Cells

15 pages, 9088 KB

Open AccessEditor’s ChoiceArticle

Fyn-T Kinase Regulates DHA-Induced Pyroptosis in Immortalized Normal Human Astrocytes

by Ai Ling Cheng, Yuek Ling Chai, Jasinda H. Lee, Clara Y. B. Low, Helen L. Ong, Gavin S. Dawe, Thiruma V. Arumugam, Deron R. Herr, Michelle G. K. Tan and Mitchell K. P. Lai

Cells 2025, 14(19), 1530; https://doi.org/10.3390/cells14191530 - 30 Sep 2025

Viewed by 1015

Abstract

Dysregulation of astroglia-mediated neuroinflammation is known to be involved in neurodegenerative diseases. Amongst multiple inflammatory pathways, pyroptosis is characterized by inflammatory cell death following inflammasome activation. Recently, the omega-3 poly-unsaturated fatty acid, DHA, has been identified as a pyroptosis inducer, although the underlying [...] Read more.

Dysregulation of astroglia-mediated neuroinflammation is known to be involved in neurodegenerative diseases. Amongst multiple inflammatory pathways, pyroptosis is characterized by inflammatory cell death following inflammasome activation. Recently, the omega-3 poly-unsaturated fatty acid, DHA, has been identified as a pyroptosis inducer, although the underlying mechanisms remain unclear. In this study, we investigated the role of the alternatively spliced T-isoform of Fyn kinase (FynT) in DHA-induced astroglial pyroptosis. Immortalized normal human astrocytes (iNHA) expressing wild-type FynT (FynT-WT), kinase-dead mutant FynT (FynT-KD), or empty vector (EV) controls were treated with DHA and assessed for pyroptotic activation. We found that DHA-treated FynT-WT cells exhibited significantly reduced cytosolic lactate dehydrogenase release, pyroptotic morphology and markers (cleaved caspase-1 and its substrates, cleaved caspase-3 and gasdermin-D N fragments) compared to either EV or FynT-KD cells. No significant differences in pyroptotic activation were observed between EV and FynT-KD cells. In addition, no differences in immunoreactivities of pro- or anti-apoptotic markers (Bax or Bcl-2) were observed across the DHA-treated cells. In summary, our study postulates a negative regulatory role of FynT kinase in DHA-induced pyroptosis in astrocytes, with implications for further understanding neuroinflammatory mechanisms in neurodegenerative diseases and identification of potential therapeutic targets. Full article

(This article belongs to the Section Cellular Neuroscience)

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18 pages, 3381 KB

Open AccessEditor’s ChoiceArticle

EPDR1 Links Fibroblast Dysfunction to Disease Severity in Idiopathic Pulmonary Fibrosis

by Jong-Uk Lee, Seung-Lee Park, Min Kyung Kim, Eunjeong Seo, Hun-Gyu Hwang, Jung Hyun Kim, Hun Soo Chang and Choon-Sik Park

Cells 2025, 14(19), 1515; https://doi.org/10.3390/cells14191515 - 28 Sep 2025

Viewed by 1779

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by aberrant fibroblast activation, lysosomal dysfunction, and cellular senescence. Transcriptomic analyses have identified ependymin-related 1 (EPDR1) as a fibroblast-enriched gene in IPF, but its biological function remains unclear. EPDR1 expression was assessed in [...] Read more.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by aberrant fibroblast activation, lysosomal dysfunction, and cellular senescence. Transcriptomic analyses have identified ependymin-related 1 (EPDR1) as a fibroblast-enriched gene in IPF, but its biological function remains unclear. EPDR1 expression was assessed in lung fibroblasts, lung tissues, bronchoalveolar lavage fluid (BALF), and serum from IPF patients and controls using qPCR, Western blotting, ELISA, and immunohistochemistry. Lysosomal function, autophagic flux, and senescence markers were analyzed in primary fibroblasts following siRNA-mediated EPDR1 knockdown. EPDR1 was significantly upregulated in IPF-derived fibroblasts and localized to fibrotic regions enriched with α-SMA⁺, COL1A1⁺, and FN1⁺ myofibroblasts of IPF-derived lung tissues. EPDR1 levels were markedly elevated in the BALF and serum of IPF patients and correlated with increased mortality. IPF fibroblasts exhibited reduced lysosomal acidification and impaired autophagic flux, indicated by p62 and LC3B accumulation. EPDR1 knockdown restored lysosomal function; enhanced autophagic degradation; and reduced senescence markers, including p21, p16, and SA-β-gal activity. EPDR1 drives lysosomal dysfunction and fibroblast senescence in IPF. Its elevated expression in lung tissue and biological fluids, together with its association with prognosis, highlights EPDR1 as a potential biomarker and therapeutic target in IPF. Full article

(This article belongs to the Special Issue Advances in Pulmonary Fibrosis)

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33 pages, 4216 KB

Open AccessEditor’s ChoiceReview

Myocardial Ischemia/Reperfusion Injury: Molecular Insights, Forensic Perspectives, and Therapeutic Horizons

by Maria Sofia Fede, Gloria Daziani, Francesco Tavoletta, Angelo Montana, Paolo Compagnucci, Gaia Goteri, Margherita Neri and Francesco Paolo Busardò

Cells 2025, 14(19), 1509; https://doi.org/10.3390/cells14191509 - 27 Sep 2025

Cited by 16 | Viewed by 5650

Abstract

Acute myocardial infarction (AMI) remains the leading cause of death worldwide, with myocardial ischemia/reperfusion injury (MIRI) emerging as a significant factor influencing patient outcomes despite timely reperfusion therapy. MIRI refers to paradoxical myocardial damage that occurs upon restoration of coronary blood flow and [...] Read more.

Acute myocardial infarction (AMI) remains the leading cause of death worldwide, with myocardial ischemia/reperfusion injury (MIRI) emerging as a significant factor influencing patient outcomes despite timely reperfusion therapy. MIRI refers to paradoxical myocardial damage that occurs upon restoration of coronary blood flow and is driven by complex inflammatory, oxidative, and metabolic mechanisms, which can exacerbate infarct size (IS), contributing to adverse outcomes. This review explores the molecular and cellular pathophysiology of MIRI, emphasizing both its clinical and forensic relevance. The principal mechanisms discussed include oxidative stress and mitochondrial dysfunction, calcium overload and ion homeostasis imbalance, inflammatory responses, with particular focus on the NLRP3 inflammasome and cytokine pathways, and multiple forms of cell death (apoptosis, necroptosis, pyroptosis, and autophagy). Additionally, the authors present original immunohistochemical findings from autopsy cases of patients who suffered ST-segment elevation myocardial infarction (STEMI) and underwent percutaneous coronary intervention (PCI), but subsequently died. These findings underscore that successful reperfusion does not completely prevent delayed complications, like arrhythmias, ventricular fibrillation (VF), and sudden cardiac death (SCD), often caused by secondary MIRI-related mechanisms. Moreover, the case series highlight the diagnostic value of inflammatory markers for pathologists in identifying MIRI as a contributing factor in such fatalities. Finally, immunotherapeutic strategies—including IL-1 and IL-6 inhibitors such as Canakinumab and Tocilizumab—are reviewed for their potential to reduce cardiovascular events and mitigate the effects of MIRI. The review advocates for continued multidisciplinary research aimed at improving our understanding of MIRI, developing effective treatments, and informing forensic investigations of reperfusion-related deaths. Full article

(This article belongs to the Special Issue Myocardial Ischemia/Reperfusion Injury: Mechanisms and New Directions in Cardioprotection)

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37 pages, 801 KB

Open AccessEditor’s ChoiceReview

Tau-Targeted Therapeutic Strategies: Mechanistic Targets, Clinical Pipelines, and Analysis of Failures

by Xinai Shen, Huan Li, Beiyu Zhang, Yunan Li and Zheying Zhu

Cells 2025, 14(19), 1506; https://doi.org/10.3390/cells14191506 - 26 Sep 2025

Cited by 9 | Viewed by 8235

Abstract

Tau protein, a neuron-enriched microtubule-associated protein encoded by the MAPT gene, plays pivotal roles in microtubule stabilisation, axonal transport, and synaptic plasticity. Aberrant post-translational modifications (PTMs), hyperphosphorylation, acetylation, ubiquitination, oxidative stress and neuroinflammation disrupt tau’s normal functions, drive its mislocalization, and promote aggregation [...] Read more.

Tau protein, a neuron-enriched microtubule-associated protein encoded by the MAPT gene, plays pivotal roles in microtubule stabilisation, axonal transport, and synaptic plasticity. Aberrant post-translational modifications (PTMs), hyperphosphorylation, acetylation, ubiquitination, oxidative stress and neuroinflammation disrupt tau’s normal functions, drive its mislocalization, and promote aggregation into neurofibrillary tangles, a hallmark of Alzheimer’s disease (AD) and related tauopathies. Over the past two decades, tau-targeted therapies have advanced into clinical development, yet most have failed to demonstrate efficacy in human trials. This review synthesises mechanistic insights into tau biology and pathology, highlighting phosphorylation and acetylation pathways, aggregation-prone motifs, and immune-mediated propagation. We analyse the current therapeutic landscape, including kinase and phosphatase modulators, O-GlcNAcase inhibitors, aggregation blockers, immunotherapies, and microtubule-stabilising agents, while examining representative clinical programs and the reasons underlying their limited success. By combining mechanistic understanding with clinical experience, this review outlines emerging opportunities for rational treatment development, aiming to inform future tau-targeted strategies for AD and other tauopathies. Full article

(This article belongs to the Special Issue Recent Advances in the Study of Tau Protein)

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18 pages, 3672 KB

Open AccessEditor’s ChoiceArticle

A Pleiotropic and Functionally Divergent RAC3 Variant Disrupts Neurodevelopment and Impacts Organogenesis

by Ryota Sugawara, Marcello Scala, Sara Cabet, Carine Abel, Louis Januel, Gaetan Lesca, Laurent Guibaud, Frédérique Le Breton, Hiroshi Ueda, Hidenori Tabata, Hidenori Ito and Koh-ichi Nagata

Cells 2025, 14(19), 1499; https://doi.org/10.3390/cells14191499 - 24 Sep 2025

Viewed by 1214

Abstract

RAC3 encodes a small Rho-family GTPase essential for cytoskeletal regulation and neurodevelopment, and de novo RAC3 variants typically act as gain-of-function alleles that cause severe neurodevelopmental disorders. In this study, we analyzed a fetus with multisystem congenital anomalies and identified a de novo [...] Read more.

RAC3 encodes a small Rho-family GTPase essential for cytoskeletal regulation and neurodevelopment, and de novo RAC3 variants typically act as gain-of-function alleles that cause severe neurodevelopmental disorders. In this study, we analyzed a fetus with multisystem congenital anomalies and identified a de novo RAC3 p.(T17R) variant by genome sequencing. To elucidate the pathogenicity of this variant, we combined in silico variant prioritization, structural and energetic modeling, and pathogenicity prediction with in vitro biochemical assays, including GDP/GTP exchange, GTP hydrolysis, effector pull-down, and luciferase reporter analyses in COS7 cells, as well as morphological analysis of primary hippocampal neurons. Furthermore, we performed in vivo analyses using a mouse in utero electroporation to assess cortical neuron migration, axon extension, and dendritic development. Our biochemical results suggest that RAC3-T17R exhibits markedly increased GDP/GTP exchange, with a preference for GDP binding, and undetectable GTP hydrolysis. The mutant displayed minimal binding to canonical RAC effectors (PAK1, MLK2, and N-WASP) and failed to activate SRF-, NFκB-, or AP1-dependent transcription. Neuronal overexpression of RAC3-T17R impaired axon formation in vitro, while in vivo expression delayed cortical neuron migration and axon extension and reduced dendritic arborization. Clinically, the fetus exhibited corpus callosum agenesis, microcephaly, organomegaly, and limb contractures. Collectively, these findings indicate that the RAC3 p.(T17R) variant may represent a signaling-deficient allele with pleiotropic, variant-specific mechanisms that disrupt corticogenesis and broader organogenesis. Our multi-tiered in silico–in vitro–in vivo approach demonstrates that noncanonical RAC3 variants can produce complex, multisystem developmental phenotypes beyond previously recognized RAC3-related neurodevelopmental disorders. Full article

(This article belongs to the Special Issue Neuronal Development and Disease: From Molecular Bases to Neurological Disorders)

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17 pages, 1030 KB

Open AccessEditor’s ChoiceReview

Next-Generation mRNA Vaccines in Melanoma: Advances in Delivery and Combination Strategies

by Stefano Zoroddu and Luigi Bagella

Cells 2025, 14(18), 1476; https://doi.org/10.3390/cells14181476 - 22 Sep 2025

Cited by 9 | Viewed by 7665

Abstract

Messenger RNA (mRNA) vaccines have redefined cancer immunotherapy, offering unparalleled flexibility to encode tumor-specific antigens and to be adapted to individual mutational landscapes. Melanoma, with its high mutational burden and responsiveness to immune checkpoint blockade, has become the leading model for translating these [...] Read more.

Messenger RNA (mRNA) vaccines have redefined cancer immunotherapy, offering unparalleled flexibility to encode tumor-specific antigens and to be adapted to individual mutational landscapes. Melanoma, with its high mutational burden and responsiveness to immune checkpoint blockade, has become the leading model for translating these advances into clinical benefit. Recent innovations in delivery—ranging from lipid nanoparticles and polymeric carriers to biomimetic hybrids and intratumoral administration—are dismantling long-standing barriers of stability, targeting, and immunogenicity. Clinical milestones, including the randomized phase IIb KEYNOTE-942, show that adding the personalized neoantigen vaccine mRNA-4157 (V940) to pembrolizumab prolonged recurrence-free survival versus pembrolizumab alone (HR 0.561, 95% CI 0.309–1.017; 18-month RFS 79% vs. 62%), with the ASCO 3-year update reporting 2.5-year RFS 74.8% vs. 55.6% and sustained distant metastasis-free survival benefit in resected high-risk melanoma. Parallel preclinical studies highlight the potential of multifunctional platforms co-delivering cytokines or innate agonists to reshape the tumor microenvironment and achieve durable systemic immunity. As artificial intelligence drives epitope selection and modular manufacturing accelerates personalization, mRNA vaccines may have the potential to transition from adjuncts to main therapies in melanoma and beyond. Full article

(This article belongs to the Special Issue mRNA Vaccines and Therapeutics in Melanoma: From Mechanisms to Cellular Impact)

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19 pages, 3243 KB

Open AccessEditor’s ChoiceArticle

PF-04691502, a PI3K/mTOR Dual Inhibitor, Ameliorates AD-like Pathology in a Mouse Model of AD

by Marika Lanza, Rossella Basilotta, Antonella Caccamo, Giovanna Casili, Alberto Repici, Salvatore Oddo and Emanuela Esposito

Cells 2025, 14(18), 1474; https://doi.org/10.3390/cells14181474 - 21 Sep 2025

Viewed by 1822

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disorder that significantly impacts the lives of patients and their families. The pathological features of AD include the accumulation of amyloid-β (Aβ) and Tau, which disrupt neuronal function and communication, ultimately leading to neuronal loss and brain [...] Read more.

