| 1. |
- Bjornholm, Katrine Dahl, et al.
(författare)
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A robust and efficient microvascular isolation method for multimodal characterization of the mouse brain vasculature
- 2023
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Ingår i: CELL REPORTS METHODS. - : Elsevier. - 2667-2375. ; 3:3
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Tidskriftsartikel (refereegranskat)abstract
- Studying disease-related changes in the brain vasculature is warranted due to its crucial role in supplying oxygen and nutrients and removing waste and due to the anticipated vascular dysfunction in brain dis-eases. To this end, we have developed a protocol for fast and simple isolation of brain vascular fragments without the use of transgenic reporters. We used it to isolate and analyze 22,515 cells by single-cell RNA sequencing. The cells distributed into 23 distinct clusters corresponding to all known vascular and perivas-cular cell types in the brain. Western blot analysis also suggested that the protocol is suitable for proteomic analysis. We further adapted it for the establishment of primary cell cultures. The protocol generated highly reproducible results. In conclusion, we have developed a simple and robust brain vascular isolation proto-col suitable for different experimental modalities, such as single-cell analyses, western blotting, and pri-mary cell culture.
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| 2. |
- Del Gaudio, Francesca, et al.
(författare)
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Left ventricular hypertrophy and metabolic resetting in the Notch3-deficient adult mouse heart
- 2023
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- The heart depends on a functional vasculature for oxygenation and transport of nutrients, and it is of interest to learn how primary impairment of the vasculature can indirectly affect cardiac function and heart morphology. Notch3-deficiency causes vascular smooth muscle cell (VSMC) loss in the vasculature but the consequences for the heart remain largely elusive. Here, we demonstrate that Notch3(-/-) mice have enlarged hearts with left ventricular hypertrophy and mild fibrosis. Cardiomyocytes were hypertrophic but not hyperproliferative, and the expression of several cardiomyocyte markers, including Tnt2, Myh6, Myh7 and Actn2, was altered. Furthermore, expression of genes regulating the metabolic status of the heart was affected: both Pdk4 and Cd36 were downregulated, indicating a metabolic switch from fatty acid oxidation to glucose consumption. Notch3(-/-) mice furthermore showed lower liver lipid content. Notch3 was expressed in heart VSMC and pericytes but not in cardiomyocytes, suggesting that a perturbation of Notch signalling in VSMC and pericytes indirectly impairs the cardiomyocytes. In keeping with this, Pdgfb(ret/ret) mice, characterized by reduced numbers of VSMC and pericytes, showed left ventricular and cardiomyocyte hypertrophy. In conclusion, we demonstrate that reduced Notch3 or PDGFB signalling in vascular mural cells leads to cardiomyocyte dysfunction.
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| 3. |
- He, Liqun, et al.
(författare)
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Single-cell RNA sequencing of mouse brain and lung vascular and vessel-associated cell types
- 2018
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Ingår i: Scientific Data. - : NATURE PUBLISHING GROUP. - 2052-4463. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Vascular diseases are major causes of death, yet our understanding of the cellular constituents of blood vessels, including how differences in their gene expression profiles create diversity in vascular structure and function, is limited. In this paper, we describe a single-cell RNA sequencing (scRNA-seq) dataset that defines vascular and vessel-associated cell types and subtypes in mouse brain and lung. The dataset contains 3,436 single cell transcriptomes from mouse brain, which formed 15 distinct clusters corresponding to cell (sub) types, and another 1,504 single cell transcriptomes from mouse lung, which formed 17 cell clusters. In order to allow user-friendly access to our data, we constructed a searchable database (http://betsholtzlab.org/VascularSingleCells/database.html). Our dataset constitutes a comprehensive molecular atlas of vascular and vessel-associated cell types in the mouse brain and lung, and as such provides a strong foundation for future studies of vascular development and diseases.
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| 4. |
- Masuda, Takahiro, et al.
(författare)
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Specification of CNS macrophage subsets occurs postnatally in defined niches
- 2022
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Ingår i: Nature. - : Springer Nature. - 0028-0836 .- 1476-4687. ; 604:7907, s. 740-
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Tidskriftsartikel (refereegranskat)abstract
- All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs(1)) such as meningeal and perivascular macrophages(2-)(7)-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma(2,8-10). It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors(11-15). However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS.
