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1.
ACS Appl Mater Interfaces ; 14(36): 40559-40568, 2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36047858

RESUMO

Piezo1 is a recently discovered Ca2+ permeable ion channel that has emerged as an integral sensor of hemodynamic forces within the cardiovascular system, contributing to vascular development and blood pressure regulation. However, how the composition of the extracellular matrix (ECM) affects the mechanosensitivity of Piezo1 in response to hemodynamic forces remains poorly understood. Using a combination of microfluidics and calcium imaging techniques, we probe the shear stress sensitivity of single HEK293T cells engineered to stably express Piezo1 in the presence of different ECM proteins. Our experiments show that Piezo1 sensitivity to shear stress is not dependent on the presence of ECM proteins. However, different ECM proteins regulate the sensitivity of Piezo1 depending on the shear stress level. Under high shear stress, fibronectin sensitizes Piezo1 response to shear, while under low shear stress, Piezo1 mechanosensitivity is improved in the presence of collagen types I and IV and laminin. Moreover, we report that α5ß1 and αvß3 integrins are involved in Piezo1 sensitivity at high shear, while αvß3 and αvß5 integrins are involved in regulating the Piezo1 response at low shear stress. These results demonstrate that the ECM/integrin interactions influence Piezo1 mechanosensitivity and could represent a mechanism whereby extracellular forces are transmitted to Piezo1 channels, providing new insights into the mechanism by which Piezo1 senses shear stress.


Assuntos
Canais Iônicos , Mecanotransdução Celular , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Integrinas/metabolismo , Mecanotransdução Celular/fisiologia
2.
Oxid Med Cell Longev ; 2022: 1135827, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36071864

RESUMO

Osteoarthritis (OA) is a frequently observed condition in aged people. OA cartilage is characterized by chondrocyte apoptosis, chondrocyte inflammation, and hyperactive catabolism of extracellular matrix. However, the specific molecular mechanisms remain unclear. Recent data has shown that Angptl4, a multifunctional cytokine, is involved in the regulation of inflammatory and apoptosis responses in different tissues. This study is aimed at defining the role of Angptl4 in the development of OA. We employed X-ray analysis, safranin O-fast green (S-O) staining, and hematoxylin staining to evaluate histomorphological characteristics in the knee joint of mice. Real-time quantitative polymerase chain reaction, Western blot assays, immunofluorescence staining, and enzyme-linked immunosorbent assays (ELISA) were performed to analyze the changes in gene and protein expression. Mechanically, our data demonstrated that Angptl4 knockdown improved the degradation of extracellular matrix and reduced TNF-α-mediated chondrocyte inflammation and apoptosis by suppressing sirtuin 1/NF-κB signaling pathway. In addition, animal studies showed that the suppression of Angptl4 expression might alleviate OA development. In conclusion, our findings revealed the underlying mechanisms of Angptl4 regulation in chondrocytes and its potential value in the treatment of OA.


Assuntos
Proteína 4 Semelhante a Angiopoietina , NF-kappa B , Osteoartrite , Proteína 4 Semelhante a Angiopoietina/genética , Animais , Apoptose , Células Cultivadas , Matriz Extracelular/metabolismo , Inativação Gênica , Humanos , Inflamação/genética , Inflamação/metabolismo , Camundongos , NF-kappa B/metabolismo , Osteoartrite/genética , Osteoartrite/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo
3.
J Cell Sci ; 135(18)2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36102918

RESUMO

The roles of the extracellular matrix molecule tenascin-C (TNC) in health and disease have been extensively reviewed since its discovery over 40 years ago. Here, we will describe recent insights into the roles of TNC in tumorigenesis, angiogenesis, immunity and metastasis. In addition to high levels of expression in tumors, and during chronic inflammation, and bacterial and viral infection, TNC is also expressed in lymphoid organs. This supports potential roles for TNC in immunity control. Advances using murine models with engineered TNC levels were instrumental in the discovery of important functions of TNC as a danger-associated molecular pattern (DAMP) molecule in tissue repair and revealed multiple TNC actions in tumor progression. TNC acts through distinct mechanisms on many different cell types with immune cells coming into focus as important targets of TNC in cancer. We will describe how this knowledge could be exploited for cancer disease management, in particular for immune (checkpoint) therapies.