Alzheimer’s disease (AD) is a neurodegenerative disorder that significantly impacts the lives of patients and their families. The pathological features of AD include the accumulation of amyloid-β (Aβ) and Tau, which disrupt neuronal function and communication, ultimately leading to neuronal loss and brain atrophy. Efforts to understand the molecular mechanisms underlying these pathological changes have led to advancements in diagnostic techniques and potential therapeutic interventions. However, the complexity of AD necessitates further research to develop more effective treatments and, ideally, preventive measures. Extensive research suggests that diminishing mTOR signaling increases lifespan and health span across various species. Increased PI3K/mTOR signaling has been linked to the progression of AD pathology, leading to neuronal degeneration and impairments in cognitive function. In this study, we explored the therapeutic potential of PF-04691502, a dual PI3K/mTOR inhibitor, in Alzheimer’s disease (AD)-like pathology using male and female B6.Cg-Tg(APPswe, PSEN1dE9)85Dbo/Mmjax mice (APP/PS1), a well-established transgenic model of AD. Eighteen-month-old APP/PS1 and wild-type mice received oral administration of PF-04691502 at a dose of 1 mg/kg for 12 weeks. Following the treatment period, spatial learning and memory were evaluated using the Morris water maze. Subsequently, the mice brains were collected for neuropathological and biochemical assessments. Our findings showed that PF-04691502 enhanced cognitive performance in APP/PS1 mice and significantly reduced insoluble Aβ accumulation in the brain. Mechanistically, these effects were associated with enhanced autophagy induction. Treatment with PF-04691502 increased the LC3-II/LC3-I ratio, upregulated Beclin-1, and elevated LAMP-2 levels, indicative of stimulated autophagosome formation and lysosomal activity. Overall, these preclinical results suggest that PF-04691502 holds promise as a potential therapeutic agent for AD and other aging-related neurodegenerative diseases involving mTOR pathway dysregulation. Full article

(This article belongs to the Special Issue Ageing and Neurodegenerative Diseases, Second Edition)

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18 pages, 4346 KB

Open AccessEditor’s ChoiceReview

Patient-Derived Tumor Organoids to Model Cancer Cell Plasticity and Overcome Therapeutic Resistance

by Roberto Coppo and Masahiro Inoue

Cells 2025, 14(18), 1464; https://doi.org/10.3390/cells14181464 - 18 Sep 2025

Cited by 2 | Viewed by 3127

Abstract

Cancer cell plasticity, defined as the ability of tumor cells to reversibly adopt distinct functional states, plays a central role in tumor heterogeneity, therapy resistance, and disease relapse. This process enables cells to enter stem-like, dormant, or drug-tolerant persister states in response to [...] Read more.

Cancer cell plasticity, defined as the ability of tumor cells to reversibly adopt distinct functional states, plays a central role in tumor heterogeneity, therapy resistance, and disease relapse. This process enables cells to enter stem-like, dormant, or drug-tolerant persister states in response to treatment or environmental stress without undergoing genetic changes. Such reversible transitions complicate and limit current treatments. Conventional cancer models often fail to capture the complexities of these adaptive states. In contrast, patient-derived tumor organoids (PDOs), which retain the cellular diversity and structure of primary tumors, provide a unique system for investigating plasticity. This review describes how PDOs can model cellular plasticity, such as the emergence of drug-tolerant persister cells and the interconversion between cancer stem cell states across multiple tumor types. We particularly focused on colorectal cancer organoids, for which research on the mechanism of plasticity is the most advanced. Combined with single-cell analysis, lineage tracing, and functional assays, PDOs can help identify the molecular pathways that control plasticity. Understanding these mechanisms is important for developing therapies to prevent treatment failure and control disease progression. Full article

(This article belongs to the Special Issue Establishment and Characterization of Novel Patient-Derived Cancer Model)

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24 pages, 3791 KB

Open AccessEditor’s ChoiceArticle

Defined Composition of Culture Media Promotes Rodent Neonatal Cardiomyocyte Maturation and Enables Functional Neuro-Cardiac Co-Culture

by Giulia Borile, Lolita Dokshokova, Nicola Moro, Antonio Campo, Valentina Prando, Jose L. Sanchez-Alonso, Julia Gorelik, Giuseppe Faggian, Marco Mongillo and Tania Zaglia

Cells 2025, 14(18), 1434; https://doi.org/10.3390/cells14181434 - 13 Sep 2025

Viewed by 4990

Abstract

Neonatal rodent cardiomyocytes (CMs) are a mainstay of in vitro cardiac research, yet their immature phenotype limits the study of key physiological processes such as excitation–contraction coupling (ECC) and sympathetic modulation. Here, we present a defined low-glucose, serum-free (LGSF) culture protocol that drives [...] Read more.

Neonatal rodent cardiomyocytes (CMs) are a mainstay of in vitro cardiac research, yet their immature phenotype limits the study of key physiological processes such as excitation–contraction coupling (ECC) and sympathetic modulation. Here, we present a defined low-glucose, serum-free (LGSF) culture protocol that drives the structural and functional maturation of neonatal CMs and supports their integration into functional neuro-cardiac co-cultures. After 15 days in LGSF conditions, CMs exhibit elongated morphology, organized sarcomeres, polarized connexin-43, mitochondrial redistribution, and sarcoplasmic reticulum (SR) development, all closely resembling features of adult cells. These structural hallmarks were paralleled by enhanced Ca²⁺ handling, with increased SR contribution and reduced spontaneous activity, indicative of a mature ECC phenotype. When co-cultured with sympathetic neurons (SN), CMs established anatomically distinct neuro-cardiac junctions. Notably, nicotine stimulation triggered spatially restricted, reversible increases in CM Ca²⁺ transients, confined to varicosity-contacted cells. Pharmacological analysis revealed subtype-specific roles for β₁- and β₂-adrenergic receptors, and uncovered evidence of functional crosstalk between them. Our study defines a reproducible culture framework that advances CM maturation and enables the high-resolution interrogation of synaptic-like sympathetic modulation. This approach opens new avenues for mechanistic studies and drug testing in developmentally relevant neuro-cardiac systems. Full article

(This article belongs to the Section Cells of the Cardiovascular System)

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16 pages, 836 KB

Open AccessEditor’s ChoiceArticle

mRNA Multipeptide-HLA Class II Immunotherapy for Melanoma

by Apostolos P. Georgopoulos, Lisa M. James and Matthew Sanders

Cells 2025, 14(18), 1430; https://doi.org/10.3390/cells14181430 - 12 Sep 2025

Cited by 3 | Viewed by 1651

Abstract

Human Leukocyte Antigen (HLA) Class II (HLA-II) molecules bind peptides of phagocytosed non-self proteins and present them on the cell surface to circulating CD4+ T lymphocytes. A successful binding of the presented peptide with the T cell receptor (TCR) activates the CD4+ T [...] Read more.

Human Leukocyte Antigen (HLA) Class II (HLA-II) molecules bind peptides of phagocytosed non-self proteins and present them on the cell surface to circulating CD4+ T lymphocytes. A successful binding of the presented peptide with the T cell receptor (TCR) activates the CD4+ T cell, leading to the production of antibodies against the peptide (and the protein of its origin) by the B cell and augmentation of the cytotoxic and memory functions of CD8+ T cells. The first and essential step in this process is the successful formation of a stable peptide-HLA-II complex (pHLA-II), which is achieved when the peptide binds with high affinity to the HLA-II molecule. Such highly antigenic non-self peptides occur in melanoma-associated proteins and could be used as antitumor agents when bound to a matching HLA-II molecule. The objective of this study was to identify such peptides from 15 melanoma-associated proteins. We determined in silico the predicted binding affinity (IC₅₀) of all pHLA-II pairs between 192 common HLA-II molecules and all possible linear 15-amino acid (15-mer) peptides (epitopes) of 15 known melanoma-associated antigens (N = 3466 epitopes) for a total of 192 × 3466 = 665,472 determinations. From this set, we identified epitopes with strong antigenicity (predicted best binding affinity [PBBA] IC₅₀ < 50 nM). Of a total of 665,472 pHLA-II tested, 5941 (0.89%) showed strong PBBA, stemming from 117 HLA-II alleles and 679 distinct epitopes. This set of 5941 pHLA-II pairs with predicted high antigenicity possesses the requisite information for devising multipeptide vaccines with those epitopes alone or in combination with the corresponding HLA-II molecules. The results obtained have a major implication for cancer therapy, namely that the administration of subsets of the 679 high antigenicity epitopes above, alone or in combination with their associated HLA-II molecules, would be successful in engaging CD4+ T helper lymphocytes to augment the cytotoxic action and memory of CD8+ T lymphocytes and induce the production of antitumor antibodies by B cells. This therapy would be effective in other solid tumors (in addition to melanoma) and would be enhanced by concomitant immunotherapy with immune checkpoint inhibitors. Full article

(This article belongs to the Special Issue mRNA Vaccines and Therapeutics in Melanoma: From Mechanisms to Cellular Impact)

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12 pages, 583 KB

Open AccessEditor’s ChoiceArticle

Circulating Serum Cell-Free Mitochondrial DNA in Amyotrophic Lateral Sclerosis

by Giada Zanini, Ilaria Martinelli, Giorgia Sinigaglia, Elisabetta Zucchi, Federico Banchelli, Cecilia Simonini, Giulia Gianferrari, Andrea Ghezzi, Jessica Mandrioli and Marcello Pinti

Cells 2025, 14(18), 1433; https://doi.org/10.3390/cells14181433 - 12 Sep 2025

Cited by 1 | Viewed by 1488

Abstract

Mitochondrial dysfunction is a key pathological hallmark in amyotrophic lateral sclerosis (ALS), yet the role of circulating cell-free mitochondrial DNA (Cf-mtDNA) as a biomarker remains unclear. This study aimed to investigate serum Cf-mtDNA levels in ALS patients compared to healthy controls and explore [...] Read more.

Mitochondrial dysfunction is a key pathological hallmark in amyotrophic lateral sclerosis (ALS), yet the role of circulating cell-free mitochondrial DNA (Cf-mtDNA) as a biomarker remains unclear. This study aimed to investigate serum Cf-mtDNA levels in ALS patients compared to healthy controls and explore its associations with disease biomarkers, clinical progression, and survival. We conducted a case–control study measuring Cf-mtDNA levels in serum samples from 54 ALS patients and 36 age- and sex-matched healthy controls using quantitative droplet digital PCR. Correlations between Cf-mtDNA levels and clinical features, neurofilament concentrations, inflammatory indices, and survival were assessed. The average Cf-mtDNA level in ALS patients was 2,426,315 copies/mL of serum (IQR: 865000–2475000), compared to 1,885,667 copies/mL of serum (IQR: 394250–2492500) in controls (p = 0.308). ROC analysis yielded an AUC of 0.595 (95% CI: 0.468–0.721), indicating very limited discriminant ability. Cf-mtDNA levels were inversely correlated with serum creatinine concentrations (r = –0.335, p = 0.018), but showed no significant associations with ALS phenotype, disease staging, neurofilaments, inflammatory indices, or survival. These findings suggest that, in a predominantly sporadic ALS cohort, serum Cf-mtDNA may not serve as a standalone diagnostic or prognostic biomarker, in contrast to previous reports. Methodological differences, cohort composition, and genetic heterogeneity may account for these discrepancies. Our results underscore the importance of further large-scale, longitudinal studies incorporating genetic stratification and multi-biomarker approaches to better elucidate the role of Cf-mtDNA in ALS pathophysiology. Full article

(This article belongs to the Special Issue Motor Neuron Diseases: From Molecular Basic Research to Diagnosis and Therapeutics Implications)

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32 pages, 2307 KB

Open AccessEditor’s ChoiceReview

The Colonic Crypt: Cellular Dynamics and Signaling Pathways in Homeostasis and Cancer

by Anh L. Nguyen, Molly A. Lausten and Bruce M. Boman

Cells 2025, 14(18), 1428; https://doi.org/10.3390/cells14181428 - 11 Sep 2025

Cited by 5 | Viewed by 6408

Abstract

The goal of this review is to expand our understanding of how the cellular organization of the normal colonic crypt is maintained and elucidate how this intricate architecture is disrupted during tumorigenesis. Additionally, it will focus on implications for new therapeutic strategies targeting [...] Read more.

The goal of this review is to expand our understanding of how the cellular organization of the normal colonic crypt is maintained and elucidate how this intricate architecture is disrupted during tumorigenesis. Additionally, it will focus on implications for new therapeutic strategies targeting Epithelial–Mesenchymal Transition (EMT). The colonic crypt is a highly structured epithelial unit that functions in maintaining homeostasis through a complex physiological function of diverse cell types: SCs, transit-amplifying (TA) progenitors, goblet cells, absorptive colonocytes, Paneth-like cells, M cells, tuft cells, and enteroendocrine cells. These cellular subpopulations are spatially organized and regulated by multiple crucial signaling pathways, including WNT, Notch, Bone Morphogenetic Protein (BMP), and Fibroblast Growth Factor (FGF). Specifically, we discuss how these regulatory networks control the precise locations and functions of crypt cell types that are necessary to achieve cellular organization and homeostasis in the normal colon crypt. In addition, we detail how the crypt’s hierarchical structure is profoundly perturbed in colorectal cancer (CRC) development. Tumorigenesis appears to be driven by LGR5+ cancer stem cells (CSCs) and the hyperproliferation of TA cells as colonocytes undergo metabolic reprogramming. Goblet cells lose their secretory phenotype, while REG4+ Paneth-like cells foster SC niches. Tumor microenvironment is also disrupted by upregulation of M cells and by tumor-immune crosstalk that is promoted by tuft cell expansion. Moreover, the presence of enteroendocrine cells in CRC has been implicated in treatment resistance due to its contribution to tumor heterogeneity. These cellular changes are caused by the disruption of homeostasis signaling whereby: overactivation of WNT/β-catenin promotes stemness, dysregulation of Notch inhibits differentiation, suppression of BMP promotes hyperproliferation, and imbalance of FGF/WNT/BMP/NOTCH enhances cellular plasticity and invasion. Further discussion of emerging therapies targeting epithelial markers and regulatory factors, emphasizing current development in novel, precision-based approaches in CRC treatment is also included. Full article

(This article belongs to the Section Tissues and Organs)

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22 pages, 1566 KB

Open AccessEditor’s ChoiceReview

Integrating Macrophages into Human-Engineered Cardiac Tissue

by Yi Peng Zhao and Barry M. Fine

Cells 2025, 14(17), 1393; https://doi.org/10.3390/cells14171393 - 6 Sep 2025

Cited by 1 | Viewed by 2892

Abstract

Heart disease remains a leading cause of morbidity and mortality worldwide, necessitating the development of in vivo models for therapeutic development. Advances in biomedical engineering in the past decade have led to the promising rise of human-based engineered cardiac tissues (hECTs) using novel [...] Read more.

