| 1. |
- Baniol, Marion, et al.
(author)
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Identification and characterization of distinct cell cycle stages in cardiomyocytes using the FUCCI transgenic system
- 2021
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In: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 408:2
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Journal article (peer-reviewed)abstract
- Understanding the regulatory mechanism by which cardiomyocyte proliferation transitions to endoreplication and cell cycle arrest during the neonatal period is crucial for identifying proproliferative factors and developing regenerative therapies. We used a transgenic mouse model based on the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system to isolate and characterize cycling cardiomyocytes at different cell cycle stages at a single-cell resolution. Single-cell transcriptome analysis of cycling and noncycling cardiomyocytes was performed at postnatal days 0 (P0) and 7 (P7). The FUCCI system proved to be efficient for the identification of cycling cardiomyocytes with the highest mitotic activity at birth, followed by a gradual decline in the number of cycling and mitotic cardiomyocytes during the neonatal period. Cardiomyocytes showed premature cell cycle exit at G1/S shortly after birth and delayed G1/S progression during endoreplication at P7. Single-cell RNA-seq confirmed previously described signaling pathways involved in cardiomyocyte proliferation (Erbb2 and Hippo/YAP), and maturation-related transcriptional changes during postnatal development, including the metabolic switch from glycolysis to fatty acid oxidation in cardiomyocytes. Importantly, we generated transcriptional profiles specific to cell division and endoreplication in cardiomyocytes at different developmental stages that may facilitate the identification of genes important for adult cardiomyocyte proliferation and heart regeneration. In conclusion, the FUCCI mouse provides a valuable system to study cardiomyocyte cell cycle activity at single cell resolution that can help to decipher the switch from cardiomyocyte proliferation to endoreplication, and to revert this process to facilitate endogenous repair.
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| 2. |
- Bonanini, Flavio, et al.
(author)
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A comparison between different human hepatocyte models reveals profound differences in net glucose production, lipid composition and metabolism in vitro
- 2024
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In: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 437:1
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Journal article (peer-reviewed)abstract
- Hepatocytes are responsible for maintaining a stable blood glucose concentration during periods of nutrient scarcity. The breakdown of glycogen and de novo synthesis of glucose are crucial metabolic pathways deeply interlinked with lipid metabolism. Alterations in these pathways are often associated with metabolic diseases with serious clinical implications. Studying energy metabolism in human cells is challenging. Primary hepatocytes are still considered the golden standard for in vitro studies and have been instrumental in elucidating key aspects of energy metabolism found in vivo. As a result of several limitations posed by using primary cells, a multitude of alternative hepatocyte cellular models emerged as potential substitutes. Yet, there remains a lack of clarity regarding the precise applications for which these models accurately reflect the metabolic competence of primary hepatocytes. In this study, we compared primary hepatocytes, stem cell-derived hepatocytes, adult donor-derived liver organoids, immortalized Upcyte-hepatocytes and the hepatoma cell line HepG2s in their response to a glucose production challenge. We observed the highest net glucose production in primary hepatocytes, followed by organoids, stem-cell derived hepatocytes, Upcyte-hepatocytes and HepG2s. Glucogenic gene induction was observed in all tested models, as indicated by an increase in G6PC and PCK1 expression. Lipidomic analysis revealed considerable differences across the models, with organoids showing the closest similarity to primary hepatocytes in the common lipidome, comprising 347 lipid species across 19 classes. Changes in lipid profiles as a result of the glucose production challenge showed a variety of, and in some cases opposite, trends when compared to primary hepatocytes.
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| 3. |
- Cracknell, Tobias, et al.
