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1.
BMC Cancer ; 23(1): 1194, 2023 Dec 06.
Article in English | MEDLINE | ID: mdl-38057796

ABSTRACT

BACKGROUND: Myxofibrosarcoma is a rare malignant soft tissue sarcoma characterised by multiple local recurrence and can become of higher grade with each recurrence. Consequently, myxofibrosarcoma represents a burden for patients, a challenge for clinicians, and an interesting disease to study tumour progression. Currently, few myxofibrosarcoma preclinical models are available. METHODS: In this paper, we present a spontaneously immortalised myxofibrosarcoma patient-derived cell line (MF-R 3). We performed phenotypic characterization through multiple biological assays and analyses: proliferation, clonogenic potential, anchorage-independent growth and colony formation, migration, invasion, AgNOR staining, and ultrastructural evaluation. RESULTS: MF-R 3 cells match morphologic and phenotypic characteristics of the original tumour as 2D cultures, 3D aggregates, and on the chorioallantoic membrane of chick embryos. Overall results show a clear neoplastic potential of this cell line. Finally, we tested MF-R 3 sensitivity to anthracyclines in 2D and 3D conditions finding a good response to these drugs. CONCLUSIONS: In conclusion, we established a novel patient-derived myxofibrosarcoma cell line that, together with the few others available, could serve as an important model for studying the molecular pathogenesis of myxofibrosarcoma and for testing new drugs and therapeutic strategies in diverse experimental settings.


Subject(s)
Fibrosarcoma , Histiocytoma, Malignant Fibrous , Sarcoma , Animals , Adult , Humans , Chick Embryo , Fibrosarcoma/drug therapy , Fibrosarcoma/pathology , Sarcoma/drug therapy , Sarcoma/pathology , Cell Line, Tumor
2.
Int J Mol Sci ; 24(7)2023 Mar 31.
Article in English | MEDLINE | ID: mdl-37047537

ABSTRACT

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure and aplastic anemia. So far, 23 genes are involved in this pathology, and their mutations lead to a defect in DNA repair. In recent years, it has been observed that FA cells also display mitochondrial metabolism defects, causing an accumulation of intracellular lipids and oxidative damage. However, the molecular mechanisms involved in the metabolic alterations have not yet been elucidated. In this work, by using lymphoblasts and fibroblasts mutated for the FANC-A gene, oxidative phosphorylation (OxPhos) and mitochondria dynamics markers expression was analyzed. Results show that the metabolic defect does not depend on an altered expression of the proteins involved in OxPhos. However, FA cells are characterized by increased uncoupling protein UCP2 expression. FANC-A mutation is also associated with DRP1 overexpression that causes an imbalance in the mitochondrial dynamic toward fission and lower expression of Parkin and Beclin1. Treatment with P110, a specific inhibitor of DRP1, shows a partial mitochondrial function recovery and the decrement of DRP1 and UCP2 expression, suggesting a pivotal role of the mitochondrial dynamics in the etiopathology of Fanconi anemia.


Subject(s)
Fanconi Anemia , Mitochondrial Dynamics , Humans , Fanconi Anemia/metabolism , Fanconi Anemia Complementation Group A Protein/genetics , Fanconi Anemia Complementation Group A Protein/metabolism , Fibroblasts/metabolism , Mitochondria/metabolism , Mitochondrial Dynamics/genetics , Proteins/metabolism , Dynamins/metabolism
3.
Int J Mol Sci ; 24(10)2023 May 10.
Article in English | MEDLINE | ID: mdl-37239904

ABSTRACT

Osteosarcoma (OS) is the most common primary malignant bone tumor and its etiology has recently been associated with osteogenic differentiation dysfunctions. OS cells keep a capacity for uncontrolled proliferation showing a phenotype similar to undifferentiated osteoprogenitors with abnormal biomineralization. Within this context, both conventional and X-ray synchrotron-based techniques have been exploited to deeply characterize the genesis and evolution of mineral depositions in a human OS cell line (SaOS-2) exposed to an osteogenic cocktail for 4 and 10 days. A partial restoration of the physiological biomineralization, culminating with the formation of hydroxyapatite, was observed at 10 days after treatment together with a mitochondria-driven mechanism for calcium transportation within the cell. Interestingly, during differentiation, mitochondria showed a change in morphology from elongated to rounded, indicating a metabolic reprogramming of OS cells possibly linked to an increase in glycolysis contribution to energy metabolism. These findings add a dowel to the genesis of OS giving new insights on the development of therapeutic strategies able to restore the physiological mineralization in OS cells.