Assuntos
Neoplasias , Tenascina , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Matriz Extracelular/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Tenascina/genética , Tenascina/metabolismo
4.
Cell Rep ; 40(11): 111362, 2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36103820

RESUMO

Obesity is associated with increased cancer incidence and progression. However, the relationship between adiposity and cancer remains poorly understood at the mechanistic level. Here, we report that adipocytes from tumor-invasive mammary fat undergo de-differentiation to fibroblast-like precursor cells during tumor progression and integrate into the tumor microenvironment. Single-cell sequencing reveals that these de-differentiated adipocytes lose their original identities and transform into multiple cell types, including myofibroblast- and macrophage-like cells, with their characteristic features involved in immune response, inflammation, and extracellular matrix remodeling. The de-differentiated cells are metabolically distinct from tumor-associated fibroblasts but exhibit comparable effects on tumor cell proliferation. Inducing de-differentiation by Xbp1s overexpression promotes tumor progression despite lower adiposity. In contrast, promoting lipid-storage capacity in adipocytes through MitoNEET overexpression curbs tumor growth despite greater adiposity. Collectively, the metabolic interplay between tumor cells and adipocytes induces adipocyte mesenchymal transition and contributes to reconfigure the stroma into a more tumor-friendly microenvironment.


Assuntos
Neoplasias da Mama , Neoplasias Mamárias Animais , Adipócitos/metabolismo , Animais , Neoplasias da Mama/patologia , Matriz Extracelular/metabolismo , Feminino , Humanos , Neoplasias Mamárias Animais/patologia , Microambiente Tumoral
5.
Sci Rep ; 12(1): 15329, 2022 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-36097150

RESUMO

Cell morphology is profoundly influenced by cellular interactions with microenvironmental factors such as the extracellular matrix (ECM). Upon adhesion to specific ECM, various cell types are known to exhibit different but distinctive morphologies, suggesting that ECM-dependent cell morphological responses may harbour rich information on cellular signalling states. However, the inherent morphological complexity of cellular and subcellular structures has posed an ongoing challenge for automated quantitative analysis. Since multi-channel fluorescence microscopy provides robust molecular specificity important for the biological interpretations of observed cellular architecture, here we develop a deep learning-based analysis pipeline for the classification of cell morphometric phenotypes from multi-channel fluorescence micrographs, termed SE-RNN (residual neural network with squeeze-and-excite blocks). We demonstrate SERNN-based classification of distinct morphological signatures observed when fibroblasts or epithelial cells are presented with different ECM. Our results underscore how cell shapes are non-random and established the framework for classifying cell shapes into distinct morphological signature in a cell-type and ECM-specific manner.


Assuntos
Matriz Extracelular , Redes Neurais de Computação , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Microscopia de Fluorescência , Fenótipo
6.
Front Immunol ; 13: 922173, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36059551

