Cells

24 pages, 5060 KiB

Open AccessArticle

CRTAP-Null Osteoblasts Have Increased Proliferation, Protein Secretion, and Skeletal Morphogenesis Gene Expression with Downregulation of Cellular Adhesion

by Aileen M. Barnes, Apratim Mitra, Marianne M. Knue, Alberta Derkyi, An Dang Do, Ryan K. Dale and Joan C. Marini

Cells 2025, 14(7), 518; https://doi.org/10.3390/cells14070518 (registering DOI) - 31 Mar 2025

Abstract

Type VII osteogenesis imperfecta (OI), caused by recessive CRTAP mutations, is predominantly lethal in the first year of life. Due to its early lethality, little is known about bone dysplasia mechanism. RNA-seq analysis of differentiated osteoblasts of siblings with a non-lethal homozygous CRTAP [...] Read more.

Type VII osteogenesis imperfecta (OI), caused by recessive CRTAP mutations, is predominantly lethal in the first year of life. Due to its early lethality, little is known about bone dysplasia mechanism. RNA-seq analysis of differentiated osteoblasts of siblings with a non-lethal homozygous CRTAP-null variant showed an enrichment of gene ontology terms involved in DNA replication and cell cycle compared to control. BrdU incorporation confirmed a ≈ 2-fold increase in proliferation in non-lethal proband osteoblasts in comparison to control cells. In addition, the expression of cyclin dependent kinase inhibitor 2A (CDKN2A), encoding a protein involved in cell cycle inhibition, was significantly reduced (>50%) in CRTAP-null osteoblasts, while cyclin B1 (CCNB1), encoding a promoter of the cell cycle, was enhanced. Ossification and bone and cartilage development gene ontology pathways were enriched among upregulated genes throughout osteoblast differentiation, as was protein secretion. Ingenuity pathway analysis indicated an upregulation of BMP2 signaling, supported by increase in both BMP2 and MSX2, an early BMP2-responsive gene, by qPCR. Throughout differentiation, CRTAP-null osteoblasts showed a decrease in transcripts related to cell adhesion and extracellular matrix organization pathways. We propose that increased proliferation and osteogenesis of type VII OI osteoblasts may be stimulated through upregulation of BMP2 signaling, altering bone homeostasis, and leading to weaker bones. Full article

(This article belongs to the Special Issue Molecular Mechanism of Bone Disease)

23 pages, 5019 KiB

Open AccessArticle

Core Molecular Clock Factors Regulate Osteosarcoma Stem Cell Survival and Behavior via CSC/EMT Pathways and Lipid Droplet Biogenesis

by Sukanya Bhoumik and Yool Lee

Cells 2025, 14(7), 517; https://doi.org/10.3390/cells14070517 (registering DOI) - 31 Mar 2025

Abstract

The circadian clock, an intrinsic 24 h cellular timekeeping system, regulates fundamental biological processes, including tumor physiology and metabolism. Cancer stem cells (CSCs), a subpopulation of cancer cells with self-renewal and tumorigenic capacities, are implicated in tumor initiation, recurrence, and metastasis. Despite growing [...] Read more.

The circadian clock, an intrinsic 24 h cellular timekeeping system, regulates fundamental biological processes, including tumor physiology and metabolism. Cancer stem cells (CSCs), a subpopulation of cancer cells with self-renewal and tumorigenic capacities, are implicated in tumor initiation, recurrence, and metastasis. Despite growing evidence for the circadian clock’s involvement in regulating CSC functions, its precise regulatory mechanisms remain largely unknown. Here, using a human osteosarcoma (OS) model (143B), we have shown that core molecular clock factors are critical for OS stem cell survival and behavior via direct modulation of CSC and lipid metabolic pathways. In single-cell-derived spheroid formation assays, 143B OS cells exhibited robust spheroid-forming capacity under 3D culture conditions. Furthermore, siRNA-mediated depletion of core clock components (i.e., BMAL1, CLOCK, CRY1/2, PER1/2)—essential positive and negative elements of the circadian clock feedback loop—significantly reduced spheroid formation in 143B CSCs isolated from in vivo OS xenografts. In contrast, knockdown of the secondary clock-stabilizing factor genes NR1D1 and NR1D2 had little effect. We also found that knockdown of BMAL1, CLOCK, or CRY1/2 markedly impaired the migration and invasion capacities of 143B CSCs. At the molecular level, silencing of BMAL1, CLOCK, or CRY1/2 distinctly altered the expression of genes associated with stem cell properties and the epithelial–mesenchymal transition (EMT) in 143B CSCs. In addition, disruption of BMAL1, CLOCK, or CRY1/2 expression significantly reduced lipid droplet formation by downregulating the expression of genes involved in lipogenesis (e.g., DGAT1, FASN, ACSL4, PKM2, CHKA, SREBP1), which are closely linked to CSC/EMT processes. Furthermore, transcriptomic analysis of human OS patient samples revealed that compared with other core clock genes, CRY1 was highly expressed in OS tumors relative to controls, and its expression exhibited strong positive correlations with patient prognosis, survival, and LD biogenesis gene expression. These findings highlight the critical role of the molecular circadian clock in regulating CSC properties and metabolism, underscoring the therapeutic potential of targeting the core clock machinery to enhance OS treatment outcomes. Full article

