| 1. |
- Ando, Koji, et al.
(författare)
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Peri-arterial specification of vascular mural cells from naive mesenchyme requires Notch signaling
- 2019
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Ingår i: Development. - : COMPANY BIOLOGISTS LTD. - 0950-1991 .- 1477-9129. ; 146:2
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Tidskriftsartikel (refereegranskat)abstract
- Mural cells (MCs) are essential for blood vessel stability and function; however, the mechanisms that regulate MC development remain incompletely understood, in particular those involved in MC specification. Here, we investigated the first steps of MC formation in zebrafish using transgenic reporters. Using pdgfrb and abcc9 reporters, we show that the onset of expression of abcc9, a pericyte marker in adult mice and zebrafish, occurs almost coincidentally with an increment in pdgfrb expression in peri-arterial mesenchymal cells, suggesting that these transcriptional changes mark the specification of MC lineage cells from naive pdgfrb(low) mesenchymal cells. The emergence of peri-arterial pdgfrb(high) MCs required Notch signaling. We found that pdgfrb-positive cells express notch2 in addition to notch3, and although depletion of notch2 or notch3 failed to block MC emergence, embryos depleted of both notch2 and notch3 lost mesoderm- as well as neural crest-derived pdgfrb(high) MCs. Using reporters that read out Notch signaling and Notch2 receptor cleavage, we show that Notch activation in the mesenchyme precedes specification into pdgfrb(high) MCs. Taken together, these results show that Notch signaling is necessary for peri-arterial MC specification.
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| 2. |
- Andrae, Johanna, et al.
(författare)
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A 1.8kb GFAP-promoter fragment is active in specific regions of theembryonic CNS
- 2001
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Ingår i: Mechanisms of Development. - 0925-4773 .- 1872-6356. ; 107:1-2, s. 181-5
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Tidskriftsartikel (refereegranskat)abstract
- The intermediate filament glial fibrillary acidic protein (GFAP) constitutes the major cytoskeletal protein in astrocytes (J. Neuroimmunol. 8 (1985) 203) and is traditionally referred to as a specific marker for astrocytes. To identify early glial precursors, we created GFAPpromoter-lacZ transgenic mice, using a 1.8kb 5' fragment of human GFAP. The expression of the transgene was first detected in the neuroepithelium at embryonic day 9.5. It was further found in the ventricular zone of the developing telencephalon, in the cerebellar primordium, trigeminal ganglia, and radial glia. Later, scattered beta-gal+ cells were seen in pons, brain stem and glia limitans. The results indicate that GFAP activity is regulated in a region-specific manner during central nervous system (CNS) development and that the gene is turned on in different cell types independently.
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| 3. |
- 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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| 4. |
- Borgegard, Tomas, et al.
(författare)
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Alzheimers Disease: Presenilin 2-Sparing gamma-Secretase Inhibition Is a Tolerable A beta Peptide-Lowering Strategy
- 2012
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Ingår i: Journal of Neuroscience. - : Society for Neuroscience. - 0270-6474 .- 1529-2401. ; 32:48, s. 17297-17305
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Tidskriftsartikel (refereegranskat)abstract
- gamma-Secretase inhibition represents a major therapeutic strategy for lowering amyloid beta (A beta) peptide production in Alzheimers disease (AD). Progress toward clinical use of gamma-secretase inhibitors has, however, been hampered due to mechanism-based adverse events, primarily related to impairment of Notch signaling. The gamma-secretase inhibitor MRK-560 represents an exception as it is largely tolerable in vivo despite displaying only a small selectivity between A beta production and Notch signaling in vitro. In exploring the molecular basis for the observed tolerability, we show that MRK-560 displays a strong preference for the presenilin 1(PS1) over PS2 subclass of gamma-secretases and is tolerable in wild-type mice but causes dose-dependent Notch-related side effect in PS2-deficient mice at drug exposure levels resulting in a substantial decrease in brain A beta levels. This demonstrates that PS2 plays an important role in mediating essential Notch signaling in several peripheral organs during pharmacological inhibition of PS1 and provide preclinical in vivo proof of concept for PS2-sparing inhibition as a novel, tolerable and efficacious gamma-secretase targeting strategy for AD.
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| 5. |
- Borgegård, Tomas, et al.
