Search Results (3,257)

Background: Eggshell membrane peptides (ESMPs) are natural bioactive compounds with reported chondroprotective properties. However, their regulatory effects on canine chondrocytes remain unclear. This study investigated ESMP in an interleukin-1β (IL-1β)-induced inflammatory model of canine chondrocytes. Methods: Chondrocytes were assigned to control (Cont), IL-1β, and ESMP + IL-1β groups. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. NF-κB p65 nuclear translocation was evaluated by immunofluorescence staining. Real-time quantitative PCR (RT-qPCR) and Western blotting (WB) were used to measure mRNA and protein expression levels, respectively. Results: ESMP inhibited IL-1β-induced NF-κB p65 nuclear translocation and reduced the IL-1β-induced increases in interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and matrix metalloproteinase-13 (MMP-13) at both mRNA and protein levels. ESMP also decreased IL-6, nitric oxide (NO), and prostaglandin E₂ (PGE₂) levels in culture supernatants. ESMP reversed the IL-1β-induced reduction in type II collagen α1 chain (COL2A1) and aggrecan (ACAN) expression at both transcriptional and protein levels. Conclusions: ESMP attenuates IL-1β-induced inflammatory responses and extracellular matrix degradation in canine chondrocytes, potentially associated with suppression of NF-κB p65 nuclear translocation. This supports its potential application in promoting joint health in dogs. Full article

Choriogenesis, the final stage of oogenesis in insects, is a highly coordinated developmental process responsible for the formation of the chorion (eggshell), a specialized multilayered extracellular matrix that protects the embryo and mediates essential physiological functions. Despite its fundamental importance for reproductive success and species survival, the mechanisms underlying chorion biogenesis remain incompletely understood across insect taxa. This review provides an updated synthesis and integrated view of choriogenesis, including cellular, molecular, biochemical, and structural perspectives. We examine the role of follicle cells in chorion formation, the regulatory mechanisms governing chorion gene expression, and the biochemical composition of the eggshell, including proteins, lipids, and carbohydrates. In addition, we compare the structural diversity of the chorion across insect taxa, highlighting both conserved multilayered organization and lineage-specific adaptations in surface morphology and internal architecture. Full article

Background: Chronic enteropathy associated with the SLCO2A1 gene (CEAS) is a rare disease characterized by multiple small intestinal ulcers whose pathogenesis remains poorly understood. This study aimed to characterize the proteomic and phosphoproteomic profiles of CEAS and to identify molecular pathways involved in its pathogenesis. Methods: Quantitative proteomics and phosphoproteomics were performed on intestinal mucosal tissues from patients with CEAS (n = 3), Crohn’s disease (CD, n = 3), and healthy controls (n = 3). Differentially expressed proteins (DEPs) and differentially phosphorylated proteins (DPPs) were analyzed using functional enrichment, gene set enrichment analysis (GSEA), protein–protein interaction (PPI) networks, and integrative analysis. Results: A total of 900 DEPs were identified in CEAS and 277 in CD relative to controls, including 717 CEAS-specific proteins. CEAS-specific alterations were strongly enriched in focal adhesion and extracellular matrix-related pathways, whereas shared proteins between CEAS and CD were primarily associated with epithelial barrier function, including tight junction and adherens junction pathways. GSEA revealed that CEAS was characterized by upregulation of tissue remodeling and focal adhesion pathways, accompanied by suppression of digestive and metabolic processes, while CD exhibited prominent adaptive immune activation. PPI network analysis identified POSTN, CDH1, TLN1, and VIM as candidate hub proteins; however, none retained significance after FDR correction, whereas brush-border components (CDHR2, MUC3A, MUC13, ALPI) and actin cytoskeletal regulators remained the most statistically robust alterations. Integrated analysis further highlighted focal adhesion-related proteins with coordinated expression and phosphorylation changes. Conclusions: This exploratory study provides the first integrative proteomic and phosphoproteomic characterization of CEAS, suggesting that impairment of the intestinal brush border and mucosal barrier, together with actin cytoskeletal reorganization, may distinguish CEAS from immune-dominant CD. These findings are hypothesis-generating and require validation in larger cohorts. Full article