(This article belongs to the Special Issue The Role of Stem Cells and Circadian Clock in Cancer Immunotherapy)

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24 pages, 3447 KiB

Open AccessArticle

Distribution and Neurochemical Characterization of Dorsal Root Ganglia (DRG) Neurons Containing Phoenixin (PNX) and Supplying the Porcine Urinary Bladder

by Paweł Janikiewicz, Urszula Mazur, Piotr Holak, Nastassia Karakina, Kamil Węglarz, Mariusz Krzysztof Majewski and Agnieszka Bossowska

Cells 2025, 14(7), 516; https://doi.org/10.3390/cells14070516 (registering DOI) - 31 Mar 2025

Abstract

The present study was designed to establish the distribution pattern and immunohistochemical characteristics of phoenixin-immunoreactive (PNX-IR) urinary bladder afferent neurons (UB-ANs) of dorsal root ganglia (DRG) in female pigs. The sensory neurons investigated were visualized with a retrograde tracing method using Fast Blue [...] Read more.

The present study was designed to establish the distribution pattern and immunohistochemical characteristics of phoenixin-immunoreactive (PNX-IR) urinary bladder afferent neurons (UB-ANs) of dorsal root ganglia (DRG) in female pigs. The sensory neurons investigated were visualized with a retrograde tracing method using Fast Blue (FB), while their chemical profile(s) were identified using double-labelling immunohistochemistry with antibodies against PNX, calcitonin gene-related peptide (CGRP), calretinin (CRT), galanin (GAL), neuronal nitric oxide synthase (nNOS), pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin (SOM) and substance P (SP). Nearly half of UB-ANs contained PNX (45%), and the majority of such encoded sensory neurons were small in size (66%). The most numerous subpopulation of FB/PNX-positive neurons were those containing SP (71%). CGRP, GAL or PACAP were observed in a smaller number of PNX-containing UB-ANs (50%, 30% or 25%, respectively), while PNX-positive sensory neurons simultaneously immunostained with nNOS, CRT or SOM constituted a small fraction of all retrogradely-traced DRG neurons (DRGs; 15%, 6.5% or 1.6%, respectively). Furthermore, the numerical analysis of neurons expressing individual antigens, performed on 10 μm-thick consecutive sections, allows us to state that studied sensory neurons can be classified as neurons “coded” either by the simultaneous presence of SP/CGRP/PACAP/GAL, SP/CGRP/PACAP/NOS, SP/CGRP/PACAP/NOS/CRT and/or SP/CGRP/GAL/PACAP, or, as a separate population, those capable of SOM synthesis (SP/CGRP/SOM/PACAP/GAL-positive neurons). The present study reveals the extensive expression of PNX in the DRGs supplying to the urinary bladder, indicating an important regulatory role of this neuropeptide in the control of physiological function(s) of this organ. Full article

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

16 pages, 2830 KiB

Open AccessArticle

Galectin-1 Inhibition as a Strategy for Malignant Peripheral Nerve Sheath Tumor Treatment

by Hsiao-Chi Wang, Keila E. Torres, Roger Xia, Marcio H. Malogolowkin, Ssu-Wei Hsu, Ching-Hsien Chen and Tsung-Chieh Shih

Cells 2025, 14(7), 515; https://doi.org/10.3390/cells14070515 (registering DOI) - 31 Mar 2025

Abstract

Neurofibromatosis type 1 (NF1) is an inherited disorder that predisposes individuals to malignant peripheral nerve sheath tumors (MPNSTs), a highly aggressive sarcoma with limited treatment options and poor prognosis. This study explores the potential of targeting the interaction between Galectin-1 and Ras as [...] Read more.

Neurofibromatosis type 1 (NF1) is an inherited disorder that predisposes individuals to malignant peripheral nerve sheath tumors (MPNSTs), a highly aggressive sarcoma with limited treatment options and poor prognosis. This study explores the potential of targeting the interaction between Galectin-1 and Ras as a novel therapeutic strategy for MPNSTs. Through molecular docking, we identified critical residues involved in the Galectin-1 and H-Ras interaction. We developed LLS30, a compound designed to target this Ras-binding pocket on Galectin-1, and tested its efficacy. LLS30 effectively disrupted the Galectin-1/Ras interaction, causing Ras delocalization from the plasma membrane and inhibiting Ras signaling. In vitro experiments showed that LLS30 significantly decreased MPNST cell proliferation and induced apoptosis. In vivo, LLS30 demonstrated potent anti-tumor effects, reducing tumor size, inhibiting metastasis, and extending survival in animal models. Transcriptome analysis further revealed the downregulation of KRAS signaling and inhibition of pathways associated with epithelial–mesenchymal transition. These findings suggest that targeting Galectin-1 with LLS30 offers therapeutic potential for MPNSTs and could be beneficial for other cancers driven by Galectin-1 and Ras signaling. Full article

(This article belongs to the Special Issue Galectins in Cancer, Fibrosis and Immune Modulation: Mechanisms, Therapeutic Strategies and Translational Advances)

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29 pages, 2247 KiB

Open AccessArticle

A Broad-Spectrum Chemokine Inhibitor Drives M2 Macrophage Polarization Through Modulation of the Myometrial Secretome

by Adam Boros-Rausch, Anna Dorogin, Lubna Nadeem, Oksana Shynlova and Stephen James Lye

Cells 2025, 14(7), 514; https://doi.org/10.3390/cells14070514 (registering DOI) - 30 Mar 2025

Abstract

The uterine smooth muscle (myometrium) is an immunomodulatory tissue capable of secreting multiple chemokines during pregnancy. We propose that before term labor, chemokines secreted as a result of mechanical stretch of the uterine walls by the growing fetus(es) induce infiltration of maternal monocytes [...] Read more.

