Cells

18 pages, 7044 KiB

Open AccessArticle

Slow Interstitial Fluid Flow Activates TGF-β Signaling and Drives Fibrotic Responses in Human Tenon Fibroblasts

by Cornelius Jakob Wiedenmann, Charlotte Gottwald, Kosovare Zeqiri, Janne Frömmichen, Emma Bungert, Moritz Gläser, Jeanne Ströble, Robert Lohmüller, Thomas Reinhard, Jan Lübke and Günther Schlunck

Cells 2023, 12(17), 2205; https://doi.org/10.3390/cells12172205 - 4 Sep 2023

Viewed by 1387

Abstract

Background: Fibrosis limits the success of filtering glaucoma surgery. We employed 2D and 3D in vitro models to assess the effects of fluid flow on human tenon fibroblasts (HTF). Methods: HTF were exposed to continuous or pulsatile fluid flow for 48 or 72 [...] Read more.

Background: Fibrosis limits the success of filtering glaucoma surgery. We employed 2D and 3D in vitro models to assess the effects of fluid flow on human tenon fibroblasts (HTF). Methods: HTF were exposed to continuous or pulsatile fluid flow for 48 or 72 h, at rates expected at the transscleral outflow site after filtering surgery. In the 2D model, the F-actin cytoskeleton and fibronectin 1 (FN1) were visualized by confocal immunofluorescence microscopy. In the 3D model, mRNA and whole cell lysates were extracted to analyze the expression of fibrosis-associated genes by qPCR and Western blot. The effects of a small-molecule inhibitor of the TGF-β receptor ALK5 were studied. Results: Slow, continuous fluid flow induced fibrotic responses in the 2D and 3D models. It elicited changes in cell shape, the F-actin cytoskeleton, the deposition of FN1 and activated the intracellular TGF-β signaling pathway to induce expression of fibrosis-related genes, such as CTGF, FN1 and COL1A1. ALK5-inhibition reduced this effect. Intermittent fluid flow also induced fibrotic changes, which decreased with increasing pause duration. Conclusions: Slow interstitial fluid flow is sufficient to induce fibrosis, could underlie the intractable nature of fibrosis following filtering glaucoma surgery and might be a target for antifibrotic therapy. Full article

(This article belongs to the Special Issue Profibrotic Mediators in Hypertrophic Scarring)

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21 pages, 1655 KiB

Open AccessReview

Combination of Genomic Landsscape and 3D Culture Functional Assays Bridges Sarcoma Phenotype to Target and Immunotherapy

by Filomena de Nigris, Concetta Meo and Wulf Palinski

Cells 2023, 12(17), 2204; https://doi.org/10.3390/cells12172204 - 4 Sep 2023

Cited by 2 | Viewed by 1858

Abstract

Genomic-based precision medicine has not only improved tumour therapy but has also shown its weaknesses. Genomic profiling and mutation analysis have identified alterations that play a major role in sarcoma pathogenesis and evolution. However, they have not been sufficient in predicting tumour vulnerability [...] Read more.

Genomic-based precision medicine has not only improved tumour therapy but has also shown its weaknesses. Genomic profiling and mutation analysis have identified alterations that play a major role in sarcoma pathogenesis and evolution. However, they have not been sufficient in predicting tumour vulnerability and advancing treatment. The relative rarity of sarcomas and the genetic heterogeneity between subtypes also stand in the way of gaining statistically significant results from clinical trials. Personalized three-dimensional tumour models that reflect the specific histologic subtype are emerging as functional assays to test anticancer drugs, complementing genomic screening. Here, we provide an overview of current target therapy for sarcomas and discuss functional assays based on 3D models that, by recapitulating the molecular pathways and tumour microenvironment, may predict patient response to treatments. This approach opens new avenues to improve precision medicine when genomic and pathway alterations are not sufficient to guide the choice of the most promising treatment. Furthermore, we discuss the aspects of the 3D culture assays that need to be improved, such as the standardisation of growth conditions and the definition of in vitro responses that can be used as a cut-off for clinical implementation. Full article

(This article belongs to the Special Issue Genomics and Novel Targeted Treatment of Soft-Tissue Sarcoma)

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14 pages, 9359 KiB

Open AccessArticle

Contribution of Oligodendrocytes, Microglia, and Astrocytes to Myelin Debris Uptake in an Explant Model of Inflammatory Demyelination in Rats

by Mariarosaria Cammarota and Francesca Boscia

Cells 2023, 12(17), 2203; https://doi.org/10.3390/cells12172203 - 3 Sep 2023

Cited by 1 | Viewed by 2213

Abstract

The internalization and degradation of myelin in glia contributes to the resolution of neuroinflammation and influences disease progression. The identification of a three-dimensional experimental model to study myelin processing under neuroinflammation will offer a novel approach for studying treatment strategies favoring inflammation resolution [...] Read more.

The internalization and degradation of myelin in glia contributes to the resolution of neuroinflammation and influences disease progression. The identification of a three-dimensional experimental model to study myelin processing under neuroinflammation will offer a novel approach for studying treatment strategies favoring inflammation resolution and neuroprotection. Here, by using a model of neuroinflammation in hippocampal explants, we show that myelin debris accumulated immediately after insult and declined at 3 days, a time point at which tentative repair processes were observed. Olig2⁺ oligodendrocytes upregulated the LRP1 receptor and progressively increased MBP immunoreactivity both at peri-membrane sites and within the cytosol. Oligodendrocyte NG2⁺ precursors increased in number and immunoreactivity one day after insult, and moderately internalized MBP particles. Three days after insult MBP was intensely coexpressed by microglia and, to a much lesser extent, by astrocytes. The engulfment of both MBP⁺ debris and whole MBP⁺ cells contributed to the greatest microglia response. In addition to improving our understanding of the spatial-temporal contribution of glial scarring to myelin uptake under neuroinflammation, our findings suggest that the exposure of hippocampal explants to LPS + IFN-γ-induced neuroinflammation may represent a valuable demyelination model for studying both the extrinsic and intrinsic myelin processing by glia under neuroinflammation. Full article

(This article belongs to the Special Issue Glial Scar: Formation and Regeneration)

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26 pages, 5238 KiB

Open AccessArticle

MicroRNA-375 Is Induced during Astrocyte-to-Neuron Reprogramming and Promotes Survival of Reprogrammed Neurons when Overexpressed

by Xuanyu Chen, Ivan Sokirniy, Xin Wang, Mei Jiang, Natalie Mseis-Jackson, Christine Williams, Kristopher Mayes, Na Jiang, Brendan Puls, Quansheng Du, Yang Shi and Hedong Li

Cells 2023, 12(17), 2202; https://doi.org/10.3390/cells12172202 - 3 Sep 2023

Cited by 4 | Viewed by 1903

Abstract

While astrocyte-to-neuron (AtN) reprogramming holds great promise in regenerative medicine, the molecular mechanisms that govern this unique biological process remain elusive. To understand the function of miRNAs during the AtN reprogramming process, we performed RNA-seq of both mRNAs and miRNAs on human astrocyte [...] Read more.

While astrocyte-to-neuron (AtN) reprogramming holds great promise in regenerative medicine, the molecular mechanisms that govern this unique biological process remain elusive. To understand the function of miRNAs during the AtN reprogramming process, we performed RNA-seq of both mRNAs and miRNAs on human astrocyte (HA) cultures upon NeuroD1 overexpression. Bioinformatics analyses showed that NeuroD1 not only activated essential neuronal genes to initiate the reprogramming process but also induced miRNA changes in HA. Among the upregulated miRNAs, we identified miR-375 and its targets, neuronal ELAVL genes (nELAVLs), which encode a family of RNA-binding proteins and were also upregulated by NeuroD1. We further showed that manipulating the miR-375 level regulated nELAVLs’ expression during NeuroD1-mediated reprogramming. Interestingly, miR-375/nELAVLs were also induced by the reprogramming factors Neurog2 and ASCL1 in HA, suggesting a conserved function to neuronal reprogramming, and by NeuroD1 in the mouse astrocyte culture and spinal cord. Functionally, we showed that miR-375 overexpression improved NeuroD1-mediated reprogramming efficiency by promoting cell survival at early stages in HA and did not appear to compromise the maturation of the reprogrammed neurons. Lastly, overexpression of miR-375-refractory ELAVL4 induced apoptosis and reversed the cell survival-promoting effect of miR-375 during AtN reprogramming. Together, we demonstrated a neuroprotective role of miR-375 during NeuroD1-mediated AtN reprogramming. Full article

(This article belongs to the Special Issue Advances in Neurogenesis: 2nd Edition)

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23 pages, 3938 KiB

Open AccessArticle

Calcineurin-Dependent Homeostatic Response of C. elegans Muscle Cells upon Prolonged Activation of Acetylcholine Receptors

by Franklin Florin, Benjamin Bonneau, Luis Briseño-Roa, Jean-Louis Bessereau and Maëlle Jospin

Cells 2023, 12(17), 2201; https://doi.org/10.3390/cells12172201 - 3 Sep 2023

Cited by 2 | Viewed by 1560

Abstract

Pharmacological adaptation is a common phenomenon observed during prolonged drug exposure and often leads to drug resistance. Understanding the cellular events involved in adaptation could provide new strategies to circumvent this resistance issue. We used the nematode Caenorhabditis elegans to analyze the adaptation [...] Read more.

Pharmacological adaptation is a common phenomenon observed during prolonged drug exposure and often leads to drug resistance. Understanding the cellular events involved in adaptation could provide new strategies to circumvent this resistance issue. We used the nematode Caenorhabditis elegans to analyze the adaptation to levamisole, an ionotropic acetylcholine receptor agonist, used for decades to treat nematode parasitic infections. Genetic screens in C. elegans identified “adapting mutants” that initially paralyze upon exposure to levamisole as the wild type (WT), but recover locomotion after a few hours whereas WT remain paralyzed. Here, we show that levamisole induces a sustained increase in cytosolic calcium concentration in the muscle cells of adapting mutants, lasting several hours and preceding a decrease in levamisole-sensitive acetylcholine receptors (L-AChR) at the muscle plasma membrane. This decrease correlated with a drop in calcium concentration, a relaxation of the animal’s body and a resumption of locomotion. The decrease in calcium and L-AChR content depends on calcineurin activation in muscle cells. We also showed that levamisole adaptation triggers homeostatic mechanisms in muscle cells including mitochondria remodeling, lysosomal tubulation and an increase in autophagic activity. Levamisole adaptation thus provides a new experimental paradigm for studying how cells cope with calcium stress. Full article

(This article belongs to the Special Issue Caenorhabditis elegans: Cell Biology and Physiology)

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35 pages, 5714 KiB

Open AccessReview

Human iPSCs as Model Systems for BMP-Related Rare Diseases

by Gonzalo Sánchez-Duffhues and Christian Hiepen

Cells 2023, 12(17), 2200; https://doi.org/10.3390/cells12172200 - 2 Sep 2023

Viewed by 2749

Abstract

Disturbances in bone morphogenetic protein (BMP) signalling contribute to onset and development of a number of rare genetic diseases, including Fibrodysplasia ossificans progressiva (FOP), Pulmonary arterial hypertension (PAH), and Hereditary haemorrhagic telangiectasia (HHT). After decades of animal research to build a solid foundation [...] Read more.

Disturbances in bone morphogenetic protein (BMP) signalling contribute to onset and development of a number of rare genetic diseases, including Fibrodysplasia ossificans progressiva (FOP), Pulmonary arterial hypertension (PAH), and Hereditary haemorrhagic telangiectasia (HHT). After decades of animal research to build a solid foundation in understanding the underlying molecular mechanisms, the progressive implementation of iPSC-based patient-derived models will improve drug development by addressing drug efficacy, specificity, and toxicity in a complex humanized environment. We will review the current state of literature on iPSC-derived model systems in this field, with special emphasis on the access to patient source material and the complications that may come with it. Given the essential role of BMPs during embryonic development and stem cell differentiation, gain- or loss-of-function mutations in the BMP signalling pathway may compromise iPSC generation, maintenance, and differentiation procedures. This review highlights the need for careful optimization of the protocols used. Finally, we will discuss recent developments towards complex in vitro culture models aiming to resemble specific tissue microenvironments with multi-faceted cellular inputs, such as cell mechanics and ECM together with organoids, organ-on-chip, and microfluidic technologies. Full article

(This article belongs to the Special Issue iPS Cells (iPSCs) for Modelling and Treatment of Human Diseases 2022)

