Extracellular Matrix Research Articles

Chronic inhalation exposure to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) alters cardiac collagen in Wistar rats.

2,4-dichlorophenoxyacetic acid (2,4-D) is one of the most used in the world and exposure to herbicides can affect animals and humans, causing toxic effects that include cardiotoxicity. This is the first study to evaluate cardiac remodeling after experimental simulation of environmental exposure by chronic inhalation (6months) to the herbicide 2,4-D. Thirty male Wistar rats were exposed to two different concentrations of the 2,4-D formulation (low - 187.17mg/m3; and high - 467.93mg/m3) and the control group exposed to nebulization of chloride solution 0.9% sodium. Inhalation exposure lasted 6months. Mice hearts were collected for histology. There was a difference between exposure concentrations in relation to the increase in cardiac collagen (P<0.0001). In mice exposed to a low dose of 2,4-D and a decrease in the fractal dimension of cardiac collagen in the high dose of 2, 4-D (P=0.010). This study shows that chronic inhalation exposure to 2,4-D induces cardiotoxicity in rats, characterized by extracellular matrix reorganization and changes in collagen levels, highlighting the impact of the exposure route on cardiac outcomes. There was no difference in relation to anatomical parameters, cardiomyocyte area, collagen types I and III and analysis of arteriole thickness. Chronic exposure at different doses to the 2,4D herbicide had the potential to cause damage to cardiac remodeling by altering cardiac collagen in rats.

Targeting SLITRK4 Restrains Proliferation and Liver Metastasis in Colorectal Cancer via Regulating PI3K/AKT/NFκB Pathway and Tumor-Associated Macrophage.

Liver metastasis is the major cause of death in colorectal cancer (CRC) due to the lack of effective treatment. To explore novel drivers of CRC liver metastasis, the transcriptomes of primary paracancerous, colorectal tumors and metastases from human patients are profiled. It is found that SLIT- and NTRK-like family member 4 (SLITRK4) is the top upregulated gene in liver metastases and is associated with worse overall survival of CRC patients. Multiple in vitro and in vivo models suggested SLITRK4 promoted CRC tumorigenesis, invasion, migration, and angiogenesis, and inhibition of it restrained CRC tumor growth and liver metastasis with a more profound effect on the tumor microenvironment (TME). Mechanistically, SLITRK4 overexpression significantly activated the PI3K/AKT/NFκB pathway, regulated extracellular matrix organization, and multiple cytokines expression. Furthermore, the results from coculture models and single-cell RNA sequencing analyses suggested SLITRK4 promoted tumor-associated macrophages (TAMs) infiltration and polarization. In addition, macrophage depletion significantly inhibited SLITRK4-induced liver metastasis in CRC. Finally, pharmacological inhibition of SLITRK4 by using lipid-polymer hybrid nanoparticles (NPs) for systemic siRNA delivery can effectively inhibit CRC liver metastasis. Taken together, these results pinpoint that SLITRK4 regulates CRC tumorigenesis and liver metastasis, and siRNA delivering NPs agents validate the therapeutic potential of targeting SLITRK4 in CRC.

Exploring Metabolic Mechanisms in Calcific Tendinopathy and Shoulder Arthrofibrosis: Insights and Therapeutic Implications