The uterine smooth muscle (myometrium) is an immunomodulatory tissue capable of secreting multiple chemokines during pregnancy. We propose that before term labor, chemokines secreted as a result of mechanical stretch of the uterine walls by the growing fetus(es) induce infiltration of maternal monocytes into myometrium, drive their differentiation into macrophages, and induce pro-inflammatory (M1) polarization, leading to labor contractions. This study used high-throughput proteomic mass-spectrometry to investigate the underlying mechanisms and explored the therapeutic potential of a broad-spectrum chemokine inhibitor (BSCI, FX125L) in modulating these effects. Primary myocytes isolated from the myometrium of term pregnant women were subjected in vitro to static mechanical stretch. Proteomic analysis of stretched myocyte-conditioned media (CM) identified significant upregulation of chemokine-related pathways and ECM degradation proteins. CM induced in vitro differentiation of human monocytes to macrophages and polarization into an M1-like phenotype characterized by elevated ROS production. BSCI treatment altered the myocyte secretome, increasing tissue-remodeling and anti-inflammatory proteins, Annexin A1 and TGF-β. BSCI-treated myocyte secretions induced Annexin A1 expression in macrophages and enhanced their phagocytic activity. We conclude that factors secreted by mechanically stretched myocytes induce pro-inflammatory M1 macrophage polarization, while BSCI modulates myocyte secretome, which reprograms macrophages to a homeostatic M2-like phenotype, thus reducing inflammation. When treated with BSCI, M2-polarized macrophages reduced myocyte-driven collagen gel contraction, whereas M1 macrophages enhanced it. This study reveals novel insights into the myocyte–macrophage interaction and identifies BSCI as a promising drug to modulate myometrial activity. We suggest that uterine macrophages may represent a therapeutic target for preventing preterm labor in women. Full article

(This article belongs to the Section Reproductive Cells and Development)

20 pages, 1972 KiB

Open AccessReview

Prostate-Specific Membrane Antigen-Targeted Antibody–Drug Conjugates: A Promising Approach for Metastatic Castration-Resistant Prostate Cancer

by Chia-Hsien Shih, Tzuo-Yi Hsieh and Wen-Wei Sung

Cells 2025, 14(7), 513; https://doi.org/10.3390/cells14070513 (registering DOI) - 30 Mar 2025

Abstract

Prostate cancer (PCa), especially metastatic castration-resistant prostate cancer (mCRPC), is a significant cancer characterized by its poor prognosis and limited treatment options. Prostate-specific membrane antigen (PSMA) has emerged as a diagnostic and therapeutic target for PCa due to its restricted expression in malignant [...] Read more.

Prostate cancer (PCa), especially metastatic castration-resistant prostate cancer (mCRPC), is a significant cancer characterized by its poor prognosis and limited treatment options. Prostate-specific membrane antigen (PSMA) has emerged as a diagnostic and therapeutic target for PCa due to its restricted expression in malignant prostate tissues. In this case, several PSMA-targeting molecules were developed for radiotherapy and immunotherapy. Antibody–drug conjugates (ADCs) are a novel therapeutic approach for various carcinomas that can selectively target PSMA-positive tumor cells and minimize off-target toxicity. ADCs have made great progress in the treatment of breast and bladder cancers, and some have received FDA approval for target therapy. However, studies on PSMA ADCs are limited, and most clinical trials are at stage I or II. Therefore, this study reviewed trials about PSMA-targeting ADCs for the treatment of PCa. Clinical trials have reported a favorable pharmacokinetic profile and antitumor activity. Toxicity studies have revealed manageable adverse effects, with no significant off-target toxicity in PSMA-negative tissues. This study highlights the therapeutic potential of PSMA ADCs for the treatment of mCRPC. However, it also emphasizes the necessity of further clinical investigation to optimize efficacy, safety, and patient outcomes. Full article

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15 pages, 3851 KiB

Open AccessArticle

Long–Term Impairment of Retinal Ganglion Cell Function After Oxygen–Induced Retinopathy

by Adam M. Schmitz, Stephanie M. Bumbaru, Laith S. Fakhouri and Dao-Qi Zhang

Cells 2025, 14(7), 512; https://doi.org/10.3390/cells14070512 (registering DOI) - 29 Mar 2025

Abstract

Premature infants with retinopathy of prematurity (ROP) have neovascularization of the retina, potentially resulting in low vision and even blindness. Some of these infants still have visual impairment, even if ROP resolves as they age. However, the mechanisms underlying the visual problems post–ROP [...] Read more.