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Full article ">Figure 1
BMP signal transduction. The interaction between BMP ligands and BMP type I receptors induces the formation of membrane heterotetrameric complexes. This interaction is influenced by the relative availability of ligands and receptors, which is regulated by soluble antagonists, co-receptors, and extracellular matrix (ECM) components. The activated BMP receptors transmit signals intracellularly through their serine/threonine kinase domain, leading to the phosphorylation of the BMP canonical mediators, SMAD1/5/9, at the C-terminal domain. Moreover, BMP receptors regulate the activation of non-canonical signalling pathways, which serve as a signalling hub integrating tissue-specific environmental cues. Canonical and non-canonical signalling pathways intersect at various levels. Once activated, SMAD1/5/9 trimerize with SMAD4 and translocate into the nucleus to bind specific gene promoters in collaboration with transcription co-factors. A number of environmental cues co-influence signalling outcomes, including cellular mechanics such as mechanical shear forces or inflammatory or hypoxic microenvironments. Due to space limitations, details on the TGF-β-specific regulation of the signalling branch are not shown. Full article ">Figure 2
iPSC generation and cell type differentiation in rare BMP diseases. So far, iPSC lines have been successfully generated for three rare BMP diseases: Fibrodysplasia ossificans progressiva (FOP), Hereditary pulmonary arterial hypertension (HPAH), and Hereditary haemorrhagic telangiectasia (HHT). Various sources of somatic tissue have been explored for this purpose. Reprogramming efficiency may be affected by the BMP gene mutation, and small molecules, like LDN-193189, that aim to restore normal BMP signalling can be utilized. BMPs are potent inducers of mesoderm. Therefore, a combination of BMP agonists and Wnt antagonists (such as CHIR-99021) is employed to induce mesodermal differentiation. In order to generate vascular cells, ALK4/5/7 inhibition is often necessary in a subsequent step. Full article ">Figure 3
Rare BMP diseases and iPSCs. Fibrodysplasia ossificans progressiva (FOP) is a musculoskeletal disease caused by heterozygous gain-of-function mutations in ACVR1/ALK2, resulting in heightened activation of downstream ALK2 signalling in response to Activin-A. Increased ALK2 signalling leads to the formation of extraskeletal bone plaques. On the right is a photograph of the skeleton of a man with FOP, from the collections of the Anatomical Museum of the Leiden University Medical Center (LUMC). This photo has been reproduced with permission from LUMC (copyright belongs to LUMC). Hereditary pulmonary arterial hypertension (HPAH) is a cardiovascular disease characterized by progressive narrowing of the pulmonary arteries, resulting in elevated pulmonary arterial pressure (mPAP > 20 mm Hg), followed by right ventricular dilatation and hypertrophy. Loss-of-function mutations in BMPR2 exacerbate disease severity and penetrance by disrupting the overall balance of TGF-β signalling in endothelial and smooth muscle cells. The right panel illustrates a cross-section of a healthy pulmonary artery (top) compared with a diseased occluded pulmonary artery (bottom). The formation of a pronounced neointima by aberrant cell growth into the lumen is shown. The PAH patient pulmonary arterial cross-section is part of a published dataset by C.H., recreated based on details mentioned in Hiepen C. et al. [<a href="#B168-cells-12-02200" class="html-bibr">168</a>]. Hereditary haemorrhagic telangiectasia (HHT) is associated with loss-of-function gene mutations in ACVRL1 (ALK1) and ENG (endoglin), among others, which impair BMP signalling. It is characterized by varying degrees of lesions due to the instability of the microvascular networks. The right panel displays a typical microvascular subdermal lesion seen in HHT. The picture was contributed with friendly permission by Prof. Dr. Urban Geisthoff (Philipps University of Marburg; Germany). Full article ">Figure 4
Current and Future Development of iPSC-based technology in BMP rare diseases. Since the inception of the first iPSC-based cell models, significant progress has been made in developing more intricate and robust systems that accurately replicate diseased tissues and organs. Of particular interest in monogenic diseases is the utilization of gene editing tools such as CRISPR/Cas9 to create isogenic cell lines. While the first culturing approaches opted for 2 dimensional methods, more complex three dimensional culture conditions, such as embryoid bodies (EBs) and organoids, are emerging. This can be combined with co-culture of different cell types differentiated from the same parental iPSC line. Culturing those cells under conditions that expose them to microenvironmental cues, such as hypoxia, biomechanics, and inflammation, allows for effective and complex disease modelling. These systems have proven to be valuable platforms for drug screening and in-depth mechanistic investigations. Further advances, such as construction of complex cellular grafts or therapeutic extracellular vesicles (EVs) derived from iPSCs, hold the potential to revolutionize tissue engineering and iPSC-based cell therapies, presenting therapeutic alternatives for patients affected by rare BMP diseases. Full article ">

18 pages, 14244 KiB

Open AccessArticle

Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy Study

by Viktoria Sergunova, Vladimir Inozemtsev, Nina Vorobjeva, Elena Kozlova, Ekaterina Sherstyukova, Snezhanna Lyapunova and Aleksandr Chernysh

Cells 2023, 12(17), 2199; https://doi.org/10.3390/cells12172199 - 2 Sep 2023

Cited by 4 | Viewed by 2214

Abstract

Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the specific characteristics of the neutrophil membrane surface, its nanostructure, and morphology. The aim of this study was to reveal the [...] Read more.

Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the specific characteristics of the neutrophil membrane surface, its nanostructure, and morphology. The aim of this study was to reveal the topography and nanosurface characteristics of neutrophils during activation and NETosis using atomic force microscopy (AFM). We showed the main stages of neutrophil activation and NETosis, which include control cell spreading, cell fragment formation, fusion of nuclear segments, membrane disruption, release of neutrophil extracellular traps (NETs), and final cell disintegration. Changes in neutrophil membrane nanosurface parameters during activation and NETosis were quantified. It was shown that with increasing activation time there was a decrease in the spectral intensity of the spatial periods. Exposure to the activator A23187 resulted in an increase in the number and average size of cell fragments over time. Exposure to the activators A23187 and PMA (phorbol 12-myristate 13-acetate) caused the same pattern of cell transformation from spherical cells with segmented nuclei to disrupted cells with NET release. A23187 induced NETosis earlier than PMA, but PMA resulted in more cells with NETosis at the end of the specified time interval (180 min). In our study, we used AFM as the main research tool. Confocal laser-scanning microscopy (CLSM) images are provided for identification and detailed analysis of the phenomena studied. In this way, we exploited the advantages of both techniques. Full article

(This article belongs to the Special Issue Advances in Scanning Probe Microscopy in Cell Biology)

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13 pages, 1629 KiB

Open AccessArticle

Identification of Unique microRNA Profiles in Different Types of Idiopathic Inflammatory Myopathy

by Sandra Muñoz-Braceras, Iago Pinal-Fernandez, Maria Casal-Dominguez, Katherine Pak, José César Milisenda, Shajia Lu, Massimo Gadina, Faiza Naz, Gustavo Gutierrez-Cruz, Stefania Dell’Orso, Jiram Torres-Ruiz, Josep Maria Grau-Junyent, Albert Selva-O’Callaghan, Julie J. Paik, Jemima Albayda, Lisa Christopher-Stine, Thomas E. Lloyd, Andrea M. Corse and Andrew L. Mammen

Cells 2023, 12(17), 2198; https://doi.org/10.3390/cells12172198 - 2 Sep 2023

Cited by 5 | Viewed by 1922

Abstract

Dermatomyositis (DM), antisynthetase syndrome (AS), immune-mediated necrotizing myopathy (IMNM), and inclusion body myositis (IBM) are four major types of idiopathic inflammatory myopathy (IIM). Muscle biopsies from each type of IIM have unique transcriptomic profiles. MicroRNAs (miRNAs) target messenger RNAs (mRNAs), thereby regulating their [...] Read more.

Dermatomyositis (DM), antisynthetase syndrome (AS), immune-mediated necrotizing myopathy (IMNM), and inclusion body myositis (IBM) are four major types of idiopathic inflammatory myopathy (IIM). Muscle biopsies from each type of IIM have unique transcriptomic profiles. MicroRNAs (miRNAs) target messenger RNAs (mRNAs), thereby regulating their expression and modulating transcriptomic profiles. In this study, 18 DM, 12 IMNM, 6 AS, 6 IBM, and 6 histologically normal muscle biopsies underwent miRNA profiling using the NanoString nCounter system. Eleven miRNAs were exclusively differentially expressed in DM compared to controls, seven miRNAs were only differentially expressed in AS, and nine miRNAs were specifically upregulated in IBM. No differentially expressed miRNAs were identified in IMNM. We also analyzed miRNA-mRNA associations to identify putative targets of differentially expressed miRNAs. In DM and AS, these were predominantly related to inflammation and cell cycle progression. Moreover, our analysis showed an association between miR-30a-3p, miR-30e-3p, and miR-199b-5p downregulation in DM and the upregulation of target genes induced by type I interferon. In conclusion, we show that muscle biopsies from DM, AS, and IBM patients have unique miRNA signatures and that these miRNAs might play a role in regulating the expression of genes known to be involved in IIM pathogenesis. Full article

(This article belongs to the Special Issue Spotlight on Neuromuscular Diseases: Pathomechanisms and New Therapeutic Targets)

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18 pages, 7506 KiB

Open AccessArticle

JNK Signalling Regulates Self-Renewal of Proliferative Urine-Derived Renal Progenitor Cells via Inhibition of Ferroptosis

by Lisa Nguyen, Leonie Thewes, Michelle Westerhoff, Wasco Wruck, Andreas S. Reichert, Carsten Berndt and James Adjaye

Cells 2023, 12(17), 2197; https://doi.org/10.3390/cells12172197 - 2 Sep 2023

Viewed by 1486

Abstract

With a global increase in chronic kidney disease patients, alternatives to dialysis and organ transplantation are needed. Stem cell-based therapies could be one possibility to treat chronic kidney disease. Here, we used multipotent urine-derived renal progenitor cells (UdRPCs) to study nephrogenesis. UdRPCs treated [...] Read more.

With a global increase in chronic kidney disease patients, alternatives to dialysis and organ transplantation are needed. Stem cell-based therapies could be one possibility to treat chronic kidney disease. Here, we used multipotent urine-derived renal progenitor cells (UdRPCs) to study nephrogenesis. UdRPCs treated with the JNK inhibitor—AEG3482 displayed decreased proliferation and downregulated transcription of cell cycle-associated genes as well as the kidney progenitor markers—SIX2, SALL1 and VCAM1. In addition, levels of activated SMAD2/3, which is associated with the maintenance of self-renewal in UdRPCs, were decreased. JNK inhibition resulted in less efficient oxidative phosphorylation and more lipid peroxidation via ferroptosis, an iron-dependent non-apoptotic cell death pathway linked to various forms of kidney disease. Our study is the first to describe the importance of JNK signalling as a link between maintenance of self-renewal and protection against ferroptosis in SIX2-positive renal progenitor cells. Full article

(This article belongs to the Section Stem Cells)

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Inhibition of JNK reduces the proliferation of UdRPCs. (A) Morphology of the three UdRPCs UM51, UM27 and UF21 with or without AEG3482 treatment after 72 h. Scale bars represent 100 µm. (B) Ki67 expression in UdRPCs treated with or not treated with AEG3482 for 72 h. Scale bars represent 50 µm. (C) Ki67 proliferation assay for JNK-inhibited UdRPCs (n = 10; * p-value < 0.05, *** p-value < 0.001). (D) Bar graph of PI-measured cell death in JNK-inhibited UdRPCs compared to untreated controls (n = 5, * p-value < 0.05, *** p-value < 0.001). Error bars indicate STDEV. (E) mRNA expression of nephron progenitor markers SIX2, SALL1, VCAM1 and Ki67. Mean values were normalised to the housekeeping gene RPL37A. (F) Gene expression of cell cycle-related genes for the time points 24 h, 72 h and 120 h depicted in a Pearson’s heatmap. Full article ">Figure 2
JNK signalling is associated with cell cycle processes in UdRPCs. (A) Representative enrichment clusters for control and JNK inhibition after 24 h depicted in a heatmap and cell cycle-related processes marked with an arrow. Venn diagram of control and JNK-inhibited UM51 cells for the time point 24 h. (B) Representative enrichment clusters for control and JNK inhibition after 72 h depicted in a heatmap and cell cycle-related processes marked with an arrow. Venn diagram of control and JNK-inhibited UM51 cells for the time point 72 h. (C) Representative enrichment clusters for control and JNK inhibition after 120 h depicted in a heatmap and cell cycle-related processes marked with an arrow. Venn diagram of control and JNK-inhibited UM51 cells for the time point 120 h. Full article ">Figure 3
Inhibition of JNK signalling affects the downstream target cJUN and SMAD proteins. (A) Representative protein expression (%) of p- and t-JNK in UdRPCs with or without JNK inhibition. (B) Representative protein expression (%) of p- and t-cJUN in UdRPCs with or without JNK inhibition. (C) Immunofluorescence stainings of cJUN and p-cJUN in UdRPCs with or without JNK inhibition. Scale bars represent 50 µm. (D) Protein expression of p-/t-SMAD1/5 and p-/t-SMAD2/3 in UdRPCs with or without JNK inhibition. Full article ">Figure 4
Inhibition of JNK signalling increases lipid peroxidation. (A) Representative histograms of measured fluorescence intensities after BODIPY staining and the respective bar plots of mean fluorescence intensity of control or JNK-inhibited UdRPCs (n = 5; * p-value < 0.05, ** p-value < 0.01). Error bars indicate SEM. (B) Representative histograms of measured fluorescence intensities after BODIPY staining and the respective bar plots of mean fluorescence intensity of control, AEG3482 and AEG3482 + Lip-1 (n = 5). Error bars indicate SEM. (C) Gene expression of iron metabolism-related genes in UM51 cells for the time points 24 h, 72 h and 120 h depicted in a Pearson’s heatmap. (D) Pearson’s heatmap depicting gene expression of glutathione metabolism-related genes in UM51 cells for the time points 24 h, 72 h and 120 h. (E) Representative protein expression (%) of GPX4, TFR1 and HO-1 in UdRPCs with or without JNK inhibition. (F) mRNA expression of glutathione metabolism-related markers GCLC, GCLM, GPX4. (G) mRNA expression of iron metabolism-related markers HMOX1, SLC11A2 and TFR1. Mean values were normalised to the housekeeping gene RPL37A. Full article ">Figure 5
JNK signalling regulates mitochondrial respiration via membrane potential. (A) Measurement of OCR in real time in UdRPCs. Basal respiration, maximal respiration and spare respiratory capacity are depicted *** p-value < 0.001). Error bars indicate SD. (a) OCR in UM51 (n = 1; N = 23). (b) OCR in UM27 (n = 1; N = 23). (c) OCR in UF21 (n = 1; N = 46). (B) Fluorescence images of MitoTracker Green and TMRM stainings in UdRPCs UM51, UM27 and UF21 with and without JNK inhibition. Scale bars depict 10 µm. (C) Measurement of mean TMRM fluorescence signal intensity in UdRPCs *** p-value < 0.001). Outliers were removed. Error bars indicate SEM. (a) Mean TMRM signal in UM51 is depicted (n = 2; ctrl. N = 1; +JNK inhibitor N= 2). (b) Mean TMRM signal in UM27 is depicted (n = 2; ctrl. N = 1; +JNK inhibitor N = 2). Error bars indicate SEM. (c) Mean TMRM signal in UF21 is depicted (n = 2; ctrl. N = 1; +JNK inhibitor N = 2). Full article ">

12 pages, 4389 KiB

Open AccessArticle

Lysophosphatidic Acid Signalling Regulates Human Sperm Viability via the Phosphoinositide 3-Kinase/AKT Pathway

by Hao-Yu Liao and Cristian O’Flaherty

Cells 2023, 12(17), 2196; https://doi.org/10.3390/cells12172196 - 2 Sep 2023

Cited by 1 | Viewed by 1360

Abstract

Lysophosphatidic acid (LPA) signalling is essential for maintaining germ cell viability during mouse spermatogenesis; however, its role in human spermatozoa is unknown. We previously demonstrated that peroxiredoxin 6 (PRDX6) calcium-independent phospholipase A₂ (iPLA₂) releases lysophospholipids such as LPA or arachidonic [...] Read more.