Rotator cuff calcific tendinopathy and arthrofibrosis of the shoulder (adhesive capsulitis) are debilitating musculoskeletal disorders that significantly impact joint function and impair quality of life. Despite its high prevalence and common clinical presentation, the metabolic mechanisms underlying these conditions characterized by pain, and reduced mobility, remain poorly understood. This review aims to elucidate the role of metabolic processes implicated in the pathogenesis of calcific tendinopathy and shoulder arthrofibrosis. We will be focusing on the mechanistic role of how these processes contribute to disease progression and can direct potential therapeutic targets. Calcific tendinopathy is marked by aberrant calcium deposition within tendons, influenced by disrupted calcium and phosphate homeostasis, and altered cellular responses. Key molecular pathways, including bone morphogenetic proteins (BMPs), Wnt signaling, and transforming growth factor-beta (TGF-β), play crucial roles in the pathophysiology of calcification, calcium imbalance, and muscle fibrosis. In contrast, shoulder arthrofibrosis involves excessive collagen deposition and fibrosis within the shoulder joint capsule, driven by metabolic dysregulation and inflammation. The TGF-β signaling pathway and inflammatory cytokines, such as interleukin-6 (IL-6), are central to the fibrotic response. A comparative analysis reveals both shared and distinct metabolic pathways between these conditions, highlighting the interplay between inflammation, cellular metabolism, extracellular matrix remodeling, calcific deposition, and calcium migration to the glenohumeral joints, resulting in adhesive capsulitis, thereby providing insights into their pathophysiology. This review discusses current therapeutic approaches and their limitations, advocating for the development of targeted therapies that address specific metabolic dysregulations. Future therapeutic strategies focus on developing targeted interventions that address the underlying metabolic dysregulation, aiming to improve patient outcomes and advance clinical management. This review offers a comprehensive overview of the metabolic mechanisms involved in calcific tendinopathy and shoulder arthrofibrosis, providing a foundation for future research and therapeutic development.

Regression of hepatic fibrosis after pharmacological therapy for nonalcoholic steatohepatitis.

The global incidence of nonalcoholic fatty liver disease (NAFLD) is escalating considerably. NAFLD covers a range of liver conditions from simple steatosis to the more severe form known as nonalcoholic steatohepatitis, which involves chronic liver inflammation and the transformation of hepatic stellate cells into myofibroblasts that generate excess extracellular matrix, leading to fibrosis. Hepatocyte ballooning is a key catalyst for fibrosis progression, potentially advancing to cirrhosis and its decompensated state. Fibrosis is a critical prognostic factor for outcomes in patients with NAFLD; therefore, those with substantial fibrosis require timely intervention. Although liver biopsy is the most reliable method for fibrosis detection, it is associated with certain risks and limitations, particularly in routine screening. Consequently, various noninvasive diagnostic techniques have been introduced. This review examines the increasing prevalence of NAFLD, evaluates the noninvasive diagnostic techniques for fibrosis, and assesses their efficacy in staging the disease. In addition, it critically appraises current and emerging antifibrotic therapies, focusing on their mechanisms, efficacy, and potential in reversing fibrosis. This review underscores the urgent need for effective therapeutic strategies, given the dire consequences of advanced fibrosis.

Application of Fetal Membranes and Natural Materials for Wound and Tissue Repair

The human fetal membrane is a globally accepted biological biomaterial for wound and tissue repair and regeneration in numerous fields, including dermatology, ophthalmology, and more recently orthopedics, maxillofacial and oral surgery, and nerve regeneration. Both cells and matrix components of amnion and chorion are beneficial, releasing a diverse range of growth factors, cytokines, peptides, and soluble extracellular matrix components. Beside fetal membranes, numerous natural materials have also been reported to promote wound healing. The biological properties of these materials may potentiate the pro-healing action of fetal membranes. Comparison of such materials with fetal membranes has been scant, and their combined use with fetal membranes has been underexplored. This review presents an up-to-date overview of (i) clinical applications of human fetal membranes in wound healing and tissue regeneration; (ii) studies comparing human fetal membranes with natural materials for promoting wound healing; and (iii) the literature on the combined use of fetal membranes and natural pro-healing materials.

Establishment of 3D Cultures of Myometrium, Leiomyoma, and Leiomyosarcoma Cells: Advantages and Disadvantages of Two Different Models.

Uterine leiomyomas are the most common benign, monoclonal, gynaecological tumors in a woman's uterus, while leiomyosarcoma is a rare but aggressive condition caused by the malignant transformation of the myometrium. To overcome the common obstacles related to the methods usually used to study these pathologies, we aimed to devise three-dimensional models of myometrium, uterine leiomyoma and leiomyosarcoma cell lines, using two different types of biocompatible scaffolds. Specifically, we exploited the agarose gel matrix in common 6-well plates and the alginate matrix using Bioprinting INKREDIBLE + (CELLINK), a pneumatic extruded base equipped with a system with double printheads, and a UV printer LED curing system. Both methods allowed the development of 3D spheroids of all three cell types, that were also suitable for morphological investigations. We showed that all cell types embedded in both agarose and alginate formed spheroids in their growth medium. The spheroids successfully proliferated and self-organized into complex structures, developing a sustainable system that emulated the condition of the tissues through the accumulation of extracellular matrix. These models could be useful for a better understanding of pathophysiology, etiopathogenesis, and testing new methods or molecules from a preventive and therapeutic point of view.