Premature infants with retinopathy of prematurity (ROP) have neovascularization of the retina, potentially resulting in low vision and even blindness. Some of these infants still have visual impairment, even if ROP resolves as they age. However, the mechanisms underlying the visual problems post–ROP are poorly understood. Because the pathological neovascularization in ROP infants can be mimicked in a mouse model with oxygen–induced retinopathy (OIR), we recapitulated post–ROP with post–OIR mice a few months after spontaneous regression of retinal neovascularization. Our pattern electroretinogram test demonstrates that post–OIR mice exhibit reduced P1–N2 responses, suggesting the impairment of retinal ganglion cells, the retina’s output neurons. However, immunohistochemistry reveals that the density of retinal ganglion cells remains unchanged in post–OIR mice, indicating that the aforementioned pattern electroretinogram changes are functional. Our data further demonstrate that both light–adapted ex vivo electroretinogram a–waves (cone responses) and in vivo electroretinogram b–waves (ON cone bipolar cell responses) were significantly impaired in post–OIR mice. These results suggest that post–OIR impairment of the retinal cone pathway appears to result in the dysfunction of retinal ganglion cells, contributing to visual problems. A similar cellular mechanism could occur in post–ROP children, which is responsible for their visual impairment. Full article

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

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42 pages, 2934 KiB

Open AccessReview

Oxidative Stress and Redox Imbalance: Common Mechanisms in Cancer Stem Cells and Neurodegenerative Diseases

by Nikhil Raj Selvaraj, Durga Nandan, Bipin G. Nair, Vipin A. Nair, Parvathy Venugopal and Rajaguru Aradhya

Cells 2025, 14(7), 511; https://doi.org/10.3390/cells14070511 (registering DOI) - 29 Mar 2025

Abstract

Oxidative stress (OS) is an established hallmark of cancer and neurodegenerative disorders (NDDs), which contributes to genomic instability and neuronal loss. This review explores the contrasting role of OS in cancer stem cells (CSCs) and NDDs. Elevated levels of reactive oxygen species (ROS) [...] Read more.

Oxidative stress (OS) is an established hallmark of cancer and neurodegenerative disorders (NDDs), which contributes to genomic instability and neuronal loss. This review explores the contrasting role of OS in cancer stem cells (CSCs) and NDDs. Elevated levels of reactive oxygen species (ROS) contribute to genomic instability and promote tumor initiation and progression in CSCs, while in NDDs such as Alzheimer’s and Parkinson’s disease, OS accelerates neuronal death and impairs cellular repair mechanisms. Both scenarios involve disruption of the delicate balance between pro-oxidant and antioxidant systems, which leads to chronic oxidative stress. Notably, CSCs and neurons display alterations in redox-sensitive signaling pathways, including Nrf2 and NF-κB, which influence cell survival, proliferation, and differentiation. Mitochondrial dynamics further illustrate these differences: enhanced function in CSCs supports adaptability and survival, whereas impairments in neurons heighten vulnerability. Understanding these common mechanisms of OS-induced redox imbalance may provide insights for developing interventions, addressing aging hallmarks, and potentially mitigating or preventing both cancer and NDDs. Full article

(This article belongs to the Special Issue Signaling in Cancer Stem Cells)

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16 pages, 2769 KiB

Open AccessArticle

Differential Effects of Hearing Loss Mutations in Homomeric P2X2 and Heteromeric P2X2/3 Receptors

by Paula-Luise Wand, Xenia Brünings, Debanjan Tewari, Stefanie Reuter, Ralf Mrowka, Klaus Benndorf, Thomas Zimmer and Christian Sattler

Cells 2025, 14(7), 510; https://doi.org/10.3390/cells14070510 (registering DOI) - 29 Mar 2025

Abstract

P2X receptors are unspecific cation channels activated by ATP. They are expressed in various tissues and found in neuronal and immune cells. In mammals, seven subunits are described, which can assemble into homomeric and heteromeric trimers. P2X2 receptors play important roles in cochlear [...] Read more.

P2X receptors are unspecific cation channels activated by ATP. They are expressed in various tissues and found in neuronal and immune cells. In mammals, seven subunits are described, which can assemble into homomeric and heteromeric trimers. P2X2 receptors play important roles in cochlear adaptation to elevated sound levels. Three mutations causing inherited progressive hearing loss have been identified. These mutations localize to the transmembrane domain 1 (V60L), the transmembrane domain 2 (G353R) and a β-sheet linking the ATP binding site to the pore (D273Y). Herein, mutations were studied in human homomeric P2X2 as well as in heteromeric P2X2/3 receptors. We measured their binding of a fluorescently labeled ATP derivative (fATP) and characterized the constructs using the patch-clamp technique. The conclusions from our results are as follows: 1. The mutations V60L and G353R show robust localization on the plasma membrane and binding of fATP, whereas the mutant D273Y has no binding to fATP. 2. The mutation V60L has an increased affinity to fATP compared with the wildtype. 3. The expression of hP2X2 V60L channels reduces cell viability, which may support its role in the pathogenesis of hearing loss. 4. All mutant P2X2 subunits can assemble into P2X2/3 heteromeric channels with distinct phenotypes. Full article

(This article belongs to the Special Issue Modulation of P2X Receptors: Emerging Implications for Cellular Signaling and Disease Management)

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27 pages, 1880 KiB

Open AccessReview

Hsp70: A Multifunctional Chaperone in Maintaining Proteostasis and Its Implications in Human Disease

by Manish Kumar Singh, Sunhee Han, Songhyun Ju, Jyotsna S. Ranbhise, Joohun Ha, Seung Geun Yeo, Sung Soo Kim and Insug Kang

Cells 2025, 14(7), 509; https://doi.org/10.3390/cells14070509 (registering DOI) - 29 Mar 2025

Abstract

Hsp70, a 70 kDa molecular chaperone, plays a crucial role in maintaining protein homeostasis. It interacts with the DnaJ family of co-chaperones to modulate the functions of client proteins involved in various cellular processes, including transmembrane transport, extracellular vesicle trafficking, complex formation, and [...] Read more.