(author)
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Proteomic resolution of IGFN1 complexes reveals a functional interaction with the actin nucleating protein COBL
- 2020
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In: Experimental Cell Research. - : ELSEVIER INC. - 0014-4827 .- 1090-2422. ; 395:2
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Journal article (peer-reviewed)abstract
- The Igfn1 gene produces multiple proteins by alternative splicing predominantly expressed in skeletal muscle. Igfn1 deficient clones derived from C2C12 myoblasts show reduced fusion index and morphological differences compared to control myotubes. Here, we first show that G:F actin ratios are significantly higher in differentiating IGFN1-deficient C2C12 myoblasts, suggesting that fusion and differentiation defects are underpinned by deficient actin remodelling. We obtained pull-downs from skeletal muscle with IGFN1 fragments and applied a proteomics approach. The proteomic composition of IGFN1 complexes identified the cytoskeleton and an association with the proteasome as the main networks. The actin nucleating protein COBL was selected for further validation. COBL is expressed in C2C12 myoblasts from the first stages of myoblast fusion but not in proliferating cells. COBL is also expressed in adult muscle and, as IGFN1, localizes to the Z-disc. We show that IGFN1 interacts, stabilizes and colocalizes with COBL and prevents the ability of COBL to form actin ruffles in COS7 cells. COBL loss of function C2C12-derived clones are able to fuse, therefore indicating that COBL or the IGFN1/COBL interaction are not essential for myoblast fusion.
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| 4. |
- Du, Feifei, et al.
(author)
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S100A9 induces reactive oxygen species-dependent formation of neutrophil extracellular traps in abdominal sepsis
- 2022
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In: Experimental Cell Research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 421:2
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Journal article (peer-reviewed)abstract
- Recent evidence suggests that targeting S100A9 reduces pathological inflammation in abdominal sepsis. Herein, we investigated the role of S100A9 in neutrophil extracellular trap (NET) formation in septic lung damage. NETs were detected by electron microscopy in the lung and by confocal microscopy in vitro. Stimulation of isolated mouse bone marrow-derived neutrophils with S100A9 triggered formation of NETs. Blocking TLR4 and RAGE reduced S100A9-induced generation of NETs and DNA-histone complexes. Moreover, S100A9 challenge increased generation of reactive oxygen species (ROS) in bone marrow neutrophils. Co-incubation with the NADPH oxidase inhibitor not only decreased ROS formation but also attenuated induction of DNA-histone complexes in S100A9-stimulated neutrophils. Abdominal sepsis was induced by cecal ligation and puncture (CLP) in male C57BL/6 mice. Administration of the S100A9 inhibitor ABR-238901 decreased CLP-induced formation of NETs in lungs and DNA-histone complexes in plasma. In addition, transmission electron microscopy revealed that S100A9 was abundantly expressed on NETs in the lungs in CLP mice. By use of intravital microscopy, we found that local injection of NETs increased leukocyte adhesion and migration in the mouse cremaster muscle microvasculature. Notably, treatment with ABR-238901 attenuated NET-induced leukocyte adhesion and extravasation in the cremaster muscle, suggesting that NET-associated S100A9 promotes leukocyte recruitment in vivo. Taken together, these novel findings suggest that S100A9 triggers ROS-dependent formation of NETs via TLR4 and RAGE signaling in neutrophils. Moreover, S100A9 regulates both formation of NETs and NET-induced leukocyte recruitment in vivo. Thus, targeting S100A9 might be useful to ameliorate lung damage in abdominal sepsis.
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| 5. |
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| 6. |
- Hikage, Fumihito, et al.