Heart disease remains a leading cause of morbidity and mortality worldwide, necessitating the development of in vivo models for therapeutic development. Advances in biomedical engineering in the past decade have led to the promising rise of human-based engineered cardiac tissues (hECTs) using novel scaffolds and pluripotent stem cell derivatives. This has led to a new frontier of human-based models for improved preclinical development. At the same time, there has been significant progress in elucidating the importance of the immune system and, in particular, macrophages, particularly during myocardial injury. This review summarizes new methods and findings for deriving macrophages from human pluripotent stem cells (hPSCs) and advances in integrating these cells into cardiac tissue. Key challenges include immune cell infiltration in 3D constructs, maintenance of tissue architecture, and modeling aged or diseased cardiac microenvironments. By integrating immune components, hECTs can serve as powerful tools to unravel the complexities of cardiac pathology and develop targeted therapeutic strategies. Full article

(This article belongs to the Special Issue Immune Cells from Pluripotent Stem Cells)

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43 pages, 4033 KB

Open AccessEditor’s ChoiceReview

Updated Applications of Stem Cells in Hypoplastic Left Heart Syndrome

by Rui Xiao, Haleema Darr, Zarif Khan and Qingzhong Xiao

Cells 2025, 14(17), 1396; https://doi.org/10.3390/cells14171396 - 6 Sep 2025

Cited by 2 | Viewed by 4811

Abstract

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease affecting 2–3 neonates every 10,000 live births. While prior research has highlighted associations of HLHS with specific chromosomal abnormalities and genetic mutations, the precise pathophysiology remains elusive. Despite early surgical intervention potentially [...] Read more.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease affecting 2–3 neonates every 10,000 live births. While prior research has highlighted associations of HLHS with specific chromosomal abnormalities and genetic mutations, the precise pathophysiology remains elusive. Despite early surgical intervention potentially allowing most HLHS patients to survive their critical heart disease with a single-ventricle physiology, patients frequently experience complications of arrhythmias and right ventricular heart failure, culminating in the need for an eventual heart transplant. Scarcity of suitable donors combined with limited understanding of mechanisms of development highlights the need for furthering our understanding of HLHS and alternative treatment options. Over the past decades, stem cell research has significantly advanced our understanding of cardiac conditions, repair, development, and therapy, opening the door for a new exciting field of regenerative medicine in cardiology with significant implications for HLHS. This review serves to provide a comprehensive overview of a much focused-on area related to HLHS. Specifically, we will first discuss the key pathophysiological basis and signalling molecules of HLHS. We then outline the emerging role of stem cell-based therapy, with a focus on adult stem cells and pluripotent stem cells (PSCs) in uncovering the pathophysiology of HLHS and optimising future treatment directions. Finally, we will also explore the latest and possible future directions of stem cell-derived techniques such as cardiac organoids and bioengineering cardiac tissues and their utility for investigating disease mechanisms, drug screening, and novel therapy for HLHF. Full article

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20 pages, 2704 KB

Open AccessFeature PaperEditor’s ChoiceArticle

Deciphering α-L-Fucosidase Activity Contribution in Human and Mouse: Tissue α-L-Fucosidase FUCA1 Meets Plasma α-L-Fucosidase FUCA2

by Hannah Bäumges, Svenja Jelinek, Heike Lange, Sandra Markmann, Emanuela Capriotti, Jan Anwar Häusser, Mai-Britt Ilse, Thomas Braulke and Torben Lübke

Cells 2025, 14(17), 1355; https://doi.org/10.3390/cells14171355 - 30 Aug 2025

Cited by 2 | Viewed by 2560

Abstract

Fucose-containing glycoproteins and glycolipids broadly occur in humans as well as in many other species and are essential for a wide range of physiological processes, such as cell adhesion, fertilization, and tumor development. In humans, the cellular degradation of various fucosylated glycoconjugates depends [...] Read more.

Fucose-containing glycoproteins and glycolipids broadly occur in humans as well as in many other species and are essential for a wide range of physiological processes, such as cell adhesion, fertilization, and tumor development. In humans, the cellular degradation of various fucosylated glycoconjugates depends on the FUCA1-encoded lysosomal tissue α-L-fucosidase (FUCA1). The crucial role of FUCA1 is reflected by the severe lysosomal storage disease fucosidosis, which causes a massive accumulation of fucosylated glycans, glycolipids, and α(1,6)-fucosylated glycoasparagines. Therefore, it is reasonable to assume that FUCA1 is predominantly responsible for the degradation of fucosylated glycoconjugates, although a second, functionally uncharacterized α-L-fucosidase, the plasma α-L-fucosidase (FUCA2), is known. To investigate the impact of both fucosidases in more detail, we generated two different monoclonal antibodies as useful tools for the detection of human and murine FUCA1 and utilized a FUCA2-specific antibody to demonstrate that FUCA2 is a bona fide lysosomal protein that is sorted in a mannose 6-phosphate (M6P)-dependent manner. We then compared FUCA1 and FUCA2 upon ectopic expression and evaluated their enzyme activity profiles under various conditions. Untagged and differently tagged versions of FUCA1 exhibited α-L-fucosidase activity, while various FUCA2 derivatives, even after affinity purification, did not show any fucosidase activity against commonly used pseudo-substrates. Our findings suggest that FUCA1 and not FUCA2 is exclusively responsible for the lysosomal de-fucosylation of glycoconjugates. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms of Lysosomal Storage Disorders)

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17 pages, 3402 KB

Open AccessEditor’s ChoiceArticle

Context-Dependent Modulation of Breast Cancer Cell E-Cadherin Expression, Mitogenesis, and Immuno-Sensitivity by Immortalized Human Mesenchymal Stem Cells In Vitro

by Bei Dai, Neha Atale, Amanda M. Clark and Alan Wells

Cells 2025, 14(17), 1316; https://doi.org/10.3390/cells14171316 - 26 Aug 2025

Viewed by 1494

Abstract

The major event that leads to death from breast cancer (BrCa) is the emergence of micrometastases into lethal growing metastases. While it is still uncertain what regulates the cell fate decision between remaining in dormancy and aggressive proliferative progression, accumulating evidence demonstrates a [...] Read more.

The major event that leads to death from breast cancer (BrCa) is the emergence of micrometastases into lethal growing metastases. While it is still uncertain what regulates the cell fate decision between remaining in dormancy and aggressive proliferative progression, accumulating evidence demonstrates a major role for the metastatic microenvironment. One area of interest is that of tissue and circulating mesenchymal stem cells (MSCs), which have been shown to alter the proliferative and metastatic potential of BrCa. Herein, we investigate how these cells impact the phenotype of metastatic BrCa. As the disseminated BrCa cells initially adopt an epithelial phenotype in ectopic organs, one that is dormant in having limited proliferation and being immune-silent, interactions that revert the disseminated metastatic BrCa to aggressive mesenchymal phenotypes, would be a driver of metastatic progression. BrCa cells exhibited phenotypic changes including increased E-cadherin expression, altered proliferation, and differential sensitivity to TRAIL-induced apoptosis when directly co-cultured with immortalized human MSCs, compared to the BrCa cells not co-cultured. These regulatory effects were dependent upon the BrCa cell’s epithelial–mesenchymal status and involved distinct juxtacrine and paracrine signaling mechanisms, as evidenced by differing responses in direct co-culture, conditioned medium, and Transwell systems. Our findings highlight the complex and context-dependent roles of MSCs in BrCa progression, improving our understanding of tumor-stroma interactions and laying groundwork for future therapeutic exploration. Full article

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18 pages, 2604 KB

Open AccessEditor’s ChoiceArticle

Calpain-1 and Calpain-2 Promote Breast Cancer Metastasis

by Danielle Harper, Jung Yeon Min, James A. MacLeod, Samantha Cockburn, Iryna Predko, Yan Gao, Peter A. Greer and Ivan Shapovalov

Cells 2025, 14(17), 1314; https://doi.org/10.3390/cells14171314 - 25 Aug 2025

Cited by 2 | Viewed by 3392

Abstract

In breast cancer, progression from localized stage I to distant metastatic stage IV disease is associated with a reduction of 5-year survival from nearly 100% to 23.2%. Expression of the calcium-activated protease isoforms calpain-1 and calpain-2 has been correlated with cell migration and [...] Read more.

In breast cancer, progression from localized stage I to distant metastatic stage IV disease is associated with a reduction of 5-year survival from nearly 100% to 23.2%. Expression of the calcium-activated protease isoforms calpain-1 and calpain-2 has been correlated with cell migration and invasion in vitro, metastatic potential in preclinical mouse models of cancer, and breast cancer prognosis in patients. It is unclear which of these two calpain isoforms is responsible for the apparent metastatic potential of cancer cells. Here, we demonstrate that while individual CRISPR-Cas9 knockouts of either CAPN1 or CAPN2 genes (encoding the catalytic subunits of calpain-1 and -2, respectively) reduce in vitro migration and marginally suppress in vivo metastasis, genetic disruption of both calpain-1 and calpain-2 through knockout of the CAPNS1 gene (encoding the common regulatory subunit of calpain-1 and -2) diminishes metastasis by 83.4 ± 13.6% in a mouse xenograft model of human triple-negative breast cancer. The effect of calpain-1/2 deficiency was replicated in vitro with a modified cell-permeable calpastatin (CAST)-based peptide inhibitor (cell migration reduced to 53.5 ± 11.0% of vehicle control). However, this peptide inhibitor was not effective in vivo at reducing metastasis under the conditions used (vehicle vs. CAST, 1.12 ± 1.35 lung metastases per mm² vs. 0.34 ± 0.20 metastases per mm²), likely due to rapid clearance, as indicated by the short serum half-life. This work demonstrates that calpain-1/2 disruption effectively abrogates metastasis and provides rationale for development of effective calpain inhibitors. Full article

(This article belongs to the Section Cellular Pathology)

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20 pages, 1149 KB

Open AccessEditor’s ChoiceReview

Calpain-1 and Calpain-2 in the Brain: What Have We Learned from 45 Years of Research?

by Michel Baudry and Xiaoning Bi

Cells 2025, 14(17), 1301; https://doi.org/10.3390/cells14171301 - 22 Aug 2025

Cited by 6 | Viewed by 3595

Abstract

Although the calcium-dependent proteases, calpains, were discovered more than 60 years ago, we still know very little regarding their functions, mostly because very few studies are addressing questions related to specific members of this relatively large family of cysteine proteases. The “classical calpains”, [...] Read more.

Although the calcium-dependent proteases, calpains, were discovered more than 60 years ago, we still know very little regarding their functions, mostly because very few studies are addressing questions related to specific members of this relatively large family of cysteine proteases. The “classical calpains”, calpain-1 and calpain-2, are ubiquitous and have received more attention because of the special roles they play in the brain. The authors have been studying the properties and functions of these two calpain isoforms in the brain for over 45 years, and this review will focus on what has been learned over this period of time. In particular, we will discuss the numerous studies that have led to the notion that calpain-1 and calpain-2 play opposite functions in the brain on processes ranging from neuronal survival or death, synaptic plasticity, and learning and memory to neurogenesis. Mechanisms underlying these opposite functions are starting to be understood and the findings support the notion that such opposite functions might be a general feature of these two isoforms in any type of cell. This review concludes with a discussion of the potential benefits of selective calpain-2 inhibitors for the treatment of a variety of neurological disorders. Full article

(This article belongs to the Special Issue Role of Calpains in Health and Diseases)

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15 pages, 2208 KB

Open AccessEditor’s ChoiceArticle

Cell Culture in a Hyperbaric Chamber: A Research Model to Study the Effects of Hyperbarism (Hyperbaric Pressure) on Bone Cell Culture

by Alessia Mariano, Valerio Consalvi, Enrico Marchetti, Angelo Rodio, Anna Scotto d’Abusco and Luigi Fattorini

Cells 2025, 14(16), 1287; https://doi.org/10.3390/cells14161287 - 19 Aug 2025

Viewed by 1570

Abstract

The hyperbaric environment, to which many categories of workers are exposed, can provoke injuries that can lead to various types of disorders. A major part of the studies aiming to explore the causes/effects leading to these injuries are conducted in vivo. In the [...] Read more.

The hyperbaric environment, to which many categories of workers are exposed, can provoke injuries that can lead to various types of disorders. A major part of the studies aiming to explore the causes/effects leading to these injuries are conducted in vivo. In the present manuscript, we describe the effects on osteoblast cell cultures stressed in a hyperbaric purpose-built chamber, using an in vitro model to analyze the affected pathways. A hyperbaric chamber for cell cultures was constructed by adapting a pressurized test chamber originally designed for technical use. The MG-63 cell line and human primary osteoblasts were placed into this chamber at different atm and exposure times, at 37 °C. After treatment, the chamber was depressurized by performing controlled decompression stops. Then, the pro-inflammatory cytokines and bone tissue biomarker expression were analyzed. The stress conditions induced the overexpression of pro-inflammatory cytokines, such as IL-6, IL-1β, and TNF-α, along with reactive oxygen species release. Moreover, the alteration of bone tissue marker production was observed. In particular, the increase in Receptor Activator of NF-κB Ligand (RANKL) and the decrease in Osteoprotegerin (OPG) were detected. Further modulation was observed regarding other biomarkers, Alkaline phosphatase, Osteocalcin, Bone Morphogenetic Protein-2, and mainly Collagen type I, all of which were downregulated by treatment. Taken together, these findings account for certain illnesses, such as dysbaric osteonecrosis, diagnosed in workers exposed to a hyperbaric environment. Inflammation induced by this kind of stress affects several factors involved in bone tissue homeostasis, leading to bone injuries, which are among the typical disorders observed in divers. Full article

(This article belongs to the Special Issue Cellular and Molecular Players in Bone Homeostasis)

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24 pages, 1526 KB

Open AccessEditor’s ChoiceReview

p38α MAPK Regulation of Energy Metabolism in Skeletal Muscle Offers a Therapeutic Path for Type 2 Diabetes

by Eyal Bengal and Sharon Aviram

Cells 2025, 14(16), 1277; https://doi.org/10.3390/cells14161277 - 18 Aug 2025

Cited by 3 | Viewed by 3871

Abstract

Type 2 diabetes (T2D), a growing global health concern, is closely linked to obesity and sedentary behavior. Central to its development are insulin resistance and impaired glucose metabolism in peripheral tissues, particularly skeletal muscle, which plays a key role in energy expenditure, glucose [...] Read more.