Transcriptomics Revealed Differentially Expressed Transcription Factors and MicroRNAs in Human Diabetic Foot Ulcers

Non-healing diabetic foot ulcers (DFUs) not only significantly increase morbidity and mortality but also cost a lot and drain healthcare resources. Persistent inflammation, decreased angiogenesis, and altered extracellular matrix remodeling contribute to delayed healing or non-healing. Recent studies suggest an increasing trend of DFUs in diabetes patients, and non-healing DFYs increase the incidence of amputation. Despite the current treatment with offloading, dressing, antibiotics use, and oxygen therapy, the risk of amputation persists. Thus, there is a need to understand the molecular and cellular factors regulating healing in DFUs. The ongoing research based on proteomics and transcriptomics has predicted multiple potential targets, but there is no definitive therapy to enhance healing in chronic DFUs. Increased or decreased expression of various proteins encoded by genes, whose expression transcriptionally and post-transcriptionally is regulated by transcription factors (TFs) and microRNAs (miRs), regulates DFU healing. For this study, RNA sequencing was conducted on 20 DFU samples of ulcer tissue and non-ulcerated nearby healthy tissues. The IPA analysis revealed various activated and inhibited transcription factors and microRNAs. Further network analysis revealed interactions between the TFs and miRs and the molecular targets of these TFs and miRs. The analysis revealed 30 differentially expressed transcription factors (21 activated and 9 inhibited), two translational regulators (RPSA and EIF4G2), and seven miRs, including mir-486, mir-324, mir-23, mir-186, mir-210, mir-199, and mir-338 in upstream regulators (p &lt; 0.05), while causal network analysis (p &lt; 0.05) revealed 28 differentially expressed TFs (19 activated and 9 inhibited), two translational regulators (RPSA and EIF4G2), and five miRs including mir-155, mir-486, mir-324, mir-210, and mir-1225. The protein–protein interaction analysis revealed the interaction of various novel proteins with the proteins involved in regulating DFU pathogenesis and healing. The results of this study highlight many activated and inhibited novel TFs and miRs not reported in the literature so far, as well as the targeted molecules. Since proteins are the functional units during biological processes, alteration of gene expression may result in different proteoforms and protein species, making the wound microenvironment a complex protein interaction (proteome complexity). Thus, investigating the effects of these TFs and miRs on protein expression using proteomics and combining these results with transcriptomics will help advance research on DFU healing and delineate potential therapeutic strategies.

Bio-orthogonal tuning of matrix properties during 3D cell culture to induce morphological and phenotypic changes.

Described herein is a protocol for producing a synthetic extracellular matrix that can be modified in situ during three-dimensional cell culture. The hydrogel platform is established using modular building blocks employing bio-orthogonal tetrazine (Tz) ligation with slow (norbornene, Nb) and fast (trans-cyclooctene, TCO) dienophiles. A cell-laden gel construct is created via the slow, off-stoichiometric Tz/Nb reaction. After a few days of culture, matrix properties can be altered by supplementing the cell culture media with TCO-tagged molecules through the rapid reaction with the remaining Tz groups in the network at the gel-liquid interface. As the Tz/TCO reaction is faster than molecular diffusion, matrix properties can be modified in a spatiotemporal fashion simply by altering the identity of the diffusive species and the diffusion time/path. Our strategy does not interfere with native biochemical processes nor does it require external triggers or a second, independent chemistry. The biomimetic three-dimensional cultures can be analyzed by standard molecular and cellular techniques and visualized by confocal microscopy. We have previously used this method to demonstrate how in situ modulation of matrix properties induces epithelial-to-mesenchymal transition, elicits fibroblast transition from mesenchymal stem cells and regulates myofibroblast differentiation. Following the detailed procedures, individuals with a bachelor's in science and engineering fields can successfully complete the protocol in 4-5 weeks. This protocol can be applied to model tissue morphogenesis and disease progression and it can also be used to establish engineered constructs with tissue-like anisotropy and tissue-specific functions.