RESUMO

Macrophage infiltration and polarization have been increasingly observed in intervertebral disc (IVD) degeneration (IDD). However, their biological roles in IDD are still unrevealed. We harvested conditioned media (CM) derived from a spectrum of macrophages induced from THP-1 cells, and examined how they affect nucleus pulposus cells (NPCs) in vitro, by studying cell proliferation, extracellular matrix (ECM) synthesis, and pro-inflammation expression; and in vivo by injection CM in a rat IDD model. Then, high-throughput sequencing was used to detect differentially expressed genes (DEGs). Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) networks were used to further analysis. Higher CCR7+ (M1 marker) and CD206+ (M2 marker) cell counts were found in the degenerated human IVD tissues as compared with the control. Furthermore, the cell co-culture model showed M1CM attenuated NPC proliferation, downregulated the expression of ECM anabolic genes encoding aggrecan and collagen IIα1, upregulated the expression of ECM catabolic genes encoding MMP-13, and inflammation-related genes encoding IL-1ß, IL-6, and IL-12, while M2CM showed contrasting trends. In IDD model, higher histological scores and lower disc height index were found following M1CM treatment, while M2CM exhibited opposite results. M1CM injection decreased ECM anabolic and increased ECM catabolic, as well as the upregulation of inflammation-related genes after 8 weeks treatment, while M2CM slowed down these trends. Finally, a total of 637 upregulated and 655 downregulated genes were detected in M1CM treated NPCs, and 975 upregulated genes and 930 downregulated genes in the M2CM groups. The top 30 GO terms were shown and the most significant KEGG pathway was cell cycle in both groups. Based on the PPI analysis, the five most significant hub genes were PLK1, KIF20A, RRM2, CDC20, and UBE2C in the M1CM groups and RRM2, CCNB1, CDC20, PLK1, and UBE2C in the M2CM groups. In conclusion, macrophage polarization exhibited diverse roles in IDD progression, with M1CM exacerbating cell proliferation suppression and IVD degeneration, while M2CM attenuated IDD development. These findings may facilitate the further elucidation of the role of macrophage polarization in IDD, and provide novel insights into the therapeutic potential of macrophages.


Assuntos
Degeneração do Disco Intervertebral , Animais , Proliferação de Células , Matriz Extracelular/metabolismo , Humanos , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Macrófagos/metabolismo , Ratos
7.
Mol Med Rep ; 26(5)2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36069233

RESUMO

Extracellular matrix tenascin­X (TNX) is the largest member of the tenascin family. Our previous study demonstrated that TNX was involved in hepatic dysfunction, including fibrosis, in mice that were administered a high­fat and high­cholesterol diet with high levels of phosphorus and calcium. The present study investigated whether overexpression of both the fibrinogen domain of TNX (TNX­FG) and integrin α11, one of the TNX cell surface receptors, induces in vitro fibrosis in LX­2 human hepatic stellate cells. Overexpression of both a 15­amino acid peptide (hTNX­FGFFFF) derived from the TNX­FG domain and integrin α11 induced the expression of type I collagen α1 chain (COL1A1). Treatment with verteporfin [YAP (Yes­associated protein) inhibitor] attenuated the elevated COL1A1 expression elicited by overexpression of both hTNX­FGFFFF and integrin α11. In addition, small interfering RNA­mediated knockdown of YAP1 resulted in a decrease in COL1A1 expression induced by overexpression of both hTNX­FGFFFF and integrin α11. These results indicated that overexpression of both hTNX­FGFFFF and integrin α11 induced COL1A1 expression via the YAP signaling pathway.


Assuntos
Integrinas , Tenascina , Aminoácidos , Animais , Matriz Extracelular/metabolismo , Fibrinogênio , Fibrose , Humanos , Cadeias alfa de Integrinas/metabolismo , Integrinas/metabolismo , Camundongos , Peptídeos , Tenascina/genética
8.
J Cell Biol ; 221(10)2022 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-36074065

RESUMO

The formation of healthy tissue involves continuous remodeling of the extracellular matrix (ECM). Whilst it is known that this requires integrin-associated cell-ECM adhesion sites (CMAs) and actomyosin-mediated forces, the underlying mechanisms remain unclear. Here, we examine how tensin3 contributes to the formation of fibrillar adhesions (FBs) and fibronectin fibrillogenesis. Using BioID mass spectrometry and a mitochondrial targeting assay, we establish that tensin3 associates with the mechanosensors such as talin and vinculin. We show that the talin R11 rod domain binds directly to a helical motif within the central intrinsically disordered region (IDR) of tensin3, whilst vinculin binds indirectly to tensin3 via talin. Using CRISPR knock-out cells in combination with defined tensin3 mutations, we show (i) that tensin3 is critical for the formation of α5ß1-integrin FBs and for fibronectin fibrillogenesis, and (ii) the talin/tensin3 interaction drives this process, with vinculin acting to potentiate it.