Lysophosphatidic acid (LPA) signalling is essential for maintaining germ cell viability during mouse spermatogenesis; however, its role in human spermatozoa is unknown. We previously demonstrated that peroxiredoxin 6 (PRDX6) calcium-independent phospholipase A₂ (iPLA₂) releases lysophospholipids such as LPA or arachidonic acid (AA) and that inhibiting PRDX6 iPLA₂ activity impairs sperm cell viability. The exogenous addition of LPA bypassed the inhibition of PRDX6 iPLA₂ activity and maintained the active phosphoinositide 3-kinase (PI3K)/AKT pathway. Here, we aimed to study PI3K/AKT pathway regulation via LPA signalling and protein kinases in maintaining sperm viability. The localization of LPARs in human spermatozoa was determined using immunocytochemistry, and P-PI3K and P-AKT substrate phosphorylations via immunoblotting. Sperm viability was determined using the hypo-osmotic swelling test. LPAR1, 3, 5 and 6 were located on the sperm plasma membrane. The inhibition of LPAR1-3 with Ki16425 promoted the impairment of sperm viability and decreased the phosphorylation of PI3K AKT substrates. Inhibitors of PKC, receptor-type PTK and PLC impaired sperm viability and the PI3K/AKT pathway. Adding 1-oleoyl-2-acetyl-snglycerol (OAG), a cell-permeable analog of diacylglycerol (DAG), prevented the loss of sperm viability and maintained the phosphorylation of PI3K. In conclusion, human sperm viability is supported by LPAR signalling and regulated by PLC, PKC and RT-PTK by maintaining phosphorylation levels of PI3K and AKT substrates. Full article

(This article belongs to the Special Issue Select Topics in Molecular and Cellular Mechanisms of Mammalian Reproduction)

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Localization of LPARs in human spermatozoa. Human spermatozoa, non-permeabilized or permeabilized with 100% methanol (Perm), were incubated with anti-LPAR antibodies. Spermatozoa incubated with the second one alone did not show any signals. The green arrow indicates the sperm midpiece; the white arrowhead indicates the sperm equatorial segment; the light blue arrow indicates the sperm acrosome; and the red head arrow indicates the post-acrosomal region. Pictures were taken at 100× using a Carl Zeiss Axiophot microscope (Oberkochen, Germany), scale bar = 15 μm. Full article ">Figure 2
The effect of Ki16425 causes the cell death of human spermatozoa. Spermatozoa were treated with Ki16425 for 3.5 h at 37 °C and a hypo-osmotic swelling buffer was added to distinguish whether the spermatozoa were non-viable or viable (<a href="#sec2-cells-12-02196" class="html-sec">Section 2</a>). The results are representative of sperm samples from different healthy donors (n = 4; *** p ≤ 0.001, ANOVA and Dunnett’s test). Full article ">Figure 3
The effect of Ki16425 on PI3K and AKT substrate phosphorylation of human spermatozoa. Spermatozoa were treated with increasing concentrations of Ki16425 for 3.5 h at 37 °C. Sperm proteins were electrophoresed, electrotransferred and immunoblotted with (A) anti-P-PI3K or (B) anti-P-AKT-S antibodies. The membrane was stripped and reblotted with the anti-Tubulin antibody to confirm equal loading for each lane. The relative intensity of P-PI3K/P-AKT-S was obtained by normalizing each band’s intensity to the respective intensity of tubulin. The results are representative of sperm samples from different healthy donors (n = 4; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, ANOVA and Dunnett’s test). Full article ">Figure 4
The effect of kinase inhibitors caused the cell death of human spermatozoa. Spermatozoa were treated with kinase inhibitors for 3.5 h at 37 °C. Then, the hypo-osmotic swelling buffer was added to distinguish whether the sperm was non-viable or viable (<a href="#sec2-cells-12-02196" class="html-sec">Section 2</a>) (n = 3, which means a significant difference within the same strain). The results are representative of sperm samples from different healthy donors (ANOVA and Dunnett’s test; * p ≤ 0.05; *** p ≤ 0.001). Full article ">Figure 5
The different kinase inhibitors affected the PI3K phosphorylation of human spermatozoa. Levels of P-PI3K were determined in spermatozoa incubated with H89, chelerythrine, PD98059, PP2 and Tyrphostin A47 for 3.5 h at 37 °C. Sperm proteins were electrophoresed, electrotransferred and immunoblotted with the anti-P-PI3K antibody. The membrane was stripped and reblotted with the anti-Tubulin antibody to confirm equal loading for each lane. The relative intensity of P-PI3K was obtained by normalizing each band’s intensity to the respective intensity of tubulin. The results are representative of sperm samples from different healthy donors (n = 4; * p ≤ 0.05, *** p ≤ 0.001, ANOVA and Dunnett’s test). Full article ">Figure 6
The different kinase inhibitors led to the effect on AKT substrates’ phosphorylation of human spermatozoa. Levels of P-AKT substrates were determined in spermatozoa incubated with H89, chelerythrine, PD98059, PP2 and Tyrphostin A47 for 3.5 h at 37 °C. Sperm proteins were electrophoresed, electrotransferred and immunoblotted with the anti-P-AKT-S antibody. The membrane was stripped and reblotted with the anti-Tubulin antibody to confirm equal loading for each lane. The relative intensity of P-AKT-S was obtained by normalizing each band’s intensity to the respective intensity of tubulin (n = 4; * p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001, ANOVA and Dunnett’s test). Full article ">Figure 7
OAG prevents the loss of human sperm viability when U-73122 is present. Spermatozoa were incubated with U-73122 and OAG for 3.5 h at 37 °C and a hypo-osmotic swelling buffer was added to distinguish whether the spermatozoa were non-viable or viable (<a href="#sec2-cells-12-02196" class="html-sec">Section 2</a>). The results are representative of sperm samples from different healthy donors (n = 3 * means a significant difference within the same strain. ANOVA and Dunnett’s test; * p ≤ 0.05). Full article ">Figure 8
OAG prevents the loss of PI3K and AKT substrates’ phosphorylation when U−73122 is present. Levels of P-PI3K and P-AKT-S in spermatozoa incubated with U-73122 and OAG for 3.5 h at 37 °C. Sperm proteins were electrophoresed, electrotransferred and immunoblotted with the anti-PI3K/P-AKT-S antibody. The membrane was stripped and reblotted with the anti-Tubulin antibody to confirm equal loading for each lane. The relative intensity of (A) P-PI3K and (B) P-AKT-S was obtained by normalizing each band’s intensity to the respective intensity of tubulin. The results are representative of sperm samples from different healthy donors (n = 4, * means significant difference within the same strain. ANOVA and Dunnett’s test; * p ≤ 0.05; *** p ≤ 0.001). Full article ">Figure 9
Lysophosphatidic signalling is essential to support viability in human spermatozoa. Lysophosphatidic acid (LPA) binds to LPAR (1, 3, 5 or 6) to activate the PI3K/AKT pathway in human spermatozoa to prevent apoptotic-like changes that could impair sperm viability. Ki6425 inhibits LPAR1 and LPAR3, thus preventing phosphorylations of PI3K and AKT substrates and leading to impairment of sperm viability. A similar fate occurs in spermatozoa treated with U−73122, TA47 or chelerythrine, inhibitors of PLC, receptor-type tyrosine-protein kinase (RT-PTK) and PKC, respectively. Full article ">

19 pages, 4753 KiB

Open AccessArticle

Lymphatic Endothelial-to-Myofibroblast Transition: A Potential New Mechanism Underlying Skin Fibrosis in Systemic Sclerosis

by Irene Rosa, Eloisa Romano, Bianca Saveria Fioretto, Khadija El Aoufy, Silvia Bellando-Randone, Marco Matucci-Cerinic and Mirko Manetti

Cells 2023, 12(17), 2195; https://doi.org/10.3390/cells12172195 - 1 Sep 2023

Cited by 8 | Viewed by 1663

Abstract

At present, only a few reports have addressed the possible contribution of the lymphatic vascular system to the pathogenesis of systemic sclerosis (SSc). Based on the evidence that blood vascular endothelial cells can undertake the endothelial-to-myofibroblast transition (EndMT) contributing to SSc-related skin fibrosis, [...] Read more.

At present, only a few reports have addressed the possible contribution of the lymphatic vascular system to the pathogenesis of systemic sclerosis (SSc). Based on the evidence that blood vascular endothelial cells can undertake the endothelial-to-myofibroblast transition (EndMT) contributing to SSc-related skin fibrosis, we herein investigated whether the lymphatic endothelium might represent an additional source of profibrotic myofibroblasts through a lymphatic EndMT (Ly-EndMT) process. Skin sections from patients with SSc and healthy donors were immunostained for the lymphatic endothelial cell-specific marker lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) in combination with α-smooth muscle actin (α-SMA) as the main marker of myofibroblasts. Commercial human adult dermal lymphatic microvascular endothelial cells (HdLy-MVECs) were challenged with recombinant human transforming growth factor-β1 (TGFβ1) or serum from SSc patients and healthy donors. The expression of lymphatic endothelial cell/myofibroblast markers was measured by quantitative real-time PCR, Western blotting and immunofluorescence. Collagen gel contraction assay was performed to assess myofibroblast-like cell contractile ability. Lymphatic endothelial cells in intermediate stages of the Ly-EndMT process (i.e., coexpressing LYVE-1 and α-SMA) were found exclusively in the fibrotic skin of SSc patients. The culturing of HdLy-MVECs with SSc serum or profibrotic TGFβ1 led to the acquisition of a myofibroblast-like morphofunctional phenotype, as well as the downregulation of lymphatic endothelial cell-specific markers and the parallel upregulation of myofibroblast markers. In SSc, the Ly-EndMT might represent a previously overlooked pathogenetic process bridging peripheral microlymphatic dysfunction and skin fibrosis development. Full article

(This article belongs to the Special Issue The Role of Epithelial Cells in Scleroderma)

