The alveolar-capillary interface is the key functional element of gas exchange in the human lung, and disruptions to this interface can lead to significant medical complications. However, it is currently challenging to adequately model this interface in vitro, as it requires not only the co-culture of human alveolar epithelial and endothelial cells but mainly the preparation of a biocompatible scaffold that mimics the basement membrane. This scaffold should support cell seeding from both sides, and maintain optimal cell adhesion, growth, and differentiation conditions. Our study investigates the use of polycaprolactone (PCL) nanofibers as a versatile substrate for such cell cultures, aiming to model the alveolar-capillary interface more accurately. We optimized nanofiber production parameters, utilized polyamide mesh UHELON as a mechanical support for scaffold handling, and created 3D-printed inserts for specialized co-cultures. Our findings confirm that PCL nanofibrous scaffolds are manageable and support the co-culture of diverse cell types, effectively enabling cell attachment, proliferation, and differentiation. Our research establishes a proof-of-concept model for the alveolar-capillary interface, offering significant potential for enhancing cell-based testing and advancing tissue-engineering applications that require specific nanofibrous matrices.
High-intensity interval training (HIIT) is considered an effective therapy strategy for improving chronic pain associated with osteoarthritis (OA). Perineuronal nets (PNNs) are specialized extracellular matrix structures in the cerebral cortex that play a crucial role in regulating chronic pain. However, little is unknown whether HIIT could alleviate OA pain sensitization by reducing PNN levels. This study aimed to determine whether HIIT could reduce sensitivity of the affected joint(s) to pain in a chronic pain model in rats with OA. A rat model of interest was induced by intra-articular injection of monosodium iodoacetate (MIA) into the right knee. Thereafter, the mechanical withdrawal thresholds (MWTs) and PNN levels in the contralateral medial prefrontal cortex (mPFC) were measured in rats in the presence or absence of HIIT alone or in combination with injection of chondroitinase-ABC (ChABC) into the contralateral mPFC (inducing the degradation of PNNs), respectively. Results indicated that rats with OA exhibited significant reductions in MWTs, but a significant increase in the PNN levels; that HIIT reversed changes in MWTs and PNN levels in rats with OA, and that pretreatment of ChABC abolished effects of HIIT on MWTs, with PNN levels not changed. We concluded that pain sensitization in rats with OA may correlate with an increase in PNN levels in the mPFC, and that HIIT may increases OA pain-sensitive threshold by reduction of the PNN levels in the mPFC. Keywords: Osteoarthritis, Chronic pain, Pain sensitization, High-intensity interval training, Perineuronal nets.
- MeSH
- chronická bolest terapie patofyziologie MeSH
- extracelulární matrix metabolismus MeSH
- kondiční příprava zvířat fyziologie metody MeSH
- krysa rodu rattus MeSH
- osteoartróza * terapie MeSH
- potkani Sprague-Dawley * MeSH
- práh bolesti * MeSH
- prefrontální mozková kůra * metabolismus MeSH
- vysoce intenzivní intervalový trénink * MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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
- extracelulární matrix metabolismus ultrastruktura MeSH
- fibronektiny * metabolismus MeSH
- kathepsin G metabolismus MeSH
- krysa rodu rattus MeSH
- lyzozomy ultrastruktura metabolismus MeSH
- matrixové metaloproteinasy metabolismus MeSH
- plíce * ultrastruktura metabolismus MeSH
- proteasy metabolismus MeSH
- stárnutí * metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- chronická lymfatická leukemie genetika MeSH
- DNA-dependentní DNA-polymerasy genetika metabolismus MeSH
- DNA-primasa genetika metabolismus MeSH
- extracelulární matrix metabolismus patologie MeSH
- geny p53 genetika MeSH
- glioblastom metabolismus patologie MeSH
- pericyty metabolismus patologie MeSH
- protein BRCA1 genetika metabolismus MeSH
- Publikační typ
- souhrny MeSH
We have recently developed a model of pancreatic islet transplantation into a decellularized pancreatic tail in rats. As the pancreatic skeletons completely lack endothelial cells, we investigated the effect of co-transplantation of mesenchymal stem cells and endothelial cells to promote revascularization. Decellularized matrix of the pancreatic tail was prepared by perfusion with Triton X-100, sodium dodecyl sulfate and DNase solution. Isolated pancreatic islets were infused into the skeletons via the splenic vein either alone, together with adipose tissue-derived mesenchymal stem cells (adMSCs), or with a combination of adMSCs and rat endothelial cells (rat ECs). Repopulated skeletons were transplanted into the subcutaneous tissue and explanted 9 days later for histological examination. Possible immunomodulatory effects of rat adMSCs on the survival of highly immunogenic green protein-expressing human ECs were also tested after their transplantation beneath the renal capsule. The immunomodulatory effects of adMSCs were also tested in vitro using the Invitrogen Click-iT EdU system. In the presence of adMSCs, the proliferation of splenocytes as a response to phytohaemagglutinin A was reduced by 47% (the stimulation index decreased from 1.7 to 0.9, P = 0.008) and the reaction to human ECs was reduced by 58% (the stimulation index decreased from 1.6 to 0.7, P = 0.03). Histological examination of the explanted skeletons seeded only with the islets showed their partial disintegration and only a rare presence of CD31-positive cells. However, skeletons seeded with a combination of islets and adMSCs showed preserved islet morphology and rich vascularity. In contrast, the addition of syngeneic rat ECs resulted in islet-cell necrosis with only few endothelial cells present. Live green fluorescence-positive endothelial cells transplanted either alone or with adMSCs were not detected beneath the renal capsule. Though the adMSCs significantly reduced in vitro proliferation stimulated by either phytohaemagglutinin A or by xenogeneic human ECs, in vivo co-transplanted adMSCs did not suppress the post-transplant immune response to xenogeneic ECs. Even in the syngeneic model, ECs co-transplantation did not lead to sufficient vascularization in the transplant area. In contrast, islet co-transplantation together with adMSCs successfully promoted the revascularization of extracellular matrix in the subcutaneous tissue.