(författare)
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Alzheimer's Disease : Presenilin 2-Sparing γ-Secretase Inhibition Is a Tolerable Aβ Peptide-Lowering Strategy
- 2012
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Ingår i: Journal of Neuroscience. - 0270-6474 .- 1529-2401. ; 32:48, s. 17297-17305
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Tidskriftsartikel (refereegranskat)abstract
- γ-Secretase inhibition represents a major therapeutic strategy for lowering amyloid β (Aβ) peptide production in Alzheimer's disease (AD). Progress toward clinical use of γ-secretase inhibitors has, however, been hampered due to mechanism-based adverse events, primarily related to impairment of Notch signaling. The γ-secretase inhibitor MRK-560 represents an exception as it is largely tolerable in vivo despite displaying only a small selectivity between Aβ production and Notch signaling in vitro. In exploring the molecular basis for the observed tolerability, we show that MRK-560 displays a strong preference for the presenilin 1 (PS1) over PS2 subclass of γ-secretases and is tolerable in wild-type mice but causes dose-dependent Notch-related side effect in PS2-deficient mice at drug exposure levels resulting in a substantial decrease in brain Aβ levels. This demonstrates that PS2 plays an important role in mediating essential Notch signaling in several peripheral organs during pharmacological inhibition of PS1 and provide preclinical in vivo proof of concept for PS2-sparing inhibition as a novel, tolerable and efficacious γ-secretase targeting strategy for AD.
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| 6. |
- Chivukula, Indira V, et al.
(författare)
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Decoding breast cancer tissue-stroma interactions using species-specific sequencing.
- 2015
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Ingår i: Breast Cancer Research. - : Springer Science and Business Media LLC. - 1465-5411 .- 1465-542X. ; 17
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Tidskriftsartikel (refereegranskat)abstract
- Decoding transcriptional effects of experimental tissue-tissue or cell-cell interactions is important; for example, to better understand tumor-stroma interactions after transplantation of human cells into mouse (xenografting). Transcriptome analysis of intermixed human and mouse cells has, however, frequently relied on the need to separate the two cell populations prior to transcriptome analysis, which introduces confounding effects on gene expression.
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| 7. |
- 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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| 8. |
- Enlund, Fredrik, 1968, et al.
(författare)
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Altered Notch signaling resulting from expression of a WAMTP1-MAML2 gene fusion in mucoepidermoid carcinomas and benign Warthin's tumors.
- 2004
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Ingår i: Experimental cell research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 292:1, s. 21-8
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Tidskriftsartikel (refereegranskat)abstract
- Chromosome translocations in neoplasia commonly result in fusion genes that may encode either novel fusion proteins or normal, but ectopically expressed proteins. Here we report the cloning of a novel fusion gene in a common type of salivary and bronchial gland tumor, mucoepidermoid carcinomas (MEC), as well as in benign Warthin's tumors (WATs). The fusion, which results from a t(11;19)(q21-22;p13) translocation, creates a chimeric gene in which exon 1 of a novel gene of unknown function, designated WAMTP1, is linked to exons 2-5 of the recently identified Mastermind-like Notch coactivator MAML2. In the fusion protein, the N-terminal basic domain of MAML2, which is required for binding to intracellular Notch (Notch ICD), is replaced by an unrelated N-terminal sequence from WAMTP1. Mutation analysis of the N-terminus of WAMTP1-MAML2 identified two regions of importance for nuclear localization (amino acids 11-20) and for colocalization with MAML2 and Notch1 ICD in nuclear granules (amino acids 21-42). Analyses of the Notch target genes HES5 and MASH1 in MEC tumors with and without the WAMTP1-MAML2 fusion revealed upregulation of HES5 and downregulation of MASH1 in fusion positive MECs compared to normal salivary gland tissue and MECs lacking the fusion. These findings suggest that altered Notch signaling plays an important role in the genesis of benign and malignant neoplasms of salivary and bronchial gland origin.
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| 9. |
- Foo, Kylie S, et al.
