INTRODUCTION: The aging process is intricately linked to alterations in cellular and tissue structures, with the respiratory system being particularly susceptible to age-related changes. Therefore, this study aimed to profile the activity of proteases using activity-based probes in lung tissues of old and young rats, focusing on the expression levels of different, in particular cathepsins G and X and matrix Metalloproteinases (MMPs). Additionally, the impact on extracellular matrix (ECM) components, particularly fibronectin, in relation to age-related histological and ultrastructural changes in lung tissues was investigated. MATERIALS AND METHODS: Lung tissues from old and young rats were subjected to activity-based probe profiling to assess the activity of different proteases. Expression levels of cathepsins G and X were quantified, and zymography was performed to evaluate matrix metalloproteinases activity. Furthermore, ECM components, specifically fibronectin, were examined for signs of degradation in the old lung tissues compared to the young ones. Moreover, histological, immunohistochemical and ultrastructural assessments of old and young lung tissue were also conducted. RESULTS: Our results showed that the expression levels of cathepsins G and X were notably higher in old rat lung tissues in contrast to those in young rat lung tissues. Zymography analysis revealed elevated MMP activity in the old lung tissues compared to the young ones. Particularly, significant degradation of fibronectin, an essential ECM component, was observed in the old lung tissues. Numerous histological and ultrastructural alterations were observed in old lung tissues compared to young lung tissues. DISCUSSION AND CONCLUSION: The findings indicate an age-related upregulation of cathepsins G and X along with heightened MMP activity in old rat lung tissues, potentially contributing to the degradation of fibronectin within the ECM. These alterations highlight potential mechanisms underlying age-associated changes in lung tissue integrity and provide insights into protease-mediated ECM remodeling in the context of aging lungs.
- MeSH
- Extracellular Matrix metabolism ultrastructure MeSH
- Fibronectins * metabolism MeSH
- Cathepsin G metabolism MeSH
- Rats MeSH
- Lysosomes ultrastructure metabolism MeSH
- Matrix Metalloproteinases metabolism MeSH
- Lung * ultrastructure metabolism MeSH
- Peptide Hydrolases metabolism MeSH
- Aging * metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
This is the first histological and molecular analysis of two chondrosarcomas with target-like chondrocytes that were compared with a group of conventional chondrosarcomas and enchondromas. The unique histological feature of target-like chondrocytes is the presence of unusual hypertrophic eosinophilic APAS-positive perichondrocytic rings (baskets). In the sections stained with Safranin O/Fast green, the outer part of the ring was blue and the material in the lacunar space stained orange, similarly to intercellular regions. Immunohistochemical examination showed strong positivity for vimentin, factor XIIIa, cyclin D1, osteonectin, B-cell lymphoma 2 apoptosis regulator (Bcl-2), p53 and p16. The S-100 protein was positive in 25 % of neoplastic cells. Antibodies against GFAP, D2-40 (podoplanin), CD99, CKAE1.3 and CD10 exhibited weak focal positivity. Pericellular rings/baskets contained type VI collagen in their peripheral part, in contrast to the type II collagen in intercellular interterritorial spaces. Ultrastructural examination revealed that pericellular rings contained an intralacunar component composed of microfibrils with abundant admixture of aggregates of dense amorphous non-fibrillar material. The outer extralacunar zone was made up of a layer of condensed thin collagen fibrils with admixture of non-fibrillar dense material. NGS sequencing identified a fusion transcript involving fibronectin 1 (FN1) and fibroblast growth factor receptor 2 (FGFR2) at the RNA level. At the DNA level, no significant variant was revealed except for the presumably germline variant in the SPTA1 gene.
- MeSH
- Chondrocytes chemistry pathology ultrastructure MeSH
- Chondrosarcoma * chemistry diagnosis pathology MeSH
- Extracellular Matrix chemistry metabolism ultrastructure MeSH
- Immunohistochemistry MeSH
- Humans MeSH
- Bone Neoplasms * diagnosis metabolism MeSH
- S100 Proteins metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Cells have developed a unique set of molecular mechanisms that allows them to probe mechanical properties of the surrounding environment. These systems are based on deformable primary mechanosensors coupled to tension transmitting proteins and enzymes generating biochemical signals. This modular setup enables to transform a mechanical load into more versatile biochemical information. Src kinase appears to be one of the central components of the mechanotransduction network mediating force-induced signalling across multiple cellular contexts. In tight cooperation with primary sensors and the cytoskeleton, Src functions as an effector molecule necessary for transformation of mechanical stimuli into biochemical outputs executing cellular response and adaptation to mechanical cues.