A bovine model of hypoxia-induced pulmonary hypertension reveals a gradient of immune and matrisome response with a complement signature found in circulation.

Pulmonary hypertension (PH) is a progressive vascular disease characterized by vascular remodeling, stiffening, and luminal obstruction, driven by dysregulated cell proliferation, inflammation, and extracellular matrix (ECM) alterations. Despite the recognized contribution of ECM dysregulation to PH pathogenesis, the precise molecular alterations in the matrisome remain poorly understood. In this study, we employed a matrisome-focused proteomics approach to map the protein composition in a young bovine calf model of acute hypoxia-induced PH. Our findings reveal distinct alterations in the matrisome along the pulmonary vascular axis, with the most prominent changes observed in the main pulmonary artery. Key alterations included a strong immune response and wound repair signature, characterized by increased levels of complement components, coagulation cascade proteins, and provisional matrix markers. Additionally, we observed upregulation of ECM-modifying enzymes, growth factors, and core ECM proteins implicated in vascular stiffening, such as collagens, periostin, tenacsin-C, and fibrin(ogen). Notably, these alterations correlated with increased mean pulmonary arterial pressure and vascular remodeling. In the plasma, we identified increased levels of complement components, indicating a systemic inflammatory response accompanying the vascular remodeling. Our findings shed light on the dynamic matrisome remodeling in early-stage PH, implicating a wound-healing trajectory with distinct patterns from the MPA to the distal vasculature. This study provides novel insights into the molecular underpinnings of PH pathogenesis and highlights potential biomarkers and therapeutic targets within the matrisome landscape.

ERO1A levels are a prognostic indicator in EGFR mutated non small cell lung cancer

We have identified endoplasmic reticulum oxidoreductase 1 alpha (ERO1A) as a poor prognostic indicator in epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (EGFRMUT-NSCLC). In addition, comparison of high versus low ERO1A expression among cohorts of EGFRMUT-NSCLC primary samples revealed that ERO1A expression correlated with increased expression of proteins that regulate secretion. Using the CPTAC proteomic data set in lung adenocarcinoma we found that high ERO1A protein expression correlated with both extracellular matrix and matrix modifying enzymes. In this report, we found that ablating ERO1A expression was a determinant of clonogenicity, tumor sphere formation, spheroid growth and growth in vivo, as well as response to Osimertinib. We validated that ERO1A-knockout EGFRMUT-LUAD cell lines demonstrated a reduction in secretion of both laminin gamma 2 (LAMC2) and the collagen modifying enzyme lysyl oxidase-like 2 (LOXL2). Our work supports the role of ERO1A in modulating the tumor microenvironment that is likely to contribute to tumor progression.

Identification of Sequential Molecular Mechanisms and Key Biomarkers in Early Glaucoma by Integrated Bioinformatics Analysis.

