Extracellular Matrix Reorganization Research Articles

Conserved regulatory switches for the transition from natal down to juvenile feather in birds

The transition from natal downs for heat conservation to juvenile feathers for simple flight is a remarkable environmental adaptation process in avian evolution. However, the underlying epigenetic mechanism for this primary feather transition is mostly unknown. Here we conducted time-ordered gene co-expression network construction, epigenetic analysis, and functional perturbations in developing feather follicles to elucidate four downy-juvenile feather transition events. We report that extracellular matrix reorganization leads to peripheral pulp formation, which mediates epithelial-mesenchymal interactions for branching morphogenesis. α-SMA (ACTA2) compartmentalizes dermal papilla stem cells for feather renewal cycling. LEF1 works as a key hub of Wnt signaling to build rachis and converts radial downy to bilateral symmetry. Novel usage of scale keratins strengthens feather sheath with SOX14 as the epigenetic regulator. We show that this primary feather transition is largely conserved in chicken (precocial) and zebra finch (altricial) and discuss the possibility that this evolutionary adaptation process started in feathered dinosaurs.

The role of ST3GAL4 in glioma malignancy, macrophage infiltration, and prognostic outcomes

BackgroundGlioma, a prevalent malignancy of the brain and spinal cord, poses a considerable threat to human health. The association between aberrant sialic acid modification and glioma progression has been suggested, but the precise mechanism is still elusive. ST3GAL4, a sialoglycosyltransferase, is implicated in increased metastatic potential and poor prognosis in various cancers; however, its specific role in glioma requires further elucidation. MethodsWe evaluated ST3GAL4 expression levels and their clinical relevance using the TCGA database, and we assessed immune infiltration via the Tumor Immune Evaluation Resource (TIMER) database. In vitro experiments were performed to determine the effects of ST3GAL4 knockdown on glioma cell malignancy, with additional co-culture assays to assess its impact on macrophage phenotype. ResultsST3GAL4 expression was markedly elevated in glioma tissues compared to normal brain tissues, with a strong correlation to glioma patient clinical characteristics. Survival analyses and receiver operating characteristic (ROC) curves suggested that ST3GAL4 is a feasible diagnostic and prognostic biomarker for glioma. Knockdown studies revealed that ST3GAL4 inhibition reduces glioma cell line proliferation, migration, and invasion, while causing G1 phase cell cycle arrest. ST3GAL4 appears to mediate glioma progression through extracellular matrix reorganization and EMT signaling pathway activation, further contributing to M2 macrophage polarization and infiltration within the tumor microenvironment. ConclusionOur research highlights the critical role of ST3GAL4 in glioma development, positioning it as a promising candidate for diagnostic and therapeutic interventions.

Role of folate receptor α in the partial rejuvenation of dentate gyrus cells: Improvement of cognitive function in 21-month-old aged mice

Neuronal aging may be, in part, related to a change in DNA methylation. Thus, methyl donors, like folate and methionine, may play a role in cognitive changes associated to neuronal aging. To test the role of these metabolites, we performed stereotaxic microinjection of these molecules into the dentate gyrus (DG) of aged mice (an average age of 21 month). Folate, but not S-Adenosyl-Methionine (SAM), enhances cognition in aged mice. In the presence of folate, we observed partial rejuvenation of DG cells, characterized by the expression of juvenile genes or reorganization of extracellular matrix. Here, we have also tried to identify the mechanism independent of DNA methylation, that involve folate effects on cognition. Our analyses indicated that folate binds to folate receptor α (FRα) and, upon folate binding, FRα is transported to cell nucleus, where it is acting as transcription factor for expressing genes like SOX2 or GluN2B. In this work, we report that a FRα binding peptide also replicates the folate effect on cognition, in aged mice. Our data suggest that such effect is not sex-dependent. Thus, we propose the use of this peptide to improve cognition since it lacks of folate-mediated side effects. The use of synthetic FRα binding peptides emerge as a future strategy for the study of brain rejuvenation.