- MeSH
- decelularizovaná extracelulární matrix MeSH
- endoteliální buňky MeSH
- fyziologická neovaskularizace * MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- Langerhansovy ostrůvky * imunologie MeSH
- lidé MeSH
- mezenchymální kmenové buňky * MeSH
- pankreas MeSH
- transplantace Langerhansových ostrůvků * metody MeSH
- transplantace mezenchymálních kmenových buněk * metody MeSH
- tuková tkáň * cytologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
In preovulatory follicles, after the endogenous gonadotropin surge, the oocyte-cumulus complexes (OCCs) produce hyaluronan (HA) in a process called "cumulus expansion". During this process, the heavy chains (HCs) of the serum-derived inter-alpha-trypsin inhibitor (IαI) family bind covalently to synthesized HA and form a unique structure of the expanded cumulus HA-rich extracellular matrix. Understanding the biochemical mechanism of the covalent linkage between HA and the HCs of the IαI family is one of the most significant discoveries in reproductive biology, since it explains basis of the cumulus expansion process running in parallel with the oocyte maturation, both essential for ovulation. Two recent studies have supported the above-mentioned findings: in the first, seven components of the extracellular matrix were detected by proteomic, evolutionary, and experimental analyses, and in the second, the essential role of serum in the process of cumulus expansion in vitro was confirmed. We have previously demonstrated the formation of unique structure of the covalent linkage of HA to HCs of IαI in the expanded gonadotropin-stimulated OCC, as well as interactions with several proteins produced by the cumulus cells: tumor necrosis factor-alpha-induced protein 6, pentraxin 3, and versican. Importantly, deletion of these genes in the mice produces female infertility due to defects in the oocyte-cumulus structure.
- MeSH
- alfa-globuliny metabolismus MeSH
- C-reaktivní protein metabolismus MeSH
- extracelulární matrix * metabolismus MeSH
- kumulární buňky * metabolismus MeSH
- kyselina hyaluronová * metabolismus MeSH
- lidé MeSH
- myši MeSH
- oocyty * metabolismus MeSH
- ovariální folikul * metabolismus MeSH
- sérový amyloidový protein metabolismus genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Crohn's disease (CD) is marked by recurring intestinal inflammation and tissue injury, often resulting in fibrostenosis and bowel obstruction, necessitating surgical intervention with high recurrence rates. To elucidate the mechanisms underlying fibrostenosis in CD, we analyzed the transcriptome of cells isolated from the transmural ileum of patients with CD, including a trio of lesions from each patient: non-affected, inflamed, and stenotic ileum samples, and compared them with samples from patients without CD. Our computational analysis revealed that profibrotic signals from a subset of monocyte-derived cells expressing CD150 induced a disease-specific fibroblast population, resulting in chronic inflammation and tissue fibrosis. The transcription factor TWIST1 was identified as a key modulator of fibroblast activation and extracellular matrix (ECM) deposition. Genetic and pharmacological inhibition of TWIST1 prevents fibroblast activation, reducing ECM production and collagen deposition. Our findings suggest that the myeloid-stromal axis may offer a promising therapeutic target to prevent fibrostenosis in CD.