Type 2 diabetes (T2D), a growing global health concern, is closely linked to obesity and sedentary behavior. Central to its development are insulin resistance and impaired glucose metabolism in peripheral tissues, particularly skeletal muscle, which plays a key role in energy expenditure, glucose uptake, and insulin sensitivity. Notably, increased accumulation of lipid metabolites in skeletal muscle is observed both in endurance exercise—associated with improved insulin sensitivity—and in high-fat diets that induce insulin resistance. The review examines the contrasting metabolic adaptations of skeletal muscle to these opposing conditions and highlights the key signaling molecules involved. The focus then shifts to the role of the stress kinase p38α mitogen-activated protein kinase (MAPK) in skeletal muscle adaptation to overnutrition and endurance exercise. p38α enhances mitochondrial oxidative capacity and regulates nutrient utilization, both critical for maintaining metabolic homeostasis. During exercise, it cooperates with AMP-activated protein kinase (AMPK) to boost glucose uptake and fatty acid oxidation, key mechanisms for improving insulin sensitivity. The co-activation of p38α and AMPK in skeletal muscle emerges as a promising therapeutic avenue to combat insulin resistance and T2D. The review explores strategies for selectively enhancing p38α activity in skeletal muscle. In conclusion, it advocates a comprehensive approach to T2D prevention and treatment, combining established caloric intake-reducing therapies, such as GLP-1 receptor agonists, with interventions aimed at increasing energy expenditure via activation of p38α and AMPK signaling pathways. Full article

(This article belongs to the Special Issue Advances in Muscle Research in Health and Disease—2nd Edition)

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26 pages, 3283 KB

Open AccessEditor’s ChoiceArticle

Expression of Toll-like Receptors on Lymphocyte Subpopulations and Their Soluble Forms in Serum and Urine of Women with Endometriosis

by Anna Sobstyl, Paulina Mertowska, Sebastian Mertowski, Rafał Tarkowski, Dominik Dudziński, Michał Kotowski, Krzysztof Bojarski, Bogusława Stelmach, Błażej Chermuła, Maciej Brązert and Ewelina Grywalska

Cells 2025, 14(16), 1273; https://doi.org/10.3390/cells14161273 - 18 Aug 2025

Viewed by 1740

Abstract

Introduction: Endometriosis is a chronic inflammatory disease affecting women of reproductive age, often accompanied by chronic pelvic pain and infertility. Despite numerous studies, its pathogenesis remains incompletely understood. Increasing evidence indicates the important role of immunological disorders, especially in the mechanisms of innate [...] Read more.

Introduction: Endometriosis is a chronic inflammatory disease affecting women of reproductive age, often accompanied by chronic pelvic pain and infertility. Despite numerous studies, its pathogenesis remains incompletely understood. Increasing evidence indicates the important role of immunological disorders, especially in the mechanisms of innate immunity and Toll-like receptors (TLRs). Study objective: This study aimed to assess the expression of selected TLRs (TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9) on peripheral blood lymphocyte subpopulations (CD4+, CD8+, and CD19+ cells) in patients diagnosed with endometriosis and to quantify the levels of their soluble forms in serum and urine. This study was conducted on patients who were not undergoing hormonal bridging therapy and were not using any form of hormonal contraception to eliminate potential confounding effects on immune parameters. Methods: Flow cytometric analysis of TLR expression on peripheral blood lymphocytes was performed, and the levels of their soluble forms in serum and urine samples were determined. Additionally, ROC curve analysis was used to evaluate the diagnostic potential of the studied parameters. Results: We found increased expression of TLRs in lymphocyte populations in patients with endometriosis compared to the control group, suggesting their involvement in both local and systemic immune responses. In addition, ROC analysis showed the diagnostic potential of TLR expression in differentiating patients with endometriosis from healthy women, and it may also identify disease subtypes. Conclusions: The findings support the role of TLRs in the immunopathogenesis of endometriosis and highlight their promise as diagnostic biomarkers and therapeutic targets. Further studies on larger patient cohorts and functional signaling analyses are warranted. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms in Gynecological Disorders)

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66 pages, 2939 KB

Open AccessEditor’s ChoiceReview

Mechanistic Insights and Clinical Implications of ELK1 in Solid Tumors: A Narrative Review

by Georgios Kalampounias, Theodosia Androutsopoulou and Panagiotis Katsoris

Cells 2025, 14(16), 1257; https://doi.org/10.3390/cells14161257 - 14 Aug 2025

Cited by 8 | Viewed by 3907

Abstract

ELK1 is a Transcription factor (TF) belonging to the ETS-domain TF family, mainly activated via RAS-RAF-MEK-ERK signaling. As a nethermost pathway molecule, ELK1 binds to Serum-response elements (SREs) and directly regulates the transcription of Immediate early genes (IEGs) including FOS and EGR1. [...] Read more.

ELK1 is a Transcription factor (TF) belonging to the ETS-domain TF family, mainly activated via RAS-RAF-MEK-ERK signaling. As a nethermost pathway molecule, ELK1 binds to Serum-response elements (SREs) and directly regulates the transcription of Immediate early genes (IEGs) including FOS and EGR1. Due to ELK1’s influence on key cellular processes such as proliferation, migration, apoptosis evasion, and Epithelial-to-mesenchymal transition (EMT), its role as a key contributor to tumorigenesis is emerging. In recent years, elevated expression and/or activation of ELK1 has been reported in various malignancies, including lung, breast, prostate, colorectal, blood, gastric, liver, cervical, thyroid and ovarian cancer. ELK1 acts primarily through direct DNA binding but also through interaction with other oncogenes, noncoding RNA molecules, TFs, and upstream kinases (other than ERK1/2), thus participating in diverse axes of transcriptional regulation. Its crucial role in IEG expression has been particularly implicated in cancer progression, metastasis, and drug resistance. Owing to its role in multiple cellular functions and its subsequent oncogenic potential, further elucidation of intracellular ELK1 interactions is of paramount importance. This review aims to summarize current evidence on ELK1’s involvement in solid tumors, dissect reported mechanistic roles, and highlight recent insights that could fuel future ventures of high translational interest. Full article

(This article belongs to the Special Issue Cell Migration and Invasion)

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16 pages, 2238 KB

Open AccessEditor’s ChoiceArticle

Gene Expression Pattern Associated with Cytoskeletal Remodeling in Lipid-Loaded Human Vascular Smooth Muscle Cells: Crosstalk Between C3 Complement and the Focal Adhesion Protein Paxillin

by Maisa Garcia-Arguinzonis, Rafael Escate, Roberta Lugano, Esther Peña, Maria Borrell-Pages, Lina Badimon and Teresa Padro

Cells 2025, 14(16), 1245; https://doi.org/10.3390/cells14161245 - 12 Aug 2025

Cited by 1 | Viewed by 1665

Abstract

Mechanical and contractile forces in the vascular wall regulate smooth muscle cell migration. We previously demonstrated the presence of C3 complement products in atherosclerotic lesions of human aortas and showed that that C3-derived fragments promote key cellular processes, such as actin cytoskeleton organization [...] Read more.

Mechanical and contractile forces in the vascular wall regulate smooth muscle cell migration. We previously demonstrated the presence of C3 complement products in atherosclerotic lesions of human aortas and showed that that C3-derived fragments promote key cellular processes, such as actin cytoskeleton organization and cell migration, in lipid-loaded human vascular smooth muscle cells (hVSMCs). In the present study, we aimed to investigate gene expression profiles related to cytoskeletal remodeling and cell adhesion in migrating hVSMCs with a particular focus on modulatory effect of the C3 complement pathway on these processes. We analyzed gene expression in migrating and non-migrating hVSMCs using real-time PCR and in silico network analysis. Additionally, we investigated cytoskeletal remodeling through Western blotting and confocal microscopy. PCR profiling revealed 30 genes with significantly altered expression in migrating hVSMCs compared to non-migrating control cells. In silico analysis identified six of these genes—PXN, AKT1, RHOA, VCL, CTNNB1, and FN1—as being associated with cytoskeletal remodeling and focal adhesion, with PXN occupying a central position in the interaction network. PXN expression was reduced at both the transcript and protein levels and showed altered subcellular localization in migrating lipid-loaded hVSMCs. Protein–protein interaction analysis using STRING predicted an association between PXN and the integrin complex αMβ2 (comprising ITGAM (CD11b) and ITGB2 (CD18)), which functions as receptors for the iC3b complement fragment. Confocal imaging of cell adhesion structures revealed that lipid-loaded hVSMCs stimulated with iC3b displayed a more diffuse PXN distribution and significantly increased PXN–F-actin colocalization in active cytoplasmic regions compared to lipid-loaded control cells. PXN–F-actin colocalization increased from 1.26% to 19.68%. Subcellular fractionation further confirmed enhanced PXN enrichment in the membrane fraction, with no significant changes observed in the cytosolic or cytoskeletal compartments. In conclusion, iC3b-mediated molecular signaling in lipid-loaded hVSMCs alters PXN distribution and enhances cytoskeletal remodeling, revealing novel molecular interactions in vascular remodeling and the progression of atherosclerotic lesions. Full article

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20 pages, 10292 KB

Open AccessEditor’s ChoiceArticle

An Ex Vivo Intervertebral Disc Slice Culture Model for Studying Disc Degeneration and Immune Cell Interactions

by Eunha G. Oh, Li Xiao, Zhiwen Xu, Yuan Xing, Yi Zhang, Parastoo Anbaei, Jialun A. Chi, Li Jin, Rebecca R. Pompano and Xudong Li

Cells 2025, 14(16), 1230; https://doi.org/10.3390/cells14161230 - 8 Aug 2025

Cited by 2 | Viewed by 2041

Abstract

Intervertebral disc degeneration is a leading cause of back and leg pain and a major contributor to disability worldwide. Despite its prevalence, treatments remain limited due to incomplete understanding of its pathology. In vivo models pose challenges for controlled conditions, while in vitro [...] Read more.

Intervertebral disc degeneration is a leading cause of back and leg pain and a major contributor to disability worldwide. Despite its prevalence, treatments remain limited due to incomplete understanding of its pathology. In vivo models pose challenges for controlled conditions, while in vitro cell cultures lack key cell–cell and cell–matrix interactions. To address these limitations, we developed a novel tissue slice culture model of mouse discs, in which intact mouse discs were sliced down to 300 μm thickness with a vibratome and cultured ex vivo at various time points. The cell viability, matrix components, structure integrity, inflammatory responses, and macrophage interactions were evaluated with biochemistry, gene expression, histology, and 3D imaging analyses. Disc slices maintained structural integrity and cell viability, with preserved extracellular matrix in the annulus fibrosus (AF) and mild degeneration in nucleus pulposus (NP) by day 5. Interleukin-1 (IL-1) induced disc degeneration manifested by increased glycosaminoglycan release in media and reduced aggrecan and collagen II mRNA levels in disc cells. Cultured disc slices promoted macrophages towards pro-inflammatory phenotype with elevated mRNA levels of il-1α, il-6, and inos. Macrophage overlay and 3D imaging demonstrated macrophage infiltration into the NP and AF tissues up to ~100 µm in depth. The disc tissue slice model captures key features of intervertebral discs and can be used for investigating mechanisms of disc degeneration and therapeutic evaluation. Full article

(This article belongs to the Special Issue Novel Insights into Mechanism and Treatment of Degenerative Disc Disease)

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16 pages, 3848 KB

Open AccessEditor’s ChoiceArticle

Reversing Preeclampsia Pathology: AXL Inhibition Restores Mitochondrial Function and ECM Balance

by Archarlie Chou, Benjamin Davidson, Paul R. Reynolds, Brett E. Pickett and Juan A. Arroyo

Cells 2025, 14(16), 1229; https://doi.org/10.3390/cells14161229 - 8 Aug 2025

Cited by 2 | Viewed by 1411

Abstract

Preeclampsia (PE) is a leading cause of maternal and fetal morbidity that affects 2–8% of pregnancies worldwide, driven by placental dysfunction and systemic inflammation. Growth arrest-specific protein 6 (Gas6) and its receptor AXL play pivotal roles in PE pathogenesis, promoting trophoblast impairment and [...] Read more.

Preeclampsia (PE) is a leading cause of maternal and fetal morbidity that affects 2–8% of pregnancies worldwide, driven by placental dysfunction and systemic inflammation. Growth arrest-specific protein 6 (Gas6) and its receptor AXL play pivotal roles in PE pathogenesis, promoting trophoblast impairment and vascular dysregulation. This study investigated the transcriptomic reversal effects of AXL Receptor Tyrosine Kinase (AXL) inhibition in a Gas6-induced rat model of PE using RNA sequencing (RNA-seq). Pregnant rats were administered Gas6 to induce PE-like symptoms such as hypertension and proteinuria; a subset also received the AXL inhibitor R428. RNA-seq of placental tissues revealed 2331 differentially expressed genes (DEGs) in Gas6-AXLi versus Gas6 (1277 upregulated, 1054 downregulated). Protein–protein interaction networks and Gene Ontology enrichment highlighted upregulated mitochondrial functions, including electron transport chain components (e.g., NDUFC2, COX5A), suggesting enhanced energy metabolism. In the secondary analysis that compared Gas6 to Control, Gas6-upregulated extracellular matrix proteins (e.g., COL4A1, LAMC1) linked to fibrosis were reversed by AXL inhibition, indicating ameliorated placental remodeling. AXL inhibition activated compensatory pathways beyond Gas6 blockade, unveiling novel mechanisms for PE resolution. These findings position AXL inhibitors as promising therapeutics, offering insights into mitochondrial and fibrotic targets to mitigate this enigmatic disorder. Full article

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32 pages, 1991 KB

Open AccessEditor’s ChoiceReview

Synthetic Small-Molecule Ligands Targeted to Adenosine Receptors: Is There Potential Towards Ischemic Heart Disease?

by Qi Xu, Yaw Nana Opoku, Kalwant S. Authi and Agostino Cilibrizzi

Cells 2025, 14(15), 1219; https://doi.org/10.3390/cells14151219 - 7 Aug 2025

Cited by 1 | Viewed by 3391

Abstract

Ischemic heart disease (IHD) represents a leading cause of global morbidity and mortality. Despite significant advances in treatment achieved over recent decades, as well as various therapeutic strategies available to manage IHD progression currently, the global incidence of this disorder remains high. This [...] Read more.