Hsp70, a 70 kDa molecular chaperone, plays a crucial role in maintaining protein homeostasis. It interacts with the DnaJ family of co-chaperones to modulate the functions of client proteins involved in various cellular processes, including transmembrane transport, extracellular vesicle trafficking, complex formation, and proteasomal degradation. Its presence in multiple cellular organelles enables it to mediate stress responses, apoptosis, and inflammation, highlighting its significance in disease progression. Initially recognized for its essential roles in protein folding, disaggregation, and degradation, later studies have demonstrated its involvement in several human diseases. Notably, Hsp70 is upregulated in multiple cancers, where it promotes tumor proliferation and serves as a tumor immunogen. Additionally, epichaperome networks stabilize protein–protein interactions in large and long-lived assemblies, contributing to both cancer progression and neurodegeneration. However, extracellular Hsp70 (eHsp70) in the tumor microenvironment can activate immune cells, such as natural killer (NK) cells, suggesting its potential in immunotherapeutic interventions, including CAR T-cell therapy. Given its multifaceted roles in cellular physiology and pathology, Hsp70 holds immense potential as both a biomarker and a therapeutic target across multiple human diseases. This review highlights the structural and functional importance of Hsp70, explores its role in disease pathogenesis, and discusses its potential in diagnostic and therapeutic applications. Full article

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29 pages, 4474 KiB

Open AccessFeature PaperReview

Role of T Follicular Helper Cells in Viral Infections and Vaccine Design

by Sohrab Ahmadivand, Robert Fux and Dušan Palić

Cells 2025, 14(7), 508; https://doi.org/10.3390/cells14070508 (registering DOI) - 29 Mar 2025

Abstract

T follicular helper (Tfh) cells are a specialized subset of CD4+ T lymphocytes that are essential for the development of long-lasting humoral immunity. Tfh cells facilitate B lymphocyte maturation, promote germinal center formation, and drive high-affinity antibody production. Our current knowledge of Tfh [...] Read more.

T follicular helper (Tfh) cells are a specialized subset of CD4+ T lymphocytes that are essential for the development of long-lasting humoral immunity. Tfh cells facilitate B lymphocyte maturation, promote germinal center formation, and drive high-affinity antibody production. Our current knowledge of Tfh interactions with the humoral immune system effectors suggests that they have a critical role in supporting the immune response against viral infections. This review discusses the mechanisms through which Tfh cells influence anti-viral immunity, highlighting their interactions with B cells and their impact on antibody quality and quantity. We explore the role of Tfh cells in viral infections and examine how vaccine design can be improved to enhance Tfh cell responses. Innovative vaccine platforms, such as mRNA vaccines and self-assembling protein nanoplatforms (SAPNs), are promising strategies to enhance Tfh cell activation. Their integration and synergistic combination could further enhance immunity and Tfh responses (SAPN-RNA vaccines). In summary, we provide a comprehensive overview of the current insights into Tfh cells’ role during viral infections, emphasizing their potential as strategic targets for innovative vaccine development. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms in Immune Regulation)

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21 pages, 8811 KiB

Open AccessArticle

Empagliflozin Plays Vasoprotective Role in Spontaneously Hypertensive Rats via Activation of the SIRT1/AMPK Pathway

by Monika Kloza, Anna Krzyżewska, Hanna Kozłowska, Sandra Budziak and Marta Baranowska-Kuczko

Cells 2025, 14(7), 507; https://doi.org/10.3390/cells14070507 (registering DOI) - 29 Mar 2025

Abstract

Empagliflozin (EMPA), a sodium-glucose co-transporter 2 (SGLT2) inhibitor, prevents endothelial dysfunction, but its effects on vascular tone in hypertension remain unclear. This study investigated whether EMPA modulates vasomotor tone via sirtuin 1 (SIRT1) and AMP-activated protein kinase (AMPK) pathways in spontaneously hypertensive rats [...] Read more.

Empagliflozin (EMPA), a sodium-glucose co-transporter 2 (SGLT2) inhibitor, prevents endothelial dysfunction, but its effects on vascular tone in hypertension remain unclear. This study investigated whether EMPA modulates vasomotor tone via sirtuin 1 (SIRT1) and AMP-activated protein kinase (AMPK) pathways in spontaneously hypertensive rats (SHR) and controls (Wistar Kyoto rats, WKY). Functional (wire myography, organ bath) and biochemical (Western blot) studies were conducted on the third-order of the superior mesenteric arteries (sMAs) and/or aortas. EMPA induced concentration-dependent relaxation of preconstricted sMAs in both groups. In SHR, EMPA enhanced acetylcholine (Ach)-induced relaxation in sMAs and aortas and reduced constriction induced by phenylephrine (Phe) and U46619 in sMAs. The SIRT1 inhibitor (EX527) abolished EMPA’s effects on Ach-mediated relaxation and U46619-induced vasoconstriction, while AMPK inhibition reduced Ach-mediated relaxation and Phe-induced vasoconstriction. SHR showed increased SGLT2 and SIRT1 expression and decreased pAMPK/AMPK levels in sMAs. In conclusion, EMPA might exert vasoprotective effects in hypertension by enhancing endothelium-dependent relaxation and reducing constriction via AMPK/SIRT1 pathways. These properties could improve vascular health in patients with hypertension and related conditions. Further studies are needed to explore new indications for SGLT2 inhibitors. Full article