Three-dimensional (3D) endothelium–stromal co-cultures were established using human endometrial cells from biopsy of healthy women (n = 13) and serum samples from both healthy and endometriotic women (n = 5). For 3D construction, stromal cells were mixed with extracellular matrix components, followed by endothelial cell seeding. Morphological analysis confirmed the organization of tissue-like structures. Immunofluorescence and flow cytometry verified the expression of specific stromal and endothelial markers (Cytokeratin, Vimentin, Insulin-like growth factor-binding protein 1, and von Willebrand factor). Cell viability and proliferation increased over time, with minimal cell death. To test functional responsiveness, these co-cultures were exposed to inflammatory serum from endometriotic patients. After 48 h, cytometric bead array showed elevated levels of IL-1β, IL-6, and IL-8 in cultures treated with inflammatory serum, indicating preserved functional activity and responsiveness. By allowing detailed investigation of functional endometrial states within a physiologically relevant cellular network, this approach provides a valuable organoid-like tool to explore conditions such as implantation failure and infertility and to study the cellular interactions underlying reproductive pathologies. Full article

Background/Objectives: Only substantial quantities of xeno-free human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) (hiPSC-CMs) with stable quality and structural and functional maturity can meet the demand for cardiac cell therapy. The use of xeno-free microcarriers can significantly increase cell yield. Co-culturing with hematopoietic stem cells (HSCs) simulates the environment in vivo and has a necessary impact on the development of CMs. However, no microcarrier-based protocol for xeno-free hiPSC-CM culture has yet been established, and the effects of HSCs on CM development and their underlying mechanisms remain unclear. Therefore, this study aims to investigate these issues. Methods: We used a xeno-free microcarrier (plastic) culture system coated by a defined xeno-free matrix (MatriClone) to expand hiPSCs and hiPSC-CMs with human hematopoietic stem cells (hHSCs). Using RNA sequencing (RNA-seq), cytokine assay, and various cellular molecular techniques, we investigated the role of hHSCs in cardiac differentiation and maturation, and underlying mechanisms. Results: hiPSCs were evenly distributed on the surface of plastic coated with 1 μg/cm² MatriClone (MatriClone-Plastic), increasing and sustaining pluripotency marker levels. Directed differentiation of hiPSCs on 1 μg/cm² MatriClone-Plastic induced a larger number of CMs, and the level of cardiac differentiation was also significantly improved. When hHSCs were co-cultured with cells at the cardiac progenitor cell stage, results from electron microscopy, electrophysiology, and qPCR showed that hiPSC-CMs significantly promoted cardiac structural and functional maturation. The co-cultured hHSCs released multiple cytokines that were changed dynamically at different time points, and that were highly likely to activate the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway to promote cardiac development and maturation. Conclusions: hHSCs can efficiently promote differentiation and maturation of xeno-free hiPSC-CMs on MatriClone-Plastic via the EGFR/MAPK/ERK signaling pathway. Full article

Extracellular vesicles (EVs), particularly small extracellular vesicles (sEVs), are lipid bilayer-delimited particles involved in intercellular communication through the transfer of proteins, lipids, and nucleic acids; many products and studies in aesthetic medicine refer to these preparations as exosomes, although endosomal origin is not always demonstrated. This review examines current evidence on the mechanisms, clinical effectiveness, safety, therapeutic applications, and regulatory challenges of EV- and sEV-based interventions, complemented by an exploratory qualitative assessment of physicians’ perceptions regarding clinical implementation. A narrative review of studies indexed in Scopus and PubMed was conducted with emphasis on skin rejuvenation, hair restoration, wound healing, pigmentation disorders, and inflammatory dermatoses, and responses from 12 aesthetic physicians in Colombia were analyzed qualitatively. Available evidence suggests that EVs/sEVs may promote extracellular matrix remodeling, angiogenesis, immunomodulation, and tissue repair, with potential benefits across several aesthetic and regenerative indications. However, the literature remains heterogeneous and limited by variability in biologic sources, isolation and administration protocols, insufficient high-quality clinical trials, and unresolved regulatory issues. Reports of adverse reactions linked to unapproved products marketed as exosome-based formulations further highlight the need for stronger oversight. EVs, particularly sEVs, often referred to as exosomes in the aesthetic literature, remain a promising therapeutic platform, but safe clinical integration requires rigorous validation, technical standardization, and robust regulatory frameworks. Full article