Abstract 5156: Is the formation of Stroma AReactive Invasion Front Areas in colorectal cancer driven by characteristic extracellular matrix reorganization processes based on the plasmin/plasminogen system

Abstract Background: As patient stratification still needs to be refined in colorectal cancer (CRC), we established SARIFA (Stroma AReactive Invasion Front Areas) as a novel H&E-based prognostic biomarker that is defined by a direct tumor-adipocyte interaction with a lack of desmoplastic reaction. Even though we could prove the high prognostic relevance of SARIFA now in multiple cohorts in gastric, colorectal, and pancreatic cancer, as well as could link an upregulation of lipid metabolism and immune alterations to this specific aggressive tumor biology, it remains an open question how SARIFAs are formed mechanistically. Materials/Methods: Based on the results of our recently established SARIFA-scores in the TCGA-CRC (TCGA-COAD & TCGA-READ cohorts; SARIFA-positive n=69, SARIFA-negative n=138) and the corresponding differential gene expression analysis, which are part of a previous study, protein levels (in ng/mL) measured by commercially available uPA and PAI1 enzyme-linked immunosorbent assays (ELISAs) were compared between SARIFA-positive and SARIFA-negative CRC patients (SARIFA-positive n=35, SARIFA-negative n=62). Additionally, immunohistochemistry for FABP4 as a key player in lipid metabolism was performed. Moreover, spatial transcriptomics (digital spatial profiling, DSP) was performed with tumor and stroma section segmentation on a subset of cases. Results: In TCGA-CRC, SERPINE1 (PAI1), as well as PLAU (uPA), are both significantly upregulated on mRNA level (LFC: 1.14 and 0.74, respectively; both adjusted p-values ≤0.001). Logically, SERPINE1 and PLAU are highly expressed in mesenchymal CMS4 CRCs, which overlap with SARIFA-positivity. This is reflected by an upregulation of matrix metalloproteinases in predicted functional protein networks (STRING analysis). In our local cohort, we could now validate this upregulation of uPA in SARIFA-positive CRCs on protein level (p=0.0027). For PAI1, a trend towards higher protein levels in SARIFA-positive CRCs could be observed (p=0.209). Moreover, SARIFA-positive CRCs show a higher number of tumor buds (p=0.013) as well as increased FABP4 expression at the invasion front (p<0.001) in our local cohort. In DSP analysis, the stroma fraction of SARIFA-positive CRCs showed significant changes compared to SARIFA-negative cases. Conclusion: Our current data shows that the formation of SARIFAs and the consecutive direct tumor-adipocyte interaction with a tumor-promoting increase in lipid metabolism may rely on alterations in the plasmin/plasminogen system, which could be a novel potential drug target in the aggressive subset of SARIFA-positive CRCs. Citation Format: Bianca Grosser, Nic G. Reitsam, Florian Sommer, Jochen Hardt, Bruno Märkl. Is the formation of Stroma AReactive Invasion Front Areas in colorectal cancer driven by characteristic extracellular matrix reorganization processes based on the plasmin/plasminogen system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5156.

ACOX1 deficiency-induced lipid metabolic disorder facilitates chronic interstitial fibrosis development in renal allografts

Chronic interstitial fibrosis presents a significant challenge to the long-term survival of transplanted kidneys. Our research has shown that reduced expression of acyl-coenzyme A oxidase 1 (ACOX1), which is the rate-limiting enzyme in the peroxisomal fatty acid β-oxidation pathway, contributes to the development of fibrosis in renal allografts. ACOX1 deficiency leads to lipid accumulation and excessive oxidation of polyunsaturated fatty acids (PUFAs), which mediate epithelial–mesenchymal transition (EMT) and extracellular matrix (ECM) reorganization respectively, thus causing fibrosis in renal allografts. Furthermore, activation of Toll-like receptor 4 (TLR4)-nuclear factor kappa-B (NF-κB) signaling induced ACOX1 downregulation in a DNA methyltransferase 1 (DNMT1)-dependent manner. Overconsumption of PUFA resulted in endoplasmic reticulum (ER) stress, which played a vital role in facilitating ECM reorganization. Supplementation with PUFAs contributed to delayed fibrosis in a rat model of renal transplantation. The study provides a novel therapeutic approach that can delay chronic interstitial fibrosis in renal allografts by targeting the disorder of lipid metabolism.

Leveraging chromatin state transitions for the identification of regulatory networks orchestrating heart regeneration.