Ischemic heart disease (IHD) represents a leading cause of global morbidity and mortality. Despite significant advances in treatment achieved over recent decades, as well as various therapeutic strategies available to manage IHD progression currently, the global incidence of this disorder remains high. This review examines essential cell biology aspects of adenosine receptors (ARs), along with the effects of known synthetic small-molecule AR ligands, to provide an up-to-date view on the therapeutic potential towards IHD treatment. In particular, we report here advancements made on a selection of AR synthetic ligands that have demonstrated efficacy in pre-clinical or clinical studies, thereby holding promise as new therapeutic candidates in the field of IHD. Although this work adds further evidence that clinically valid small-molecule therapeutic agents targeting ARs exist, their use represents an emerging area, with most drug prototypes still in the pre-clinical developmental stage and many lacking large-scale clinical trials. The future lies in identifying improved AR synthetic ligands with enhanced efficacy and selectivity, as well as reduced adverse side effects, along with establishing a platform of specific and diversified pre-clinical tests, to inform in turn the resulting clinical investigations. Full article

(This article belongs to the Special Issue Molecular and Cellular Mechanisms of Cardiovascular and Metabolic Diseases)

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13 pages, 1941 KB

Open AccessEditor’s ChoiceArticle

When Two Worlds Collide: The Contribution and Association Between Genetics (APOEε4) and Neuroinflammation (IL-1β) in Alzheimer’s Neuropathogenesis

by Jagadeesh Narasimhappagari, Ling Liu, Meenakshisundaram Balasubramaniam, Srinivas Ayyadevara and W. Sue T. Griffin

Cells 2025, 14(15), 1216; https://doi.org/10.3390/cells14151216 - 7 Aug 2025

Cited by 1 | Viewed by 1543

Abstract

Introduction: Here we consider the collision of a genetic factor and an essential instigator in Alzheimer’s neuropathogenesis: (i) the Alzheimer’s gene (APOEε4), which downregulates lysosomal autophagy and induces synthesis of (ii) the instigator, interleukin-1β (IL-1β), which drives synthesis of βAPP for Aβ plaques [...] Read more.

Introduction: Here we consider the collision of a genetic factor and an essential instigator in Alzheimer’s neuropathogenesis: (i) the Alzheimer’s gene (APOEε4), which downregulates lysosomal autophagy and induces synthesis of (ii) the instigator, interleukin-1β (IL-1β), which drives synthesis of βAPP for Aβ plaques and of MAPKp38 for phosphorylation of tau for formation of neurofibrillary tangles (NFTs), the two cardinal features of AD. Methods: RT-PCR, immunoblotting and immunohistochemistry techniques were used to assess the levels of IL-1β and its signaling cascade in ADε4,4, ε3,3, and age-matched controls (AMC3,3) in hippocampal regions of the brain. Results: IL-1β and its downstream signaling proteins TLR-2, MyD88, NFκB, COX-1, and COX-2 were greater in tissues from ADε4,4 than ADε3,3 or AMC3,3. Cathepsin B, D, and L levels, which play a pivotal role and are necessary for lysosomal autophagy, were lower in ADε4,4 than in ADε3,3 or AMC ε3,3. IL-1β and its downstream signaling cascade TLR-2, MyD88, NFκB, COX-1, and COX-2 expression levels were high in SH-SY5Y and T98G cells transfected with APOεE4. Conclusions: APOEε4 causes Alzheimer’s by downregulating autophagy, thus inducing IL-1β for Aβ plaque and neurofibrillary tangle formation. Full article

(This article belongs to the Special Issue Advanced Research in Neurogenesis and Neuroinflammation)

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16 pages, 2608 KB

Open AccessEditor’s ChoiceArticle

MicroRNA210 Suppresses Mitochondrial Metabolism and Promotes Microglial Activation in Neonatal Hypoxic–Ischemic Brain Injury

by Shirley Hu, Yanelly Lopez-Robles, Guofang Shen, Elena Liu, Lubo Zhang and Qingyi Ma

Cells 2025, 14(15), 1202; https://doi.org/10.3390/cells14151202 - 5 Aug 2025

Cited by 3 | Viewed by 1625

Abstract

Neuroinflammation is the major contributor to the pathology of neonatal hypoxic–ischemic (HI) brain injury. Our previous studies have demonstrated that microRNA210 (miR210) inhibition with antisense locked nucleic acid (LNA) inhibitor mitigates neuroinflammation and provides neuroprotection after neonatal HI insult. However, the underlying mechanisms [...] Read more.

Neuroinflammation is the major contributor to the pathology of neonatal hypoxic–ischemic (HI) brain injury. Our previous studies have demonstrated that microRNA210 (miR210) inhibition with antisense locked nucleic acid (LNA) inhibitor mitigates neuroinflammation and provides neuroprotection after neonatal HI insult. However, the underlying mechanisms remain elusive. In the present study, using miR210 knockout (KO) mice and microglial cultures, we tested the hypothesis that miR210 promotes microglial activation and neuroinflammation through suppressing mitochondrial function in microglia after HI. Neonatal HI brain injury was conducted on postnatal day 9 (P9) wild-type (WT) and miR210 knockout (KO) mouse pups. We found that miR210 KO significantly reduced brain infarct size at 48 h and improved long-term locomotor functions assessed by an open field test three weeks after HI. Moreover, miR210 KO mice exhibited reduced IL1β levels, microglia activation and immune cell infiltration after HI. In addition, in vitro studies of microglia exposed to oxygen–glucose deprivation (OGD) revealed that miR210 inhibition with LNA reduced OGD-induced expression of Il1b and rescued OGD-mediated downregulation of mitochondrial iron–sulfur cluster assembly enzyme (ISCU) and mitochondrial oxidative phosphorylation activity. To validate the link between miR210 and microglia activation, isolated primary murine microglia were transfected with miR210 mimic or negative control. The results showed that miR210 mimic downregulated the expression of mitochondrial ISCU protein abundance and induced the expression of proinflammatory cytokines similar to the effect observed with ISCU silencing RNA. In summary, our results suggest that miR210 is a key regulator of microglial proinflammatory activation through reprogramming mitochondrial function in neonatal HI brain injury. Full article

(This article belongs to the Special Issue Non-Coding RNAs as Regulators of Cellular Function and Disease)

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22 pages, 4087 KB

Open AccessEditor’s ChoiceArticle

Intranasal Administration of Extracellular Vesicles Derived from Adipose Mesenchymal Stem Cells Has Therapeutic Effect in Experimental Autoimmune Encephalomyelitis

by Barbara Rossi, Federica Virla, Gabriele Angelini, Ilaria Scambi, Alessandro Bani, Giulia Marostica, Mauro Caprioli, Daniela Anni, Roberto Furlan, Pasquina Marzola, Raffaella Mariotti, Gabriela Constantin, Bruno Bonetti and Ermanna Turano

Cells 2025, 14(15), 1172; https://doi.org/10.3390/cells14151172 - 30 Jul 2025

Cited by 6 | Viewed by 3378

Abstract

Adipose stem cells (ASCs) are a subset of mesenchymal stem cells with validated immunomodulatory and regenerative capabilities that make them attractive tools for treating neurodegenerative disorders, such as multiple sclerosis (MS). Several studies conducted on experimental autoimmune encephalomyelitis (EAE), the animal model of [...] Read more.

Adipose stem cells (ASCs) are a subset of mesenchymal stem cells with validated immunomodulatory and regenerative capabilities that make them attractive tools for treating neurodegenerative disorders, such as multiple sclerosis (MS). Several studies conducted on experimental autoimmune encephalomyelitis (EAE), the animal model of MS, have clearly shown a therapeutic effect of ASCs. However, controversial data on their efficacy were obtained from I- and II-phase clinical trials in MS patients, highlighting standardization issues and limited data on long-term safety. In this context, ASC-derived extracellular vesicles from (ASC-EVs) represent a safer, more reproducible alternative for EAE and MS treatment. Moreover, their physical characteristics lend themselves to a non-invasive, efficient, and easy handling of intranasal delivery. Using an in vitro setting, we first verified ASC-EVs’ ability to cross the human nasal epithelium under an inflammatory milieu. Magnetic resonance corroborated these data in vivo in intranasally treated MOG35-55-induced EAE mice, showing a preferential accumulation of ASC-EVs in brain-inflamed lesions compared to a stochastic distribution in healthy control mice. Moreover, intranasal treatment of ASC-EVs at the EAE onset led to a long-term therapeutic effect using two different experimental protocols. A marked reduction in T cell infiltration, demyelination, axonal damage, and cytokine production were correlated to EAE amelioration in ASC-EV-treated mice compared to control mice, highlighting the immunomodulatory and neuroprotective roles exerted by ASC-EVs during EAE progression. Overall, our study paves the way for promising clinical applications of self-administered ASC-EV intranasal treatment in CNS disorders, including MS. Full article

(This article belongs to the Section Cellular Neuroscience)

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23 pages, 8937 KB

Open AccessEditor’s ChoiceArticle

Neuro-Cells Mitigate Amyloid Plaque Formation and Behavioral Deficits in the APPswe/PS1dE9 Model of Alzheimer Disease While Also Reducing IL-6 Production in Human Monocytes

by Johannes de Munter, Kirill Chaprov, Ekkehard Lang, Kseniia Sitdikova, Erik Ch. Wolters, Evgeniy Svirin, Aliya Kassenova, Andrey Tsoy, Boris W. Kramer, Sholpan Askarova, Careen A. Schroeter, Daniel C. Anthony and Tatyana Strekalova

Cells 2025, 14(15), 1168; https://doi.org/10.3390/cells14151168 - 29 Jul 2025

Cited by 2 | Viewed by 1814

Abstract

Neuroinflammation is a key feature of Alzheimer’s disease (AD), and stem cell therapies have emerged as promising candidates due to their immunomodulatory properties. Neuro-Cells (NC), a combination of unmodified mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs), have demonstrated therapeutic potential in [...] Read more.

Neuroinflammation is a key feature of Alzheimer’s disease (AD), and stem cell therapies have emerged as promising candidates due to their immunomodulatory properties. Neuro-Cells (NC), a combination of unmodified mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs), have demonstrated therapeutic potential in models of central nervous system (CNS) injury and neurodegeneration. Here, we studied the effects of NC in APPswe/PS1dE9 mice, an AD mouse model. Twelve-month-old APPswe/PS1dE9 mice or their wild-type littermates were injected with NC or vehicle into the cisterna magna. Five to six weeks post-injection, cognitive, locomotor, and emotional behaviors were assessed. The brain was stained for amyloid plaque density using Congo red, and for astrogliosis using DAPI and GFAP staining. Gene expression of immune activation markers (Il-1β, Il-6, Cd45, Tnf) and plasticity markers (Tubβ3, Bace1, Trem2, Stat3) was examined in the prefrontal cortex. IL-6 secretion was measured in cultured human monocytes following endotoxin challenge and NC treatment. Untreated APPswe/PS1dE9 mice displayed impaired learning in the conditioned taste aversion test, reduced object exploration, and anxiety-like behavior, which were improved in the NC-treated mutants. NC treatment normalized the expression of several immune and plasticity markers and reduced the density of GFAP-positive cells in the hippocampus and thalamus. NC treatment decreased amyloid plaque density in the hippocampus and thalamus, targeting plaques of <100 μm². Additionally, NC treatment suppressed IL-6 secretion by human monocytes. Thus, NC treatment alleviated behavioral deficits and reduced amyloid plaque formation in APPswe/PS1dE9 mice, likely via anti-inflammatory mechanisms. The reduction in IL-6 production in human monocytes further supports the potential of NC therapy for the treatment of AD. Full article

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32 pages, 10235 KB

Open AccessEditor’s ChoiceArticle

Estradiol Downregulates MicroRNA-193a to Mediate Its Anti-Mitogenic Actions on Human Coronary Artery Smooth Muscle Cell Growth

by Lisa Rigassi, Marinella Rosselli, Brigitte Leeners, Mirel Adrian Popa and Raghvendra Krishna Dubey

Cells 2025, 14(15), 1132; https://doi.org/10.3390/cells14151132 - 23 Jul 2025

Viewed by 1350

Abstract

The abnormal growth of smooth muscle cells (SMCs) contributes to the vascular remodeling associated with coronary artery disease, a leading cause of death in women. Estradiol (E2) mediates cardiovascular protective actions, in part, by inhibiting the abnormal growth (proliferation and migration) of SMCs [...] Read more.

The abnormal growth of smooth muscle cells (SMCs) contributes to the vascular remodeling associated with coronary artery disease, a leading cause of death in women. Estradiol (E2) mediates cardiovascular protective actions, in part, by inhibiting the abnormal growth (proliferation and migration) of SMCs through various mechanism. Since microRNAs (miRNAs) play a major role in regulating cell growth and vascular remodeling, we hypothesize that miRNAs may mediate the protective actions of E2. Following preliminary leads from E2-regulated miRNAs, we found that platelet-derived growth factor (PDGF)-BB-induced miR-193a in SMCs is downregulated by E2 via estrogen receptor (ER)α, but not the ERβ or G-protein-coupled estrogen receptor (GPER). Importantly, miR-193a is actively involved in regulating SMC functions. The ectopic expression of miR-193a induced vascular SMC proliferation and migration, while its suppression with antimir abrogated PDGF-BB-induced growth, effects that were similar to E2. Importantly, the restoration of miR-193a abrogated the anti-mitogenic actions of E2 on PDGF-BB-induced growth, suggesting a key role of miR-193a in mediating the growth inhibitory actions of E2 in vascular SMCs. E2-abrogated PDGF-BB, but not miR-193a, induced SMC growth, suggesting that E2 blocks the PDGF-BB-induced miR-193a formation to mediate its anti-mitogenic actions. Interestingly, the PDGF-BB-induced miR-193a formation in SMCs was also abrogated by 2-methoxyestradiol (2ME), an endogenous E2 metabolite that inhibits SMC growth via an ER-independent mechanism. Furthermore, we found that miR-193a induces SMC growth by activating the phosphatidylinositol 3-kinases (PI3K)/Akt signaling pathway and promoting the G1 to S phase progression of the cell cycle, by inducing Cyclin D1, Cyclin Dependent Kinase 4 (CDK4), Cyclin E, and proliferating-cell-nuclear-antigen (PCNA) expression and Retinoblastoma-protein (RB) phosphorylation. Importantly, in mice, treatment with miR-193a antimir, but not its control, prevented cuff-induced vascular remodeling and significantly reducing the vessel-wall-to-lumen ratio in animal models. Taken together, our findings provide the first evidence that miR-193a promotes SMC proliferation and migration and may play a key role in PDGF-BB-induced vascular remodeling/occlusion. Importantly, E2 prevents PDGF-BB-induced SMC growth by downregulating miR-193a formation in SMCs. Since, miR-193a antimir prevents SMC growth as well as cuff-induced vascular remodeling, it may represent a promising therapeutic molecule against cardiovascular disease. Full article

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31 pages, 4221 KB

Open AccessEditor’s ChoiceArticle

Estradiol Downregulates MicroRNA-193a to Mediate Its Angiogenic Actions

by Lisa Rigassi, Mirel Adrian Popa, Ruth Stiller, Brigitte Leeners, Marinella Rosselli and Raghvendra Krishna Dubey

Cells 2025, 14(15), 1134; https://doi.org/10.3390/cells14151134 - 23 Jul 2025

Cited by 1 | Viewed by 1563

Abstract

Estrogens regulate many physiological processes in the human body, including the cardiovascular system. Importantly, Estradiol (E2) exerts its vascular protective actions, in part, by promoting endothelial repair via induction of endothelial cell (EC) proliferation, migration and angiogenesis. Recent evidence that microRNAs (miRNAs) play [...] Read more.