Vein graft restenosis is a leading cause of long-term failure after coronary artery bypass grafting (CABG), driven by maladaptive vascular smooth muscle cell (VSMC) responses to arterialization-induced inflammation. The key molecular mediators of this pathological remodeling, however, remain incompletely defined. Here, we integrated multi-omics analyses of human and canine vein graft specimens with in vitro functional assays to identify tenascin-C (TNC)—a matricellular extracellular matrix protein—as a critical regulator of VSMC dysfunction. TNC was specifically enriched in a synthetic, pro-inflammatory VSMC subpopulation. Pro-inflammatory stimuli potently induced TNC expression, which was functionally linked to VSMC phenotypic modulation, hyperproliferation, and enhanced migration. Mechanistically, TNC acts upstream of NF-κB signaling; siRNA-mediated TNC knockdown significantly reduced nuclear p65 protein levels and attenuated inflammatory responses. Our integrated computational and experimental data suggest that TNC, NF-κB, and TNF-α function within a sequential pro-inflammatory signaling cascade that sustains vascular inflammation and promotes neointimal hyperplasia. These findings reposition TNC from a passive structural component to an active driver of vascular pathology and highlight the TNC–NF-κB axis as a candidate target for therapeutic intervention to improve vein graft patency. Full article

Regenerative medicine integrates interdisciplinary approaches towards restoring the function of diseased organs. This study examined alterations that occurred in the liver under a high-fat diet (HFD) with the development of obesity and fatty liver, and changes in metabolic homeostasis and glucose levels, in mice. HFD nutrition causes hyperglycemia, leading to the formation and accumulation of advanced glycation end-products (AGEs) promoting protein post-translational modifications (PTMs) and introducing crosslinking in the extracellular matrix (ECM). Using histological and gene expression analyses, we detected an increase in adiposity, as well as in ECM protein deposition in the liver. Further, decellularization of the liver yielded the isolated ECM organ scaffold, allowing us to analyze the chemical modification in proteins by various imaging methods combined with spectroscopy. The measurements of intrinsic protein fluorescence are consistent with increased AGE-associated levels. SEM allows for the visualization of ECM fiber thickening as a result of protein crosslinking. Using cathodoluminescence, a label-free imaging method, we confirmed the protein modifications. The combination of innovative technologies highlights the ECM structural alterations associated with impaired glucose regulation and liver adiposity. These findings provide novel views on liver-scaffold ECM structure under metabolic diseases that will play a significant role in accelerating the understanding of effective regenerative therapies. Full article