(author)
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NF-kappa B activation in retinal microglia is involved in the inflammatory and neovascularization signaling in laser-induced choroidal neovascularization
- 2021
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In: Experimental Cell Research. - : ELSEVIER INC. - 0014-4827 .- 1090-2422. ; 403:1
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Journal article (peer-reviewed)abstract
- Purpose: To evaluate Nuclear Factor NF-kappa B (NF-kappa B) signaling on microglia activation, migration, and angiogenesis in laser-induced choroidal neovascularization (CNV). Methods: Nine-week-old C57BL/6 male mice were randomly assigned to IMD-0354 treated or untreated groups (5 mice, 10 eyes per group). CNV was induced with a 532-nm laser. Laser spots (power 250 mW, spot size 100 mu m, time of exposure 50 ms) were created in each eye using a slit-lamp delivery system. Selective inhibitor of nuclear factor kappa-B kinase subunit beta (IKK2) inhibitor IMD-0354 (10 mu g) was delivered subconjunctivally; vehicle-treated mice were the control. The treatment effect on CNV development was assessed at five days post-CNV induction in vivo in C57BL/6 and Cx3cr1(gfp/wt) mice by fluorescent angiography, fundus imaging, and ex vivo by retinal flatmounts immunostaining and Western blot analysis of RPE/Choroidal/Scleral complexes (RCSC) lysates. In vitro evaluations of IMD-0354 effects were performed in the BV-2 microglial cell line using lipopolysaccharide (LPS) stimulation. Results: IMD-0354 caused a significant reduction in the fluorescein leakage and size of the laser spot, as well as a reduction in microglial cell migration and suppression of phospho-I kappa B alpha, Vascular endothelial growth factor (VEGF-A), and Prostaglandin-endoperoxide synthase 2 (COX-2). In vivo and ex vivo observations demonstrated reduced lesion size in mice, CD68, and Allograft inflammatory factor 1 (IBA-1) positive microglia cells migration to the laser injury site in IMD-0354 treated eyes. The data further corroborate with GFP-positive cells infiltration of the CNV site in Cx3cr1(wt/gfp) mice. In vitro IMD-0354 (10-25 ng/ml) treatment reduced NF-kappa B activation, expression of COX-2, caused decreased Actin-F presence and organization, resulting in reduced BV-2 cells migration capacity. Conclusion: The present data indicate that NF-kappa B activation in microglia and its migration capacity is involved in the development of laser CNV in mice. Its suppression by NF-kappa B inhibition might be a promising therapeutic strategy for wet AMD.
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| 7. |
- Jamalpour, Maria, 1981-, et al.
(author)
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Absence of the Shb gene in mixed-lineage leukemia MLL-AF9 cells increases latency in mice despite higher proliferation rates in vitro
- 2020
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In: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 397:2
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Journal article (peer-reviewed)abstract
- Mixed lineage leukemia (MLL) arises from several KMT2A-gene chromosomal translocations. Shb gene deficiency has been found to exhibit pleiotropic effects in different models of leukemia, and consequently, this study aimed to investigate MLL-AF9-induced leukemia in Shb deficiency. Bone marrow cells from wild type and Shb knockout (KO) mice were transduced with the MLL-AF9 gene. Shb KO MLL-AF9 cells proliferated at an increased rate, exhibited altered expression of certain cytokine genes (Kitl, Csf3, IL6, IL1b) and higher expression of cell cycle genes (Ccnd2, Ccne1). Mice receiving Shb KO MLL-AF9 cells showed longer latency without displaying any difference in rates of leukemic cell proliferation, indicating a dichotomy between the in vitro and in vivo phenotypes. The mice with Shb deficient MLL-AF9 cells had a lower content of leukemic bone marrow cells allowing elevated normal hematopoiesis, explaining the longer latency. Finally, Shb knockout GFP-positive bone marrow cells showed a higher percentage of cells expressing myeloid markers. The result suggests a role of Shb in the progression of leukemia and that the relevance of the Shb gene is context-dependent as inferred from the differences between the in vivo and in vitro responses. These findings help to obtain an increased understanding of human MLL-AF9 leukemia.
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| 8. |
- Lennikov, Anton, et al.