Estrogens regulate many physiological processes in the human body, including the cardiovascular system. Importantly, Estradiol (E2) exerts its vascular protective actions, in part, by promoting endothelial repair via induction of endothelial cell (EC) proliferation, migration and angiogenesis. Recent evidence that microRNAs (miRNAs) play an important role in vascular health and disease as well as in regulating Estrogen actions in many cell types. We hypothesize that E2 may mediate its vascular protective actions via the regulation of miRNAs. Following initial screening, we found that E2 downregulates the levels of miR-193a-3p in ECs. Moreover, miR-193a-3p downregulation by miR-193a-3p-antimir mimicked the effects as E2 on EC growth, migration, and capillary formation. Restoring miR-193a-3p levels with mimics after E2 treatment abrogated the vasculogenic actions of E2, suggesting a key role of miR-193a-3p in E2-mediated EC-growth-promoting effects. We further investigated the cellular mechanisms involved and found that miR-193a-3p inhibits angiogenesis by blocking phosphoinositide-3-kinase (PI3K)/Akt-vascular endothelial growth factor (VEGF) and Activin receptor-like kinase 1 (ALK1)/SMAD1/5/8 signaling in ECs, both pathways that are important in E2-mediated vascular protection. Additionally, using reverse transcription polymerase chain reaction (RT-PCR), we demonstrate that E2 downregulates miR-193a-3p in ECs via Estrogen Receptor (ER)α, but not ERβ or G protein-coupled estrogen receptor (GPER). Moreover, these actions occur post-transcriptionally, as the expression of pri-miR-193a-3p was not affected. The anti-angiogenic actions of miR-193a-3p were also observed in in vivo Matrigel implant-based capillary formation studies in ovariectomized mice where E2 induced capillary formation, and these effects were abrogated in the presence of miR-193a-3p, but not in the control mimic. Assessment of miR-193a-3p levels in plasma collected from in vitro fertilization (IVF) subjects with low and high E2 levels showed significantly lower miR-193a-3p levels in responders during the high E2 period. Hence, our findings provide the first evidence that miR-193a-3p mimic inhibits angiogenesis whereas its antimir is angiogenic. Importantly, E2 mediates its regenerative actions on ECs/capillary formation by downregulating endogenous miR-193a-3p expression. Both miR-193a-3p mimic or antimir may represent important therapeutic molecules to prevent or to induce endothelial function in treating pathophysiologies associated with capillary growth. Full article

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17 pages, 2227 KB

Open AccessEditor’s ChoiceArticle

Divergent Mechanisms of H2AZ.1 and H2AZ.2 in PRC1-Mediated H2A Ubiquitination

by Xiangyu Shen, Chunxu Chen, Amanda E. Jones, Xiaokun Jian, Gengsheng Cao and Hengbin Wang

Cells 2025, 14(15), 1133; https://doi.org/10.3390/cells14151133 - 23 Jul 2025

Viewed by 1710

Abstract

The histone H2A variant H2AZ plays pivotal roles in shaping chromatin architecture and regulating gene expression. We recently identified H2AZ.2 in histone H2A lysine 119 ubiquitination (H2AK119ub)-enriched nucleosomes, but it is not known whether its highly related isoform H2AZ.1 also regulates this modification. [...] Read more.

The histone H2A variant H2AZ plays pivotal roles in shaping chromatin architecture and regulating gene expression. We recently identified H2AZ.2 in histone H2A lysine 119 ubiquitination (H2AK119ub)-enriched nucleosomes, but it is not known whether its highly related isoform H2AZ.1 also regulates this modification. In this study, we employed isoform-specific epitope-tagged knock-in mouse embryonic stem cell (ESC) lines to dissect the roles of each isoform in Polycomb Repressive Complex 1 (PRC1)-mediated H2AK119ub. Our results show that H2AZ.1 and H2AZ.2 share highly overlapping genomic binding profiles, both co-localizing extensively with H2AK119ub-enriched loci. The knockdown of either isoform led to reduced H2AK119ub levels; however, the two isoforms appear to function through distinct mechanisms. H2AZ.1 facilitates the recruitment of Ring1B, the catalytic subunit of PRC1, thereby promoting the deposition of H2AK119ub. In contrast, H2AZ.2 does not significantly affect Ring1B recruitment but instead functions as a structural component that stabilizes H2AK119ub-modified nucleosomes. In vitro ubiquitination assays indicate that H2AZ.1-containing nucleosomes serve as more efficient substrates for PRC1-mediated ubiquitination compared to those containing H2AZ.2. Thus, these findings define the distinct mechanisms of the two H2AZ variants in regulated PRC1-mediated H2AK119 ubiquitination and highlight a functional division of labor in epigenetic regulation. Full article

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16 pages, 2512 KB

Open AccessEditor’s ChoiceArticle

Optimizing PH Domain-Based Biosensors for Improved Plasma Membrane PIP₃ Measurements in Mammalian Cells

by Amir Damouni, Dániel J. Tóth, Aletta Schönek, Alexander Kasbary, Adél P. Boros and Péter Várnai

Cells 2025, 14(14), 1125; https://doi.org/10.3390/cells14141125 - 21 Jul 2025

Cited by 1 | Viewed by 1570

Abstract

Phosphoinositide-binding pleckstrin homology (PH) domains interact with both phospholipids and proteins, often complicating their use as specific lipid biosensors. In this study, we introduced specific mutations into the phosphatidylinositol 3,4,5-trisphosphate (PIP₃)-specific PH domains of protein kinase B (Akt) and general receptor [...] Read more.

Phosphoinositide-binding pleckstrin homology (PH) domains interact with both phospholipids and proteins, often complicating their use as specific lipid biosensors. In this study, we introduced specific mutations into the phosphatidylinositol 3,4,5-trisphosphate (PIP₃)-specific PH domains of protein kinase B (Akt) and general receptor for phosphoinositides 1 (GRP1) that disrupt protein-mediated interactions while preserving lipid binding, in order to enhance biosensor specificity for PIP₃, and evaluated their impact on plasma membrane (PM) localization and lipid-tracking ability. Using bioluminescence resonance energy transfer (BRET) and confocal microscopy, we assessed the localization of PH domains in HEK293A cells under different conditions. While Akt-PH mutants showed minimal deviations from the wild type, GRP1-PH mutants exhibited significantly reduced PM localization both at baseline and after stimulation with epidermal growth factor (EGF), insulin, or vanadate. We further developed tandem mutant GRP1-PH domain constructs to enhance PM PIP₃ avidity. Additionally, our investigation into the influence of ADP ribosylation factor 6 (Arf6) activity on GRP1-PH-based biosensors revealed that while the wild-type sensors were Arf6- dependent, the mutants operated independently of Arf6 activity level. These optimized GRP1-PH constructs provide a refined biosensor system for accurate and selective detection of dynamic PIP₃ signaling, expanding the toolkit for dissecting phosphoinositide-mediated pathways. Full article

(This article belongs to the Section Cell Signaling)

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28 pages, 2988 KB

Open AccessEditor’s ChoiceReview

Circular RNAs as Targets for Developing Anticancer Therapeutics

by Jaewhoon Jeoung, Wonho Kim, Hyein Jo and Dooil Jeoung

Cells 2025, 14(14), 1106; https://doi.org/10.3390/cells14141106 - 18 Jul 2025

Cited by 7 | Viewed by 4206

Abstract

Circular RNA (CircRNA) is a single-stranded RNA arising from back splicing. CircRNAs interact with mRNA, miRNA, and proteins. These interactions regulate various life processes, including transcription, translation, cancer progression, anticancer drug resistance, and metabolism. Due to a lack of cap and poly(A) tails, [...] Read more.

Circular RNA (CircRNA) is a single-stranded RNA arising from back splicing. CircRNAs interact with mRNA, miRNA, and proteins. These interactions regulate various life processes, including transcription, translation, cancer progression, anticancer drug resistance, and metabolism. Due to a lack of cap and poly(A) tails, circRNAs show exceptional stability and resistance to RNase degradation. CircRNAs exhibit dysregulated expression patterns in various cancers and influence cancer progression. Stability and regulatory roles in cancer progression make circRNAs reliable biomarkers and targets for the development of anticancer therapeutics. The dysregulated expression of circRNAs is associated with resistance to anticancer drugs. Enhanced glycolysis by circRNAs leads to resistance to anticancer drugs. CircRNAs have been known to regulate the response to chemotherapy drugs and immune checkpoint inhibitors. Exogenous circRNAs can encode antigens that can induce both innate and adaptive immunity. CircRNA vaccines on lipid nanoparticles have been shown to enhance the sensitivity of cancer patients to immune checkpoint inhibitors. In this review, we summarize the roles and mechanisms of circRNAs in anticancer drug resistance and glycolysis. This review discusses clinical applications of circRNA vaccines to overcome anticancer drug resistance and enhance the efficacy of immune checkpoint inhibitors. The advantages and disadvantages of circRNA vaccines are also discussed. Overall, this review stresses the potential value of circRNAs as new therapeutic targets and diagnostic/prognostic biomarkers for cancer Full article

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21 pages, 3512 KB

Open AccessEditor’s ChoiceArticle

IP3R2-Mediated Astrocytic Ca²⁺ Transients Are Critical to Sustain Modulatory Effects of Locomotion on Neurons in Mouse Somatosensory Cortex

by Mario Fernández de la Puebla, Xiaoyi Zhang, Erlend A. Nagelhus, Magnar Bjørås and Wannan Tang

Cells 2025, 14(14), 1103; https://doi.org/10.3390/cells14141103 - 18 Jul 2025

Cited by 2 | Viewed by 2436

Abstract

Accumulating studies have shown that astrocytes are essential for regulating neurons at both synaptic and circuit levels. The main mechanism of brain astrocytic intracellular Ca²⁺ activity is through the release of Ca²⁺ via the inositol 1,4,5-trisphosphate receptor type 2 (IP3R2) from [...] Read more.

Accumulating studies have shown that astrocytes are essential for regulating neurons at both synaptic and circuit levels. The main mechanism of brain astrocytic intracellular Ca²⁺ activity is through the release of Ca²⁺ via the inositol 1,4,5-trisphosphate receptor type 2 (IP3R2) from the endoplasmic reticulum (ER). Studies using IP3R2 knockout mouse models (Itpr2^−/−) have shown that eliminating IP3R2 leads to a significant reduction in astrocytic Ca²⁺ activity However, there is ongoing controversy regarding the effect of this IP3R2-dependent reduction in astrocytic Ca²⁺ transients on neuronal activity. In our study, we employed dual-color two-photon Ca²⁺ imaging to study astrocytes and neurons simultaneously in vibrissa somatosensory cortex (vS1) in awake-behaving wild-type and Itpr2^−/− mice. We systematically characterized and compared both recorded astrocytic and neuronal Ca²⁺ activities in wild-type and Itpr2^−/− mice during various animal behaviors, particularly during the transition period from stillness to locomotion. We report that vS1 astrocytic Ca²⁺ elevation in both wild-type and Itpr2^−/− mice was significantly modulated by free whisking and locomotion. However, vS1 neurons were only significantly modulated by locomotion in wild-type mice, but not in Itpr2^−/− mice. Our study suggests a non-synaptic modulatory mechanism on functions of astrocytic IP3R2-dependent Ca²⁺ transients to local neurons. Full article

(This article belongs to the Section Cellular Neuroscience)

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39 pages, 1388 KB

Open AccessEditor’s ChoiceReview

Neuroprotective Effects of Metformin Through the Modulation of Neuroinflammation and Oxidative Stress

by Sarah Reed, Equar Taka, Selina Darling-Reed and Karam F. A. Soliman

Cells 2025, 14(14), 1064; https://doi.org/10.3390/cells14141064 - 11 Jul 2025

Cited by 39 | Viewed by 7188

Abstract

Epidemiological studies have shown that individuals with type 2 diabetes have an increased risk of developing neurodegenerative diseases. These diseases and type 2 diabetes share several risk factors. Meanwhile, the antidiabetic drug metformin offers promising neuroprotective effects by reducing oxidative stress and neuroinflammation, [...] Read more.

Epidemiological studies have shown that individuals with type 2 diabetes have an increased risk of developing neurodegenerative diseases. These diseases and type 2 diabetes share several risk factors. Meanwhile, the antidiabetic drug metformin offers promising neuroprotective effects by reducing oxidative stress and neuroinflammation, two significant factors in neurodegenerative diseases. This review examines the mechanisms by which metformin mitigates neuronal damage. Metformin reduces neuroinflammation by inhibiting microglial activation and suppressing proinflammatory cytokines. It also triggers the nuclear factor erythroid-2-related factor-2 (Nrf2) pathway to combat oxidative stress, an essential regulator of antioxidant defenses. These outcomes support the possible neuroprotective roles of metformin in type 2 diabetes-related cognitive decline and conditions like Alzheimer’s disease. Metformin’s therapeutic potential is further supported by its capacity to strengthen the blood–brain barrier’s (BBB’s) integrity and increase autophagic flux. Metformin also offers several neuroprotective effects by targeting multiple pathological pathways. Moreover, metformin is being studied for its potential benefits beyond glycemic control, particularly in the areas of cognition, Alzheimer’s disease, aging, and stroke management. Evidence from both clinical and preclinical studies indicates a complex and multifaceted impact, with benefits varying among populations and depending on underlying disease conditions, making it an appealing candidate for managing several neurodegenerative diseases. Full article

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16 pages, 3666 KB

Open AccessEditor’s ChoiceArticle

Horse Meat Hydrolysate Ameliorates Dexamethasone-Induced Muscle Atrophy in C57BL/6 Mice via the AKT/FoxO3a/mTOR Pathway

by Hee-Jeong Lee, Dongwook Kim, Yousung Jung, Soomin Oh, Cho Hee Kim and Aera Jang

Cells 2025, 14(14), 1050; https://doi.org/10.3390/cells14141050 - 9 Jul 2025

Cited by 2 | Viewed by 1788

Abstract

As life expectancy increases, muscle atrophy, characterized by a decline in muscle mass and strength that can impair mobility, has become a growing concern, highlighting the potential of protein supplementation as a promising intervention strategy. A horse meat hydrolysate, with a molecular weight [...] Read more.