The limited regenerative capacity of the human heart contributes to high morbidity and mortality worldwide. In contrast, zebrafish exhibit robust regenerative capacity, providing a powerful model for studying how to overcome intrinsic epigenetic barriers maintaining cardiac homeostasis and initiate regeneration. Here, we present a comprehensive analysis of the histone modifications H3K4me1, H3K4me3, H3K27me3and H3K27ac during various stages of zebrafish heart regeneration. We found a vast gain of repressive chromatin marks one day after myocardial injury, followed by the acquisition of active chromatin characteristics on day four and a transition to a repressive state on day 14, and identified distinct transcription factor ensembles associated with these events. The rapid transcriptional response involves the engagement of super-enhancers at genes implicated in extracellular matrix reorganization and TOR signaling, while H3K4me3 breadth highly correlates with transcriptional activity and dynamic changes at genes involved in proteolysis, cell cycle activity, and cell differentiation. Using loss- and gain-of-function approaches, we identified transcription factors in cardiomyocytes and endothelial cells influencing cardiomyocyte dedifferentiation or proliferation. Finally, we detected significant evolutionary conservation between regulatory regions that drive zebrafish and neonatal mouse heart regeneration, suggesting that reactivating transcriptional and epigenetic networks converging on these regulatory elements might unlock the regenerative potential of adult human hearts.

The cycling and aging mouse female reproductive tract at single-cell resolution

The female reproductive tract (FRT) undergoes extensive remodeling during reproductive cycling. This recurrent remodeling and how it shapes organ-specific aging remains poorly explored. Using single-cell and spatial transcriptomics, we systematically characterized morphological and gene expression changes occurring in ovary, oviduct, uterus, cervix, and vagina at each phase of the mouse estrous cycle, during decidualization, and into aging. These analyses reveal that fibroblasts play central-and highly organ-specific-roles in FRT remodeling by orchestrating extracellular matrix (ECM) reorganization and inflammation. Our results suggest a model wherein recurrent FRT remodeling over reproductive lifespan drives the gradual, age-related development of fibrosis and chronic inflammation. This hypothesis was directly tested using chemical ablation of cycling, which reduced fibrotic accumulation during aging. Our atlas provides extensive detail into how estrus, pregnancy, and aging shape the organs of the female reproductive tract and reveals the unexpected cost of the recurrent remodeling required for reproduction.

The Neurovascular Unit as a Locus of Injury in Low-Level Blast-Induced Neurotrauma.

Blast-induced neurotrauma has received much attention over the past decade. Vascular injury occurs early following blast exposure. Indeed, in animal models that approximate human mild traumatic brain injury or subclinical blast exposure, vascular pathology can occur in the presence of a normal neuropil, suggesting that the vasculature is particularly vulnerable. Brain endothelial cells and their supporting glial and neuronal elements constitute a neurovascular unit (NVU). Blast injury disrupts gliovascular and neurovascular connections in addition to damaging endothelial cells, basal laminae, smooth muscle cells, and pericytes as well as causing extracellular matrix reorganization. Perivascular pathology becomes associated with phospho-tau accumulation and chronic perivascular inflammation. Disruption of the NVU should impact activity-dependent regulation of cerebral blood flow, blood-brain barrier permeability, and glymphatic flow. Here, we review work in an animal model of low-level blast injury that we have been studying for over a decade. We review work supporting the NVU as a locus of low-level blast injury. We integrate our findings with those from other laboratories studying similar models that collectively suggest that damage to astrocytes and other perivascular cells as well as chronic immune activation play a role in the persistent neurobehavioral changes that follow blast injury.

Hippo Signaling Mediates TGFβ-Dependent Transcriptional Inputs in Cardiac Cushion Mesenchymal Cells to Regulate Extracellular Matrix Remodeling.