Assuntos
Fibronectinas , Adesões Focais , Talina , Tensinas , Adesão Celular , Matriz Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Adesões Focais/genética , Adesões Focais/metabolismo , Integrinas/metabolismo , Talina/genética , Talina/metabolismo , Tensinas/genética , Tensinas/metabolismo , Vinculina/genética , Vinculina/metabolismo
9.
Int J Mol Sci ; 23(17)2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-36076905

RESUMO

Tumor cell infiltrative ability into surrounding brain tissue is a characteristic of diffusely infiltrative astrocytoma and is strongly associated with extracellular matrix (ECM) stiffness. Collagens are the most abundant ECM scaffolding proteins and contribute to matrix organization and stiffness. LOX family members, copper-dependent amine oxidases, participate in the collagen and elastin crosslinking that determine ECM tensile strength. Common IDH mutations in lower-grade gliomas (LGG) impact prognosis and have been associated with ECM stiffness. We analyzed the expression levels of LOX family members and matrisome-associated genes in astrocytoma stratified by malignancy grade and IDH mutation status. A progressive increase in expression of all five LOX family members according to malignancy grade was found. LOX, LOXL1, and LOXL3 expression correlated with matrisome gene expressions. LOXL1 correlations were detected in LGG with IDH mutation (IDHmut), LOXL3 correlations in LGG with IDH wild type (IDHwt) and strong LOX correlations in glioblastoma (GBM) were found. These increasing correlations may explain the increment of ECM stiffness and tumor aggressiveness from LGG-IDHmut and LGG-IDHwt through to GBM. The expression of the mechanosensitive transcription factor, ß-catenin, also increased with malignancy grade and was correlated with LOXL1 and LOXL3 expression, suggesting involvement of this factor in the outside-in signaling pathway.


Assuntos
Astrocitoma , Neoplasias Encefálicas , Glioblastoma , Glioma , Astrocitoma/genética , Neoplasias Encefálicas/metabolismo , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Expressão Gênica , Glioblastoma/genética , Glioma/metabolismo , Humanos , Isocitrato Desidrogenase/genética , Mutação
10.
Int J Mol Sci ; 23(17)2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-36076910

RESUMO

Matrix metalloproteinases (MMPs) are critical enzymes involved in a variety of cellular processes. MMPs are well known for their ability to degrade the extracellular matrix (ECM) and their extracellular role in cell migration. Recently, more research has been conducted on investigating novel subcellular localizations of MMPs and their intracellular roles at their respective locations. In this review article, we focus on the subcellular localization and novel intracellular roles of two closely related MMPs: membrane-type-1 matrix metalloproteinase (MT1-MMP) and matrix metalloproteinase-2 (MMP-2). Although MT1-MMP is commonly known to localize on the cell surface, the protease also localizes to the cytoplasm, caveolae, Golgi, cytoskeleton, centrosome, and nucleus. At these subcellular locations, MT1-MMP functions in cell migration, macrophage metabolism, invadopodia development, spindle formation and gene expression, respectively. Similar to MT1-MMP, MMP-2 localizes to the caveolae, mitochondria, cytoskeleton, nucleus and nucleolus and functions in calcium regulation, contractile dysfunction, gene expression and ribosomal RNA transcription. Our particular interest lies in the roles MMP-2 and MT1-MMP serve within the nucleus, as they may provide critical insights into cancer epigenetics and tumor migration and invasion. We suggest that targeting nuclear MT1-MMP or MMP-2 to reduce or halt cell proliferation and migration may lead to the development of new therapies for cancer and other diseases.


Assuntos
Metaloproteinase 2 da Matriz , Neoplasias , Matriz Extracelular/metabolismo , Humanos , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinases da Matriz/metabolismo , Metaloendopeptidases/metabolismo , Neoplasias/metabolismo
11.
Int J Mol Sci ; 23(17)2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36077382