Subject(s)
Bone Neoplasms , Osteosarcoma , Humans , Osteogenesis , Biomineralization , Cell Line, Tumor , Osteosarcoma/metabolism , Cell Differentiation/physiology , Mitochondria/metabolism , Bone Neoplasms/metabolism , Cell Proliferation/physiology
4.
Int J Mol Sci ; 24(19)2023 Sep 29.
Article in English | MEDLINE | ID: mdl-37834212

ABSTRACT

Osteochondral lesions, when not properly treated, may evolve into osteoarthritis (OA), especially in the elderly population, where altered joint function and quality are usual. To date, a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold (OC) has demonstrated good clinical results, although suboptimal subchondral bone regeneration still limits its efficacy. This study was aimed at evaluating the in vitro osteogenic potential of this scaffold, functionalized with two different strategies: the addition of Bone Morphogenetic Protein-2 (BMP-2) and the incorporation of strontium (Sr)-ion-enriched amorphous calcium phosphate (Sr-ACP) granules. Human osteoblasts were seeded on the functionalized scaffolds (OC+BMP-2 and OC+Sr-ACP, compared to OC) under stress conditions reproduced with the addition of H2O2 to the culture system, as well as in normal conditions, and evaluated in terms of morphology, metabolic activity, gene expression, and matrix synthesis. The OC+BMP-2 scaffold supported a better osteoblast morphology and stimulated scaffold colonization, cell activity, and extracellular matrix secretion, especially in the stressed culture environment but also in normal culture conditions, with increased expression of genes related to osteoblast differentiation. In conclusion, the incorporation of BMP-2 into the Col/Col-Mg-HAp scaffold also represents an improvement of the osteochondral scaffold in more challenging conditions, supporting further preclinical studies to optimize it for use in clinical practice.


Subject(s)
Biocompatible Materials , Tissue Scaffolds , Aged , Humans , Biocompatible Materials/pharmacology , Hydrogen Peroxide , Bone Regeneration , Osteogenesis/physiology , Collagen , Durapatite , Osteoblasts
5.
Am J Hum Genet ; 105(4): 689-705, 2019 10 03.
Article in English | MEDLINE | ID: mdl-31495489

ABSTRACT

Sphingomyelinases generate ceramide from sphingomyelin as a second messenger in intracellular signaling pathways involved in cell proliferation, differentiation, or apoptosis. Children from 12 unrelated families presented with microcephaly, simplified gyral pattern of the cortex, hypomyelination, cerebellar hypoplasia, congenital arthrogryposis, and early fetal/postnatal demise. Genomic analysis revealed bi-allelic loss-of-function variants in SMPD4, coding for the neutral sphingomyelinase-3 (nSMase-3/SMPD4). Overexpression of human Myc-tagged SMPD4 showed localization both to the outer nuclear envelope and the ER and additionally revealed interactions with several nuclear pore complex proteins by proteomics analysis. Fibroblasts from affected individuals showed ER cisternae abnormalities, suspected for increased autophagy, and were more susceptible to apoptosis under stress conditions, while treatment with siSMPD4 caused delayed cell cycle progression. Our data show that SMPD4 links homeostasis of membrane sphingolipids to cell fate by regulating the cross-talk between the ER and the outer nuclear envelope, while its loss reveals a pathogenic mechanism in microcephaly.


Subject(s)
Arthrogryposis/genetics , Microcephaly/genetics , Neurodevelopmental Disorders/genetics , Sphingomyelin Phosphodiesterase/genetics , Arthrogryposis/pathology , Cell Lineage , Child , Endoplasmic Reticulum/metabolism , Female , Gene Expression Profiling , HEK293 Cells , Humans , Male , Microcephaly/pathology , Mitosis , Neurodevelopmental Disorders/pathology , Pedigree , RNA Splicing
6.
Cell Mol Life Sci ; 75(5): 889-903, 2018 03.
Article in English | MEDLINE | ID: mdl-28975370

ABSTRACT

We evaluated the energy metabolism of human mesenchymal stem cells (MSC) isolated from umbilical cord (UC) of preterm (< 37 weeks of gestational age) and term (≥ 37 weeks of gestational age) newborns, using MSC from adult bone marrow as control. A metabolic switch has been observed around the 34th week of gestational age from a prevalently anaerobic glycolysis to the oxidative phosphorylation. This metabolic change is associated with the organization of mitochondria reticulum: preterm MSCs presented a scarcely organized mitochondrial reticulum and low expression of proteins involved in the mitochondrial fission/fusion, compared to term MSCs. These changes seem governed by the expression of CLUH, a cytosolic messenger RNA-binding protein involved in the mitochondria biogenesis and distribution inside the cell; in fact, CLUH silencing in term MSC determined a metabolic fingerprint similar to that of preterm MSC. Our study discloses novel information on the production of energy and mitochondrial organization and function, during the passage from fetal to adult life, providing useful information for the management of preterm birth.