As life expectancy increases, muscle atrophy, characterized by a decline in muscle mass and strength that can impair mobility, has become a growing concern, highlighting the potential of protein supplementation as a promising intervention strategy. A horse meat hydrolysate, with a molecular weight of less than 3 kDa, derived from m. biceps femoris and produced using the food-grade enzyme Alcalase^® (A4 < 3kDa) was evaluated for its efficacy in mitigating dexamethasone-induced muscle atrophy, a widely accepted model for studying catabolic muscle loss. Administered orally to C57BL/6 mice at dosages of 200 mg/kg or 500 mg/kg body weight for 35 days, A4 < 3kDa effectively countered the weight loss induced by dexamethasone in the whole body, quadriceps, tibialis anterior, and gastrocnemius muscles. Moreover, it increased muscle fiber cross-sectional area and grip strength. These effects were attributed to increased protein synthesis via the protein kinase B (AKT)/forkhead box O3 (FoxO3a)/mammalian target of rapamycin (mTOR) signaling pathway. A4 < 3kDa augmented the phosphorylation of key components of the signaling pathways associated with muscle atrophy, resulting in reduced mRNA expression of Atrogin-1 and MuRF-1. These findings demonstrate the potential of A4 < 3kDa as a functional food ingredient for preventing muscle atrophy. Full article

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23 pages, 2571 KB

Open AccessEditor’s ChoiceCommunication

Duchenne Muscular Dystrophy Patient iPSCs—Derived Skeletal Muscle Organoids Exhibit a Developmental Delay in Myogenic Progenitor Maturation

by Urs Kindler, Lampros Mavrommatis, Franziska Käppler, Dalya Gebrehiwet Hiluf, Stefanie Heilmann-Heimbach, Katrin Marcus, Thomas Günther Pomorski, Matthias Vorgerd, Beate Brand-Saberi and Holm Zaehres

Cells 2025, 14(13), 1033; https://doi.org/10.3390/cells14131033 - 7 Jul 2025

Cited by 3 | Viewed by 3087

Abstract

Background: Duchenne muscular dystrophy (DMD), which affects 1 in 3500 to 5000 newborn boys worldwide, is characterized by progressive skeletal muscle weakness and degeneration. The reduced muscle regeneration capacity presented by patients is associated with increased fibrosis. Satellite cells (SCs) are skeletal muscle [...] Read more.

Background: Duchenne muscular dystrophy (DMD), which affects 1 in 3500 to 5000 newborn boys worldwide, is characterized by progressive skeletal muscle weakness and degeneration. The reduced muscle regeneration capacity presented by patients is associated with increased fibrosis. Satellite cells (SCs) are skeletal muscle stem cells that play an important role in adult muscle maintenance and regeneration. The absence or mutation of dystrophin in DMD is hypothesized to impair SC asymmetric division, leading to cell cycle arrest. Methods: To overcome the limited availability of biopsies from DMD patients, we used our 3D skeletal muscle organoid (SMO) system, which delivers a stable population of myogenic progenitors (MPs) in dormant, activated, and committed stages, to perform SMO cultures using three DMD patient-derived iPSC lines. Results: The results of scRNA-seq analysis of three DMD SMO cultures versus two healthy, non-isogenic, SMO cultures indicate reduced MP populations with constant activation and differentiation, trending toward embryonic and immature myotubes. Mapping our data onto the human myogenic reference atlas, together with primary SC scRNA-seq data, indicated a more immature developmental stage of DMD organoid-derived MPs. DMD fibro-adipogenic progenitors (FAPs) appear to be activated in SMOs. Conclusions: Our organoid system provides a promising model for studying muscular dystrophies in vitro, especially in the case of early developmental onset, and a methodology for overcoming the bottleneck of limited patient material for skeletal muscle disease modeling. Full article

(This article belongs to the Special Issue The Current Applications and Potential of Stem Cell-Derived Organoids)

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28 pages, 933 KB

Open AccessEditor’s ChoiceReview

Therapeutic Horizons: Gut Microbiome, Neuroinflammation, and Epigenetics in Neuropsychiatric Disorders

by Shabnam Nohesara, Hamid Mostafavi Abdolmaleky, Ahmad Pirani and Sam Thiagalingam

Cells 2025, 14(13), 1027; https://doi.org/10.3390/cells14131027 - 4 Jul 2025

Cited by 10 | Viewed by 3695

Abstract

Neuroinflammation is a hallmark of many neuropsychiatric disorders (NPD), which are among the leading causes of disability worldwide. Emerging evidence highlights the significant role of the gut microbiota (GM)–immune system–brain axis in neuroinflammation and the pathogenesis of NPD, primarily through epigenetic mechanisms. Gut [...] Read more.

Neuroinflammation is a hallmark of many neuropsychiatric disorders (NPD), which are among the leading causes of disability worldwide. Emerging evidence highlights the significant role of the gut microbiota (GM)–immune system–brain axis in neuroinflammation and the pathogenesis of NPD, primarily through epigenetic mechanisms. Gut microbes and their metabolites influence immune cell activity and brain function, thereby contributing to neuroinflammation and the development and progression of NPD. The enteric nervous system, the autonomic nervous system, neuroendocrine signaling, and the immune system all participate in bidirectional communication between the gut and the brain. Importantly, the interaction of each of these systems with the GM influences epigenetic pathways. Here, we first explore the intricate relationship among intestinal microbes, microbial metabolites, and immune cell activity, with a focus on epigenetic mechanisms involved in NPD pathogenesis. Next, we provide background information on the association between inflammation and epigenetic aberrations in the context of NPD. Additionally, we review emerging therapeutic strategies—such as prebiotics, probiotics, methyl-rich diets, ketogenic diet, and medications—that may modulate the GM–immune system–brain axis via epigenetic regulation for the prevention or treatment of NPD. Finally, we discuss the challenges and future directions in investigating the critical role of this axis in mental health. Full article

(This article belongs to the Special Issue Interplay Between Intestinal Bacteria and Epigenetic Markers in Pathogenesis of Metabolic and Neuropsychiatric Disorders)

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34 pages, 6837 KB

Open AccessEditor’s ChoiceArticle

Porcine Single-Eye Retinal Pigment Epithelium Cell Culture for Barrier and Polarity Studies

by Philipp Dörschmann, Sina von der Weppen, Emi Koyama, Johann Roider and Alexa Klettner

Cells 2025, 14(13), 1007; https://doi.org/10.3390/cells14131007 - 1 Jul 2025

Cited by 3 | Viewed by 1873

Abstract

Age-related macular degeneration (AMD) is the main cause of blindness in Western nations. AMD models addressing specific pathological pathways are desired. Through this study, a best-practice protocol for polarized porcine single-eye retinal pigment epithelium (RPE) preparation for AMD-relevant models of RPE barrier and [...] Read more.

Age-related macular degeneration (AMD) is the main cause of blindness in Western nations. AMD models addressing specific pathological pathways are desired. Through this study, a best-practice protocol for polarized porcine single-eye retinal pigment epithelium (RPE) preparation for AMD-relevant models of RPE barrier and polarity is established. Single-eye porcine primary RPE cells (from one eye for one well) were prepared in 12-well plates including Transwell inserts. Different coatings (laminin (Lam), Poly-ᴅ-Lysine (PDL), fibronectin (Fn) and collagens) and varying serum contents (1%, 5% and 10%) were investigated to determine optimal culture parameters for this model. Success rates of cultures, cell number (trypan-blue exclusion assay), morphology/morphometry (light and fluorescence microscopy), protein secretion/expression (ELISA, Western blot), gene expression (qPCR), transepithelial electric resistance (TEER) and polar location of bestrophin 1 (BEST1) by cryosectioning (IHC-Fr) were assessed. Cells seeded on Lam exhibited the highest level of epithelial cells and confluence properties. Fn resulted in the highest cell number growth. Lam and Fn exhibited the highest culture success rates. TEER values and vascular endothelial growth factor secretion were highest when Lam was used. For the first time, polar (Transwell) porcine single-eye RPE morphometry parameters were determined. RPE on Lam showed bigger cells with a higher variety of cell shapes. CIV displayed the lowest claudin 19 expression. The highest basolateral expression of BEST1 was achieved with Lam coating. The higher the serum, the better the cell number increase and confluence success. A reduction in serum on Lam showed positive results for RPE morphology, while morphometry remained stable. A five percent serum on Lam showed the highest culture success rate and best barrier properties. RPE65 expression was reduced by using 10% serum. Altogether, the most suitable coating of Transwell inserts was Lam, and a reduction in serum to 5% is recommended, as well as a cultivation time of 28 days. A protocol for the use of polar porcine single-eye cultures with validated parameters was established and is provided herein. Full article

(This article belongs to the Special Issue Retinal Pigment Epithelium in Degenerative Retinal Diseases)

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39 pages, 2145 KB

Open AccessEditor’s ChoiceReview

NLRP3 Inflammasome and Inflammatory Response in Aging Disorders: The Entanglement of Redox Modulation in Different Outcomes

by Bhavana Chhunchha, Eri Kubo, Deepali Lehri and Dhirendra P. Singh

Cells 2025, 14(13), 994; https://doi.org/10.3390/cells14130994 - 29 Jun 2025

Cited by 13 | Viewed by 7667

Abstract

Increasing evidence reveals that the deregulation of cellular antioxidant response with advancing age, resulting in the continuing amplification of oxidative stress-induced inflammatory response, is a pre-eminent cause for the onset of aging-related disease states, including blinding diseases. However, several safeguards, like an antioxidant [...] Read more.

Increasing evidence reveals that the deregulation of cellular antioxidant response with advancing age, resulting in the continuing amplification of oxidative stress-induced inflammatory response, is a pre-eminent cause for the onset of aging-related disease states, including blinding diseases. However, several safeguards, like an antioxidant defense system, are genetically in place to maintain redox homeostasis. Nonetheless, if the homeostatic capacity of such systems fails (like in aging), an inflammatory pathway elicited by excessive oxidative stress-evoked aberrant NLRP3 (NOD, LRR- and pyrin domain-containing protein 3) inflammasome activation can become pathogenic and lead to disease states. Among all known inflammasomes, NLRP3 is the most studied and acts as an intracellular sensor to detect danger(s). Upon activation, NLRP3 recruits apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization and facilitates the recruitment of activated Caspase-1 (Cas-1), which results in the release of inflammatory cytokines, IL-1β and IL-18 and the activation of GasderminD, an executor of pyroptosis. NLRP3 inflammasome is tightly regulated in favor of cell health. However, when and how the activation of NLRP3 and its inflammatory components goes awry, leading to cellular derangement, and what regulatory factors are involved in the normal physiological and aging/oxidative conditions will be included in this review. Also, we address the latest findings to highlight the connection between oxidative stress, antioxidants, and NLRP3 activation as this begets aging diseases and explore the cellular pathways that are in place to regulate oxidative-induced inflammations and the pathobiological consequences of dysregulated inflammatory responses and vice versa. Full article

(This article belongs to the Special Issue New Insights into Inflammasome: Mechanisms of Activation, Cell Death, and Diseases)

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23 pages, 8906 KB

Open AccessEditor’s ChoiceArticle

9-cis-Retinoic Acid Improves Disease Modelling in iPSC-Derived Liver Organoids

by Mina Kazemzadeh Dastjerd, Vincent Merens, Ayla Smout, Rebeca De Wolf, Christophe Chesné, Catherine Verfaillie, Stefaan Verhulst and Leo A. van Grunsven

Cells 2025, 14(13), 983; https://doi.org/10.3390/cells14130983 - 26 Jun 2025

Cited by 5 | Viewed by 2739

Abstract

Liver fibrosis majorly impacts global health, necessitating the development of in vitro models to study disease mechanisms and develop drug therapies. Relevant models should at least include hepatocytes and hepatic stellate cells (HSCs) and ideally use three-dimensional cultures to mimic in vivo conditions. [...] Read more.

Liver fibrosis majorly impacts global health, necessitating the development of in vitro models to study disease mechanisms and develop drug therapies. Relevant models should at least include hepatocytes and hepatic stellate cells (HSCs) and ideally use three-dimensional cultures to mimic in vivo conditions. Induced pluripotent stem cells (iPSCs) allow for patient-specific liver modelling, but current models based on iPSC-derived hepatocytes (iHepatocytes) and HSCs (iHSCs) still lack key functions. We developed organoids of iHepatocytes and iHSCs and compared them to HepaRG and primary HSC organoids. RNA sequencing analysis comparison of these cultures identified a potential role for the transcription factor RXRA in hepatocyte differentiation and HSC quiescence. Treating cells with the RXRA ligand 9-cis-retinoic acid (9CRA) promoted iHepatocyte metabolism and iHSC quiescence. In organoids, 9CRA enhanced fibrotic response to TGF-β and acetaminophen, highlighting its potential for refining iPSC-based liver fibrosis models to more faithfully replicate human drug-induced liver injury and fibrotic conditions. Full article

(This article belongs to the Special Issue Organoids as an Experimental Tool)

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21 pages, 4834 KB

Open AccessEditor’s ChoiceArticle

Neuroprotective Effect of Mixed Mushroom Mycelia Extract on Neurotoxicity and Neuroinflammation via Regulation of ROS-Induced Oxidative Stress in PC12 and BV2 Cells

by Sang-Seop Lee, Da-Hyun Ko, Ga-Young Lee, So-Yeon Kim, Seung-Yun Han, Jong-Yea Park, MiNa Park, Hyun-Min Kim, Ya-El Kim and Yung-Choon Yoo

Cells 2025, 14(13), 977; https://doi.org/10.3390/cells14130977 - 25 Jun 2025

Cited by 2 | Viewed by 2869

Abstract

In this study, we investigated the potential of a three-mushroom complex extract (GMK) to inhibit neuronal cell death induced by the activation of AMPA and NMDA receptors following glutamate treatment in NGF-differentiated PC12 neuronal cells. GMK significantly mitigated glutamate-induced excitotoxic neuronal apoptosis by [...] Read more.