Protease-activated receptor 1 (PAR1), a G protein-coupled receptor, plays a central role in coordinating multiple phases of cutaneous wound healing, including hemostasis, cell proliferation, migration, and extracellular matrix remodeling. Despite its therapeutic potential, PAR1-selective positive allosteric modulators (PAMs) remain limited. Here, we characterized the wound healing efficacy of gestodene, a third-generation progestin previously identified as a selective PAM of PAR1. Gestodene exhibited no intrinsic agonist activity but selectively potentiated PAR1-activating peptide (PAR1-AP)-induced calcium signaling without affecting PAR2 or PAR4 responses. Consistently, gestodene induced a concentration-dependent leftward shift in the PAR1-AP dose–response curve. Notably, gestodene enhanced PAR1-dependent cell proliferation, migration, and ERK1/2 activation, effects abolished by PAR1 knockout or pharmacological inhibition with vorapaxar in human keratinocytes (HaCaT) and dermal fibroblasts (HDF). Gestodene also potentiated the expression of wound healing-associated genes, including matrix metalloproteinases (MMP-1, -2, -3, -10), fibronectin, and type I collagen (COL1A1). In a murine wound model, topical administration of gestodene accelerated wound closure, achieving complete re-epithelialization by Day 8 and significantly enhancing collagen deposition, effects reversed by vorapaxar. Collectively, these findings demonstrate that gestodene accelerates cutaneous wound healing through PAR1-selective positive allosteric modulation and supports its potential as a drug repositioning candidate for wound repair. Full article

Calcium hydroxyapatite (CaHA)-based dermal fillers have been shown to help counteract and potentially reverse certain aspects of skin aging. By applying isolated CaHA microspheres, we investigated the importance of the direct contact of dermal cells to microspheres and their role for the expression of extracellular matrix (ECM) components. To this end, human dermal fibroblasts were cultured in the presence of CaHA microspheres. Cell migration, cell–microsphere interaction, and CaHA dose-dependent effects on the expression of ECM proteins were examined using microscopy, mRNA and protein expression analysis. Our results indicated that fibroblasts established direct and close contact to CaHA microspheres. This interaction was associated with a time- and dose-dependent increase in ECM protein expression, including collagen-1, emilin-1, elastin, fibulin-5, fibronectin, and the proteoglycans—lumican and versican. These observations indicate that direct contact between fibroblasts and CaHA microspheres promotes ECM protein expression, suggesting a role for this interaction in supporting skin regeneration and counteracting age-related changes, potentially augmented in vivo by immunomodulatory effects. Full article

Background and Objectives: Temporomandibular disorders (TMDs) encompass a broad spectrum of functional and structural abnormalities of the temporomandibular joint (TMJ). Conventional diagnostic tools, although essential, often fail to capture the underlying biochemical mechanisms driving disease progression. Synovial fluid (SF), by virtue of its direct proximity to intra-articular tissues, represents an accessible biological matrix for identifying molecular signatures of inflammation, cartilage degradation, lubrication failure, oxidative stress, and angiogenic activation. The objective of this review is to synthesize current evidence on SF proteomics in TMD and evaluate its potential translational value in precision medicine. Materials and Methods: A narrative review of the literature was conducted on PubMed to identify human studies focused on SF proteomic and biochemical biomarkers in TMD. Eligible studies included original research articles assessing SF composition in relation to specific TMJ pathologies, diagnostic categories, or clinical phenotypes. Extracted data included study design, sample characteristics, analytic methodology, biomarkers investigated, and key findings. Google Gemini (Google LLC, Mountain View, CA, USA) was used as an AI-assisted tool to support language editing and manuscript writing during the preparation of this article. The use of this tool was limited to linguistic refinement; all scientific content, data interpretation, and conclusions were formulated and verified by the authors. Results: Across the analyzed studies, TMD phenotypes—particularly disc displacement with or without reduction (DDwR, DDwoR) and osteoarthritis (OA)—were characterized by consistent alterations in cytokines (IL-1β, IL-6, IL-8, TNF-α), extracellular matrix (ECM) components (aggrecan, glycosaminoglycans (GAGs), decorin, MMP-2, MMP-9), lubrication molecules (lubricin/PRG4), oxidative stress mediators (myeloperoxidase (MPO), nitric oxide (NO), glutathione peroxidase (GPX)), adipokines (chemerin, resistin, adiponectin), and angiogenic factors (vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2)). Recent liquid chromatography–tandem mass spectrometry (LC–MS/MS) analyses further revealed phenotype-specific protein clusters and pathways related to inflammation, ferroptosis, hypoxia signaling, and proteoglycan metabolism. Conclusions: Current evidence suggests that SF proteomics and multi-analyte biomarker profiling offer a promising, hypothesis-generating approach for understanding the biological mechanisms underlying TMD. The integration of proteomic, metabolic, and inflammatory markers holds future potential for diagnostic panel development; however, prospective clinical validation is still required before SF-based molecular profiling can be implemented as a precision medicine tool in TMJ disorders. Full article