RESUMO

Myocilin is an enigmatic glaucoma-associated glycoprotein whose biological role remains incompletely understood. To gain novel insight into its normal function, we used transposon-mediated transgenesis to generate the first zebrafish line stably overexpressing myocilin [Tg(actb1:myoc-2A-mCherry)]. qPCR showed an approximately four-fold increased myocilin expression in transgenic zebrafish embryos (144 hpf). Adult (13 months old) transgenic animals displayed variable and age-dependent ocular anterior segment alterations. Almost 60% of two-year-old male, but not female, transgenic zebrafish developed enlarged eyes with severe asymmetrical and variable abnormalities in the anterior segment, characterized by corneal limbus hypertrophy, and thickening of the cornea, iris, annular ligament and lens capsule. The most severe phenotype presented small or absent ocular anterior chamber and pupils, due to iris overgrowth along with dysplastic retinal growth and optic nerve hypertrophy. Immunohistochemistry revealed increased presence of myocilin in most altered ocular tissues of adult transgenic animals, as well as signs of retinal gliosis and expanded ganglion cells and nerve fibers. The preliminary results indicate that these cells contributed to retinal dysplasia. Visual impairment was demonstrated in all old male transgenic zebrafish. Transcriptomic analysis of the abnormal transgenic eyes identified disrupted expression of genes involved in lens, muscular and extracellular matrix activities, among other processes. In summary, the developed transgenic zebrafish provides a new tool to investigate this puzzling protein and provides evidence for the role of zebrafish myocilin in ocular anterior segment and retinal biology, through the influence of extracellular matrix organization and cellular proliferation.


Assuntos
Anormalidades do Olho , Peixe-Zebra , Animais , Proteínas do Citoesqueleto , Matriz Extracelular/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Hipertrofia , Masculino , Camundongos , Camundongos Transgênicos , Retina/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
12.
Arthritis Res Ther ; 24(1): 216, 2022 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-36068644

RESUMO

BACKGROUND: Osteoarthritis (OA) is a chronic degenerative joint disease. Extracellular matrix (ECM) degradation is essential for OA progression. Previous studies have shown that circular RNAs (circRNAs) are involved in the pathological process of OA. CircPRKCH has been shown to be upregulated in OA chondrocytes. The present study was aimed to explore the roles of circPRKCH in vivo and in vitro models of OA and its underlying molecular mechanisms. METHODS: IL-1ß-induced chondrocytes and mice injected with monosodium iodoacetate were used as OA models in vitro and in vivo, respectively. RT-qPCR was performed to measure the expression of circPRKCH, miR-145, and HGF in cartilage tissues and chondrocytes. The interaction between miR-145 and circPRKCH or HGF was verified by a dual-luciferase reporter assay. Chondrocyte apoptosis, viability, and ECM-related proteins were examined by flow cytometry, MTT assay, and Western blotting, respectively. Histopathological changes were detected by HE and Safranin O-fast green staining. RESULTS: The expression of circPRKCH and HGF was increased in OA cartilage tissues and IL-1ß-treated chondrocytes, while miR-145 expression was decreased. IL-1ß induced chondrocyte apoptosis and ECM degradation in chondrocytes. Moreover, circPRKCH promoted HGF expression and activated HGF/c-MET by directly binding to miR-145. miR-145 knockdown or HGF overexpression significantly reversed circPRKCH knockdown-mediated inhibition of apoptosis and ECM degradation in IL-1ß-induced chondrocytes. Besides, miR-145 overexpression alleviated IL-1ß-induced chondrocyte apoptosis and ECM degradation by inhibiting HGF/c-MET. Finally, circPRKCH knockdown reduced ECM degradation by regulating the miR-145/HGF axis in an experimental OA model in mice. CONCLUSION: Our study demonstrated that circPRKCH promoted chondrocyte apoptosis and ECM degradation via the miR-145/HGF axis in OA, which may provide a novel target for OA treatment.