Glaucoma is a neurodegenerative disease characterized by progressive optic nerve degeneration and retinal ganglion cell (RGC) loss. In early glaucoma, before obvious axon loss, highly organized pathological processes in RGCs occur sequentially, involving axons, dendrites and synaptic terminals. The optic nerve head (ONH) is the critical structure of early glaucomatous neurodegeneration. Taking advantage of high-throughput data from the ONH and the weighted gene coexpression network analysis (WGCNA) method, the current study aims to gain insight into the full scope of pathological events in early glaucoma and define their chronological sequence. The expression profiles of GSE26299, GSE110019, and GSE139605, which measure ONH gene expression in different glaucoma models, were downloaded from the Gene Expression Omnibus (GEO) database. In GSE26299, which uses 10.5-month-old DBA/2J mice, WGCNA was utilized to construct a gene coexpression network, and the most significant modules of early (NOE), moderate (MOD) and severe (SEV) glaucoma were identified. The differentially expressed genes (DEGs) of GSE110019 and GSE139605 significantly overlapped with the correlated module of the MOD group, so the 3 gene sets were analyzed together. Pathway enrichment analysis via the GO, KEGG, and Reactome pathways was subsequently performed, followed by protein‒protein interaction (PPI) analysis to screen key genes associated with each stage. Several hub gene expression patterns were identified in a glucocorticoid-induced glaucoma (GIG) model via quantitative PCR and immunostaining. The pink module was positively correlated with the NOE group (r = 0.48, p = 4e-04) and negatively correlated with the glaucoma stage (r = -0.88, p = 3e-17). The genes in the pink module were enriched in the synaptic transmission and axonal transport pathways. The tan module was negatively correlated with the NOE group (r = -0.43, p = 0.002) and positively correlated with the glaucoma stage (r = 0.77, p = 7e-11). The genes in the tan module were associated with pathways such as tight junctions, retinol metabolism, and linoleic acid metabolism. The purple module was positively correlated with the MOD group (r = 0.64, p = 5e-07). The common genes among the purple module and the DEGs of the two other datasets were enriched in pathways related to mitotic cell division, cytokine activity, and the extracellular matrix (ECM). The hub genes identified by PPI included Nrn1, Cplx1, Timp1, and Cdk1. Quantitative PCR and immunostaining confirmed that Limk1 expression was increased in the ONH of GIG mice. In early glaucomatous neuropathy, intrinsic changes in RGCs precede the activation of glial cells and ECM remodeling. These latter events are common pathological changes observed in the ONH in both cats and mice. Our study may provide new targets for the early detection and treatment of glaucoma.

Molecular Lineages and Spatial Distributions of Subplate Neurons in the Human Fetal Cerebral Cortex.

The expansion of neural progenitors and production of distinct neurons are crucial for architectural assembly and formation of connectivity in human brains. Subplate neurons (SPNs) are among the firstborn neurons in the human fetal cerebral cortex, and play a critical role in establishing intra- and extracortical connections. However, little is known about SPN origin and developmental lineages. In this study, spatial landscapes and molecular trajectories of SPNs in the human fetal cortices from gestational weeks (GW) 10 to 25 are created by performing spatial transcriptomics and single-cell RNA sequencing. Genes known to be evolutionarily human-specific and genes associated with extracellular matrices (ECMs) are found to maintain stable proportions of subplate neurons among other neuronal types. Enriched ECM gene expression in SPNs varies in distinct cortical regions, with the highest level in the frontal lobe of human fetal brains. This study reveals molecular origin and lineage specification of subplate neurons in the human fetal cerebral cortices, and highlights underpinnings of SPNs to cortical neurogenesis and early structural folding.

Cumulative learning based segmentation aided cell mixtures classification in digital holographic microscopy

The tumor microenvironment consists of several components like tumor cells, normal healthy cells, extracellular matrix, secreted factors, and other non-cellular components. The interaction among these components is crucial for tumor progression and metastasis. Identifying the presence of tumor cells among the normal healthy cells during the initial stages of cancer will aid in its early detection and treatment. One of the main features that differentiates a cancer cell from a normal healthy cell distinctively is its morphology. In this study, the effectiveness of machine learning algorithms is evaluated in classifying cells co-cultured in a Petri dish. To achieve this, digital holographic microscopy is used to capture the quantitative phase images of human dermal fibroblast and A375 human melanoma cancer cells co-cultured at 1:1, 1:2, and 2:1 ratios. Cell segmentation was performed using a cumulative learning approach. Subsequently, morphological and phase-based features were extracted to train the machine learning algorithms for binary classification of cells. Phase-based features in addition to the morphological features were found to provide improved performance of the classifier. The classifier demonstrated an average F1-score of 0.9 during testing.

The Inhibitory Effect and Mechanism of the Histidine-Rich Peptide rAj-HRP from Apostichopus japonicus on Human Colon Cancer HCT116 Cells.