Background: Systemic sclerosis (SSc)-associated interstitial lung disease (SSc-ILD) is the leading cause of morbidity and mortality in patients with SSc, with an unmet need for validated, minimally invasive biomarkers for early detection. Extracellular vesicles (EVs) present underexplored pathogenic players and potential biomarker sources in SSc-ILD. We performed a proteomic shotgun study aiming to identify disease-specific protein signatures and potential biomarker candidates. Methods: The study included 30 SSc patients divided into SSc-ILD and SSc w/o ILD groups and 20 matched controls. Plasma-derived EV-enriched fractions were analysed using liquid chromatography–mass spectrometry. Bioinformatic analysis, including differentially expressed proteins (DEPs), functional enrichment, protein–protein interaction network and Markov Cluster (MCL) analysis was performed. Results: Analysis of DEPs showed 14 significantly upregulated and 1 downregulated protein when comparing the SSc-ILD to the SSc w/o ILD group, 222 upregulated and 257 downregulated proteins between the SSc-ILD and control group, as well as 362 upregulated and 492 downregulated proteins between the SSc w/o ILD and control group. Functional enrichment analysis and MCL analysis pointed to disease-specific processes of extracellular matrix (ECM) and immune dysregulation, which largely overlapped between SSc-ILD and SSc w/o ILD groups. Among identified DEPs, SP-B, Cav-1 and Siglec-5 emerged as potential candidate biomarkers for SSc-ILD. Conclusions: Proteomic analysis of plasma-derived EV-enriched fractions shows potential EV involvement in pathogenic SSc processes, mainly related to ECM and immune dysregulation, as well as potential candidate biomarkers for SSc-ILD. Further studies are required to validate these results and assess biomarker potential and translational applicability of identified proteins. Full article

Introduction: The 3 Rs principle advocates developing alternative, biologically relevant models. Thus, 3D fish liver in vitro models have been increasingly used for ecotoxicological studies. We previously optimized spheroids from the rainbow trout non-tumoral liver cell line RTL-W1 and employed them to assess the effects of aquatic pollutants. Although they demonstrated potential for assessing ecotoxicological effects, further optimization is warranted to enhance their physiological relevance. Incorporating an extracellular matrix (ECM), such as collagen, has been shown to be a promising strategy to improve spheroids’ structural organization and functionality. Objective: This study aimed to optimize 3D culturing conditions of RTL-W1 spheroids by evaluating the effects of collagen supplementation on viability, morphology, and functional response. Methodology: Spheroids from the RTL-W1 cell line (60,000 cells per well) were cultured in 96-well ultra-low attachment (ULA) plates at 18 °C. After spheroids’ formation, rat tail collagen was supplemented at concentrations of 15 (C15), 30 (C30), and 60 (C60) µg/mL at culture days 7, 8, and 9. Spheroids were collected at two sampling days (10 and 14). Viability was assessed using alamarBlue and lactate dehydrogenase (LDH) assays, while morphology was assessed by optical microscopy. Collagen penetration was evaluated using Masson’s trichrome staining technique. Protein expression of cytochrome P450(CYP)1A was assessed by quantifying immunocytochemistry staining using an anti-CYP1A antibody. Results: On day 10, LDH leakage decreased in C15 and C60, compared with the control, whilst C15 spheroids showed lower absorbance levels in the alamarBlue assay. On day 14, LDH showed no significant differences; however, C30 and C60 had higher alamarBlue absorbance, indicating greater metabolic capacity. Spheroid morphology appeared intact in all conditions. Masson trichrome revealed collagen fibrils at the periphery of the spheroids, especially in C30 and C60, indicating that spheroids incorporated collagen. CYP1A immunostain was present in all conditions, localized in the spheroids’ border, and tended to be higher when supplementation occurred in earlier days. Conclusions: Our results suggest that RTL-W1 spheroids interacted with the collagen matrix and appeared to functionally improve. Data suggest that incorporating ECM may increase the complexity and physiological relevance of RTL-W1 spheroids, thereby better supporting mechanistic and ecotoxicological applications. Full article