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| 5. |
- Oliveira, Daniel V, et al.
(författare)
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Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model.
- 2022
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Ingår i: EMBO molecular medicine. - : EMBO. - 1757-4684 .- 1757-4676. ; 15:2
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Tidskriftsartikel (refereegranskat)abstract
- Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic form of familial small vessel disease; 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 4months 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, the number and integrity of vascular smooth muscle cells in the retina, neuronal survival, or inflammation or the renal system, suggesting that the therapy is tolerable. This is the first therapeutic study reporting a successful reduction of NOTCH3 accumulation in a CADASIL mouse model supporting further development towards clinical application for the benefit of CADASIL patients.
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| 6. |
- Pietilä, Riikka, et al.
(författare)
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Molecular anatomy of adult mouse leptomeninges
- 2023
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Ingår i: Neuron. - : Elsevier. - 0896-6273 .- 1097-4199. ; 111:23
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Tidskriftsartikel (refereegranskat)abstract
- Leptomeninges, consisting of the pia mater and arachnoid, form a connective tissue investment and barrier enclosure of the brain. The exact nature of leptomeningeal cells has long been debated. In this study, we iden-tify five molecularly distinct fibroblast-like transcriptomes in cerebral leptomeninges; link them to anatomically distinct cell types of the pia, inner arachnoid, outer arachnoid barrier, and dural border layer; and contrast them to a sixth fibroblast-like transcriptome present in the choroid plexus and median eminence. Newly identified transcriptional markers enabled molecular characterization of cell types responsible for adherence of arach-noid layers to one another and for the arachnoid barrier. These markers also proved useful in identifying the molecular features of leptomeningeal development, injury, and repair that were preserved or changed after traumatic brain injury. Together, the findings highlight the value of identifying fibroblast transcriptional subsets and their cellular locations toward advancing the understanding of leptomeningeal physiology and pathology.
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| 7. |
- Vanlandewijck, Michael, et al.
(författare)
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A molecular atlas of cell types and zonation in the brain vasculature
- 2018
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Ingår i: Nature. - : NATURE PUBLISHING GROUP. - 0028-0836 .- 1476-4687. ; 554:7693, s. 475-480
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Tidskriftsartikel (refereegranskat)abstract
- Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature.
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| 8. |
- Wu, Dan, et al.
(författare)
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The infantile myofibromatosis NOTCH3 L1519P mutation leads to hyperactivated ligand-independent Notch signaling and increased PDGFRB expression
- 2021
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Ingår i: Disease Models and Mechanisms. - : COMPANY BIOLOGISTS LTD. - 1754-8403 .- 1754-8411. ; 14:2
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Tidskriftsartikel (refereegranskat)abstract
- Infantile myofibromatosis (IMF) is a benign tumor form characterized by the development of nonmetastatic tumors in skin, bone, muscle and sometimes viscera. Autosomal-dominant forms of IMF are caused by mutations in the PDGFRB gene, but a family carrying a L1519P mutation in the NOTCH3 gene has also recently been identified. In this study, we address the molecular consequences of the NOTCH3L1519P mutation and the relationship between Notch and PDGFRB signaling in IMF. The NOTCH3L1519P receptor generates enhanced downstream signaling in a ligand-independent manner. Despite the enhanced signaling, the NOTCH3L1519P receptor is absent from the cell surface and instead accumulates in the endoplasmic reticulum. Furthermore, the localization of the NOTCH3L1519P receptor in the bipartite, heterodimeric state is altered, combined with avid secretion of the mutated extracellular domain from the cell. Chloroquine treatment strongly reduces the amount of secreted NOTCH3L1519P extracellular domain and decreases signaling. Finally, NOTCH3L1519P upregulates PDGFRB expression in fibroblasts, supporting a functional link between Notch and PDGF dysregulation in IMF. Collectively, our data define a NOTCH3-PDGFRB axis in IMF, in which an IMF-mutated NOTCH3 receptor elevates PDGFRB expression. The functional characterization of a ligand-independent gain-of-function NOTCH3 mutation is important for Notch therapy considerations for IMF, including strategies aimed at altering lysosome function.
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