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Figure 1
Presence of lymphatic endothelial-to-myofibroblast transition (Ly-EndMT) in dermal lymphatic capillaries of SSc patients. Representative fluorescence microphotographs of skin sections from healthy donors (n = 3) and patients with SSc (n = 5) double immunostained for the lymphatic endothelial cell-specific marker LYVE-1 (green) and the myofibroblast marker α-SMA (red). The nuclei are stained blue with 4′,6-diamidino-2-phenylindole (DAPI). In healthy skin, the expression of α-SMA is detectable exclusively in the pericytes and vascular smooth muscle cells of dermal blood microvessels and in the arrector pili muscles. In the fibrotic skin of SSc patients, colocalized LYVE-1 and α-SMA is evident as yellow staining in the transitional Ly-EndMT cells of dermal lymphatic capillaries (arrowheads). Arrows point to α-SMA+ stromal myofibroblasts detected in SSc skin. Scale bar = 50 μm. Bars represent the mean ± SEM of the percentage of dermal lymphatic vessels displaying transitional endothelial cells per high-power field (40× original magnification). Unpaired Student’s t-test was used for statistical analysis. *** p < 0.001 vs. healthy skin. LYVE-1: lymphatic vessel endothelial hyaluronan receptor-1; α-SMA: α-smooth muscle actin; SSc: systemic sclerosis; ECs, endothelial cells. Full article ">Figure 2
Treatment with serum from SSc patients leads to the acquisition of myofibroblast-like morphological features in healthy dermal lymphatic microvascular endothelial cells (HdLy-MVECs). The representative phase-contrast microphotographs of HdLy-MVECs (n = 3 cell lines) at basal condition and after 72 h culture with serum from healthy controls (n = 6), serum from SSc patients (n = 6), or recombinant human TGFβ1 are shown in the upper panels. Scale bar = 400 μm. The representative fluorescence microphotographs of HdLy-MVECs at the same experimental conditions and stained for filamentous actin (F-actin) with Alexa 488-conjugated phalloidin (green) are shown in the middle panels. The nuclei are stained blue with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar = 50 μm. When compared to the basal condition, HdLy-MVEC morphology does not change in cultures challenged with serum from healthy donors. Upon treatment with SSc serum or TGFβ1, HdLy-MVECs lose their characteristic polygonal cobblestone-like morphology and exhibit an elongated spindle-shaped morphology. Bars in the lower panels represent the mean ± SEM of the percentage of cells with an elongated spindle-shaped morphology (left) or cells with stress fibers (right) per high-power field (40× original magnification). One-way ANOVA with post hoc Tukey’s test was used for statistical analysis. *** p < 0.001 vs. basal condition; $$$ p < 0.001 vs. healthy serum. TGFβ1: transforming growth factor-β1; SSc: systemic sclerosis. Full article ">Figure 3
Culture with serum from SSc patients induces changes in gene expression levels of lymphatic endothelial cell-specific markers and myofibroblast markers in healthy dermal lymphatic microvascular endothelial cells (HdLy-MVECs). HdLy-MVECs were subjected to 48 h treatment with serum from healthy controls (n = 6), serum from SSc patients (n = 6), or recombinant human TGFβ1, and then examined for the expression levels of PROX1, LYVE1, PDPN, S100A4, ACTA2, COL1A1, COL1A2, and SNAI1 genes by quantitative SYBR Green real-time PCR. For each gene, the basal expression level was set to 1 for the normalization of the other results. As a reference gene, 18S ribosomal RNA was used in all real-time PCR assays. Bars represent the mean ± SEM of triplicate gene expression determinations from three cell lines. One-way ANOVA with post hoc Tukey’s test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. basal condition; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 vs. healthy serum. TGFβ1: transforming growth factor-β1; SSc: systemic sclerosis. Full article ">Figure 4
Culture with SSc serum induces changes in protein expression of lymphatic endothelial cell markers and myofibroblast markers in healthy dermal microvascular endothelial cells (HdLy-MVECs). HdLy-MVECs were subjected to 72 h challenge with serum from healthy controls (n = 6), serum from patients with SSc (n = 6), or recombinant human TGFβ1, and then analyzed by Western blotting for the protein expression of Prox1 (A), LYVE-1 (B), PDPN (C), S100A4 (D), α-SMA (E), and COL1A1 (F). Representative immunoblots are shown. The molecular weight in kDa is reported on the right of the bands. α-tubulin and GAPDH were used for normalization (loading controls). Bars represent the mean ± SEM of optical density in arbitrary units (a.u.). One-way ANOVA with post hoc Tukey’s test was used for statistical analysis. * p < 0.05, *** p < 0.001 vs. basal condition; $ p < 0.05, $$$ p < 0.001 vs. healthy serum. Prox1: prospero-related homeobox protein 1; LYVE-1: lymphatic vessel endothelial hyaluronan receptor-1; PDPN: podoplanin; α-SMA: α-smooth muscle actin; COL1A1: α-1 chain of type I collagen; TGFβ1: transforming growth factor-β1; SSc: systemic sclerosis. Full article ">Figure 5
Culture with SSc serum induces a myofibroblast-like immunophenotype in healthy dermal lymphatic microvascular endothelial cells (HdLy-MVECs). Representative fluorescence microphotographs of HdLy-MVECs at basal condition and after treatment for 72 h with healthy serum (n = 6), SSc serum (n = 6), or recombinant human TGFβ1 immunostained for Prox1, LYVE-1, S100A4, α-SMA, and Snail1. The nuclei are stained blue with 4′,6-diamidino-2-phenylindole (DAPI). In HdLy-MVECs at basal condition and cultured in the presence of healthy serum, the expression of α-SMA, S100A4, and Snail1 is negligible. HdLy-MVECs challenged with SSc serum or TGFβ1 are characterized by the strong downregulation of nuclear Prox1 and cell surface LYVE-1, and by the parallel upregulation of α-SMA, partly assembled into stress fibers, as well as S100A4 and nuclear Snail1. Scale bar = 50 μm. Bars represent the mean ± SEM of the percentage of immunopositive cells or nuclei per high-power field (40× original magnification). One-way ANOVA with post hoc Tukey’s test was used for statistical analysis. *** p < 0.001 vs. basal condition; $$$ p < 0.001 vs. healthy serum. Prox1: prospero-related homeobox protein 1; LYVE-1: lymphatic vessel endothelial hyaluronan receptor-1; α-SMA: α-smooth muscle actin; TGFβ1: transforming growth factor-β1; SSc: systemic sclerosis. Full article ">Figure 6
Healthy dermal lymphatic microvascular endothelial cells (HdLy-MVECs) acquire a myofibroblast-like functional phenotype after culture with serum from SSc patients. The upper panel shows representative pictures of collagen gels with HdLy-MVECs at basal condition and after treatment for 72 h with serum from healthy donors (n = 6), serum from SSc patients (n = 6), or recombinant human TGFβ1. Gel size in the presence of unstimulated HdLy-MVECs (basal) was assumed to be 100% for the normalization of the other results. Negative control consisted of collagen gel without embedded cells. Bars represent the mean ± SEM of triplicate determinations from three cell lines. One-way ANOVA with post hoc Tukey’s test was used for statistical analysis. ** p < 0.01, *** p < 0.001 vs. basal condition; $ p < 0.05 vs. healthy serum. TGFβ1: transforming growth factor-β1; SSc: systemic sclerosis. Full article ">Figure 7
Culture with SSc serum activates Smad-dependent TGFβ signaling in healthy dermal lymphatic microvascular endothelial cells (HdLy-MVECs). HdLy-MVECs were subjected to 72 h challenge with serum from healthy controls (n = 6), serum from patients with SSc (n = 6), or recombinant human TGFβ1, and then analyzed by Western blotting for the protein expression of p-Smad3, Smad3 and α-tubulin (loading control). Representative immunoblots are shown. Molecular weight values (kDa) are indicated on the right of the bands. Bars represent the mean ± SEM of optical density in arbitrary units (a.u.). One-way ANOVA with post hoc Tukey’s test was used for statistical analysis. *** p < 0.001 vs. basal condition; $$$ p < 0.001 vs. healthy serum. p-Smad3: phosphorylated-Smad3; TGFβ1: transforming growth factor-β1; SSc: systemic sclerosis. Full article ">

24 pages, 12072 KiB

Open AccessArticle

ODF2 Negatively Regulates CP110 Levels at the Centrioles/Basal Bodies to Control the Biogenesis of Primary Cilia

by Madeline Otto and Sigrid Hoyer-Fender

Cells 2023, 12(17), 2194; https://doi.org/10.3390/cells12172194 - 1 Sep 2023

Cited by 3 | Viewed by 1209

Abstract

Primary cilia are essential sensory organelles that develop when an inhibitory cap consisting of CP110 and other proteins is eliminated. The degradation of CP110 by the ubiquitin-dependent proteasome pathway mediated by NEURL4 and HYLS1 removes the inhibitory cap. Here, we investigated the suitability [...] Read more.

Primary cilia are essential sensory organelles that develop when an inhibitory cap consisting of CP110 and other proteins is eliminated. The degradation of CP110 by the ubiquitin-dependent proteasome pathway mediated by NEURL4 and HYLS1 removes the inhibitory cap. Here, we investigated the suitability of rapamycin-mediated dimerization for centriolar recruitment and asked whether the induced recruitment of NEURL4 or HYLS1 to the centriole promotes primary cilia development and CP110 degradation. We used rapamycin-mediated dimerization with ODF2 to induce their targeted recruitment to the centriole. We found decreased CP110 levels in the transfected cells, but independent of rapamycin-mediated dimerization. By knocking down ODF2, we showed that ODF2 controls CP110 levels. The overexpression of ODF2 is not sufficient to promote the formation of primary cilia, but the overexpression of NEURL4 or HYLS1 is. The co-expression of ODF2 and HYLS1 resulted in the formation of tube-like structures, indicating an interaction. Thus, ODF2 controls primary cilia formation by negatively regulating the concentration of CP110 levels. Our data suggest that ODF2 most likely acts as a scaffold for the binding of proteins such as NEURL4 or HYLS1 to mediate CP110 degradation. Full article

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24 pages, 2552 KiB

Open AccessReview

Macrophage Implication in IPF: Updates on Immune, Epigenetic, and Metabolic Pathways

by Deepak Pokhreal, Bruno Crestani and Doumet Georges Helou

Cells 2023, 12(17), 2193; https://doi.org/10.3390/cells12172193 - 1 Sep 2023

Cited by 7 | Viewed by 4352

Abstract

Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease of unknown etiology with a poor prognosis. It is a chronic and progressive disease that has a distinct radiological and pathological pattern from common interstitial pneumonia. The use of immunosuppressive medication was shown [...] Read more.

Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease of unknown etiology with a poor prognosis. It is a chronic and progressive disease that has a distinct radiological and pathological pattern from common interstitial pneumonia. The use of immunosuppressive medication was shown to be completely ineffective in clinical trials, resulting in years of neglect of the immune component. However, recent developments in fundamental and translational science demonstrate that immune cells play a significant regulatory role in IPF, and macrophages appear to be among the most crucial. These highly plastic cells generate multiple growth factors and mediators that highly affect the initiation and progression of IPF. In this review, we will provide an update on the role of macrophages in IPF through a systemic discussion of various regulatory mechanisms involving immune receptors, cytokines, metabolism, and epigenetics. Full article

(This article belongs to the Special Issue Idiopathic Pulmonary Fibrosis: Pathogenic Pathways and Treatment Strategies)

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The pathogenic potential of pro-fibrotic macrophages that participate in the onset of pulmonary fibrosis. Repetitive epithelial cell injury caused by a variety of risk factors results in dysregulated epithelial function. AECs release coagulation factors and inflammatory mediators, leading to the activation/recruitment of various immune cells to the site of microinjury. Among these cells, pulmonary macrophages release profibrotic mediators such as TGF-β, IL-1β, PDGF, and CCL18, which lead to the activation of fibroblasts and their differentiation into myofibroblasts to heal the wounded area in the lung interstitium. Defective repair mechanisms and macrophage alternation cause excessive ECM production. Gaseous exchange is significantly decreased due to uncontrolled scarification, which eventually ends up causing respiratory distress. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 27 August 2023). Full article ">Figure 2
Macrophage polarization. Classically activated macrophages (M1) are generally considered to be pro-inflammatory/anti-fibrotic, and alternatively activated macrophages (M2) are generally anti-inflammatory/pro-fibrotic. M2 macrophages are further classified as M2a, M2b, M2c, and M2d subtypes. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 27 August 2023). Full article ">Figure 3
Schematic representation of macrophage-related mechanisms in IPF. Injurious and pro-fibrotic factors are shown in red. Protective /anti-fibrotic factors are shown in green. (A) Surface receptor-dependent mechanisms—Above-shown receptors were found to be upregulated in macrophages and implicated in lung fibrosis. (B) Metabolism-related mechanisms—Illustrating various fibrosis-related metabolites and metabolic regulators in macrophages. (C) Transcriptional and epigenetic mechanisms—Illustrating various transcriptional regulators and epigenetic modifications related to macrophages in lung fibrosis. (D) Subsets and macrophage-derived cytokines—Illustrating different cytokines and distinct subsets of lung macrophages in lung fibrosis. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 27 August 2023). Full article ">

25 pages, 1413 KiB

Open AccessReview

Systemic Inflammatory Disorders, Immunosuppressive Treatment and Increase Risk of Head and Neck Cancers—A Narrative Review of Potential Physiopathological and Biological Mechanisms

by Nuno Vale, Mariana Pereira and Rui Amaral Mendes

Cells 2023, 12(17), 2192; https://doi.org/10.3390/cells12172192 - 1 Sep 2023

Cited by 1 | Viewed by 1612

Abstract

Head and neck cancers (HNCs) are known to present multiple factors likely to influence their development. This review aims to provide a comprehensive overview of the current scientific literature on the interplay between systemic inflammatory disorders, immunosuppressive treatments and their synergistic effect on [...] Read more.

Head and neck cancers (HNCs) are known to present multiple factors likely to influence their development. This review aims to provide a comprehensive overview of the current scientific literature on the interplay between systemic inflammatory disorders, immunosuppressive treatments and their synergistic effect on HNC risk. Both cell-mediated and humoral-mediated systemic inflammatory disorders involve dysregulated immune responses and chronic inflammation and these inflammatory conditions have been associated with an increased risk of HNC development, primarily in the head and neck region. Likewise, the interaction between systemic inflammatory disorders and immunosuppressive treatments appears to amplify the risk of HNC development, as chronic inflammation fosters a tumor-promoting microenvironment, while immunosuppressive therapies further compromise immune surveillance and anti-tumor immune responses. Understanding the molecular and cellular mechanisms underlying this interaction is crucial for developing targeted prevention strategies and therapeutic interventions. Additionally, the emerging field of immunotherapy provides potential avenues for managing HNCs associated with systemic inflammatory disorders, but further research is needed to determine its efficacy and safety in this specific context. Future studies are warranted to elucidate the underlying mechanisms and optimize preventive strategies and therapeutic interventions. Full article

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26 pages, 6636 KiB

Open AccessArticle

Changes in the Proteome of Platelets from Patients with Critical Progression of COVID-19

by Monika Wolny, Svitlana Rozanova, Cornelius Knabbe, Kathy Pfeiffer, Katalin Barkovits, Katrin Marcus and Ingvild Birschmann

Cells 2023, 12(17), 2191; https://doi.org/10.3390/cells12172191 - 1 Sep 2023

Cited by 2 | Viewed by 1552

Abstract

Platelets, the smallest cells in human blood, known for their role in primary hemostasis, are also able to interact with pathogens and play a crucial role in the immune response. In severe coronavirus disease 2019 (COVID-19) cases, platelets become overactivated, resulting in the [...] Read more.

Platelets, the smallest cells in human blood, known for their role in primary hemostasis, are also able to interact with pathogens and play a crucial role in the immune response. In severe coronavirus disease 2019 (COVID-19) cases, platelets become overactivated, resulting in the release of granules, exacerbating inflammation and contributing to the cytokine storm. This study aims to further elucidate the role of platelets in COVID-19 progression and to identify predictive biomarkers for disease outcomes. A comparative proteome analysis of highly purified platelets from critically diseased COVID-19 patients with different outcomes (survivors and non-survivors) and age- and sex-matched controls was performed. Platelets from critically diseased COVID-19 patients exhibited significant changes in the levels of proteins associated with protein folding. In addition, a number of proteins with isomerase activity were found to be more highly abundant in patient samples, apparently exerting an influence on platelet activity via the non-genomic properties of the glucocorticoid receptor (GR) and the nuclear factor κ-light-chain-enhancer of activated B cells (NFκB). Moreover, carbonic anhydrase 1 (CA-1) was found to be a candidate biomarker in platelets, showing a significant increase in COVID-19 patients. Full article

(This article belongs to the Special Issue Platelet Biology and Functions)

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17 pages, 6144 KiB

Open AccessArticle

Attenuation of Oxidative Damage via Upregulating Nrf2/HO-1 Signaling Pathway by Protease SH21 with Exerting Anti-Inflammatory and Anticancer Properties In Vitro

by Hasan Tarek, Seung Sik Cho, Md. Selim Hossain and Jin Cheol Yoo

Cells 2023, 12(17), 2190; https://doi.org/10.3390/cells12172190 - 1 Sep 2023

Cited by 4 | Viewed by 1314

Abstract

Oxidative damage and inflammation are among the very significant aspects interrelated with cancer and other degenerative diseases. In this study, we investigated the biological activities of a 25 kDa protease (SH21) that was purified from Bacillus siamensis. SH21 exhibited very powerful antioxidant [...] Read more.