In this study, we investigated the potential of a three-mushroom complex extract (GMK) to inhibit neuronal cell death induced by the activation of AMPA and NMDA receptors following glutamate treatment in NGF-differentiated PC12 neuronal cells. GMK significantly mitigated glutamate-induced excitotoxic neuronal apoptosis by reducing the elevated expression of BAX, a critical regulator of apoptosis, and restoring BCL2 levels. These neuroprotective effects were associated with redox regulation, as evidenced by the upregulation of SOD, CAT, and GSH levels, and the downregulation of MDA levels. Mechanistic studies further revealed that GMK effectively scavenged ROS by downregulating NOX1, NOX2, and NOX4, while upregulating NRF1, P62, NRF2, HO1, and NQO1. Additionally, in the same model, GMK treatment increased acetylcholine, choline acetyltransferase, and GABA levels while reducing acetylcholinesterase activity. These effects were also attributed to the regulation of redox balance. Furthermore, we investigated the antioxidant and anti-inflammatory mechanisms of GMK in LPS-stimulated BV2 microglia. GMK inhibited the activation of IκB and MAPK pathways, positively regulated the BCL2/BAX ratio, suppressed TXNIP activity, and upregulated NQO1 and NOX1. In conclusion, GMK improved neuronal excitotoxicity and microglial inflammation through the positive modulation of the redox regulatory system, demonstrating its potential as a natural resource for pharmaceutical applications and functional health foods. Full article

(This article belongs to the Special Issue Natural Products in Neurodegenerative Diseases: Current Trends and Future Perspectives)

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25 pages, 2485 KB

Open AccessEditor’s ChoiceArticle

Epigenetic Changes Regulating Epithelial–Mesenchymal Plasticity in Human Trophoblast Differentiation

by William E. Ackerman IV, Mauricio M. Rigo, Sonia C. DaSilva-Arnold, Catherine Do, Mariam Tariq, Martha Salas, Angelica Castano, Stacy Zamudio, Benjamin Tycko and Nicholas P. Illsley

Cells 2025, 14(13), 970; https://doi.org/10.3390/cells14130970 - 24 Jun 2025

Cited by 3 | Viewed by 2562

Abstract

The phenotype of human placental extravillous trophoblast (EVT) at the end of pregnancy reflects both differentiation from villous cytotrophoblast (CTB) and later gestational changes, including loss of proliferative and invasive capacity. Invasion abnormalities are central to major obstetric pathologies, including placenta accreta spectrum, [...] Read more.

The phenotype of human placental extravillous trophoblast (EVT) at the end of pregnancy reflects both differentiation from villous cytotrophoblast (CTB) and later gestational changes, including loss of proliferative and invasive capacity. Invasion abnormalities are central to major obstetric pathologies, including placenta accreta spectrum, early onset preeclampsia, and fetal growth restriction. Characterization of the normal differentiation processes is, thus, essential for the analysis of these pathologies. Our gene expression analysis, employing purified human CTB and EVT cells, demonstrates a mechanism similar to the epithelial–mesenchymal transition (EMT), which underlies CTB–EVT differentiation. In parallel, DNA methylation profiling shows that CTB cells, already hypomethylated relative to non-trophoblast cell lineages, show further genome-wide hypomethylation in the transition to EVT. A small subgroup of genes undergoes gains of methylation (GOM), associated with differential gene expression (DE). Prominent in this GOM-DE group are genes involved in epithelial–mesenchymal plasticity (EMP). An exemplar is the transcription factor RUNX1, for which we demonstrate a functional role in regulating the migratory and invasive capacities of trophoblast cells. This analysis highlights epigenetically regulated genes acting to underpin the epithelial–mesenchymal plasticity characteristic of human trophoblast differentiation. Identification of these elements provides important information for the obstetric disorders in which these processes are dysregulated. Full article

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

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11 pages, 647 KB

Open AccessEditor’s ChoiceReview

Understanding the Role of Epithelial Cells in the Pathogenesis of Systemic Sclerosis

by Lydia Nagib, Anshul Sheel Kumar and Richard Stratton

Cells 2025, 14(13), 962; https://doi.org/10.3390/cells14130962 - 24 Jun 2025

Cited by 1 | Viewed by 2214

Abstract

Systemic sclerosis (SSc) is an autoimmune fibrotic disorder affecting the skin and internal organs, categorized as either limited cutaneous SSc, where distal areas of skin are involved, or diffuse cutaneous SSc, where more extensive proximal skin involvement is seen. Vascular remodelling and internal [...] Read more.

Systemic sclerosis (SSc) is an autoimmune fibrotic disorder affecting the skin and internal organs, categorized as either limited cutaneous SSc, where distal areas of skin are involved, or diffuse cutaneous SSc, where more extensive proximal skin involvement is seen. Vascular remodelling and internal organ involvement are frequent complications in both subsets. Multiple pathogenic mechanisms have been demonstrated, including production of disease-specific autoantibodies, endothelial cell damage at an early stage, infiltration of involved tissues by immune cells, as well as environmental factors triggering the onset such as solvents and viruses. Although not strongly familial, susceptibility to SSc is associated with multiple single nucleotide polymorphisms in immunoregulatory genes relevant to antigen presentation, T cell signalling and adaptive immunity, as well as innate immunity. In addition, several lines of evidence demonstrate abnormalities within the epithelial cell layer in SSc. Macroscopically, the SSc epidermis is pigmented, thickened and stiff and strongly promotes myofibroblasts in co-culture. Moreover, multiple activating factors and pathways have been implicated in the disease epidermis, including wound healing responses, induction of damage associated molecular patterns (DAMPS) and the release of pro-fibrotic growth factors and cytokines. Similar to SSc, data from studies of cutaneous wound healing indicate a major role for epidermal keratinocytes in regulating local fibroblast responses during repair of the wound defect. Since the epithelium is strongly exposed to environmental factors and richly populated with protective immune cells, it is possible that disease-initiating mechanisms in SSc involve dysregulated immunity and tissue repair within this cell layer. Treatments designed to restore epithelial homeostasis or else disrupt epithelial–fibroblast cross-talk could be of benefit in this severe and resistant disease. Accordingly, single cell analysis has confirmed an active signature in SSc keratinocytes, which was partially reversed following a period of JAK inhibitor therapy. Full article

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

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27 pages, 7507 KB

Open AccessEditor’s ChoiceArticle

High-Speed-Ventral-Plane Videography Identifies Specific Gait Pattern Changes in Cuprizone-Induced Demyelination in Mice

by Paula Giesler, Markus Kipp and Alexander Hawlitschka

Cells 2025, 14(13), 969; https://doi.org/10.3390/cells14130969 - 24 Jun 2025

Viewed by 1328

Abstract

Gait disturbances are among the most prominent motor symptoms in multiple sclerosis (MS), yet their functional characterization in preclinical models remains limited. In this study, we used high-speed ventral plane videography (DigiGait™) to analyze locomotor behavior during 5 weeks of cuprizone-induced demyelination in [...] Read more.

Gait disturbances are among the most prominent motor symptoms in multiple sclerosis (MS), yet their functional characterization in preclinical models remains limited. In this study, we used high-speed ventral plane videography (DigiGait™) to analyze locomotor behavior during 5 weeks of cuprizone-induced demyelination in 10 male C57BL/6 mice. Gait analysis revealed significant alterations in stride time (left front paw from 0.303 ± 0.01 s to 0.257 ± 0.007 s; p = 0.003), paw angle (right fore paw from −13.78 ± 0.928° to 5.456 ± 2.146°; p = 0.003), and midline distance (right hind paw from 1.889 ± 0.099 cm to 1.216 ± 0.096 cm; p = 0.013), particularly in the hind limbs. These behavioral impairments correlated with histopathological findings of reduced myelination and elevated microglial activation in motor-relevant brain regions, including the corpus callosum, caudate-putamen, and motor cortex. Notably, specific gait parameters showed strong correlations with the degree of demyelination, supporting their relevance as functional biomarkers. Our data demonstrate that high-resolution gait analysis provides a sensitive, non-invasive tool to monitor functional deficits in demyelinating models and may aid in evaluating therapeutic efficacy in future studies. Full article

(This article belongs to the Section Cellular Neuroscience)

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19 pages, 6401 KB

Open AccessEditor’s ChoiceArticle

Identification of Transcriptomic Differences in Induced Pluripotent Stem Cells and Neural Progenitors from Amyotrophic Lateral Sclerosis Patients Carrying Different Mutations: A Pilot Study

by Chiara Sgromo, Martina Tosi, Cristina Olgasi, Fabiola De Marchi, Francesco Favero, Giorgia Venturin, Beatrice Piola, Alessia Cucci, Lucia Corrado, Letizia Mazzini, Sandra D’Alfonso and Antonia Follenzi

Cells 2025, 14(13), 958; https://doi.org/10.3390/cells14130958 - 23 Jun 2025

Viewed by 1841

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting motor neurons with a phenotypic and genetic heterogeneity and elusive molecular mechanisms. With the present pilot study, we investigated different genetic mutations (C9orf72, TARDBP, and KIF5A) associated with ALS [...] Read more.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting motor neurons with a phenotypic and genetic heterogeneity and elusive molecular mechanisms. With the present pilot study, we investigated different genetic mutations (C9orf72, TARDBP, and KIF5A) associated with ALS by generating induced pluripotent stem cells (iPSCs) from peripheral blood of ALS patients and healthy donors. iPSCs showed the typical morphology, expressed stem cell markers both at RNA (OCT4, SOX2, KLF4, and c-Myc) and protein (Oct4, Sox2, SSEA3, and Tra1-60) levels. Moreover, embryoid bodies expressing the three germ-layer markers and neurospheres expressing neural progenitor markers were generated. Importantly, the transcriptomic profiles of iPSCs and neurospheres were analyzed to highlight the differences between ALS patients and healthy controls. Interestingly, the differentially expressed genes (DEGs) shared across all ALS iPSCs are linked to extracellular matrix, highlighting its importance in ALS progression. In contrast, ALS neurospheres displayed widespread deficits in neuronal pathways, although these DEGs were varied among patients, reflecting the disease’s heterogeneity. Overall, we generated iPSC lines from ALS patients with diverse genetic backgrounds offering a tool for unravelling the intricate molecular landscape of ALS, paving the way for identifying key pathways implicated in pathogenesis and the disease’s phenotypic variability. Full article

(This article belongs to the Collection Molecular Insights into Neurodegenerative Diseases)

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21 pages, 4336 KB

Open AccessEditor’s ChoiceArticle

Humanized scFv Molecule Specific to an Extracellular Epitope of P2X4R as Therapy for Chronic Pain Management

by Adinarayana Kunamneni and Karin N. Westlund

Cells 2025, 14(13), 953; https://doi.org/10.3390/cells14130953 - 22 Jun 2025

Cited by 2 | Viewed by 1703

Abstract

Chronic pain affects a significant portion of the population, with fewer than 30% achieving adequate relief from existing treatments. This study describes the humanization methodology and characterization of an effective non-opioid single-chain fragment variable (scFv) biologic that reverses pain-related behaviors, in this case [...] Read more.

Chronic pain affects a significant portion of the population, with fewer than 30% achieving adequate relief from existing treatments. This study describes the humanization methodology and characterization of an effective non-opioid single-chain fragment variable (scFv) biologic that reverses pain-related behaviors, in this case by targeting P2X4. After nerve injury, ATP release activates/upregulates P2X4 receptors (P2X4R) sequestered in late endosomes, triggering a cascade of chronic pain-related events. Nine humanized scFv (hscFv) variants targeting a specific extracellular 13-amino-acid peptide fragment of human P2X4R were generated via CDR grafting. ELISA analysis revealed nanomolar binding affinities, with most humanized molecules exhibiting comparable or superior affinity compared to the original murine antibody. Octet measurements confirmed that the lead, HC3-LC3, exhibited nanomolar binding kinetics (KD = 2.5 × 10⁻⁹ M). In vivo functional validation with P2X4R hscFv reversed nerve injury-induced chronic pain-related behaviors with a single dose (0.4 mg/kg, intraperitoneal) within two weeks. The return to naïve baseline remained durably reduced > 100 days. In independent confirmation, the spared nerve injury (SNI) model was similarly reduced. This constitutes an original method whereby durable reversals of chronic nerve injury pain, anxiety and depression measures are accomplished. Full article

(This article belongs to the Special Issue Mechanisms and Therapies in Chronic Pain)

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25 pages, 1161 KB

Open AccessEditor’s ChoiceReview

Biological Aging and Uterine Fibrosis in Cattle: Reproductive Trade-Offs from Enhanced Productivity

by Yuta Matsuno and Kazuhiko Imakawa

Cells 2025, 14(13), 955; https://doi.org/10.3390/cells14130955 - 22 Jun 2025

Cited by 4 | Viewed by 3236

Abstract

Reproductive efficiency in cattle remains sub-optimal, with pregnancy rates often below 50%, despite fertilization rates approaching 100%, indicating that implantation failure and/or early embryonic loss are major limiting factors. This disparity highlights the need to understand the biological and physiological mechanisms underlying implantation [...] Read more.

Reproductive efficiency in cattle remains sub-optimal, with pregnancy rates often below 50%, despite fertilization rates approaching 100%, indicating that implantation failure and/or early embryonic loss are major limiting factors. This disparity highlights the need to understand the biological and physiological mechanisms underlying implantation failure. This review elucidates the cellular and molecular mechanisms underlying reduced pregnancy rates, with a particular focus on biological aging and fibrosis in the reproductive organs as emerging contributors to uterine dysfunction. Accumulated evidence suggests that metabolic demands associated with intensive breeding strategies aimed at maximizing meat and milk productivity may induce multiple forms of stress, including oxidative stress, metabolic stress, and inflammation, which accelerate biological aging and fibrosis in the female reproductive tract. However, the direct molecular mechanisms remain poorly characterized. We hypothesize that biological aging and fibrosis are interconnected mechanisms contributing to impaired uterine function, resulting in reduced implantation rates. By summarizing recent findings and adopting a comparative perspective, this review explores the extent to which insights from human and mouse models can be applied to cattle, considering species-specific reproductive physiology and metabolic adaptations. It explores their relevance to reproductive inefficiencies and discusses potential strategies to enhance fertility and extend bovine reproductive longevity. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms in Gynecological Disorders)

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