The transforming growth factor beta (TGFβ) and Hippo signaling pathways are evolutionarily conserved pathways that play a critical role in cardiac fibroblasts during embryonic development, tissue repair, and fibrosis. TGFβ signaling and Hippo signaling are also important for cardiac cushion remodeling and septation during embryonic development. Loss of TGFβ2 in mice causes cardiac cushion remodeling defects resulting in congenital heart disease. In this study, we used in vitro molecular and pharmacologic approaches in the cushion mesenchymal cell line (tsA58-AVM) and investigated if the Hippo pathway acts as a mediator of TGFβ2 signaling. Immunofluorescence staining showed that TGFβ2 induced nuclear translocation of activated SMAD3 in the cushion mesenchymal cells. In addition, the results indicate increased nuclear localization of Yes-associated protein 1 (YAP1) following a similar treatment of TGFβ2. In collagen lattice formation assays, the TGFβ2 treatment of cushion cells resulted in an enhanced collagen contraction compared to the untreated cushion cells. Interestingly, verteporfin, a YAP1 inhibitor, significantly blocked the ability of cushion cells to contract collagen gel in the absence or presence of exogenously added TGFβ2. To confirm the molecular mechanisms of the verteporfin-induced inhibition of TGFβ2-dependent extracellular matrix (ECM) reorganization, we performed a gene expression analysis of key mesenchymal genes involved in ECM remodeling in heart development and disease. Our results confirm that verteporfin significantly decreased the expression of α-smooth muscle actin (Acta2), collagen 1a1 (Col1a1), Ccn1 (i.e., Cyr61), and Ccn2 (i.e., Ctgf). Western blot analysis indicated that verteporfin treatment significantly blocked the TGFβ2-induced activation of SMAD2/3 in cushion mesenchymal cells. Collectively, these results indicate that TGFβ2 regulation of cushion mesenchymal cell behavior and ECM remodeling is mediated by YAP1. Thus, the TGFβ2 and Hippo pathway integration represents an important step in understanding the etiology of congenital heart disease.

Instructive Interaction between Myelodysplastic Hematopoiesis and the Bone Marrow Microenvironment at Single Cell Level

Myelodysplastic Neoplasms (MDS) are hypothesized to re-model their bone marrow (BM) microenvironment to re-inforce conditions for their propagation. In this study, we investigated interactions between MDS cells and the BM niche at single cell level in a MDS patient-derived xenograft model (PDX) as well as primary human MDS and healthy (HY) bone marrow (BM samples in order to identify cues of disturbed crosstalk between MDS hematopoiesis and the BM microenvironment. In the PDX model, we analyzed >13,000 cells of n=15 different murine niche cell populations after long-term (>24 weeks) exposure to n=3 MDS patient samples versus n=2 age-matched HY human grafts. Subsequently, we analyzed over 24,000 primary human BM cells enriched for the non-hematopoietic compartment by using whole bone fragments from n=8 MDS patients and n=7 HY age-matched donors. All cells were analyzed with combined 10x single cell RNA sequencing and human “Cellular Indexing of the Transcriptome and Epitopes by Sequencing” (CITE Seq). Results were validated by flow cytometry and gene expression analyses. Mesenchymal cell (MSC) subpopulations from PDX mice exposed to MDS versus HY hematopoiesis could be subdivided into distinct “Cxcl12+ abundant reticular” (CAR) cells with osteogenic and adipogenic differentiation trajectories. Various CAR cell populations displayed an increased frequency in MDS transplanted mice as compared to PDX transplanted with HY grafts ( Panel A). Upon contact with hematopoietic MDS cells, CAR cell populations overexpressed numerous genes involved in extracellular matrix reorganization and hematopoietic supporting factors, such as Cxcl12, Il7, Lox and Loxl1 as compared to mice bearing HY grafts. In situ, BM cells from MDS patients showed highly heterogeneous MSC subpopulations with similar differentiation trajectories to the ones observed in our PDX models on a patient-individual level. We detected two non-hematopoietic cell populations, designated as endothelial cells (ECs) and MSCs characterized by expression of CXCL12, IGFBP5 or COL1A2 and quantification with epitope sequencing (CITE Seq) with mutually exclusive detection of CD31 and CD271 expression (n=581 cells in total, Panel B). In n=3 MDS patients, we identified an MDS-specific MSC subpopulation with high IGFPB4 and CXCL12 expression as well as overexpression of KITLG and IL7. This suggests that this altered specialized secretory mesenchymal state is present in a subset of MDS patients and may support the malignant hematopoietic clones by overexpression of key hematopoietic factors. Interactome analyses using spatial receptor:ligand bioinformatic tools such as CellphoneDB, RNAMagnet and omnipath further confirmed that stromal cells alter their cellular profile and secretory behavior in response to MDS hematopoiesis. In conclusion, we show that murine BM stroma is re-programmed by exposure to human MDS xenografts to upregulate extracellular matrix (ECM) re-modeling and HSC supporting factors. In native human MDS BM in vivo, rare primary mesenchymal cells are re-programmed to increase hematopoietic support in a patient-individual manner pointing to altered MSC subpopulations with increased growth factor expression profiles in a subgroup of MDS patients.