Oxidative damage and inflammation are among the very significant aspects interrelated with cancer and other degenerative diseases. In this study, we investigated the biological activities of a 25 kDa protease (SH21) that was purified from Bacillus siamensis. SH21 exhibited very powerful antioxidant and reactive oxygen species (ROS) generation inhibition activity in a dose-dependent approach. The mRNA and protein levels of antioxidant enzymes such as superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione peroxidase 1 (GPx-1) were enhanced in the SH21-treated sample. SH21 also increased the transcriptional and translational activities of NF-E2-related factor 2 (Nrf2) with the subsequent development of detoxifying enzyme heme oxygenase-1 (HO-1). In addition, SH21 showed potential anti-inflammatory activity via inhibition of nitric oxide (NO) and proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β, production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. At concentrations of 60, 80, and 100 μg/mL, SH21 potentially suppressed nitric oxide synthase (iNOS) and cytokine gene expressions. Furthermore, SH21 significantly released lactate dehydrogenase (LDH) enzyme in cancer cell supernatant in a concentration-dependent manner and showed strong activity against three tested cancer cell lines, including HL-60, A549, and Hela. Our results suggest that SH21 has effective antioxidant, anti-inflammatory, and anticancer effects and could be an excellent therapeutic agent against inflammation-related diseases. Full article

(This article belongs to the Special Issue Reactive Oxygen Species and Oxidative Stress in Cellular Homeostasis and Pathophysiology)

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26 pages, 1146 KiB

Open AccessReview

Current Advances in Cellular Approaches for Pathophysiology and Treatment of Polycystic Ovary Syndrome

by Yi-Ru Tsai, Yen-Nung Liao and Hong-Yo Kang

Cells 2023, 12(17), 2189; https://doi.org/10.3390/cells12172189 - 31 Aug 2023

Cited by 2 | Viewed by 3605

Abstract

Polycystic ovary syndrome (PCOS) is a prevalent gynecological and endocrine disorder that results in irregular menstruation, incomplete follicular development, disrupted ovulation, and reduced fertility rates among affected women of reproductive age. While these symptoms can be managed through appropriate medication and lifestyle interventions, [...] Read more.

Polycystic ovary syndrome (PCOS) is a prevalent gynecological and endocrine disorder that results in irregular menstruation, incomplete follicular development, disrupted ovulation, and reduced fertility rates among affected women of reproductive age. While these symptoms can be managed through appropriate medication and lifestyle interventions, both etiology and treatment options remain limited. Here we provide a comprehensive overview of the latest advancements in cellular approaches utilized for investigating the pathophysiology of PCOS through in vitro cell models, to avoid the confounding systemic effects such as in vitro fertilization (IVF) therapy. The primary objective is to enhance the understanding of abnormalities in PCOS-associated folliculogenesis, particularly focusing on the aberrant roles of granulosa cells and other relevant cell types. Furthermore, this article encompasses analyses of the mechanisms and signaling pathways, microRNA expression and target genes altered in PCOS, and explores the pharmacological approaches considered as potential treatments. By summarizing the aforementioned key findings, this article not only allows us to appreciate the value of using in vitro cell models, but also provides guidance for selecting suitable research models to facilitate the identification of potential treatments and understand the pathophysiology of PCOS at the cellular level. Full article

(This article belongs to the Special Issue Recent Advances in Mammalian Reproductive Biology—a Focus on Development of Gametes and Embryos)

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13 pages, 3756 KiB

Open AccessArticle

A Protocol for Organoids from the Urine of Bladder Cancer Patients

by Simon Walz, Paul Pollehne, Ruizhi Geng, Johannes Schneider, Moritz Maas, Wilhelm K. Aicher, Arnulf Stenzl, Bastian Amend and Niklas Harland

Cells 2023, 12(17), 2188; https://doi.org/10.3390/cells12172188 - 31 Aug 2023

Cited by 4 | Viewed by 1792

Abstract

This study investigates the feasibility of establishing urine-derived tumor organoids from bladder cancer (BC) patients as an alternative to tissue-derived organoids. BC is one of the most common cancers worldwide and current diagnostic methods involve invasive procedures. Here, we investigated the potential of [...] Read more.

This study investigates the feasibility of establishing urine-derived tumor organoids from bladder cancer (BC) patients as an alternative to tissue-derived organoids. BC is one of the most common cancers worldwide and current diagnostic methods involve invasive procedures. Here, we investigated the potential of using urine samples, which contain exfoliated tumor cells, to generate urine-derived BC organoids (uBCOs). Urine samples from 29 BC patients were collected and cells were isolated and cultured in a three-dimensional matrix. The establishment and primary expansion of uBCOs were successful in 83% of the specimens investigated. The culturing efficiency of uBCOs was comparable to cancer tissue-derived organoids. Immunohistochemistry and immunofluorescence to characterize the uBCOs exhibited similar expressions of BC markers compared to the parental tumor. These findings suggest that urine-derived BC organoids hold promise as a non-invasive tool for studying BC and evaluating therapeutic responses. This approach could potentially minimize the need for invasive procedures and provide a platform for personalized drug screening. Further research in this area may lead to improved diagnostic and treatment strategies for BC patients. Full article

(This article belongs to the Collection Advances in 3D Cell Culture)

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Figure 1
Method and workflow of the culturing process of uBCOs. The materials and methods section contains a comprehensive and detailed step-by-step workflow, along with the materials and reagents used in the study. Abbreviations: BC, bladder cancer; uBCO, urine-derived bladder cancer organoid. Created with biorender.com (accessed 12 May 2023). Full article ">Figure 2
Culturing efficiency of uBCOs (indicated in black) and tBCOs (indicated in grey hatched) (A) with respect to the individual passages (efficiency rate). Culturing efficiency according to patient characteristics and histopathological features with regard to the maximum achieved passage (B–I). Dots represent data from individual organoids. Boxes indicate median and 25th and 75th percentiles with min/max whiskers. p-values indicate differences between two groups (A: two-sided Fischer exact test; B: Mann–Whitney-U test). The ρ-values indicate linear correlations between two parameters (Spearman correlation). Abbreviations: uBCO, urine-derived bladder cancer organoid; tBCO, tissue-derived bladder cancer organoid; NMIBC, non-muscle- invasive bladder cancer; MIBC, muscle-invasive bladder cancer. Full article ">Figure 3
Light microscopic images of uBCO #027 during cultivation in basal membrane extract in the upper row on the first, fourth, and thirteenth day of the first passage, in the middle and lower row on the first and seventh day of the third and fourth passage, respectively. Size bars indicate 100 mm. Abbreviations: uBCO, urine-derived bladder cancer organoid; P, passage; D, day. Full article ">Figure 4
Immunohistochemical images from parental primary BC (left column) and autologous, uBCO #027 (middle column) in the fifth passage. The left column displays fluorescence staining of relevant antigens in urothelial cancer or tumor stem cell antigens in the corresponding uBCO #027 (passage 5). The tBCO only reached the first passage and could, therefore, not be implemented in the analysis. Abbreviations: BC, bladder cancer; uBCO, urine-derived bladder cancer organoid; tBCO, tissue-derived bladder cancer organoid HE, hematoxylin-eosin; GATA, glutamyl amino tranverase A; CK, cytoceratine; p, protein; CD, cluster of differentiation. Full article ">

16 pages, 951 KiB

Open AccessReview

MT1-MMP as a Key Regulator of Metastasis

by Noritaka Tanaka and Takeharu Sakamoto

Cells 2023, 12(17), 2187; https://doi.org/10.3390/cells12172187 - 31 Aug 2023

Cited by 9 | Viewed by 2356

Abstract

Membrane type1-matrix metalloproteinase (MT1-MMP) is a member of metalloproteinases that is tethered to the transmembrane. Its major function in cancer progression is to directly degrade the extracellular matrix components, which are mainly type I–III collagen or indirectly type IV collagen through the activation [...] Read more.

Membrane type1-matrix metalloproteinase (MT1-MMP) is a member of metalloproteinases that is tethered to the transmembrane. Its major function in cancer progression is to directly degrade the extracellular matrix components, which are mainly type I–III collagen or indirectly type IV collagen through the activation of MMP-2 with a cooperative function of the tissue inhibitor of metalloproteinase-2 (TIMP-2). MT1-MMP is expressed as an inactive form (zymogen) within the endoplasmic reticulum (ER) and receives truncation processing via furin for its activation. Upon the appropriate trafficking of MT1-MMP from the ER, the Golgi apparatus to the cell surface membrane, MT1-MMP exhibits proteolytic activities to the surrounding molecules such as extracellular matrix components and cell surface molecules. MT1-MMP also retains a non-proteolytic ability to activate hypoxia-inducible factor 1 alpha (HIF-1A) via factors inhibiting the HIF-1 (FIH-1)-Mint3-HIF-1 axis, resulting in the upregulation of glucose metabolism and oxygen-independent ATP production. Through various functions of MT1-MMP, cancer cells gain motility on migration/invasion, thus causing metastasis. Despite the long-time efforts spent on the development of MT1-MMP interventions, none have been accomplished yet due to the side effects caused by off-target effects. Recently, MT1-MMP-specific small molecule inhibitors or an antibody have been reported and these inhibitors could potentially be novel agents for cancer treatment. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms of Cancer Invasion and Metastasis)

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5 pages, 785 KiB

Open AccessComment

βIV-Spectrin in Cardiac Fibroblasts: Implications for Fibrosis and Therapeutic Targeting in Cardiac Diseases. Comment on Nassal et al. Spectrin-Based Regulation of Cardiac Fibroblast Cell-Cell Communication. Cells 2023, 12, 748

by Wenjing Xiang, Ning Zhou, Lei Li, Faming Chen, Lei Li and Ying Wang

Cells 2023, 12(17), 2186; https://doi.org/10.3390/cells12172186 - 31 Aug 2023

Viewed by 872

Abstract

Fibroblasts in the heart, traditionally recognized as interstitial cells, have long been overlooked in the study of cardiac physiology and pathology [...] Full article

(This article belongs to the Special Issue Research Advances Related to Cardiovascular System)

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12 pages, 1894 KiB

Open AccessArticle

Aquaglyceroporins in Human Breast Cancer

by Teresa Kirkegaard, Andreas Riishede, Trine Tramm and Lene N. Nejsum

Cells 2023, 12(17), 2185; https://doi.org/10.3390/cells12172185 - 31 Aug 2023

Cited by 3 | Viewed by 1465

Abstract

Aquaporins are water channels that facilitate passive water transport across cellular membranes following an osmotic gradient and are essential in the regulation of body water homeostasis. Several aquaporins are overexpressed in breast cancer, and AQP1, AQP3 and AQP5 have been linked to spread [...] Read more.

Aquaporins are water channels that facilitate passive water transport across cellular membranes following an osmotic gradient and are essential in the regulation of body water homeostasis. Several aquaporins are overexpressed in breast cancer, and AQP1, AQP3 and AQP5 have been linked to spread to lymph nodes and poor prognosis. The subgroup aquaglyceroporins also facilitate the transport of glycerol and are thus involved in cellular metabolism. Transcriptomic analysis revealed that the three aquaglyceroporins, AQP3, AQP7 and AQP9, but not AQP10, are overexpressed in human breast cancer. It is, however, unknown if they are all expressed in the same cells or have a heterogeneous expression pattern. To investigate this, we employed immunohistochemical analysis of serial sections from human invasive ductal and lobular breast cancers. We found that AQP3, AQP7 and AQP9 are homogeneously expressed in almost all cells in both premalignant in situ lesions and invasive lesions. Thus, potential intervention strategies targeting cellular metabolism via the aquaglyceroporins should consider all three expressed aquaglyceroporins, namely AQP3, AQP7 and AQP9. Full article

(This article belongs to the Special Issue Advances in Aquaporins II)