- MeSH
- Adaptor Proteins, Signal Transducing genetics metabolism MeSH
- Mechanotransduction, Cellular genetics MeSH
- Cytoskeleton metabolism pathology ultrastructure MeSH
- Extracellular Matrix metabolism pathology ultrastructure MeSH
- Integrins genetics metabolism MeSH
- Humans MeSH
- Stress, Mechanical MeSH
- Neoplasms genetics metabolism pathology MeSH
- Protein Serine-Threonine Kinases genetics metabolism MeSH
- Gene Expression Regulation MeSH
- src-Family Kinases genetics metabolism MeSH
- Crk-Associated Substrate Protein genetics metabolism MeSH
- Transcription Factors genetics metabolism MeSH
- Receptor-Like Protein Tyrosine Phosphatases, Class 4 genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
RATIONALE: Cardiac ECM (extracellular matrix) comprises a dynamic molecular network providing structural support to heart tissue function. Understanding the impact of ECM remodeling on cardiac cells during heart failure (HF) is essential to prevent adverse ventricular remodeling and restore organ functionality in affected patients. OBJECTIVES: We aimed to (1) identify consistent modifications to cardiac ECM structure and mechanics that contribute to HF and (2) determine the underlying molecular mechanisms. METHODS AND RESULTS: We first performed decellularization of human and murine ECM (decellularized ECM) and then analyzed the pathological changes occurring in decellularized ECM during HF by atomic force microscopy, 2-photon microscopy, high-resolution 3-dimensional image analysis, and computational fluid dynamics simulation. We then performed molecular and functional assays in patient-derived cardiac fibroblasts based on YAP (yes-associated protein)-transcriptional enhanced associate domain (TEAD) mechanosensing activity and collagen contraction assays. The analysis of HF decellularized ECM resulting from ischemic or dilated cardiomyopathy, as well as from mouse infarcted tissue, identified a common pattern of modifications in their 3-dimensional topography. As compared with healthy heart, HF ECM exhibited aligned, flat, and compact fiber bundles, with reduced elasticity and organizational complexity. At the molecular level, RNA sequencing of HF cardiac fibroblasts highlighted the overrepresentation of dysregulated genes involved in ECM organization, or being connected to TGFβ1 (transforming growth factor β1), interleukin-1, TNF-α, and BDNF signaling pathways. Functional tests performed on HF cardiac fibroblasts pointed at mechanosensor YAP as a key player in ECM remodeling in the diseased heart via transcriptional activation of focal adhesion assembly. Finally, in vitro experiments clarified pathological cardiac ECM prevents cell homing, thus providing further hints to identify a possible window of action for cell therapy in cardiac diseases. CONCLUSIONS: Our multiparametric approach has highlighted repercussions of ECM remodeling on cell homing, cardiac fibroblast activation, and focal adhesion protein expression via hyperactivated YAP signaling during HF.
- MeSH
- Adaptor Proteins, Signal Transducing genetics metabolism MeSH
- Mechanotransduction, Cellular MeSH
- Cardiomyopathy, Dilated genetics metabolism pathology physiopathology MeSH
- Extracellular Matrix genetics metabolism ultrastructure MeSH
- Fibroblasts metabolism ultrastructure MeSH
- Ventricular Function, Left * MeSH
- Myocardial Infarction genetics metabolism pathology physiopathology MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Disease Models, Animal MeSH
- Myocardium metabolism ultrastructure MeSH
- Mice, Inbred C57BL MeSH
- Cell Movement MeSH
- Ventricular Remodeling * MeSH
- Heart Failure genetics metabolism pathology physiopathology MeSH
- Case-Control Studies MeSH
- Transcription Factors genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Video-Audio Media MeSH
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
INTRODUCTION: Multilamellar bodies (MLBs) are concentric cytoplasmic membranes which form through an autophagy-dependent mechanism. In the cornea, the presence of MLBs is associated with Schnyder corneal dystrophy (SCD). Ex vivo 3D modelling of the corneal stroma and SCD can help study pathogenesis and resolution of the disorder. METHODS: Corneal stroma explants were isolated from cadavers and cultivated long-term for more than 3 months to achieve spontaneous 3D outgrowth of corneal stroma-derived mesenchymal stem-like cells (CSMSCs). The 3D tissues were then examined by transmission electron microscopy (TEM) for presence of MLBs, and by immunofluorescent labelling against markers for autophagy (p62, LC3). Autophagy was induced by classical serum starvation or rapamycin (RAP) treatment (50 nM), and inhibited by the autophagy inhibitor 3-methyladenine (3-MA, 10 mM) for 24 hours. RESULTS: CSMSCs can form spontaneously 3D outgrowths over a 3-4 weeks period, depositing their own extracellular matrix containing collagen I. TEM confirmed the presence of MLBs in the long-term (>3 months) 3D cultures, which became more abundant under starvation and RAP treatment, and decreased in number under autophagy inhibition with 3-MA. The presence of autophagy and its disappearance could be confirmed by an inversely related increase and decrease in the expression of LC3 and p62, respectively. CONCLUSIONS: MLB formation in long-standing CSMSC cultures could serve as a potential ex vivo model for studying corneal stroma diseases, including SCD. Inhibition of autophagy can decrease the formation of MLBs, which may lead to a novel treatment of the disease in the future.