Comparative Proteomics Reveals Prolonged Corneal Preservation Impaired Ocular Surface Immunity Accompanied by Fibrosis in Human Stroma.

Cornea transplantation is one of the most commonly performed allotransplantations worldwide. Prolonged storage of donor corneas leads to decreased endothelial cell viability, severe stromal edema, and opacification, significantly compromising the success rate of corneal transplantation. Corneal stroma, which constitutes the majority of the cornea, plays a crucial role in maintaining its shape and transparency. In this study, we conducted proteomic analysis of corneal stroma preserved in Optisol-GS medium at 4 °C for 7 or 14 days to investigate molecular changes during storage. Among 1923 identified proteins, 1634 were quantifiable and 387 were significantly regulated with longer preservation. Compared to stroma preserved for 7 days, proteins involved in ocular surface immunomodulation were largely downregulated while proteins associated with extracellular matrix reorganization and fibrosis were upregulated in those preserved for 14 days. The increase in extracellular matrix structural proteins together with upregulation of growth factor signaling implies the occurrence of stromal fibrosis, which may compromise tissue clarity and cause vision impairments. This study is the first to provide insights into how storage duration affects corneal stroma from a proteomic perspective. Our findings may contribute to future research efforts aimed at developing long-term preservation techniques and improving the quality of preserved corneas, thus maximizing their clinical application.

EPCO-04. SPATIAL TRANSCRIPTOMIC ANALYSIS OF MALIGNANT PERIPHERAL NERVE SHEATH TUMORS REVEALS THERAPEUTICALLY TARGETABLE MOLECULAR SIGNATURES IN REGIONS UNDERGOING HISTOPATHOLOGIC TRANSFORMATION

Abstract Malignant peripheral nerve sheath tumors (MPNSTs) evolve from plexiform neurofibromas (pNF) in patients with neurofibromatosis type-1 (NF-1) yet the cellular and transcriptomic mechanisms underlying this transformation remain unclear. Here, we perform spatial gene expression profiling on fifteen MPNSTs to correlate histologic observations with transcriptomic programs and identify mechanisms underlying malignant transformation. METHODS: Fifteen MPNSTs, including tumors with a histopathologically defined transition zone between adjacent low-grade and high-grade areas (n=3), were retrospectively identified. Spatial transcriptomic profiling was performed using Visium followed by downstream processing with SpaceRanger, Seurat, Harmony, and monocle. RESULTS: 50,807 unique spatial transcriptomes were distributed across 17 unique clusters, of which 12 were enriched for tumor cells and 5 for non-tumor microenvironment cells based on histologic evaluation, unsupervised cell-type assignment, marker gene expression, and cell signature gene sets. Transcriptomic clusters in low-grade areas were enriched for genes regulating mononuclear cell migration and cell-cell adhesion, while high-grade regions were enriched for Ras/Raf/MEK/ERK target genes, anti-apoptotic programs, and extracellular matrix reorganization genes. Trajectory inference beginning in low grade regions revealed spatially organized molecular signatures of malignant transformation enriched for cell cycle progression within histopathologically homogeneous regions. Moreover, the intermediate transformation zone between low-grade neurofibromatous and high-grade MPNST histopathologic regions was marked by loss of cell differentiation genes and increased cell cycle progression. CONCLUSIONS: Spatial transcriptomic analysis elucidates the cellular and gene expression patterns underlying progression from pNF to MPNST. Distinct cell populations exist across low-grade, transformation zone, and high-grade regions in MPNSTs highlighted by alterations in Ras/Raf/MEK/ERK signaling, cell differentiation and cell cycle programs, shedding light on targets for combinatorial molecular therapy to decrease the risk of malignant transformation and serving as a biomarker of aggressive peripheral nerve tumors to complement conventional histopathology.