(författare)
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Human ISL1+ ventricular progenitors self-assemble into an in vivo functional heart patch and preserve cardiac function post infarction
- 2018
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Ingår i: Molecular Therapy. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 1525-0016. ; 26:7, s. 1644-1659
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Tidskriftsartikel (refereegranskat)abstract
- The generation of human pluripotent stem cell (hPSC)-derived ventricular progenitors and their assembly into a 3-dimensional in vivo functional ventricular heart patch has remained an elusive goal. Herein, we report the generation of an enriched pool of hPSC-derived ventricular progenitors (HVPs), which can expand, differentiate, self-assemble, and mature into a functional ventricular patch in vivo without the aid of any gel or matrix. We documented a specific temporal window, in which the HVPs will engraft in vivo. On day 6 of differentiation, HVPs were enriched by depleting cells positive for pluripotency marker TRA-1-60 with magnetic-activated cell sorting (MACS), and 3 million sorted cells were sub-capsularly transplanted onto kidneys of NSG mice where, after 2 months, they formed a 7 mm x 3 mm x 4 mm myocardial patch resembling the ventricular wall. The graft acquired several features of maturation: expression of ventricular marker (MLC2v), desmosomes, appearance of T-tubule-like structures, and electrophysiological action potential signature consistent with maturation, all this in a non-cardiac environment. We further demonstrated that HVPs transplanted into un-injured hearts of NSG mice remain viable for up to 8 months. Moreover, transplantation of 2 million HVPs largely preserved myocardial contractile function following myocardial infarction. Taken together, our study reaffirms the promising idea of using progenitor cells for regenerative therapy. Correction in Mol Ther. 2021 Jan 6;29(1):409, DOI: 10.1016/j.ymthe.2020.11.015
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| 10. |
- Gudey, Shyam Kumar, 1982-
(författare)
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TRAF6 stimulates TGFβ-induced oncogenic signal transduction in cancer cells
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Prostate cancer is one of the leading causes of cancer-related deaths in men worldwide, with 10,000 new cases/year diagnosed in Sweden. In this context, there is an urgent need to identify new biomarkers to detect prostate cancer at an initial stage for earlier treatment intervention. Although how prostate cancer develops has not been fully established, the male sex hormone testosterone is a known prerequisite for prostate cancer development. High levels of transforming growth factor-β (TGFβ) are prognostically unfavorable in prostate cancer patients.TGFβ is a multifunctional cytokine that regulates a broad range of cellular responses. TGFβ signals through either the canonical Smad or the non-Smad signaling cascade. Cancerous cells develop different strategies to evade defense mechanisms and metastasize to different parts of the body. This thesis unveils one such novel mechanism related to TGFβ signaling.The first two articles provide evidence that TGFβ receptor type I (TβRI) is ubiquitinated by tumor necrosis factor receptor-associated factor 6 (TRAF6) and is cleaved at the ectodomain region by tumor necrosis factor alpha converting enzyme (TACE) in a protein kinase C zeta type-dependent manner. After TβRI is shed from the ectodomain, it undergoes a second cleavage by presenilin 1 (PS1), a γ-secretase catalytic subunit, which liberates the TβRI intracellular domain (TβRI-ICD) from the cell membrane. TRAF6 promotes TGFβ-dependent Lys63-linked polyubiquitination and recruitment of PS1 to the TβRI complex, and facilitates the cleavage of TβRI by PS1 to generate a TβRI-ICD. The TβRI-ICD then translocates to the nucleus, where it binds with the transcriptional co-activator p300 and regulates the transcription of pro-invasive target genes such as Snail1. Moreover, the nuclear translocated TβRI-ICD cooperates with the Notch intracellular domain (NICD), a core component in the Notch signaling pathway, to drive the expression of invasive genes. Interestingly, treatment with g-secretase inhibitors was able to inhibit cleavage of TβRI and inhibit the TGFβ-induced oncogenic pathway in an in vivo prostate cancer xenograft model.In the third article, we identified that Lysine 178 is the acceptor lysine in TβRI that is ubiquitinated by TRAF6. The TβRI K178R mutant was neither ubiquitinated nor translocated to the nucleus, and prevented transcriptional regulation of invasive genes in a dominant negative manner.In the fourth article, we show that TGFβ utilizes the E3-ligase TRAF6 and the p38 mitogen-activated protein kinase to phosphorylate c-Jun. In turn, the phosphorylated c-Jun activates p21 and Snail1 in a non-canonical Smad-independent pathway, and thereby promotes invasion in cancerous cells.In summary, we elucidate a new mechanism of TGFβ-induced oncogenic signal transduction in cancer cells in which TRAF6 plays a fundamental role. This opens a new avenue in the field of TGFβ signaling.
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