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Graphical illustration of workflow. Formalin-fixed, paraffin-embedded samples from invasive female breast cancer tumors were obtained in fully anonymized form from surgical specimens from the Department of Pathology, Aarhus University Hospital, Denmark. Following retrieval, the anonymized tissue samples were sectioned. For diagnostic assessment, hematoxylin and eosin (H&E) stainings, as well as immunohistochemical stainings for E-cadherin, were performed. Samples representing invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) were further analyzed. Serial sections were processed for immunohistochemical stainings in the following order: #1 AQP7, #2 AQP9, #3 AQP3 and #4 SMMS-1. The SMMS-1 staining identified myoepithelial cells, allowing a distinction between in situ and invasion lesions. Images of all samples were captured by brightfield light microscopy, and select slides for publication figures were scanned on a NanoZoomer 2.0HT (Hamamatsu) with the 40× objective. Part of this figure was generated in BioRender.com; accessed on August 11th 2023. Full article ">Figure 2
AQP7, AQP9 and AQP3 localize to epithelial cells of lobules and extralobular ducts in benign regions adjacent to tumor tissue. Formalin-fixed, paraffin-embedded samples from invasive female breast cancer tumors were obtained in fully anonymized form from surgical specimens and processed for immunohistochemistry with antibodies against AQP7, AQP9, AQP3 and SMMS-1. The figure depicts representative images from immunohistochemical stainings of serial sections from the normal/benign part of samples from invasive lobular carcinoma. (A) Lobule and (B) extralobular duct. Immune cells in the surrounding connective tissue also stain positive (red asterisks in B). Arrowheads in B point to myoepithelial cells, and the arrows in B point to the apical region of extralobular ducts. Slides were scanned in a NanoZoomer 2.0HT (Hamamatsu) using the 40× objective. White balance was adjusted in Adobe Photoshop CS4. Scale bars are 100 µm. Full article ">Figure 3
AQP7, AQP9 and AQP3 localize to neoplastic cells of lobular carcinoma in situ and invasive lobular carcinoma. Formalin-fixed, paraffin-embedded samples from invasive female breast cancer tumors were obtained in fully anonymized form from surgical specimens and processed for immunohistochemistry with antibodies against AQP7, AQP9, AQP3 and SMMS-1. The figure depicts representative images from immunohistochemical stainings of serial sections from patients with invasive lobular carcinoma (ILC) and enlarged images (inserts) of the marked areas. (A) Images are from a region of lobular carcinoma in situ (LCIS) from an ILC patient, and (B) is ILC. Slides were scanned in a NanoZoomer 2.0HT (Hamamatsu) using the 40× objective. White balance was adjusted in Adobe Photoshop CS4. Scale bars are 100 µm (A,B) and 20 µm ((A,B), inserts). Full article ">Figure 4
AQP7, AQP9 and AQP3 localize to neoplastic cells of ductal carcinoma in situ and invasive ductal carcinoma. Formalin-fixed, paraffin-embedded samples from invasive female breast cancer tumors were obtained in fully anonymized form from surgical specimens and processed for immunohistochemistry with antibodies against AQP7, AQP9, AQP3 and SMMS-1. The figure depicts representative images from immunohistochemical stainings of serial sections from patients with invasive ductal carcinoma (IDC) and enlarged images (inserts) of the marked areas. (A) Images are from a region of ductal carcinoma in situ (DCIS) from an IDC patient, and (B) is IDC. Slides were scanned in a NanoZoomer 2.0HT (Hamamatsu) using the 40x objective. White balance was adjusted in Adobe Photoshop CS4. Scale bars are 175 µm (A,B) and 50 µm ((A,B), inserts). Full article ">

16 pages, 6947 KiB

Open AccessArticle

miR-369-3p Modulates Intestinal Inflammatory Response via BRCC3/NLRP3 Inflammasome Axis

by Viviana Scalavino, Emanuele Piccinno, Anna Maria Valentini, Nicolò Schena, Raffaele Armentano, Gianluigi Giannelli and Grazia Serino

Cells 2023, 12(17), 2184; https://doi.org/10.3390/cells12172184 - 31 Aug 2023

Cited by 7 | Viewed by 1399

Abstract

Inflammasomes are multiprotein complexes expressed by immune cells in response to distinct stimuli that trigger inflammatory responses and the release of pro-inflammatory cytokines. Evidence suggests a different role of inflammasome NLRP3 in IBD. NLRP3 inflammasome activation can be controlled by post-translational modifications such [...] Read more.

Inflammasomes are multiprotein complexes expressed by immune cells in response to distinct stimuli that trigger inflammatory responses and the release of pro-inflammatory cytokines. Evidence suggests a different role of inflammasome NLRP3 in IBD. NLRP3 inflammasome activation can be controlled by post-translational modifications such as ubiquitination through BRCC3. The aim of this study was to investigate the effect of miR-369-3p on the expression and activation of NLRP3 inflammasomes via BRCC3 regulation. After bioinformatics prediction of Brcc3 as a gene target of miR-369-3p, in vitro, we validated its modulation in bone marrow-derived macrophages (BMDM). The increase in miR-369-3p significantly reduced BRCC3 gene and protein expression. This modulation, in turn, reduced the expression of NLRP3 and blocked the recruitment of ASC adaptor protein by NLRP3. As a result, miR-369-3p reduced the activity of Caspase-1 by the inflammasome, decreasing the cleavage of pro-IL-1β and pro-IL-18. These results support a novel mechanism that seems to act on post-translational modification of NLRP3 inflammasome activation by BRCC3. This may be an interesting new target in the personalized treatment of inflammatory disorders, including IBD. Full article

(This article belongs to the Special Issue Non-coding RNA Regulation of Stem Cell Regenerative Mechanisms: Advances, Challenges, and Perspectives)

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miR-369-3p targets Brrc3. (A) Sequence alignments of miR-369-3p and binding sites in 3′ UTR of Brcc3 mRNA. (B) The mRNA expression levels of Brcc3 evaluated by qRT-PCR in BMDM cells transfected with 30 nM and 50 nM of miR-369-3p mimic both in the basal condition and after inflammasome activation. (C) Western blot analysis of BRCC3 protein expression after miR-369-3p mimic transfection in unstimulated BMDM cells as well as following inflammasome activation of the BMDM cell line. To normalize data, GAPDH was used as housekeeping protein. Data are representative of four independent experiments. Raw data of the independent experiments of Western blot were reported in <a href="#app1-cells-12-02184" class="html-app">Supplementary File S1</a>. The histograms correspond to mean ± SEM. (* p < 0.05; ** p < 0.01; *** p < 0.001). Full article ">Figure 2
miR-369-3p regulates Nlrp3 mRNA and protein expression. (A) The mRNA expression levels of Nlrp3 evaluated by qRT-PCR in BMDM cells transfected with 30 nM and 50 nM of miR-369-3p mimic both in the basal condition and after inflammasome-activating stimulations. (B) Western blot analysis of NLRP3 protein expression after miR-369-3p mimic transfection in unstimulated BMDM cells as well as following inflammasome activation in the BMDM cell line. To normalize data, GAPDH was used as housekeeping protein. Data are representative of four independent experiments. Raw data of the independent experiments of Western blot were reported in <a href="#app1-cells-12-02184" class="html-app">Supplementary File S1</a>. The histograms correspond to mean ± SEM. (* p < 0.05; ** p < 0.01). Full article ">Figure 3
Immunofluorescence staining of ASC in BMDM cell cultures after miR-369-3p mimic transfection. Representative images of BMDM transfected with miR-369-3p mimic and stimulated with LPS 1 μg/mL for 4 h then Nigericin 20 μM for 30 min. Mock condition and transfected conditions were acquired by fluorescence microscopy. Red spots represent the expression of ASC, while DAPI (blue) correspond to cells’ nuclei. White arrows point to ASC expression. Original magnification, ×20. Scale bar presents 50 μm. Full article ">Figure 4
miR-369-3p modulated the deubiquitination of NLRP3 by BRCC3. (A) Endogenous NLRP3 immunoprecipitates were analyzed for ubiquitination. miR-369-3p modulated the deubiquitination of NLRP3 by BRCC3 after miR-369-3p transient transfection and LPS and Nigericin stimulation. (B) Expression of NLRP3 and BRCC3 in BMDM lysates after miR-369-3p transient transfection and LPS and Nigericin stimulation (input samples). (C) Histograms of NLRP3 and BRCC3 expression from lysates of BMDM treated with miR-369-3p mimic and stimulated with LPS and Nigericin. To normalize data, GAPDH was used as housekeeping protein. Raw data of the independent experiments of Western blot were reported in <a href="#app1-cells-12-02184" class="html-app">Supplementary File S1</a>. The histograms correspond to mean ± SEM. Data are representative of four independent experiments. (* p < 0.05; ** p < 0.01; *** p < 0.001). Full article ">Figure 5
miR-369-3p reduced the activation and release of caspase-1. (A) Western blot analysis of pro-CASP1 protein expression after miR-369-3p mimic transfection following LPS and Nigericin stimulations in the BMDM cell line. To normalize data, GAPDH was used as housekeeping protein. Raw data of the independent experiments of Western blot were reported in <a href="#app1-cells-12-02184" class="html-app">Supplementary File S1</a>. (B) Caspase-1 activity after LPS and Nigericin stimulations in the mimic transfected BMDM cell line. (C) In vitro cell viability assay of BMDM cells transiently transfected with miR-369-3p mimic and stimulated with LPS and Nigericin. (* p < 0.05; ** p < 0.01; *** p < 0.0001). Full article ">Figure 6
miR-369-3p modulated the release of pro-inflammatory cytokines, NLRP3 inflammasome-related. miR-369-3p induction in BMDM after mimic transfection led to a significant decrease in IL-1β (A), IL-18 (B) production in response to LPS and LPS and Nigericin stimulation. Data are representative of four independent experiments. (* p < 0.05; ** p < 0.01). Full article ">Figure 7
BRCC3 and NLRP3 and ASC expression in UC patients. (A) BRCC3, NLRP3, and ASC protein expression in formalin-fixed, paraffin-embedded tissues obtained from healthy controls and ulcerative colitis (UC) patients. Original magnification, ×4. (B) Inflammatory score representing the expression levels of BRCC3, NLRP3, and ASC proteins in the immune infiltrate (* p < 0.01; *** p < 0.001). Full article ">

38 pages, 821 KiB

Open AccessReview

Mitochondrial Properties in Skeletal Muscle Fiber

by Han Dong and Shih-Yin Tsai

Cells 2023, 12(17), 2183; https://doi.org/10.3390/cells12172183 - 30 Aug 2023

Cited by 15 | Viewed by 10287

Abstract

Mitochondria are the primary source of energy production and are implicated in a wide range of biological processes in most eukaryotic cells. Skeletal muscle heavily relies on mitochondria for energy supplements. In addition to being a powerhouse, mitochondria evoke many functions in skeletal [...] Read more.

Mitochondria are the primary source of energy production and are implicated in a wide range of biological processes in most eukaryotic cells. Skeletal muscle heavily relies on mitochondria for energy supplements. In addition to being a powerhouse, mitochondria evoke many functions in skeletal muscle, including regulating calcium and reactive oxygen species levels. A healthy mitochondria population is necessary for the preservation of skeletal muscle homeostasis, while mitochondria dysregulation is linked to numerous myopathies. In this review, we summarize the recent studies on mitochondria function and quality control in skeletal muscle, focusing mainly on in vivo studies of rodents and human subjects. With an emphasis on the interplay between mitochondrial functions concerning the muscle fiber type-specific phenotypes, we also discuss the effect of aging and exercise on the remodeling of skeletal muscle and mitochondria properties. Full article

(This article belongs to the Special Issue Mitochondria: New Findings from Single Cells to Organs)

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13 pages, 634 KiB

Open AccessPerspective

Metformin: A New Inhibitor of the Wnt Signaling Pathway in Cancer

by Domenico Conza, Paola Mirra, Francesca Fiory, Luigi Insabato, Antonella Nicolò, Francesco Beguinot and Luca Ulianich

Cells 2023, 12(17), 2182; https://doi.org/10.3390/cells12172182 - 30 Aug 2023

Cited by 3 | Viewed by 1976

Abstract

The biguanide drug metformin is widely used in type 2 diabetes mellitus therapy, due to its ability to decrease serum glucose levels, mainly by reducing hepatic gluconeogenesis and glycogenolysis. A considerable number of studies have shown that metformin, besides its antidiabetic action, can [...] Read more.

The biguanide drug metformin is widely used in type 2 diabetes mellitus therapy, due to its ability to decrease serum glucose levels, mainly by reducing hepatic gluconeogenesis and glycogenolysis. A considerable number of studies have shown that metformin, besides its antidiabetic action, can improve other disease states, such as polycystic ovary disease, acute kidney injury, neurological disorders, cognitive impairment and renal damage. In addition, metformin is well known to suppress the growth and progression of different types of cancer cells both in vitro and in vivo. Accordingly, several epidemiological studies suggest that metformin is capable of lowering cancer risk and reducing the rate of cancer deaths among diabetic patients. The antitumoral effects of metformin have been proposed to be mainly mediated by the activation of the AMP-activated protein kinase (AMPK). However, a number of signaling pathways, both dependent and independent of AMPK activation, have been reported to be involved in metformin antitumoral action. Among these, the Wingless and Int signaling pathway have recently been included. Here, we will focus our attention on the main molecular mechanisms involved. Full article

(This article belongs to the Special Issue From Mechanisms to Therapeutics: Wnt Signaling in Cancer)

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21 pages, 1551 KiB

Open AccessReview

New Views of the DNA Repair Protein Ataxia–Telangiectasia Mutated in Central Neurons: Contribution in Synaptic Dysfunctions of Neurodevelopmental and Neurodegenerative Diseases

by Sabrina Briguglio, Clara Cambria, Elena Albizzati, Elena Marcello, Giovanni Provenzano, Angelisa Frasca and Flavia Antonucci

Cells 2023, 12(17), 2181; https://doi.org/10.3390/cells12172181 - 30 Aug 2023

Cited by 2 | Viewed by 2273

Abstract

Ataxia–Telangiectasia Mutated (ATM) is a serine/threonine protein kinase principally known to orchestrate DNA repair processes upon DNA double-strand breaks (DSBs). Mutations in the Atm gene lead to Ataxia–Telangiectasia (AT), a recessive disorder characterized by ataxic movements consequent to cerebellar atrophy or dysfunction, along [...] Read more.