Assuntos
MicroRNAs , Osteoartrite , Animais , Apoptose/genética , Cartilagem/metabolismo , Condrócitos/metabolismo , Matriz Extracelular/metabolismo , Interleucina-1beta/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Osteoartrite/metabolismo
13.
Mol Med Rep ; 26(5)2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36069235

RESUMO

Platyconic acid A (PA), the active component of Platycodi radix­derived saponin, exerts ameliorating effects on liver fibrosis. Platycodon grandiflorum is used to treat lung disease. Therefore, the present study evaluated the effects of PA on pulmonary fibrosis. Transforming growth factor­ß1 (TGF­ß1) was used to induce MRC­5 cells to establish an in vitro pulmonary fibrosis model. The viability of MRC­5 cells in the presence or absence of TGF­ß1 induction was examined using a Cell Counting Kit­8 assay and the results demonstrated that PA markedly decreased viability of TGF­ß1­induced MRC­5 cells in a dose­dependent manner. Wound healing analysis, immunofluorescent staining and western blotting were performed to determine the levels of cell migration and expression of α­smooth muscle actin and extracellular matrix (ECM)­associated proteins. The results of the present study demonstrated that PA significantly suppressed the migration and ECM deposition of TGF­ß1­induced MRC­5 cells. Furthermore, results obtained from ELISA and western blotting demonstrated that PA exerted suppressive effects on the inflammation of MRC­5 cells following TGF­ß1 stimulation. The mRNA and protein expression levels of protein phosphatase Mg2+/Mn2+­dependent 1A (PPM1A) before and after transfection were assessed using reverse transcription­quantitative PCR and western blotting and the results demonstrated that the mRNA and protein expression levels of PPM1A were significantly decreased following transfection with small interfering RNA targeting PPM1A. Moreover, following PPM1A knockdown, PA significantly inhibited the proliferation, migration, inflammation and ECM deposition of TGF­ß1­induced MRC­5 cells via activation of the SMAD/ß­catenin signaling pathway. In conclusion, PA activated PPM1A to ameliorate TGF­ß1­elicited lung fibroblast injury via modulating SMAD/ß­catenin signaling.


Assuntos
Fibrose Pulmonar , Saponinas , Proliferação de Células , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Inflamação/metabolismo , Pulmão/metabolismo , Proteína Fosfatase 2C/genética , Proteína Fosfatase 2C/metabolismo , Fibrose Pulmonar/metabolismo , RNA Mensageiro/metabolismo , Saponinas/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Triterpenos , Regulação para Cima , beta Catenina/metabolismo
14.
Biomed Pharmacother ; 153: 113519, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36076604

RESUMO

Secreted protein acidic and rich in cysteine (SPARC), also known as osteonectin or BM-40, is a matricellular protein involved in several biological processes including cell adhesion, growth factor availability, extracellular matrix remodeling and immune-regulation. SPARC has also been associated with a variety of diseases including diabetes, colon cancer, and leukemia. The expression of SPARC in different diseases exhibits some degree of ambiguity, especially in hemopathies. Herein, we review the current expression and effects of SPARC in various hematologic disorders with respect to nanoparticle albumin bound innovative therapies and related diagnostic research, providing a clinical perspective on the use of NAB technology in the frontier treatment of hematologic diseases.


Assuntos
Neoplasias Hematológicas , Osteonectina , Albuminas , Adesão Celular , Matriz Extracelular/metabolismo , Neoplasias Hematológicas/metabolismo , Humanos , Osteonectina/genética , Osteonectina/metabolismo
15.
Methods Mol Biol ; 2579: 197-207, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36045208

RESUMO

Three-dimensional (3D) cell culture technology is a powerful tool in cancer research and drug development. It retains several critical components of the in vivo environment, including cell-cell and cell-extracellular matrix (ECM) interactions in a 3D fashion, gradients of oxygen, nutrients and metabolic waste, and it is thus more physiologically relevant than traditional two-dimensional (2D) cell culture. Here, we describe a simple and versatile method using commercially available chamber slides and Matrigel, a surrogate ECM hydrogel, to set up a 3D culture model for breast cancer cells. In this 3D culture model, cells form aggregates or spheroids on top of a thin layer of Matrigel, which can be fixed directly onto the chamber slides for cell imaging and immunofluorescence staining. Alternatively, RNA and protein can be extracted from the cells for further investigation. This 3D cell culture model provides a useful platform for cancer research and drug development, in which the effects of novel compounds or genetic modifications can be tested.