(author)
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Synergistic interactions of PlGF and VEGF contribute to blood-retinal barrier breakdown through canonical NF kappa B activation
- 2020
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In: Experimental Cell Research. - : ELSEVIER INC. - 0014-4827 .- 1090-2422. ; 397:2
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Journal article (peer-reviewed)abstract
- To investigate the role of placental growth factor/vascular endothelial growth factor (PlGF-VEGF) hetemdimers are involved in the blood-retinal barrier (BRB) breakdown and the associated mechanism, human retinal endothelial cells (HRECs) were treated with recombinant human (rh)PlGF-VEGF hetemdimers and rhPlGF and studied in normal and high-glucose conditions. HREC barrier function was evaluated by the measurement of trans-endothelial electrical resistance (TEER). Adeno-Associated Virus Type 5 (AAV5) vectors overexpressed PlGF in the retina by intravitreal injection into the C57BL6 mouse eye. AAV5-GFP vector and naive animals were used as controls. Immunofluorescence (IF) and western blots examined the protein expression of PlGF-VEGF hetemdimers, VEGF, PlGF, NF kappa B, p-I kappa B alpha, ZO-1, and VE-cadherin in HREC and mouse retina. PlGF-VEGF heterodimers were detected predominantly in the HREC cell nuclei based on IF and cytoplasmic and nuclear fractionation experiments. High glucose treatment increased PlGF-VEGF nuclear abundance. Dot immunoblotting demonstrated a strong affinity of the 5D11D4 antibody to PlGF-VEGF heterodimers. rhPlGF-VEGF disrupted the barrier function of HREC, which was prevented by the neutralization of PlGF-VEGF by the 5D11D4 antibody. Stimulation of HRECs with rhPlGF also led to an increase in the nuclear signals for PlGF-VEGF, p-I kappa B alpha, and colocalization of NF kappa B p65 and PlGF-VEGF in the nuclei. The selective IKK2 inhibitor IMD0354 disrupted the nuclear colocalization. Treatment with IMD0354 restored the barrier function of HREC, as indicated by the ZO-1 and VE-cadherin expression. In the mouse retinas, P1GF overexpression by AAV5 vector reduced ZO-1 expression and increased abundance of pI kappa B alpha. PIGF/VEGF heterodimers mediate BRB breakdown potentially through the canonical NF kappa B activation.
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| 9. |
- Lewerentz, Jacob, 1992-, et al.
(author)
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The path to immortalization of cells starts by managing stress through gene duplications
- 2023
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In: Experimental Cell Research. - : Elsevier. - 0014-4827 .- 1090-2422. ; 422:1
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Journal article (peer-reviewed)abstract
- The genomes of immortalized cell lines (and cancer cells) are characterized by multiple types of aberrations, ranging from single nucleotide polymorphisms (SNPs) to structural rearrangements that have accumulated over time. Consequently, it is difficult to estimate the relative impact of different aberrations, the order of events, and which gene functions were under selective pressure at the early stage towards cellular immortalization. Here, we have established novel cell cultures derived from Drosophila melanogaster embryos that were sampled at multiple time points over a one-year period. Using short-read DNA sequencing, we show that copy-number gain in preferentially stress-related genes were acquired in a dominant fraction of cells in 300-days old cultures. Furthermore, transposable elements were active in cells of all cultures. Only a few (<1%) SNPs could be followed over time, and these showed no trend to increase or decrease. We conclude that the early cellular responses of a novel culture comprise sequence duplication and transposable element activity. During immortalization, positive selection first occurs on genes that are related to stress response before shifting to genes that are related to growth.
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| 10. |
- Liu, Jianping, et al.
(author)
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A human cell type similar to murine central nervous system perivascular fibroblasts
- 2021
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In: Experimental Cell Research. - : Elsevier. - 0014-4827 .- 1090-2422. ; 402:2
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Journal article (peer-reviewed)abstract
- The brain vasculature has several specific features, one of them being the blood-brain barrier (BBB), which supports and protects the brain by allowing for the passage of oxygen and nutrients, while at the same time preventing passage of pathogens and toxins. The BBB also prevents efficient delivery of drugs to the brain, e.g. for treatment of brain tumors. In the murine brain, perivascular fibroblasts were recently identified as a novel potential constituent of the BBB. Here we present the existence of human cells that could be the equivalent to the murine brain perivascular fibroblasts. Using RNA sequencing, we show a similar transcriptomic profile of cultured human brain cells and murine perivascular fibroblasts. These data open up a window for new hypotheses on cell types involved in human CNS diseases.
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