Ataxia–Telangiectasia Mutated (ATM) is a serine/threonine protein kinase principally known to orchestrate DNA repair processes upon DNA double-strand breaks (DSBs). Mutations in the Atm gene lead to Ataxia–Telangiectasia (AT), a recessive disorder characterized by ataxic movements consequent to cerebellar atrophy or dysfunction, along with immune alterations, genomic instability, and predisposition to cancer. AT patients show variable phenotypes ranging from neurologic abnormalities and cognitive impairments to more recently described neuropsychiatric features pointing to symptoms hardly ascribable to the canonical functions of ATM in DNA damage response (DDR). Indeed, evidence suggests that cognitive abilities rely on the proper functioning of DSB machinery and specific synaptic changes in central neurons of ATM-deficient mice unveiled unexpected roles of ATM at the synapse. Thus, in the present review, upon a brief recall of DNA damage responses, we focus our attention on the role of ATM in neuronal physiology and pathology and we discuss recent findings showing structural and functional changes in hippocampal and cortical synapses of AT mouse models. Collectively, a deeper knowledge of ATM-dependent mechanisms in neurons is necessary not only for a better comprehension of AT neurological phenotypes, but also for a higher understanding of the pathological mechanisms in neurodevelopmental and degenerative disorders involving ATM dysfunctions. Full article

(This article belongs to the Topic Animal Models of Human Disease)

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25 pages, 16017 KiB

Open AccessSystematic Review

Arginine, Transsulfuration, and Folic Acid Pathway Metabolomics in Chronic Obstructive Pulmonary Disease: A Systematic Review and Meta-Analysis

by Angelo Zinellu and Arduino A. Mangoni

Cells 2023, 12(17), 2180; https://doi.org/10.3390/cells12172180 - 30 Aug 2023

Cited by 4 | Viewed by 1895

Abstract

There is an increasing interest in biomarkers of nitric oxide dysregulation and oxidative stress to guide management and identify new therapeutic targets in patients with chronic obstructive pulmonary disease (COPD). We conducted a systematic review and meta-analysis of the association between circulating metabolites [...] Read more.

There is an increasing interest in biomarkers of nitric oxide dysregulation and oxidative stress to guide management and identify new therapeutic targets in patients with chronic obstructive pulmonary disease (COPD). We conducted a systematic review and meta-analysis of the association between circulating metabolites within the arginine (arginine, citrulline, ornithine, asymmetric, ADMA, and symmetric, SDMA dimethylarginine), transsulfuration (methionine, homocysteine, and cysteine) and folic acid (folic acid, vitamin B₆, and vitamin B₁₂) metabolic pathways and COPD. We searched electronic databases from inception to 30 June 2023 and assessed the risk of bias and the certainty of evidence. In 21 eligible studies, compared to healthy controls, patients with stable COPD had significantly lower methionine (standardized mean difference, SMD = −0.50, 95% CI −0.95 to −0.05, p = 0.029) and folic acid (SMD = −0.37, 95% CI −0.65 to −0.09, p = 0.009), and higher homocysteine (SMD = 0.78, 95% CI 0.48 to 1.07, p < 0.001) and cysteine concentrations (SMD = 0.34, 95% CI 0.02 to 0.66, p = 0.038). Additionally, COPD was associated with significantly higher ADMA (SMD = 1.27, 95% CI 0.08 to 2.46, p = 0.037), SDMA (SMD = 3.94, 95% CI 0.79 to 7.08, p = 0.014), and ornithine concentrations (SMD = 0.67, 95% CI 0.13 to 1.22, p = 0.015). In subgroup analysis, the SMD of homocysteine was significantly associated with the biological matrix assessed and the forced expiratory volume in the first second to forced vital capacity ratio, but not with age, study location, or analytical method used. Our study suggests that the presence of significant alterations in metabolites within the arginine, transsulfuration, and folic acid pathways can be useful for assessing nitric oxide dysregulation and oxidative stress and identifying novel treatment targets in COPD. (PROSPERO registration number: CRD42023448036.) Full article

(This article belongs to the Special Issue Metabolomics and Proteomics in Chronic Obstructive Pulmonary Disease (COPD))

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13 pages, 3147 KiB

Open AccessArticle

Comparative Shotgun Proteomics Reveals the Characteristic Protein Signature of Osteosarcoma Subtypes

by Maram Alaa, Nouran Al-Shehaby, Ali Mostafa Anwar, Nesma Farid, Mustafa Shaban Shawky, Manal Zamzam, Iman Zaky, Ahmed Elghounimy, Shahenda El-Naggar and Sameh Magdeldin

Cells 2023, 12(17), 2179; https://doi.org/10.3390/cells12172179 - 30 Aug 2023

Cited by 3 | Viewed by 1750

Abstract

Osteosarcoma is a primary malignant bone tumor affecting adolescents and young adults. This study aimed to identify proteomic signatures that distinguish between different osteosarcoma subtypes, providing insights into their molecular heterogeneity and potential implications for personalized treatment approaches. Using advanced proteomic techniques, we [...] Read more.

Osteosarcoma is a primary malignant bone tumor affecting adolescents and young adults. This study aimed to identify proteomic signatures that distinguish between different osteosarcoma subtypes, providing insights into their molecular heterogeneity and potential implications for personalized treatment approaches. Using advanced proteomic techniques, we analyzed FFPE tumor samples from a cohort of pediatric osteosarcoma patients representing four various subtypes. Differential expression analysis revealed a significant proteomic signature that discriminated between these subtypes, highlighting distinct molecular profiles associated with different tumor characteristics. In contrast, clinical determinants did not correlate with the proteome signature of pediatric osteosarcoma. The identified proteomics signature encompassed a diverse array of proteins involved in focal adhesion, ECM-receptor interaction, PI3K-Akt signaling pathways, and proteoglycans in cancer, among the top enriched pathways. These findings underscore the importance of considering the molecular heterogeneity of osteosarcoma during diagnosis or even when developing personalized treatment strategies. By identifying subtype-specific proteomics signatures, clinicians may be able to tailor therapy regimens to individual patients, optimizing treatment efficacy and minimizing adverse effects. Full article

(This article belongs to the Topic Bone-Related Diseases: From Molecular Mechanisms to Therapy Development)

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Full article ">Figure 1
Histological response to chemotherapy and years-to-event predict the outcome of osteosarcoma patients. (A) Kaplan–Meier plot of overall (OS) and event-free survival (EFS) for 30 OS patients stratified according to histological response to chemotherapy (≥90% and <90%). (B) Kaplan–Meier plot of OS for patients stratified according to years-to-event (≤1 year and >1 year). Survival probability (y-axis) and time indicated in months (x-axis). p-values were calculated using the log-rank test. Full article ">Figure 2
Clinical features do not affect the proteome signature of osteosarcoma patients. (A) Heat map and hierarchical clustering based on the total proteome identified. (B) Volcano plots of all proteins significantly altered by the (top) histological response to chemotherapy and (bottom) years-to-event (log2-fold-change threshold = 1, Benjamini–Hochberg corrected p-value threshold = 0.1). (C) Heat map and hierarchical clustering based on the top 20 genes differentiated between groups of (top) Histological response to chemotherapy and (bottom) years-to-event. Heat map colors are based on the z-scored (log2) intensity values. Grey and red correspond to decreased and increased expression levels, respectively. Full article ">Figure 3
Proteome signatures of osteosarcoma samples segregate patients according to the pathological subtypes. (A) Venn diagram showing protein distribution among pathological subtypes. (B) Anova diagram showing differentially expressed proteins (DEPs) among pathological subtypes (p-value < 0.05). (C) Partial least squares discrimination analysis (PLS-DA) based on DEPs showing segregation of pathological subtypes (left) and PLS permutation plot showing PLS-DA significance (right). (D) Heat map and hierarchical clustering based on DEPs differentiated between pathological subtypes. Heat map colors are based on the z-scored (log2) intensity values. Grey and red correspond to decreased and increased expression levels, respectively. (E) KEGG pathways according to DEPs among pathological subtypes. (F) PPI networks and molecular functions of DEPs unique to fibroblastic osteosarcoma. (G) PPI networks and molecular functions of DEPs unique to chondroblastic osteosarcoma, respectively. Full article ">

18 pages, 4427 KiB

Open AccessArticle

The Preventive Effect of Melatonin on Radiation-Induced Oral Mucositis

by Reiko Tokuyama-Toda, Hirochika Umeki, Mitsuru Okubo, Chika Terada-Ito, Toshio Yudo, Shinji Ide, Susumu Tadokoro, Masashi Shimozuma and Kazuhito Satomura

Cells 2023, 12(17), 2178; https://doi.org/10.3390/cells12172178 - 30 Aug 2023

Cited by 2 | Viewed by 1497

Abstract

Melatonin exerts various physiological effects through melatonin receptors and their ability to scavenge free radicals. Radiotherapy is a common treatment for head and neck tumors, but stomatitis, a side effect affecting irradiated oral mucosa, can impact treatment outcomes. This study investigated the preventive [...] Read more.

Melatonin exerts various physiological effects through melatonin receptors and their ability to scavenge free radicals. Radiotherapy is a common treatment for head and neck tumors, but stomatitis, a side effect affecting irradiated oral mucosa, can impact treatment outcomes. This study investigated the preventive effect of melatonin, a potent free radical scavenger, on radiation-induced oral mucositis. Mice were irradiated with 15 Gy of X-ray radiation to the head and neck, and the oral mucosa was histologically compared between a melatonin-administered group and a control group. The results showed that radiation-induced oral mucositis was suppressed in mice administered melatonin before and after irradiation. It was suggested that the mechanism involved the inhibition of apoptosis and the inhibition of DNA damage. From these findings, we confirmed that melatonin has a protective effect against radiation-induced oral mucositis. Full article

(This article belongs to the Special Issue Cellular and Molecular Biology of Melatonin)

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Histology of the tongue in each group. (a,b) Control group (no irradiation). (c,d) IR group (vehicle intraperitoneal administration + 15 Gy irradiation + normal water feeding). (e,f) IR + Mel group (intraperitoneal administration of melatonin + 15 Gy irradiation + melatonin-containing water breeding). H–E staining. The basal layer exhibited cell enlargement, dense nuclei, an increased nucleus-to-cytoplasm ratio, and disordered arrangement. In addition, polygonal cells, degenerated cells, and keratohyalin-like granules were observed sporadically. The entire epithelium showed atrophy, dilation of capillaries in the lamina propria, and edematous space in the tongue muscle. In contrast, the tongues of mice in the IR + Mel group exhibited partial radiation damage. However, the thickness of the epithelial layer was maintained, and only slight disturbance was observed in the basal layer. Compared with the IR group, radiation damage was suppressed in the IR + Mel group, and the histological images resembled those of the nonirradiated control group. (a,c,e) Bars are 50 μm. (b,d,f) Bars are 20 μm. (g) Graph of the length of the epithelial leg in the tongue. * p < 0.05, ** p < 0.01. In the IR group, the epithelial leg flattened significantly compared with the control group, whereas in the IR + Mel group, the flattening of the epithelial leg was suppressed, and the length was maintained. Full article ">Figure 2
Histology of the buccal mucosa in each group. (a,b) Control group (no irradiation). (c,d) IR group (vehicle intraperitoneal administration + 15 Gy irradiation + normal water feeding). (e,f) IR + Mel group (intraperitoneal administration of melatonin + 15 Gy irradiation + melatonin-containing water breeding). H–E staining. As in the tongue, radiation damage was observed in both the epithelium and lamina propria in the IR group, whereas radiation damage was suppressed in the IR + Mel group. Histological images similar to those in the control group were observed. (a,c,e) Bars are 50 μm. (b,d,f) Bars are 20 μm. (g) Graph of the number of nucleated cells observed in the epithelium in the buccal mucosa. * p < 0.05, ** p < 0.01. The IR group showed a significant decrease in cell number compared to the control group. In contrast, the IR + Mel group suppressed the decrease in cell number and maintained the number of nucleated cells. Full article ">Figure 3
TUNEL staining image in the tongue. (a–c) Control group. (d–f) IR group. (g–i) IR + Mel group. (a,d,g) FITC. (b,e,h) DAPI. (c,f,i) merge. The scale bar is 50 μm. All images have the same magnification. The white dashed line represents the boundary between the epithelium and subepithelial tissue. (j) Graph of the number of TUNEL-positive cells in the epithelium, subepithelial tissue, and full thickness of the tongue mucosa. * p < 0.05, ** p < 0.01. Significantly, more positive cells were observed in the IR group than in the control group in all regions of the epithelium, subepithelial tissue, and mucosa. On the other hand, the number of positive cells in the RT + Mel group was higher than that in the control group, but it was significantly lower than that in the IR group. Full article ">Figure 4
Immunohistochemical staining image of active caspase-3 in the tongue. (a–c) Control group. (d–f) IR group. (g–i) IR + Mel group. (a,d,g) Texas red for cleaved caspase-3. (b,e,h) DAPI. (c,f,i) merge. Scale bar is 50 μm. All images have the same magnification. The white dashed line represents the boundary between the epithelium and subepithelial tissue. (j) Graph of the number of cleaved caspase-3-positive cells in the epithelium, subepithelial tissue, and full thickness of the tongue mucosa. * p < 0.05, ** p < 0.01. Significantly more positive cells were observed in the IR group than in the control group in all regions of the epithelium, subepithelial tissue, and mucosa. On the other hand, although the RT + Mel group had more positive cells than the control group, it tended to decrease compared with the IR group. Full article ">Figure 5
Immunohistochemical staining image of HMGB1 in the tongue. (a–c) Control group. (d–f) IR group. (g–i) IR + Mel group. (a,d,g) Texas red for HMGB1. (b,e,h) DAPI. (c,f,i) merge. The scale bar is 50 μm. All images have the same magnification. The white dashed line represents the boundary between the epithelium and subepithelial tissue. (j) Graph of the number of HMGB1-positive cells in the epithelium, subepithelial tissue, and full thickness of the tongue mucosa. * p < 0.05. Although there was a significant difference between the control group and IR group in the whole mucosa, there was no significant difference between the other groups. Full article ">Figure 6
Immunohistochemical staining image of 8-OHdG in the tongue. (a–c) Control group. (d–f) IR group. (g–i) IR + Mel group. (a,d,g) Texas red for 8-OHdG. (b,e,h) DAPI. (c,f,i) merge. Scale bar is 50 μm. All images have the same magnification. The white dashed line represents the boundary between the epithelium and subepithelial tissue. (j) Graph of the number of 8-OHdG-positive cells in the epithelium, subepithelial tissue, and full thickness of the tongue mucosa. * p < 0.05, ** p < 0.01. Significantly more positive cells were observed in the IR group than in the control group in all regions of the epithelium, subepithelial tissue, and mucosa. On the other hand, it was significantly decreased in the RT + Mel group compared with that in the IR group. The number of positive cells tended to be higher than in the control group, but the difference was not significant. Full article ">Figure 7
Side effects due to melatonin administration. (a) Weight gain. (b–e) Peripheral blood biochemistry test results. (b) AST. (c) ALT. (d) BUN. (e) Creatinine. Changes in body weight (A) and serum levels of AST (B), ALT (C), BUN (D), and creatinine (E) in the experimental period. Generally, none of these laboratory parameters were significantly affected by melatonin application throughout the experimental period. Values are mean ± SE from 10 animals per group. Full article ">Figure 8
Effect of melatonin on the proliferation of RT7 cells. (a) Effect of the addition of various melatonin concentrations on cell proliferation in a normal culture sample. * p < 0.05, ** p < 0.01. (b) Effect of various melatonin concentrations on the proliferation of RT7 cells after irradiation with X-ray 10 Gy. * p < 0.05, ** p < 0.01. (c) Effects of addition and non-addition of melatonin on cell proliferation with and without X-ray irradiation. The transition of cell proliferation is shown in the graph. It was revealed that melatonin has a cytostatic effect on RT7 cells under normal culture conditions. After irradiation, RT7 cells were influenced by irradiation, so the proliferation was slower than that in a normal culture sample, but cell proliferation was suppressed depending on the melatonin concentration. Among these results, the effect of melatonin was examined in nonX-ray irradiation and 10 Gy irradiation. In the absence of melatonin, cell proliferation was suppressed in the irradiation 10 Gy group compared with the control group, but when 1000 μM melatonin was added, there was no significant difference in proliferation between the control group and irradiation 10 Gy group. Full article ">