Assuntos
Técnicas de Cultura de Células , Matriz Extracelular , Técnicas de Cultura de Células/métodos , Proliferação de Células , Matriz Extracelular/metabolismo , Hidrogéis/metabolismo , Esferoides Celulares
16.
J Biomech ; 142: 111260, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36027637

RESUMO

The intervertebral disc (IVD) provides flexibility, acts as a shock absorber, and transmits load. Degeneration of the IVD includes alterations in the biomechanics, extracellular matrix (ECM), and cellular activity. These changes are not always perceived, however, IVD degeneration can lead to severe health problems including long-term disability. To understand the pathogenesis of IVD degeneration and suitable testing methods for emerging treatments and therapies, this review documents in-vitro models of IVD degeneration including physical disruption, hyperphysiological loading, ECM degradation by enzyme digestion, or a combination of these methods. This paper reviews and critically analyses the models of degeneration published since the year 2000 in either in human or animal specimens. The results are categorised in terms of the IVD biomechanics, physical attributes, ECM composition, tissue damage and cellularity to evaluate the models with respect to natural human degeneration, and to provide recommendations for clinically relevant models for the various stages of degeneration. There is no one model that replicates the wide range of degenerative changes that occur as part of normal degeneration. However, cyclic overloading replicates many aspects of degeneration, with the advantage of a dose-response allowing the tuning of damage initiated. Models of severe degeneration are currently lacking, but there is potential that combining cyclic overloading and enzymatic digestion will provide model that closely resembles human IVD degeneration. This will provide an effective way to investigate the effects of severe degeneration, and the evaluation of treatments for the IVD, which would generally be indicated at this advanced stage of degeneration.


Assuntos
Degeneração do Disco Intervertebral , Disco Intervertebral , Animais , Fenômenos Biomecânicos , Matriz Extracelular/metabolismo , Humanos , Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/metabolismo
17.
Neuropharmacology ; 218: 109210, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-35985392

RESUMO

Perineuronal nets (PNNs) are cartilage-like structures of extracellular matrix molecules that enwrap in a net-like manner the cell-body and proximal dendrites of special subsets of neurons. PNNs stabilize their incoming connections and restrict plasticity. Consequently, they have been proposed as a candidate mechanism for drug-induced learning and memory. In the cerebellum, PNNs surround Golgi inhibitory interneurons and both inhibitory and excitatory neurons in the deep cerebellar nuclei (DCN). Previous studies from the lab showed that cocaine-induced conditioned memory increased PNN expression in the granule cell layer of the posterior vermis. The present research aimed to investigate the role of cerebellar PNNs in cocaine-induced conditioned preference. For this purpose, we use the enzyme chondroitinase ABC (ChABC) to digest PNNs at different time points of the learning process to ascertain whether their removal can affect drug-induced memory. Our results show that PNN digestion using ChABC in the posterior vermis (Lobule VIII) did not affect the acquisition of cocaine-induced conditioned preference. However, the removal of PNNs in Lobule VIII -but not in the DCN- disrupted short-term memory of conditioned preference. Moreover, although PNN digestion facilitated the formation of extinction, reinstatement of cocaine-induced conditioned preference was encouraged under PNN digestion. The present findings suggests that PNNs around Golgi interneurons are needed to maintain cocaine-induced Pavlovian memory but also to stabilize extinction memory. Conversely, PNN degradation within the DCN did not affect stability of cocaine-induced memories. Therefore, degradation of PNNs in the vermis might be used as a promising tool to manipulate drug-induced memory.


Assuntos
Cocaína , Córtex Cerebelar , Cerebelo/metabolismo , Condroitina ABC Liase/metabolismo , Condroitina ABC Liase/farmacologia , Cocaína/metabolismo , Cocaína/farmacologia , Matriz Extracelular/metabolismo , Neurônios/metabolismo
18.
Nano Lett ; 22(17): 6877-6887, 2022 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-36036792