Colon cancer is a common and lethal malignancy, ranking second in global cancer-related mortality, highlighting the urgent need for novel targeted therapies. The sea cucumber (Apostichopus japonicus) is a marine organism known for its medicinal properties. After conducting a bioinformatics analysis of the cDNA library of Apostichopus japonicus, we found and cloned a cDNA sequence encoding histidine-rich peptides, and the recombinant peptide was named rAj-HRP. Human histidine-rich peptides are known for their anti-cancer properties, raising questions as to whether rAj-HRP might exhibit similar effects. To investigate whether rAj-HRP can inhibit colon cancer, we used human colon cancer HCT116 cells as a model and studied the tumor suppressive activity in vitro and in vivo. The results showed that rAj-HRP inhibited HCT116 cell proliferation, migration, and adhesion to extracellular matrix (ECM) proteins in vitro. It also disrupted the cytoskeleton and induced apoptosis in these cells. In vivo, rAj-HRP significantly inhibited the growth of HCT116 tumors in BALB/c mice, reducing tumor volume and weight without affecting the body weight of the tumor-bearing mice. Western blot analysis showed that rAj-HRP inhibited HCT116 cell proliferation and induced apoptosis by upregulating BAX and promoting PARP zymogen degradation. Additionally, rAj-HRP inhibited HCT116 cell adhesion and migration by reducing MMP2 levels. Further research showed that rAj-HRP downregulated EGFR expression in HCT116 cells and inhibited key downstream molecules, including AKT, P-AKT, PLCγ, P38 MAPK, and c-Jun. In conclusion, rAj-HRP exhibits significant inhibitory effects on HCT116 cells in both in vitro and in vivo, primarily through the EGFR and apoptosis pathways. These findings suggest that rAj-HRP has the potential as a novel targeted therapy for colon cancer.

YAP signaling orchestrates the endothelin-1-guided invadopodia formation in high-grade serous ovarian cancer.

The high-grade serous ovarian cancer (HG-SOC) is a notoriously challenging disease, characterized by a rapid peritoneal dissemination. HG-SOC cells leverage actin-rich membrane protrusions, known as invadopodia, to degrade the surrounding extracellular matrix (ECM) and invade, initiating the metastatic cascade. In HG-SOC, the endothelin-1 (ET-1)/endothelin A receptor (ETAR)-driven signaling coordinates invadopodia activity, however how this axis integrates pro-oncogenic signaling routes, as YAP-driven one, impacting on the invadopodia-mediated ECM degradation and metastatic progression, deserves a deeper investigation. Herein, we observed that downstream of the ET-1/ET-1R axis, the RhoC and Rac1 GTPases, acting as signaling intermediaries, promote the de-phosphorylation and nuclear accumulation of YAP. Conversely, the treatment with the dual ETA/ETB receptor antagonist, macitentan, inhibits the ET-1-driven YAP activity. Similarly, RhoC silencing, or cell transfection with a dominant inactive form of Rac1, restore the YAP phosphorylated and inhibited state. Mechanistically, the ET-1R/YAP signal alliance coordinates invadopodia maturation into ECM-degrading structures, indicating how such ET-1R-guided protein network represents a route able to enhance the HG-SOC invasive potential. At functional level, we found that the interconnection between the ET-1R/RhoC and YAP signals is required for MMP-2 and MMP-9 proteolytic functions, cell invasion, and cytoskeleton architecture changes, supporting the HG-SOC metastatic strength. In HG-SOC patient-derived xenografts (PDX) macitentan, turning-off the invadopodia regulators RhoC/YAP, halt the metastatic colonization. ET-1R targeting, hindering the YAP activity, weakens the invadopodia machinery, embodying a promising therapeutic avenue to prevent peritoneal dissemination in HG-SOC.

Sex Differences in Response to Diet Enriched with Glutathione Precursors in the Aging Heart.

Common features of the aging heart are dysregulated metabolism, inflammation, and fibrosis. Elevated oxidative stress is another hallmark of cardiac aging that can exacerbate each of these conditions. We hypothesize that by increasing natural antioxidant levels (glutathione), we will improve cardiac function. Twenty-one-month-old mice were fed Glycine and N-Acetyl Cysteine (GlyNAC) (glutathione precursors)-supplemented or control diets for 12 weeks. Heart function was monitored longitudinally, and the exercise performance was determined at the end of the study. We found that the GlyNAC diet was beneficial for old male but not old female mice, leading to an increase of Ndufb8 expression (a subunit of the mitochondrial respiratory chain complex), and higher enzymatic activity for CPT1b and CrAT, two carnitine acyltransferases that are critical to cardiomyocyte metabolism. Although no quantifiable change of collagen turnover was detected, hearts from GlyNAC-fed old males exhibited a slight but significant enrichment in Fmod, a protein that can inhibit collagen fibril formation, possibly reducing extracellular matrix (ECM) stiffness and thus improving diastolic function. Cardiac diastolic function was modestly improved in males but not females, and surprisingly GlyNAC-fed female mice showed a decline in exercise performance. In summary, our work supports the concept that aged male and female hearts are phenotypically different. These basic differences may affect the response to pharmacological and diet interventions, including antioxidants.