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15 pages, 886 KiB

Open AccessFeature PaperArticle

Lipocalin-2, Matrix Metalloproteinase-9, and MMP-9/NGAL Complex in Upper Aerodigestive Tract Carcinomas: A Pilot Study

by Luca Cavalcanti, Silvia Francati, Giampiero Ferraguti, Francesca Fanfarillo, Daniele Peluso, Christian Barbato, Antonio Greco, Antonio Minni and Carla Petrella

Cells 2025, 14(7), 506; https://doi.org/10.3390/cells14070506 (registering DOI) - 29 Mar 2025

Abstract

Upper aerodigestive tract (UADT) carcinomas have a high and rapidly increasing incidence, particularly in industrialized countries. The identification of diagnostic and prognostic biomarkers remains a key objective in oncological research. However, conflicting data have been reported regarding Lipocalin-2 (LCN-2 or NGAL), Matrix Metalloproteinase-9 [...] Read more.

Upper aerodigestive tract (UADT) carcinomas have a high and rapidly increasing incidence, particularly in industrialized countries. The identification of diagnostic and prognostic biomarkers remains a key objective in oncological research. However, conflicting data have been reported regarding Lipocalin-2 (LCN-2 or NGAL), Matrix Metalloproteinase-9 (MMP-9), and the MMP-9/NGAL complex in UADT carcinomas. For this reason, the primary aim of this study was to investigate the involvement and modulation of the LCN-2 system in UADT cancer by selecting patients at first diagnosis and excluding any pharmacological or interventional treatments that could act as confounding factors. In this clinical retrospective pilot study, we investigated LCN-2 and MMP-9 tissue gene expression, as well as circulating levels of LCN-2, MMP-9, and the MMP-9/NGAL complex. Our findings revealed a downregulation of LCN-2 and an upregulation of MMP-9 gene expression in tumor tissues compared to healthy counterparts. A similar trend was observed in circulating levels, with decreased LCN-2 and increased MMP-9 in cancer patients compared to healthy controls. Additionally, serum levels of the MMP-9/NGAL complex were significantly elevated in UADT cancer patients relative to controls. Our study suggests a potentially distinct role for the free form of LCN-2 and its conjugated form (MMP-9/NGAL complex) in UADT tumors. These findings not only provide new insights into the molecular mechanisms underlying tumor progression but also highlight the potential clinical relevance of these biomarkers. The differential expression patterns observed suggest that the LCN-2 and MMP-9/NGAL complex could serve as valuable tools for improving early diagnosis, monitoring disease progression, and potentially guiding therapeutic strategies. Further research is needed to validate their utility in clinical settings and to explore their prognostic and predictive value in personalized treatment approaches. Full article

(This article belongs to the Special Issue New Trends and Advances in Diagnosis and Prognosis of Head and Neck Cancer from Carcinogenesis to Future Molecular Targeted Therapies: 2nd Edition)

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32 pages, 1765 KiB

Open AccessReview

Preclinical Models for Studying Fuchs Endothelial Corneal Dystrophy

by Fancheng Sun, Lexie W. Q. Xi, Wesley Luu, Myagmartsend Enkhbat, Dawn Neo, Jodhbir S. Mehta, Gary S. L. Peh and Evelyn K. F. Yim

Cells 2025, 14(7), 505; https://doi.org/10.3390/cells14070505 (registering DOI) - 28 Mar 2025

Abstract

Fuchs Endothelial Corneal Dystrophy (FECD) is a corneal endothelial disease that causes microenvironment alterations and endothelial cell loss, which leads to vision impairment. It has a high global prevalence, especially in elderly populations. FECD is also one of the leading indications of corneal [...] Read more.

Fuchs Endothelial Corneal Dystrophy (FECD) is a corneal endothelial disease that causes microenvironment alterations and endothelial cell loss, which leads to vision impairment. It has a high global prevalence, especially in elderly populations. FECD is also one of the leading indications of corneal transplantation globally. Currently, there is no clearly defined canonical pathway for this disease, and it has been proposed that the combinatorial effects of genetic mutations and exogenous factors cause FECD. Clinical studies and observations have provided valuable knowledge and understanding of FECD, while preclinical studies are essential for gaining insights into disease progression and mechanisms for the development and testing of regenerative medicine therapies. In this review, we first introduce the proposed genetic and molecular pathologies of FECD. Notably, we discuss the impact of abnormal extracellular matrix deposition (guttata), endothelial-to-mesenchymal transition, cell senescence, and oxidative stress on the pathology and etiology of FECD. We review and summarize the in vitro cell models, ex vivo tissues, and in vivo animal models used to study FECD. The benefits and challenges of each model are also discussed. Full article

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16 pages, 3447 KiB

Open AccessArticle

Preserving Life: How Retinoic Acid (RA) Enhances Cell Viability and Reduces Apoptosis in Cryopreserved Blastocyst Cells of Pudong Chickens

by Lingwei Sun, Fuqin Liu, Mengqian He, Jiehuan Xu, Caifeng Wu, Shushan Zhagn, Jun Gao and Jianjun Dai

Cells 2025, 14(7), 504; https://doi.org/10.3390/cells14070504 - 28 Mar 2025

Abstract

The preservation of chicken embryonic cells is essential for protecting avian genetic resources and enhancing breeding programs. This study investigates the effects of retinoic acid (RA) on the viability, functionality, and adhesion of thawed chicken blastoderm cells (BCs) following cryopreservation. After thawing and [...] Read more.