Proteometabolomics of initial and recurrent glioblastoma highlights an increased immune cell signature with altered lipid metabolism.

There is an urgent need to better understand the mechanisms associated with the development, progression, and onset of recurrence after initial surgery in glioblastoma (GBM). The use of integrative phenotype-focused -omics technologies such as proteomics and lipidomics provides an unbiased approach to explore the molecular evolution of the tumor and its associated environment. We assembled a cohort of patient-matched initial (iGBM) and recurrent (rGBM) specimens of resected GBM. Proteome and metabolome composition were determined by mass spectrometry-based techniques. We performed neutrophil-GBM cell coculture experiments to evaluate the behavior of rGBM-enriched proteins in the tumor microenvironment. ELISA-based quantitation of candidate proteins was performed to test the association of their plasma concentrations in iGBM with the onset of recurrence. Proteomic profiles reflect increased immune cell infiltration and extracellular matrix reorganization in rGBM. ASAH1, SYMN, and GPNMB were highly enriched proteins in rGBM. Lipidomics indicates the downregulation of ceramides in rGBM. Cell analyses suggest a role for ASAH1 in neutrophils and its localization in extracellular traps. Plasma concentrations of ASAH1 and SYNM show an association with time to recurrence. We describe the potential importance of ASAH1 in tumor progression and development of rGBM via metabolic rearrangement and showcase the feedback from the tumor microenvironment to plasma proteome profiles. We report the potential of ASAH1 and SYNM as plasma markers of rGBM progression. The published datasets can be considered as a resource for further functional and biomarker studies involving additional -omics technologies.

Systemic Type 2 Inflammation-Associated Atopic Dermatitis Exacerbates Periodontitis

Introduction: Atopic dermatitis (AD) is a prevalent and chronic inflammatory skin disease characterized by Th2 cell-mediated type 2 inflammation. Emerging evidence indicated that AD patients exhibit an increased incidence of oral disorders. In the present study, we sought mechanistic insights into how AD affects periodontitis. Methods: Onset of AD was induced by 2,4-dinitrochlorobenzene (DNCB). Furthermore, we induced periodontitis (P) in AD mice. The effect of AD in promoting inflammation and bone resorption in gingiva was evaluated. Hematoxylin and eosin staining, tartrate-resistant acid phosphatase staining, immunofluorescence assay, and flow cytometry were used to investigate histomorphology and cytology analysis, respectively. RNA sequencing of oral mucosa is used tissues to further understand the dynamic transcriptome changes. 16S rRNA microbial analysis is used to profile oral microbial composition. Results: Compared to control group, mice in AD group showed inflammatory signatures and infiltration of a proallergic Th2 (Th2A)-like subset in oral mucosa but not periodontitis, as identified by not substantial changes in mucosa swelling, alveolar bone loss, and TRAP⁺ osteoclasts infiltration. Similarly, more Th2A-like cell infiltration and interleukin-4 levels were significantly elevated in the oral mucosa of DNCB-P mice compared to P mice. More importantly, AD exacerbates periodontitis when periodontitis has occurred and the severity of periodontitis increased with aggravation of dermatitis. Transcriptional analysis revealed that aggravated periodontitis was positively correlated with more macrophage infiltration and abundant CCL3 secreted. AD also altered oral microbiota, indicating the re-organization of extracellular matrix. Conclusions: These data provide solid evidence about exacerbation of periodontitis caused by type 2 dermatitis, advancing our understanding in cellular and microbial changes during AD-periodontitis progression.

A Proteomics-Based Identification of the Biological Networks Mediating the Impact of Epigallocatechin-3-Gallate on Trophoblast Cell Migration and Invasion, with Potential Implications for Maternal and Fetal Health.

