evidence of ACE2 expression and replicative infection by SARS-CoV-2 in human endothelial cells

A striking feature of severe COVID-19 is thrombosis in large as well as small vessels of multiple organs. This has led to the assumption that SARS-CoV-2 virus directly infects and damages the vascular endothelium. However, endothelial expression of ACE2, the cellular receptor for SARS-CoV-2, has not been convincingly demonstrated. Interrogating human bulk and single-cell transcriptomic data, we found ACE2 expression in endothelial cells to be extremely low or absent in vivo and not upregulated by exposure to inflammatory agents in vitro. Also, the endothelial chromatin landscape at the ACE2 locus showed presence of repressive and absence of activation marks, suggesting that the gene is inactive in endothelial cells. Finally, we failed to achieve infection and replication of SARS-CoV-2 in cultured human endothelial cells, which were permissive to productive infection by coronavirus 229E that uses CD13 as the receptor. Our data suggest that SARS-Cov-2 is unlikely to infect endothelial cells directly; these findings are consistent with a scenario where endothelial injury is indirectly caused by the infection of neighbouring epithelial cells and/or due to systemic effects mediated by immune cells, platelets, complement activation, and/or proinflammatory cytokines.

pathology↗

bioRxiv2020

Neural Plate Targeting by in Utero NanoinjEction (NEPTUNE) reveals a role for Sptbn2 in neurulation and abdominal wall closure

Gene variants associated with disease are efficiently identified with whole genome sequencing or GWAS, but validation in vivo lags behind. We developed NEPTUNE (neural plate targeting by in utero nanoinjection), to rapidly and flexibly introduce gene expression-modifying viruses to the embryonic murine neural plate prior to neurulation, to target the future adult nervous system. Stable integration in >95% of cells in the brain enabled long-term gain- or loss-of-function, and conditional expression was achieved using mini-promotors for cell types of interest. Using NEPTUNE, we silenced Sptbn2, a gene associated with Spinocerebellar ataxia type 5 (SCA5) in humans. Silencing of Sptbn2 induced severe neural tube defects and embryo resorption, suggesting that SPTBN2 in-frame and missense deletions in SCA5 reflect hypomorphic or neomorphic functions, not loss of function. In conclusion, NEPTUNE offers a novel, rapid and cost-effective technique to test gene function in brain development, and can reveal loss of function phenotypes incompatible with life.

developmental biology↗

bioRxiv2020

Pericyte-specific vascular expression of SARS-CoV-2 receptor ACE2 - implications for microvascular inflammation and hypercoagulopathy in COVID-19 patients

Accumulating clinical observations implicate vascular inflammation as an underlying cause of coagulopathy in severely ill COVID-19 patients and it was recently suggested that SARS-CoV-2 virus particles infect endothelial cells. Here, we show that endothelial cells do not express angiotensin-converting enzyme-2 (ACE2), the SARS-CoV-2 receptor. Instead, pericytes and microvascular smooth muscle cells express ACE2 in an organotypic manner. Pericyte deficiency leads to increased endothelial expression and release of Von Willebrand factor and intravascular platelet and fibrin aggregation, suggesting that pericytes limit endothelial pro-thrombotic responses. That pericytes and not endothelial cells express ACE2 may provide important clues to the pathology of COVID-19, as pericytes are normally shielded behind an endothelial barrier and may get infected only when this barrier is compromised by COVID-19 risk factors.

pathology↗

bioRxiv2019

DoUble resin Casting micro computed Tomography (DUCT) reveals biliary and vascular pathology in a mouse model of Alagille syndrome

BACKGROUND AND AIMSAlagille syndrome, like several other liver diseases, is characterized by malformation of lumenized structures, such as the circulatory or biliary systems. Liver architecture has typically been studied through 2D sections and, more recently, using thick tissue sections combined with immunofluorescence. We aimed to develop a robust method to image, digitalize and quantify 3D architecture of the biliary and vascular systems in tandem. METHODSThe biliary and portal vein trees of the mouse liver were injected with Microfil resin, followed by microCT scanning. Tomographic data was segmented and analyzed using a MATLAB script we wrote to investigate length, volume, tortuosity, branching, and the relation between the vascular and biliary systems. Double resin casting micro computed tomography (DUCT) was applied to a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice), in which the biliary system is absent at postnatal stages, but regenerates by adulthood. Phenotypes discovered using DUCT were validated with cumbersome consecutive liver sections from mouse and human liver including patients with Alagille syndrome. RESULTSDUCT revealed tortuous bile ducts either placed further from portal veins, or ectopically traversing the parenchyma and connecting two portal areas, in Jag1Ndr/Ndr mice. Furthermore, bile ducts either ended abruptly, or branched independently of portal vein branching, with bifurcations placed hilar or peripheral to portal vein branches. The branching defects, parenchymal bile ducts, and blunt endings were confirmed in patient samples. CONCLUSIONDUCT is a powerful technique, which provides computerized 3D reconstruction of casted networks. It exposes and quantifies previously unknown vascular and biliary phenotypes in mouse models, revealing new phenotypes in patients. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/848481v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1565121org.highwire.dtl.DTLVardef@1e86cfcorg.highwire.dtl.DTLVardef@4b4cc3org.highwire.dtl.DTLVardef@19cb20e_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