Prognostic value of EMT-related genes and immune cell infiltration in thyroid carcinoma

BackgroundThe Epithelial–Mesenchymal Transition (EMT) is a very important process involved in cancer invasion and metastasis. Additionally, the Cathepsin K (CTSK) gene is closely related to the degradation of the extracellular matrix, which is a critical component of the EMT. The purpose of this study was to determine the relationships between EMT-related genes and immune cell infiltration and their prognostic value in Thyroid carcinoma (THCA). The effect of the CTSK gene on the aggressive biological features of THCA was assessed.MethodsWithin the framework of the present study, the THCA cohort was analyzed in detail based on data obtained from The TCGA database in the context of the EMT. The TCGA-THCA cohort was then divided into two groups, namely, high- and low-risk groups, based on the calculated EMT scores. Finally, based on the findings from the Weighted Gene Co-Expression Network Analysis (WGCNA) algorithm, LASSO regression analysis, and Kaplan−Meier plotter, we selected five genes (CTSK, C3ORF80, FBLN2, PRELP and SRPX2) associated with patient prognosis. Furthermore, this study examined the presence of various immune cells within the THCA samples using three distinct algorithms, namely ssGSEA, xCell, and MCPcounter. Additional studies have been conducted to establish the roles of CTSK in THCA cell proliferation and migration using various assays, such as CCK8, colony formation, EdU proliferation, Transwell migration and wound healing assays. Additionally, the involvement of CTSK in the regulation of various EMT-related markers was confirmed using Western blot analysis.ResultsBased on EMT scores, TCGA-THCA patients were further divided into two groups, and the study revealed that patients in the high-risk group had a worse prognosis than those in the low-risk group. Among the five genes linked to the prognostic value of EMT (CTSK, C3ORF80, FBLN2, PRELP, and SRPX2), CTSK exhibited notably elevated expression in the high-risk cohort. This group also exhibited pronounced immune cell infiltration, with a marked correlation observed between CTSK expression and the levels of macrophages, MDSCs, and various T-cell subtypes. Furthermore, in vitro studies demonstrated that reducing CTSK expression led to significant reductions in THCA cell viability; clonogenic, proliferative, motility and migratory capacities; and the expression of key EMT-related proteins, including N-cadherin, vimentin, slug, and snail.ConclusionOur results suggest that the expression of CTSK, a gene associated with the EMT, may be associated with THCA onset and progression and thus may serve as a promising prognostic biomarker.

Obesity, white adipose tissue and cancer.

White adipose tissue (WAT) is crucial for whole-body energy homeostasis and plays an important role in metabolic and hormonal regulation. While healthy WAT undergoes controlled expansion and contraction to meet the body's requirements, dysfunctional WAT in conditions like obesity is characterized by excessive tissue expansion, alterations in lipid homeostasis, inflammation, hypoxia, and fibrosis. Obesity is strongly associated with an increased risk of numerous cancers, with obesity-induced WAT dysfunction influencing cancer development through various mechanisms involving both systemic and local interactions between adipose tissue and tumors. Unhealthy obese WAT affects circulating levels of free fatty acids and factors like leptin, adiponectin, and insulin, altering systemic lipid metabolism and inducing inflammation that supports tumor growth. Similar mechanisms are observed locally in an adipose-rich tumor microenvironment (TME), where WATcells can also trigger extracellular matrix remodeling, thereby enhancing the TME's ability to promote tumor growth. Moreover, tumors reciprocally interact with WAT, creating a bidirectional communication that further enhances tumorigenesis. This review focuses on the complex interplay between obesity, WAT dysfunction, and primary tumor growth, highlighting potential targets for therapeutic intervention.