Trophoblast migration and invasion play crucial roles in placental development. However, the effects of (-)-epigallocatechin-3-gallate (EGCG) on trophoblast cell functions remain largely unexplored. In this study, we investigated the impact of EGCG on the survival of trophoblast cells and employed a proteomics analysis to evaluate its influence on trophoblast cell migration and invasion. Be-Wo trophoblast cells were treated with EGCG, and a zone closure assay was conducted to assess the cell migration and invasion. Subsequently, a proteomics analysis was performed on the treated and control groups, followed by a bioinformatics analysis to evaluate the affected biological pathways and protein networks. A quantitative real-time PCR and Western blot analysis were carried out to validate the proteomics findings. Our results showed that EGCG significantly suppressed the trophoblast migration and invasion at a concentration not affecting cell survival. The proteomics analysis revealed notable differences in the protein expression between the EGCG-treated and control groups. Specifically, EGCG downregulated the signaling pathways related to EIF2, mTOR, and estrogen response, as well as the processes associated with the cytoskeleton, extracellular matrix, and protein translation. Conversely, EGCG upregulated the pathways linked to lipid degradation and oxidative metabolism. The quantitative PCR showed that EGCG modulated protein expression by regulating gene transcription, and the Western blot analysis confirmed its impact on cytoskeleton and extracellular matrix reorganization. These findings suggest EGCG may inhibit trophoblast migration and invasion through multiple signaling pathways, highlighting the potential risks associated with consuming EGCG-containing products during pregnancy. Future research should investigate the impact of EGCG intake on maternal and fetal proteoforms.

Airway wall extracellular matrix changes induced by bronchial thermoplasty in severe asthma

Airway remodeling is a prominent feature of asthma, including increased airway smooth muscle (ASM) mass and altered extracellular matrix (ECM) composition. Bronchial thermoplasty (BT), a bronchoscopic treatment for severe asthma, targets this airway remodeling. To investigate the effect of BT on ECM composition and its association with clinical outcomes. This is a sub-study of the TASMA trial. Thirty severe asthma patients were BT treated, of which 13 patients prior to BT were treated by six months of standard therapy (control group). Demographic data, clinical data including pulmonary function and bronchial biopsies were collected. Biopsies at BT treated and non-treated locations were analyzed by histological and immune-histochemical staining. Associations between histology and clinical outcomes were explored. Six months after treatment, reticular basement membrane (RBM) thickness was reduced from 7.28μm to 5.74μm (21% relative reduction) and the percentage area of tissue positive for collagen increased from 26.3% to 29.8% (13% relative increase). Collagen structure analysis revealed a reduction in the curvature frequency of fibers. The percentage area positive for fibulin-1 and fibronectin increased with 2.5% and 5.9% respectively (relative increase of 124% and 15%). No changes were found for elastin. The changes in collagen and fibulin-1 negatively associated with changes in FEV1-reversibility. Next to ASM reduction, BT impacts RBM thickness and the ECM arrangement characterized by an increase in tissue area occupied by collagen with a less dense fiber organization. Both collagen and fibulin-1 are negatively associated with the change in FEV1-reversibility. BT induces ECM reorganization and airway wall stability.

Characterization of the TRPV6 calcium channel-specific phenotype by RNA-seq in castration-resistant human prostate cancer cells.

Background: Transient receptor potential vanilloid subfamily member 6 (TRPV6), a highly calcium-selective channel, has been shown to play a significant role in calcium homeostasis and to participate both in vitro and in vivo in growth, cell survival, and drug resistance of prostate cancer. Its role and the corresponding calcium-dependent pathways were mainly studied in hormone-dependent human prostate cancer cell lines, often used as a model of early-stage prostate cancers. The goal of the present study was to describe the TRPV6-specific phenotype and signaling pathways it is involved in, using castration-resistant prostate cancer cell lines. Methods: RNA sequencing (RNA-seq) was used to study the gene expression impacted by TRPV6 using PC3Mtrpv6-/- versus PC3Mtrpv6+/+ and its derivative PC3M-luc-C6trpv6+/+ cell line in its native and TRPV6 overexpressed form. In addition to the whole-cell RNA sequencing, immunoblotting, quantitative PCR, and calcium imaging were used to validate trpv6 gene status and functional consequences, in both trpv6 -/- and TRPV6 overexpression cell lines. Results: trpv6 -/- status was validated using both immunoblotting and quantitative PCR, and the functional consequences of either trpv6 gene deletion or TRPV6 overexpression were shown using calcium imaging. RNA-seq analysis demonstrated that the calcium channel TRPV6, being a crucial player of calcium signaling, significantly impacts the expression of genes involved in cancer progression, such as cell cycle regulation, chemotaxis, migration, invasion, apoptosis, ferroptosis as well as drug resistance, and extracellular matrix (ECM) re-organization. Conclusion: Our data suggest that the trpv6 gene is involved in and regulates multiple pathways related to tumor progression and drug resistance in castration-resistant prostate cancer cells.