The preservation of chicken embryonic cells is essential for protecting avian genetic resources and enhancing breeding programs. This study investigates the effects of retinoic acid (RA) on the viability, functionality, and adhesion of thawed chicken blastoderm cells (BCs) following cryopreservation. After thawing and culturing the cells for 24 h, RA treatment resulted in significantly higher cell viability and adhesion rates compared to the control group, with the 2.0 μM RA group demonstrating the best outcomes. After 48 and 72 h of culture, similar trends were observed, with the 2.0 μM RA group consistently maintaining the highest cell viability and adhesion rates. Furthermore, immunofluorescence TUNEL assays revealed that RA significantly reduced both early and late apoptosis rates, particularly at a concentration of 2.0 μM, which exhibited a strong protective effect. Flow cytometry analysis indicated that RA treatment enhanced the mitochondrial membrane potential (MMP), reflecting improved cellular health. Analysis of the apoptosis-related genes BAX, BCL-2, and Caspase-3 revealed that moderate RA concentrations promoted the expression of anti-apoptotic factors while also upregulating pro-apoptotic factors, with the 2.0 μM RA group exhibiting the highest expression levels. Cell cycle analysis showed that RA significantly influenced the distribution of BCs across different phases, with the 4.0 μM RA group exhibiting the highest proportion of cells in the G1/G0 phase, suggesting an enhanced tolerance to cryopreservation stress. Conversely, the S phase cell population was notably reduced at higher RA concentrations, indicating potential inhibition of cell proliferation. These results suggest that RA not only significantly enhances the survival rates and mitochondrial function of BCs, but also regulates the cell cycle, providing better conditions for BC cryopreservation. Overall, the addition of RA represents a valuable strategy for optimizing cryopreservation techniques in chicken embryonic cells, with implications for avian genetic resource conservation and breeding strategies. Full article

(This article belongs to the Section Reproductive Cells and Development)

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13 pages, 2027 KiB

Open AccessArticle

Cigarette Smoke Exposure Leads to Organic and Mineral Bone Component Changes: The Importance of Rho Kinase Function in These Events

by Alex Ferreira da Silva, Franciele Jesus Lima, Alyne Riani Moreira, Cintia do Nascimento Silva, Ivone Braga de Oliveira, Alexandra Fernandes Callera, Ana Luiza Porfirio, Luan Henrique Vasconcelos Alves, Iolanda de Fátima Lopes Calvo Tibério, Ana Paula Pereira Velosa, Vanda Jorgetti, Walcy Rosolia Teodoro and Fernanda Degobbi Tibério Quirino Dos Santos Lopes

Cells 2025, 14(7), 503; https://doi.org/10.3390/cells14070503 - 28 Mar 2025

Abstract

Aberrant Rho-associated kinase function could be associated with increased bone fragility. Since cigarette smoke (CS) exposure promotes the increase in bone fragility due to changes in bone tissue components, this study aimed to investigate how CS exposure could modulate the Rho kinase-associated bone [...] Read more.

Aberrant Rho-associated kinase function could be associated with increased bone fragility. Since cigarette smoke (CS) exposure promotes the increase in bone fragility due to changes in bone tissue components, this study aimed to investigate how CS exposure could modulate the Rho kinase-associated bone structural changes. Mice were assigned to four groups: control; smoke; control with Rho kinase inhibitor administration; and smoke with a Rho kinase inhibitor. Bone samples were obtained to assess bone histomorphometry analysis, type I collagen composition, and MEPE expression in trabeculae. We observed that CS exposure induced decreased trabecular and osteoid thickness. A concomitant increase in the osteoclastic and erosion surfaces and a decrease in the mineralization surface were observed. Additionally, CS exposure decreased the type I collagen and MEPE expression. Rho kinase inhibitor administration recovered the bone mineralization and the collagen type I deposition. Conclusions: CS exposure increases Rho kinase activity in bone cells, leading to structural changes. The administration of a Rho GTPases inhibitor partially reverses these effects, likely due to the recovery in osteoblast activity. Full article

(This article belongs to the Topic Osteoimmunology and Bone Biology)

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25 pages, 3911 KiB

Open AccessArticle

Genetic and Molecular Characterization of H9c2 Rat Myoblast Cell Line

by Thomas Liehr, Stefanie Kankel, Katharina S. Hardt, Eva M. Buhl, Heidi Noels, Diandra T. Keller, Sarah K. Schröder-Lange and Ralf Weiskirchen

Cells 2025, 14(7), 502; https://doi.org/10.3390/cells14070502 - 28 Mar 2025

Abstract

This study presents a comprehensive genetic characterization of the H9c2 cell line, a widely used model for cardiac myoblast research. We established a short tandem repeat (STR) profile for H9c2 that is useful to confirm the identity and stability of the cell line. [...] Read more.