Branched-chain amino acids and their metabolites decrease human and rat hepatic stellate cell activation

BackgroundEnd-stage liver diseases (ESLDs) are a significant global health challenge due to their high prevalence and severe health impacts. Despite the severe outcomes associated with ESLDs, therapeutic options remain limited. Targeting the activation of hepatic stellate cells (HSCs), key drivers of extracellular matrix accumulation during liver injury presents a novel therapeutic approach. In ESLDs patients, branched-chain amino acids (BCAAs, leucine, isoleucine and valine) levels are decreased, and supplementation has been proposed to attenuate liver fibrosis and improve regeneration. However, their effects on HSCs require further investigation.ObjectiveTo evaluate the efficacy of BCAAs and their metabolites, branched-chain α-keto acids (BCKAs), in modulating HSCs activation in human and rat models.MethodsPrimary HSCs from rats and cirrhotic and non-cirrhotic human livers, were cultured and treated with BCAAs or BCKAs to assess their effects on both preventing (from day 1 of isolation) and reversing (from day 7 of isolation) HSCs activation.ResultsIn rat HSCs, leucine and BCKAs significantly reduced fibrotic markers and cell proliferation. In human HSCs, the metabolite of isoleucine decreased cell proliferation around 85% and increased the expression of branched-chain ketoacid dehydrogenase. The other metabolites also showed antifibrotic effects in HSCs from non-cirrhotic human livers.ConclusionBCAAs and their respective metabolites inhibit HSC activation with species-specific responses. Further research is needed to understand how BCAAs influence liver fibrogenesis. BCKAs supplementation could be a strategic approach for managing ESLDs, considering the nutritional status and amino acid profiles of patients.Graphical abstractThe antifibrotic effects of BCAAs and BCKAs in various conditions are depicted for human HSCs (left) and rat HSCs (right) The symbol ‘↓’ indicates a downregulation or a decrease. α-SMA alpha-smooth muscle actin, BCAAs branched-chain amino acids, BCKAs branched-chain keto acids, HSCs hepatic stellate cells, KMV α-keto-β-methylvalerate. Figure created with Biorender.com

Nuclear translocation of SIRT4 mediates deacetylation of U2AF2 to modulate renal fibrosis through alternative splicing-mediated upregulation of CCN2.

TGF-β stimulates CCN2 expression which in turn amplifies TGF-β signaling. This process promotes extracellular matrix production and accelerates the pathological progression of fibrotic diseases. Alternative splicing plays an important role in multiple disease development, while U2 small nuclear RNA auxiliary factor 2 (U2AF2) is an essential factor in the early steps of pre-mRNA splicing. However, the molecular mechanism underlying abnormal CCN2 expression upon TGF-β stimulation remains unclear. This study elucidates that SIRT4 acts as a master regulator for CCN2 expression in response to TGF-β by modulating U2AF2-mediated alternative splicing. Analyses of renal biopsy specimens from patients with CKD and mouse fibrotic kidney tissues revealed marked nuclear accumulation of SIRT4. The tubulointerstitial fibrosis was alleviated by global deletion or tubular epithelial cell (TEC)-specific knockout of Sirt4, and aggravated by adeno-associated virus-mediated SIRT4 overexpression in TECs. Furthermore, SIRT4 was found to translocate from the mitochondria to the cytoplasm through the BAX/BAK pore under TGF-β stimulation. In the cytoplasm, TGF-β activated the ERK pathway and induced the phosphorylation of SIRT4 at Ser36, which further promoted its interaction with importin α1 and subsequent nuclear translocation. In the nucleus, SIRT4 was found to deacetylate U2AF2 at K413, facilitating the splicing of CCN2 pre-mRNA to promote CCN2 protein expression. Importantly, exosomes containing anti-SIRT4 antibodies were found to effectively mitigate the UUO-induced kidney fibrosis in mice. Collectively, these findings indicated that SIRT4 plays a role in kidney fibrosis by regulating CCN2 expression via the pre-mRNA splicing.