24 pages, 12458 KiB

Open AccessArticle

Human Mast Cells Upregulate Cathepsin B, a Novel Marker of Itch in Psoriasis

by Peter W. West, Chiara Tontini, Haris Atmoko, Orsolya Kiss, Terence Garner, Rajia Bahri, Richard B. Warren, Christopher E. M. Griffiths, Adam Stevens and Silvia Bulfone-Paus

Cells 2023, 12(17), 2177; https://doi.org/10.3390/cells12172177 - 30 Aug 2023

Viewed by 1834

Abstract

Mast cells (MCs) contribute to skin inflammation. In psoriasis, the activation of cutaneous neuroimmune networks commonly leads to itch. To dissect the unique contribution of MCs to the cutaneous neuroinflammatory response in psoriasis, we examined their density, distribution, relation to nerve fibres and [...] Read more.

Mast cells (MCs) contribute to skin inflammation. In psoriasis, the activation of cutaneous neuroimmune networks commonly leads to itch. To dissect the unique contribution of MCs to the cutaneous neuroinflammatory response in psoriasis, we examined their density, distribution, relation to nerve fibres and disease severity, and molecular signature by comparing RNA-seq analysis of MCs isolated from the skin of psoriasis patients and healthy volunteers. In involved psoriasis skin, MCs and Calcitonin Gene-Related Peptide (CGRP)-positive nerve fibres were spatially associated, and the increase of both MC and nerve fibre density correlated with disease severity. Gene set enrichment analysis of differentially expressed genes in involved psoriasis skin showed significant representation of neuron-related pathways (i.e., regulation of neuron projection along with dendrite and dendritic spine morphogenesis), indicating MC engagement in neuronal development and supporting the evidence of close MC–nerve fibre interaction. Furthermore, the analysis of 208 identified itch-associated genes revealed that CTSB, TLR4, and TACR1 were upregulated in MCs in involved skin. In both whole-skin published datasets and isolated MCs, CTSB was found to be a reliable indicator of the psoriasis condition. Furthermore, cathepsin B+ cells were increased in psoriasis skin and cathepsin B+ MC density correlated with disease severity. Therefore, our study provides evidence that cathepsin B could serve as a common indicator of the MC-dependent itch signature in psoriasis. Full article

(This article belongs to the Special Issue Mast Cells in Immunity and Inflammation)

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Figure 1

Figure 1
Mast cell and nerve fiber density, spatial association, and correlation with psoriasis severity. Immunofluorescence microscopy was used to identify and measure the density of (a) dermal MC tryptase and (b) PGP 9.5+ nerve fibres in normal (n = 8) and involved psoriasis skin (n = 11). Each subject is represented by a different symbol. (c,d) The distance in µm between MCs and PGP9.5+ nerves and the normalised frequency distribution of MCs within a given distance of a PGP9.5+ nerve fibre. (e,f) Representative photomicrographs of normal (e) and involved (f) skin show MC tryptase (cyan) and PGP9.5 (magenta). Discrete areas of PGP9.5 immunofluorescence in the dermis are identified (arrowheads). Scale bar = 20 µm. The tissue density of tryptase-positive MCs (g) and PGP9.5-positive nerve fibres (h) was correlated with the psoriasis severity index (PASI). * p < 0.05, ** p < 0.01, **** p < 0.0001, (unpaired t-test (a,b) Mann–Whitney U test (c), Pearson correlation (g,h)). Full article ">Figure 2
Calcitonin gene-related peptide density, spatial association with MCs, and correlation with severity of psoriasis. Immunofluorescence microscopy was used to identify and measure the density of MCs (tryptase), nerve fibres (PGP 9.5), and CGRP in normal (n = 6) and involved psoriasis skin (n = 10). (a) CGRP nerve fibre density and (b) proportion of CGRP+ nerve fibres in skin sections. (c) Distance between MC and CGRP+ nerve fibres and (d) normalised frequency distribution of MCs within a given distance of a CGRP+ nerve fibres in skin. (e) Correlation between CGRP density and severity index (PASI). (f,g) Representative photomicrographs of normal (f) and involved (g) skin show MC tryptase (cyan), PGP9.5 (yellow), and CGRP (magenta). The inset panel shows a magnified area with colocalized fluorescence. Scale bar = 20µm. Data are median ± IQR of n = 6 and n = 10 donors. * p < 0.05, **** p < 0.0001, ns: not significant (Mann–Whitney U test (a,c), unpaired t-test (b), Pearson correlation (e)). Full article ">Figure 3
RNA-seq analysis of mast cells isolated from normal and psoriasis skin. Mast cells (MCs) were isolated from normal (n = 7) and psoriasis-involved skin (n = 6, 2 donors pooled): (a) 3D PCA plot of log-transformed normalized matrix showing confidence ellipses (PCA1: 25.5%, PCA2: 12.42%, PCA3: 9.5% of explained variance) and (b) volcano plot of DEGs. Significantly different genes based on FDR-corrected p values and log2 fold change ± 2 are highlighted in red. (c) Z-score heatmap of the top 50 DEGs (q < 0.05) ordered by log2 fold change. Full article ">Figure 4
Canonical pathway (CP) analysis revealed functional enrichment of the CLEAR and Autophagy pathways in mast cells, with an upstream role for IL1B. Canonical pathway analysis was carried out on 203/249 differentially expressed genes (q < 0.05) using IPA. (a) CP analysis of DEGs, showing a Fisher exact test p-value < 0.05. Activation z-scores are displayed as orange for predicted activation. (b) Venn diagram of shared differentially-expressed genes identified in the CLEAR, Autophagy, and PI3K Signaling in B Lymphocytes (PI3K) canonical pathways. (c) Upstream regulator analysis of isolated MCs based on gene expression. Upregulated/downregulated genes (log2 fold change > 0/< 0) are respectively marked in magenta/cyan. Activation z-scores are displayed as orange arrows for predicted activation, blue for predicted inhibition, and grey for no prediction. Inconsistent findings are highlighted in yellow. Full article ">Figure 5
Neuron-associated ontology terms in psoriasis mast cells in gene set enrichment analysis. Gene set enrichment analysis of RNA isolated from MCs from healthy (n = 7) and psoriasis lesional skin (n = 6, 2 donors pooled). (a) Scatterplot of significantly enriched ontology terms obtained through the gseGO, gseKEGG, and gsePathway functions of ClusterProfiler/ReactomePA R tools. The cut-off for significance was set to the false discovery rate-corrected p value of < 0.05, and the analyses returned a total of 29 enriched ontology descriptions (23 GO, 2 KEGG, 4 REACTOME), as shown on the left of the plot. The colour of the dots represents the adjusted p-value, while the size of the dots represents the gene counts for each ontology term. The gene ratio is a measure of the total enrichment based on positive hits out of the total number of genes in that pathway. (b) Plot of enrichment scores and rank in the ordered dataset of the neuron-associated GO ontology terms GO:0048814, GO:0061001 and GO:0010975, with the highest-ranked genes showing the most enrichment. Full article ">Figure 6
Psoriasis mast cells share half of their differentially expressed genes with whole skin, while mast cells and neuronal signatures are unequally represented across studies and conditions. (a) Venn diagram of the number of overlapping differentially expressed genes between healthy donors and psoriasis patients in isolated MCs and microarray datasets. (b) Log2 fold change compared to healthy controls of 15 overlapping genes across datasets. (c) Cell type enrichment analysis of publicly available datasets and of MCs isolated from normal skin and psoriasis lesions, performed using xCell. The proportion of samples in which significant (p < 0.05) MC and neuron enrichment was detected is displayed per study. Isolated MCs were added as internal control for MC-specific signatures. Full article ">Figure 7
Random forest analysis of itch-associated genes and cathepsin B expression in mast cells. Random forest plot of differentially expressed genes in (a) psoriasis whole skin and (b) isolated MCs. Attributes that were significant (green bars), tentative, (yellow bars), or rejected (red bars) as indicative of the psoriasis condition are listed in order of importance. Worst, average and best shadow in each iteration are shown (blue bars). Gene names of significantly indicative attributes are enlarged below each plot. (c) Cathepsin B measured in the cell-free supernatant from human MCs stimulated with SP (concentrations shown) for 1 h. (d,e) Representative photomicrographs of the dermis of healthy (d) and psoriasis skin (e), showing cathepsin B (magenta) and MC tryptase (cyan) with nuclei stained with DAPI. Scale bar = 20 µm. Cathepsin B+ MCs are indicated by arrows. Examples of cathepsin-stained mast cells have been enlarged in each panel. (f,g) Number of total cathepsin B+ cells (f) and cathepsin B+ MCs (g) in normal skin (blue dots) and psoriasis skin (red dots). Data are mean± SEM (f) and median± IQR (g) of n = 9 donors. **** = p < 0.0001, ns: not significant (unpaired t-test). (h,i) total number of cathepsin B+ cells and cathepsin B+ MCs in psoriasis skin correlated with PASI. Significant positive correlation indicated by * p = 0.0225 (Pearson correlation). Full article ">

23 pages, 8513 KiB

Open AccessArticle

Adverse Crosstalk between Extracellular Matrix Remodeling and Ferroptosis in Basal Breast Cancer

by Christophe Desterke, Emma Cosialls, Yao Xiang, Rima Elhage, Clémence Duruel, Yunhua Chang and Ahmed Hamaï

Cells 2023, 12(17), 2176; https://doi.org/10.3390/cells12172176 - 30 Aug 2023

Viewed by 2013

Abstract

(1) Background: Breast cancer is a frequent heterogeneous disorder diagnosed in women and causes a high number of mortality among this population due to rapid metastasis and disease recurrence. Ferroptosis can inhibit breast cancer cell growth, improve the sensitivity of chemotherapy and radiotherapy, [...] Read more.

(1) Background: Breast cancer is a frequent heterogeneous disorder diagnosed in women and causes a high number of mortality among this population due to rapid metastasis and disease recurrence. Ferroptosis can inhibit breast cancer cell growth, improve the sensitivity of chemotherapy and radiotherapy, and inhibit distant metastases, potentially impacting the tumor microenvironment. (2) Methods: Through data mining, the ferroptosis/extracellular matrix remodeling literature text-mining results were integrated into the breast cancer transcriptome cohort, taking into account patients with distant relapse-free survival (DRFS) under adjuvant therapy (anthracyclin + taxanes) with validation in an independent METABRIC cohort, along with the MDA-MB-231 and HCC338 transcriptome functional experiments with ferroptosis activations (GSE173905). (3) Results: Ferroptosis/extracellular matrix remodeling text-mining identified 910 associated genes. Univariate Cox analyses focused on breast cancer (GSE25066) selected 252 individual significant genes, of which 170 were found to have an adverse expression. Functional enrichment of these 170 adverse genes predicted basal breast cancer signatures. Through text-mining, some ferroptosis-significant adverse-selected genes shared citations in the domain of ECM remodeling, such as TNF, IL6, SET, CDKN2A, EGFR, HMGB1, KRAS, MET, LCN2, HIF1A, and TLR4. A molecular score based on the expression of the eleven genes was found predictive of the worst prognosis breast cancer at the univariate level: basal subtype, short DRFS, high-grade values 3 and 4, and estrogen and progesterone receptor negative and nodal stages 2 and 3. This eleven-gene signature was validated as regulated by ferroptosis inductors (erastin and RSL3) in the triple-negative breast cancer cellular model MDA-MB-231. (4) Conclusions: The crosstalk between ECM remodeling-ferroptosis functionalities allowed for defining a molecular score, which has been characterized as an independent adverse parameter in the prognosis of breast cancer patients. The gene signature of this molecular score has been validated to be regulated by erastin/RSL3 ferroptosis activators. This molecular score could be promising to evaluate the ECM-related impact of ferroptosis target therapies in breast cancer. Full article

(This article belongs to the Special Issue Novel Mechanisms and Therapeutic Opportunities of Ferroptosis)

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