SEMA7a primes integrin α5β1 engagement instructing fibroblast mechanotransduction, phenotype and transcriptional programming

Integrins are cellular receptors that bind the extracellular matrix (ECM) and facilitate the transduction of biochemical and biophysical microenvironment cues into cellular responses. Upon engaging the ECM, integrin heterodimers must rapidly strengthen their binding with the ECM, resulting in the assembly of force-resistant and force-sensitive integrin associated complexes (IACs). The IACs constitute an essential apparatus for downstream signaling and fibroblast phenotypes. During wound healing, integrin signaling is essential for fibroblast motility, proliferation, ECM reorganization and, ultimately, restoration of tissue homeostasis. Semaphorin 7A (SEMA7a) has been previously implicated in post-injury inflammation and tissue fibrosis, yet little is known about SEMA7a's role in directing stromal cell, particularly fibroblast, behaviors. We demonstrate that SEMA7a regulates integrin signaling through cis-coupling with active integrin α5β1 on the plasma membrane, enabling rapid integrin adhesion strengthening to fibronectin (Fn) and normal downstream mechanotransduction. This molecular function of SEMA7a potently regulates fibroblast adhesive, cytoskeletal, and migratory phenotype with strong evidence of downstream alterations in chromatin structure resulting in global transcriptomic reprogramming such that loss of SEMA7a expression is sufficient to impair the normal migratory and ECM assembly phenotype of fibroblasts resulting in significantly delayed tissue repair in vivo.

Mesenchymal stem cells: An efficient celltherapy for tendon repair (Review).

Tendon injury is a common disorder of the musculoskeletal system caused by overuse or trauma. With increasing incidence of tendon injuries, it is necessary to find an effective treatment. Mesenchymal stem cells (MSCs) are attracting attention because of their high proliferative and self‑renewal capacity. These functions of MSCs show promise in treating a variety of diseases, including immune and musculoskeletal system disorder and cardiovascular disease, and show especially satisfactory effects in the treatment of tendon injury. First, since MSCs have multidirectional differentiation potential, they differentiate into specific cells after induction invivo and invitro. Furthermore, MSCs have paracrine functions and can secrete biologically active molecules and exosomes such as cytokines, growth factors and chemokines to promote tissue repair and regeneration. In tendon injury, MSCs promote tendon repair through four mechanisms: Decreasing inflammation and promoting neovascularization and cell proliferation and differentiation. They are also involved in extracellular matrix reorganization by promoting collagen production and transforming typeIII collagen fibers to typeI collagen fibers. The present review summarized preclinical experiments with different sources of MSCs and their mechanisms in tendon repair, as well as the limitations of MSCs in current clinical applications and directions that need to be explored in the future.

Experience in complex therapy of lower limb trophic ulcers of venous and diabetic origin

Trophic ulcers (TU) associated with venous circulation pathology or diabetes mellitus are considered chronic wounds with all the attributes of the complex extracellular matrix (ECM) reorganization and tissue remodelling of the edges and bed of these wounds. It was therefore determined that only a comprehensive step-wise approach to the treatment of TU can produce a stable clinical result. The article presents the experience of comprehensive management of venous and diabetic TUs. Three clinical cases of management of patients with lower limb TUs are described. The treatment of patients was planned taking into account the accepted algorithms for the management of TU associated venous circulation pathology or diabetes mellitus. The topical therapy included advanced wound coverings. The choice of dressing was determined by the clinical course of wound process. Alginate dressings were used on stages I and II wounds; hydrocolloid dressings were used on stages II and III wounds. Effects resulting from treatment included almost complete healing or readiness of a TU for skin grafting. The topical therapy of venous and diabetic TUs is part of the comprehensive treatment. During phase 1 wound process, the topical drugs should have antimicrobial, necrolytic, draining, osmotic and analgesic action. During phase 2, the granulation tissue should be induced and the balanced moist environment should be created to promote tissue regeneration. The wound coverings specified in the above clinical examples are fully consistent with the principles of the TIME concept, their ease of use and comfort for the patient are pointed out.