- MeSH
- Crohnova nemoc * metabolismus patologie imunologie MeSH
- dospělí MeSH
- endopeptidasy metabolismus genetika MeSH
- extracelulární matrix metabolismus patologie MeSH
- fibroblasty * metabolismus patologie MeSH
- fibróza * MeSH
- ileum patologie metabolismus imunologie MeSH
- jaderné proteiny metabolismus genetika MeSH
- lidé MeSH
- mezibuněčná komunikace MeSH
- monocyty * metabolismus patologie imunologie MeSH
- myši MeSH
- receptory buněčného povrchu metabolismus genetika MeSH
- transkripční faktor Twist * metabolismus genetika MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Recent research has identified that miR-539-3p impedes chondrogenic differentiation, yet its specific role and underlying mechanisms in childhood-onset osteoarthritis (OA) remain unclear. This study found that miR-539-3p levels were considerably lower in cartilage samples derived from childhood-onset OA patients compared to the control group. Enhancing miR-539-3p expression or suppressing RUNX2 expression notably reduced apoptosis, inflammation, and extracellular matrix (ECM) degradation in OA chondrocytes. In contrast, reducing miR-539-3p or increasing RUNX2 had the opposite effects. RUNX2 was confirmed as a direct target of miR-539-3p. Further experiments demonstrated that miR-539-3p targeting RUNX2 effectively lessened apoptosis, inflammation, and ECM degradation in OA chondrocytes, accompanied by changes in key molecular markers like reduced caspase-3 and matrix etallopeptidase 13 (MMP-13) levels, and increased B-cell lymphoma 2 (Bcl-2) and collagen type X alpha 1 chain (COL2A1). This study underscores the pivotal role of miR-539-3p in alleviating inflammation and ECM degradation in childhood-onset OA through targeting RUNX2, offering new insights for potential therapeutic strategies against this disease.
- MeSH
- apoptóza * MeSH
- chondrocyty * metabolismus patologie MeSH
- dítě MeSH
- extracelulární matrix * metabolismus patologie MeSH
- kultivované buňky MeSH
- lidé MeSH
- mikro RNA * metabolismus genetika MeSH
- mladiství MeSH
- osteoartróza * metabolismus patologie genetika MeSH
- protein PEBP2alfaA * metabolismus genetika MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Gliomagenesis induces profound changes in the composition of the extracellular matrix (ECM) of the brain. In this study, we identified a cellular population responsible for the increased deposition of collagen I and fibronectin in glioblastoma. Elevated levels of the fibrillar proteins collagen I and fibronectin were associated with the expression of fibroblast activation protein (FAP), which is predominantly found in pericyte-like cells in glioblastoma. FAP+ pericyte-like cells were present in regions rich in collagen I and fibronectin in biopsy material and produced substantially more collagen I and fibronectin in vitro compared to other cell types found in the GBM microenvironment. Using mass spectrometry, we demonstrated that 3D matrices produced by FAP+ pericyte-like cells are rich in collagen I and fibronectin and contain several basement membrane proteins. This expression pattern differed markedly from glioma cells. Finally, we have shown that ECM produced by FAP+ pericyte-like cells enhances the migration of glioma cells including glioma stem-like cells, promotes their adhesion, and activates focal adhesion kinase (FAK) signaling. Taken together, our findings establish FAP+ pericyte-like cells as crucial producers of a complex ECM rich in collagen I and fibronectin, facilitating the dissemination of glioma cells through FAK activation.
- MeSH
- endopeptidasy MeSH
- extracelulární matrix * metabolismus patologie MeSH
- fibronektiny * metabolismus MeSH
- glioblastom patologie metabolismus MeSH
- gliom * patologie metabolismus MeSH
- kolagen typu I metabolismus MeSH
- lidé MeSH
- membránové proteiny metabolismus MeSH
- nádorové buněčné linie MeSH
- nádorové mikroprostředí fyziologie MeSH
- nádory mozku * patologie metabolismus MeSH
- pericyty * metabolismus patologie MeSH
- pohyb buněk fyziologie MeSH
- serinové endopeptidasy metabolismus MeSH
- želatinasy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Lately, the need for three-dimensional (3D) cell culture has been recognized in order to closely mimic the organization of native tissues. Thus, 3D scaffolds started to be employed to facilitate the 3D cell organization and enable the artificial tissue formation for the emerging tissue engineering applications. 3D scaffolds can be prepared by various techniques, each with certain advantages and disadvantages. Decellularization is an easy method based on removal of cells from native tissue sample, yielding extracellular matrix (ECM) scaffold with preserved architecture and bioactivity. This chapter provides a detailed protocol for decellularization of pig lung and also some basic assays for evaluation of its effectivity, such as determination of DNA content and histological verification of the selected ECM components. Such decellularized scaffold can subsequently be used for various tissue engineering applications, for example, for recellularization with cells of interest, for natural ECM hydrogel preparation, or as a bioink for 3D bioprinting.
- MeSH
- extracelulární matrix MeSH
- hydrogely MeSH
- plíce * MeSH
- prasata MeSH
- tkáňové inženýrství * metody MeSH
- tkáňové podpůrné struktury * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