- MeSH
- Adenine analogs & derivatives pharmacology MeSH
- Models, Anatomic MeSH
- Autophagy * drug effects MeSH
- Inclusion Bodies pathology ultrastructure MeSH
- Corneal Dystrophies, Hereditary pathology physiopathology MeSH
- Adult MeSH
- Extracellular Matrix metabolism ultrastructure MeSH
- Fluorescent Antibody Technique MeSH
- Cells, Cultured MeSH
- Middle Aged MeSH
- Humans MeSH
- Mesenchymal Stem Cells MeSH
- Cadaver MeSH
- Cornea metabolism MeSH
- Aged MeSH
- Corneal Stroma pathology physiopathology ultrastructure MeSH
- Microscopy, Electron, Transmission MeSH
- Imaging, Three-Dimensional MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Aged MeSH
- Publication type
- Journal Article MeSH
In this study we present an innovative method for the preparation of fully hydrated samples of microbial biofilms of cultures Staphylococcus epidermidis, Candida parapsilosis and Candida albicans. Cryo-scanning electron microscopy (cryo-SEM) and high-pressure freezing (HPF) rank among cutting edge techniques in the electron microscopy of hydrated samples such as biofilms. However, the combination of these techniques is not always easily applicable. Therefore, we present a method of combining high-pressure freezing using EM PACT2 (Leica Microsystems), which fixes hydrated samples on small sapphire discs, with a high resolution SEM equipped with the widely used cryo-preparation system ALTO 2500 (Gatan). Using a holder developed in house, a freeze-fracturing technique was applied to image and investigate microbial cultures cultivated on the sapphire discs. In our experiments, we focused on the ultrastructure of the extracellular matrix produced during cultivation and the relationships among microbial cells in the biofilm. The main goal of our investigations was the detailed visualization of areas of the biofilm where the microbial cells adhere to the substrate/surface. We show the feasibility of this technique, which is clearly demonstrated in experiments with various freeze-etching times.
- MeSH
- Biofilms MeSH
- Candida albicans ultrastructure MeSH
- Candida parapsilosis ultrastructure MeSH
- Cryoelectron Microscopy methods MeSH
- Extracellular Matrix ultrastructure MeSH
- Microscopy, Electron, Scanning methods MeSH
- Freeze Fracturing methods MeSH
- Staphylococcus epidermidis ultrastructure MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The formation and development of bacterial as well as yeast or filamentous fungi biofilms, which is connected with dangerous or even fatal human infections, represents a relatively new and poorly studied problem in current medicine. The understanding of the processes running in biofilms of pathogenic microorganisms and finding of new possibilities of the biofilm prevention or eradication and also the regulation of biofilm resistance development is necessary for proposing advanced treatment procedures for wide range of illnesses.