This study presents a comprehensive genetic characterization of the H9c2 cell line, a widely used model for cardiac myoblast research. We established a short tandem repeat (STR) profile for H9c2 that is useful to confirm the identity and stability of the cell line. Additionally, we prepared H9c2 metaphase chromosomes and performed karyotyping and molecular cytogenetics to further investigate chromosomal characteristics. The genetic analysis showed that H9c2 cells exhibit chromosomal instability, which may impact experimental reproducibility and data interpretation. Next-generation sequencing (NGS) was performed to analyze the transcriptome, revealing gene expression patterns relevant to cardiac biology. Western blot analysis further validated the expression levels of selected cardiac genes identified through NGS. Additionally, Phalloidin staining was used to visualize cytoskeletal organization, highlighting the morphological features of these cardiac myoblasts. Our findings collectively support that H9c2 cells are a reliable model for studying cardiac myoblast biology, despite some genetic alterations identified resembling sarcoma cells. The list of genes identified through NGS analysis, coupled with our comprehensive genetic analysis, will serve as a valuable resource for future studies utilizing this cell line in cardiovascular medicine. Full article

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23 pages, 6875 KiB

Open AccessArticle

The Impact of TRPM8 on Prostate Cancer Transcriptomic Dynamics

by Swapna Asuthkar, Susovon Bayen, Erick B. Saldes, Benny Tom, Jai Velpula, Sarangi Siddharth, Timothy E. Koeltzow and Donald J. Vander Griend

Cells 2025, 14(7), 501; https://doi.org/10.3390/cells14070501 - 27 Mar 2025

Abstract

Prostate cancer (PC) remains a significant health challenge, with androgen receptor (AR) signaling playing a pivotal role in its progression. This study investigates the expression and functional implications of the transient receptor potential melastatin 8 (TRPM8) channel in PC, focusing on its interaction [...] Read more.

Prostate cancer (PC) remains a significant health challenge, with androgen receptor (AR) signaling playing a pivotal role in its progression. This study investigates the expression and functional implications of the transient receptor potential melastatin 8 (TRPM8) channel in PC, focusing on its interaction with AR and its impact on oncogenic pathways. We analyzed mRNA expression levels of TRPM8 and AR in PC tissues, revealing that TRPM8 is upregulated in benign and early-stage tumors but significantly downregulated in metastatic samples. This decline correlates with increased AR expression, suggesting a compensatory mechanism that enhances AR-driven tumorigenesis. RNA sequencing and pathway enrichment analyses demonstrated that TRPM8 knockout (KO) prostates exhibited significant alterations in gene expression, particularly in pathways related to extracellular matrix (ECM) remodeling, cell proliferation, and survival signaling. Notably, genes associated with metastasis, such as MMP2 and FAP, were upregulated in TRPM8 KO samples, indicating a potential role for TRPM8 in inhibiting tumor invasion. Furthermore, Gene Set Enrichment Analysis (GSEA) revealed positive enrichment of androgen response, angiogenesis, and epithelial–mesenchymal transition (EMT) pathways in TRPM8 KO prostates, reinforcing the notion that TRPM8 loss creates a pro-tumorigenic environment. Our findings suggest that TRPM8 functions as a molecular brake on PC progression, and its loss may contribute to the development of aggressive disease phenotypes. This study underscores the importance of TRPM8 as a potential therapeutic target and biomarker in PC, warranting further investigation into its role in cancer biology and treatment response. Full article

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18 pages, 717 KiB

Open AccessReview

Platelet Reactive Oxygen Species, Oxidised Lipid Stress, Current Perspectives, and an Update on Future Directions

by Lih T. Cheah, Matthew S. Hindle, Jawad S. Khalil, Cedric Duval, Amanda J. Unsworth and Khalid M. Naseem

Cells 2025, 14(7), 500; https://doi.org/10.3390/cells14070500 - 27 Mar 2025

Abstract

Blood platelets are anucleate cells that play a vital role in haemostasis, innate immunity, angiogenesis, and wound healing. However, the inappropriate activation of platelets also contributes to vascular inflammation, atherogenesis, and thrombosis. Platelet activation is a highly complex receptor-mediated process that involves a [...] Read more.

Blood platelets are anucleate cells that play a vital role in haemostasis, innate immunity, angiogenesis, and wound healing. However, the inappropriate activation of platelets also contributes to vascular inflammation, atherogenesis, and thrombosis. Platelet activation is a highly complex receptor-mediated process that involves a multitude of signalling intermediates in which Reactive Oxygen Species (ROS) are proposed to play an important role. However, like for many cells, changes in the balance of ROS generation and/or scavenging in disease states may lead to the adoption of maladaptive platelet phenotypes. Here, we review the diverse roles of ROS in platelet function and how ROS are linked to specific platelet activation pathways. We also examine how changes in disease, particularly the plasma oxidised low-density lipoprotein (oxLDL), affect platelet ROS generation and platelet function. Full article

(This article belongs to the Special Issue Molecular and Cellular Insights into Platelet Function)

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