RESUMO

Intracellular transcytosis can enhance the penetration of nanomedicines to deep avascular tumor tissues, but strategies that can improve transcytosis are limited. In this study, we discovered that pyknomorphic extracellular matrix (ECM) is a shield that impairs endocytosis of nanoparticles and their movement between adjacent cells and thus limits their active transcytosis in tumors. We further showed that degradation of pivotal constituent of ECM (i.e., collagen) effectively enhances intracellular transcytosis of nanoparticles. Specifically, a collagenase conjugating transcytosis nanoparticle (Col-TNP) can dissociate into collagenase and cationized gold nanoparticles in response to tumor acidity, which enables their ECM tampering ability and active transcytosis in tumors. The breakage of ECM further enhances the active transcytosis of cationized nanoparticles into deep tumor tissues as well as radiosensitization efficacy of pancreatic adenocarcinoma. Our study opens up new paths to enhance the active transcytosis of nanomedicines for the treatment of cancers and other diseases.


Assuntos
Adenocarcinoma , Nanopartículas Metálicas , Nanopartículas , Neoplasias Pancreáticas , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/radioterapia , Colagenases/metabolismo , Matriz Extracelular/metabolismo , Ouro/metabolismo , Humanos , Nanopartículas Metálicas/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/radioterapia , Transcitose
19.
Mech Ageing Dev ; 207: 111715, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35952859

RESUMO

Intervertebral disc degeneration (IDD) is highly ubiquitous in the aged population and is an essential factor for low back pain and spinal disability. Because of the association between IDD and senescence, we investigated the ability of the anti-aging drug Klotho to inhibit age-dependent advancement of nucleus pulposus cell (NPC) degeneration. The results indicated that 400 pM exogenous Klotho significantly ameliorated extracellular matrix degradation and angiogenesis. Moreover, we demonstrated that the suppression of angiogenesis and extracellular matrix catabolism was related to inhibition of the Ras-related C3 botulinum toxin substrate 1 (Rac1)/PAK1 axis and matrix metalloproteinase 2 protein expression by exogenous Klotho cotreatment with a Rac1 inhibitor, gene overexpression in NPCs, and stimulation of human umbilical vein endothelial cells with conditioned medium from NPCs. The treatment also preserved the NPC phenotype, viability, and matrix content. In conclusion, these results suggest that the new anti-aging drug Klotho is a potential treatment strategy to mitigate IDD, and thus, provides an innovative understanding of the molecular mechanism of IDD. DATA AVAILABILITY: All data supporting the findings of this study are available from the corresponding authors upon reasonable request.


Assuntos
Degeneração do Disco Intervertebral , Disco Intervertebral , Idoso , Meios de Cultivo Condicionados , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Humanos , Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/genética , Metaloproteinase 2 da Matriz/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
20.
EMBO Mol Med ; 14(9): e15829, 2022 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-35916241

RESUMO

Whole-exome sequencing of two patients with idiopathic complex neurodevelopmental disorder (NDD) identified biallelic variants of unknown significance within FIBCD1, encoding an endocytic acetyl group-binding transmembrane receptor with no known function in the central nervous system. We found that FIBCD1 preferentially binds and endocytoses glycosaminoglycan (GAG) chondroitin sulphate-4S (CS-4S) and regulates GAG content of the brain extracellular matrix (ECM). In silico molecular simulation studies and GAG binding analyses of patient variants determined that such variants are loss-of-function by disrupting FIBCD1-CS-4S association. Gene knockdown in flies resulted in morphological disruption of the neuromuscular junction and motor-related behavioural deficits. In humans and mice, FIBCD1 is expressed in discrete brain regions, including the hippocampus. Fibcd1 KO mice exhibited normal hippocampal neuronal morphology but impaired hippocampal-dependent learning. Further, hippocampal synaptic remodelling in acute slices from Fibcd1 KO mice was deficient but restored upon enzymatically modulating the ECM. Together, we identified FIBCD1 as an endocytic receptor for GAGs in the brain ECM and a novel gene associated with an NDD, revealing a critical role in nervous system structure, function and plasticity.


Assuntos
Transtornos do Neurodesenvolvimento , Receptores de Superfície Celular , Animais , Endocitose , Matriz Extracelular/metabolismo , Humanos , Camundongos , Transtornos do Neurodesenvolvimento/genética , Receptores de Superfície Celular/metabolismo
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