- Keywords
- extracelulární polymerní látky,
- MeSH
- Drug Resistance, Microbial drug effects MeSH
- Anti-Infective Agents MeSH
- Aspergillus fumigatus pathogenicity MeSH
- Aspergillosis, Allergic Bronchopulmonary microbiology MeSH
- Bacterial Adhesion physiology MeSH
- Biofilms * growth & development drug effects MeSH
- Candida classification pathogenicity MeSH
- Cystic Fibrosis microbiology MeSH
- Disinfection MeSH
- Escherichia coli pathogenicity MeSH
- Extracellular Matrix Proteins MeSH
- Extracellular Matrix physiology ultrastructure MeSH
- Prosthesis-Related Infections etiology microbiology MeSH
- Catheters microbiology MeSH
- Otitis Media microbiology MeSH
- Periodontitis microbiology MeSH
- Pseudomonas aeruginosa pathogenicity MeSH
- Staphylococcus aureus pathogenicity MeSH
- Staphylococcus epidermidis pathogenicity MeSH
- Streptococcus mutans pathogenicity MeSH
- Trichosporon pathogenicity MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
The use of non-standard low-temperature conditions in environmental scanning electron microscopy might be promising for the observation of coniferous tissues in their native state. This study is aimed to analyse and evaluate the method based on the principle of low-temperature sample stabilization. We demonstrate that the upper mucous layer is sublimed and a microstructure of the sample surface can be observed with higher resolution at lower gas pressure conditions, thanks to a low-temperature method. An influence of the low-temperature method on sample stability was also studied. The results indicate that high-moisture conditions are not suitable for this method and often cause the collapse of samples. The potential improvement of stability to beam damage has been demonstrated by long-time observation at different operation parameters. We finally show high applicability of the low-temperature method on different types of conifers and Oxalis acetosella.
In organized tissues, the precise geometry and the overall shape are critical for the specialized functions that the cells carry out. Odontoblasts are major matrix-producing cells of the tooth and have also been suggested to participate in sensory transmission. However, refined morphologic data on these important cells are limited, which hampers the analysis and understanding of their cellular functions. We took advantage of fluorescent color-coding genetic tracing to visualize and reconstruct in 3 dimensions single odontoblasts, pulp cells, and their assemblages. Our results show distinct structural features and compartments of odontoblasts at different stages of maturation, with regard to overall cellular shape, formation of the main process, orientation, and matrix deposition. We demonstrate previously unanticipated contacts between the processes of pulp cells and odontoblasts. All reported data are related to mouse incisor tooth. We also show that odontoblasts express TRPM5 and Piezo2 ion channels. Piezo2 is expressed ubiquitously, while TRPM5 is asymmetrically distributed with distinct localization to regions proximal to and within odontoblast processes.
- MeSH
- Ameloblasts cytology ultrastructure MeSH
- Cell Nucleus ultrastructure MeSH
- Cell Surface Extensions ultrastructure MeSH
- Dentin ultrastructure MeSH
- Extracellular Matrix ultrastructure MeSH
- Fluorescent Antibody Technique MeSH
- Ion Channels ultrastructure MeSH
- TRPM Cation Channels ultrastructure MeSH
- Cell Compartmentation MeSH
- Mesenchymal Stem Cells cytology ultrastructure MeSH
- Microscopy, Electron, Scanning methods MeSH
- Mice, Transgenic MeSH
- Mice MeSH
- Odontoblasts cytology ultrastructure MeSH
- Incisor cytology ultrastructure MeSH
- Cell Shape MeSH
- Imaging, Three-Dimensional methods MeSH
- Dental Pulp cytology ultrastructure MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Invadopodia and podosomes have been intensively studied because of their involvement in the degradation of extracellular matrix. As both structures have been studied mostly on thin matrices, their commonly reported shapes and characteristics may differ from those in vivo. To assess the morphology of invadopodia in a complex 3D environment, we observed invadopodial formation in cells grown on a dense matrix based on cell-free dermis. We have found that invadopodia differ in morphology when cells grown on the dermis-based matrix and thin substrates are compared. The cells grown on the dermis-based matrix display invadopodia which are formed by a thick protruding base rich in F-actin, phospho-paxillin, phospho-cortactin and phosphotyrosine signal, from which numerous thin filaments protrude into the matrix. The protruding filaments are composed of an F-actin core and are free of phospho-paxillin and phospho-cortactin but capped by phosphotyrosine signal. Furthermore, we found that a matrix-degrading activity is localized to the base of invadopodia and not along the matrix-penetrating protrusions. Our description of invadopodial structures on a dermis-based matrix should greatly aid the development of new criteria for the identification of invadopodia in vivo, and opens up the possibility of studying the invadopodia-related signaling in a more physiological environment.
- MeSH
- Actins metabolism MeSH
- Cell Culture Techniques MeSH
- Cell Surface Extensions metabolism ultrastructure MeSH
- Cytoskeleton metabolism MeSH
- Microscopy, Electron MeSH
- Sarcoma, Experimental metabolism ultrastructure MeSH
- Extracellular Matrix metabolism physiology ultrastructure MeSH
- Fluorescent Antibody Technique MeSH
- Cortactin metabolism MeSH
- Rats MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Swine MeSH
- Signal Transduction MeSH
- Imaging, Three-Dimensional MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