The lysosomal enzyme α-galactosidase A (AGAL) degrades globotriaosylceramide (Gb₃). While this enzymatic function in lysosomal metabolism is well characterized, interaction partners and alternative functions are unknown. This study aims to identify new potential AGAL-interacting proteins. AGAL was fused to the mutated biotin ligase BirA from E. coli (TurboID). Expression of the fusion protein was confirmed by Western blot and immunofluorescence, while enzymatic activity was verified by functional assays. In three experimental settings (AGAL wild-type (WT), AGAL missense variant (p.N215S), and the control cell line), TurboID-biotinylated proximal proteins were enriched by streptavidin pull-down and analyzed by mass spectrometry. Gene Ontology (GO) terms were subsequently evaluated to characterize biological functions and localizations of the identified proteins. Selected candidates were co-immunoprecipitated with AGAL to confirm direct interactions. The AGAL-TurboID fusion protein was successfully expressed in AB8/13 podocytes. Immunofluorescence and enzyme activity assays confirmed the presence and functionality of the fusion protein. Subsequent functional analysis (GO term analysis) showed enrichment of driver terms, including extracellular matrix organization (ECM), multicellular organism development, and protein metabolic process, in the biological process category. The identified top-hit proteins were predominantly involved in the organization of ECM, cell proliferation and cytokinesis, unfolded protein response during endoplasmic reticulum stress, and protein ubiquitination. Co-immunoprecipitation confirmed the interaction between AGAL and the candidate Galectin-3-binding protein (Gal-3BP). Our results suggest that AGAL may play a role in other pathways and/or the ECM organization beyond its lysosomal function. The confirmed interaction with Gal-3BP can now be functionally investigated in further studies. Full article

Cervical cancer is a common gynecological malignancy, with a 5-year survival rate of only 17% for recurrent or metastatic cases. Increased extracellular matrix stiffness, a key change in the tumor mechanical microenvironment, promotes tumor metastasis via mechanotransduction. Piezo1, a mechanosensitive cation channel, senses matrix stiffness and converts mechanical signals into intracellular chemical signals. Neutrophil extracellular traps (NETs) are overformed in tumors, but the mechanism by which matrix stiffness regulates NETs in cervical cancer remains unclear. We detected matrix stiffness and related protein expression in cervical cancer tissues using atomic force microscopy and histochemical staining. Polyacrylamide gel models were used to culture HeLa/SiHa cells, with transcriptome sequencing and ELISA to analyze IL-8 expression. NETs were induced from human peripheral blood neutrophils, and their effect on lymphatic endothelial cells was evaluated. A TC-1 mouse model was used to verify in vivo effects, and Western blot/ELISA explored the Piezo1/NF-κB pathway. Higher Young’s modulus, increased α-SMA/Collagen I expression and collagen content in metastatic cervical cancer tissues. High matrix stiffness activated Piezo1/NF-κB, upregulated IL-8, induced NETs, and enhanced lymphatic endothelial cell tube formation/migration. BAPN reduced tumor stiffness, inhibited metastasis, and decreased NETs in mice. Knocking down Piezo1 blocked NF-κB activation and IL-8 upregulation. High matrix stiffness activates Piezo1/NF-κB to promote IL-8 secretion and NETs formation, enhancing lymphangiogenesis and cervical cancer metastasis, providing a new target for advanced cervical cancer treatment. Full article