Subject(s)
Energy Metabolism/physiology , Glycolysis/physiology , Mesenchymal Stem Cells/metabolism , Oxidative Phosphorylation , Premature Birth/metabolism , Term Birth/metabolism , Anaerobiosis , Cells, Cultured , Humans , Infant, Newborn , Infant, Premature , Mesenchymal Stem Cells/cytology , Umbilical Cord/cytology , Umbilical Cord/metabolism
7.
J Cell Physiol ; 233(2): 1736-1751, 2018 Feb.
Article in English | MEDLINE | ID: mdl-28681917

ABSTRACT

Metformin (MET) is the drug of choice for patients with type 2 diabetes and has been proposed for use in cancer therapy and for treating other metabolic diseases. More than 14,000 studies have been published addressing the cellular mechanisms affected by MET. However, several in vitro studies have used concentrations of the drug 10-100-fold higher than the plasmatic concentration measured in patients. Here, we evaluated the biochemical, metabolic, and morphologic effects of various concentrations of MET. Moreover, we tested the effect of MET on Fanconi Anemia (FA) cells, a DNA repair genetic disease with defects in energetic and glucose metabolism, as well as on human promyelocytic leukemia (HL60) cell lines. We found that the response of wild-type cells to MET is concentration dependent. Low concentrations (15 and 150 µM) increase both oxidative phosphorylation and the oxidative stress response, acting on the AMPK/Sirt1 pathway, while the high concentration (1.5 mM) inhibits the respiratory chain, alters cell morphology, becoming toxic to the cells. In FA cells, MET was unable to correct the energetic/respiratory defect and did not improve the response to oxidative stress and DNA damage. By contrast, HL60 cells appear sensitive also at 150 µM. Our findings underline the importance of the MET concentration in evaluating the effect of this drug on cell metabolism and demonstrate that data obtained from in vitro experiments, that have used high concentrations of MET, cannot be readily translated into improving our understanding of the cellular effects of metformin when used in the clinical setting.


Subject(s)
Energy Metabolism/drug effects , Fanconi Anemia/drug therapy , Leukemia/drug therapy , Lymphocytes/drug effects , Metformin/pharmacology , AMP-Activated Protein Kinases/metabolism , Case-Control Studies , Cell Survival/drug effects , DNA Damage , Dose-Response Relationship, Drug , Enzyme Activation , Fanconi Anemia/metabolism , Fanconi Anemia/pathology , HL-60 Cells , Humans , Leukemia/metabolism , Leukemia/pathology , Lymphocytes/metabolism , Lymphocytes/pathology , Metformin/toxicity , Oxidative Phosphorylation/drug effects , Oxidative Stress/drug effects , Sirtuin 1/metabolism
8.
Int J Mol Sci ; 19(11)2018 Nov 20.
Article in English | MEDLINE | ID: mdl-30463350

ABSTRACT

Osteosarcoma therapy might be moving toward nanotechnology-based drug delivery systems to reduce the cytotoxicity of antineoplastic drugs and improve their pharmacokinetics. In this paper, we present, for the first time, an extensive chemical and in vitro characterization of dual-loaded photo- and chemo-active keratin nanoparticles as a novel drug delivery system to treat osteosarcoma. The nanoparticles are prepared from high molecular weight and hydrosoluble keratin, suitably functionalized with the photosensitizer Chlorin-e6 (Ce6) and then loaded with the chemotherapeutic drug Paclitaxel (PTX). This multi-modal PTX-Ce6@Ker nanoformulation is prepared by both drug-induced aggregation and desolvation methods, and a comprehensive physicochemical characterization is performed. PTX-Ce6@Ker efficacy is tested on osteosarcoma tumor cell lines, including chemo-resistant cells, using 2D and 3D model systems. The single and combined contributions of PTX and Ce6 is evaluated, and results show that PTX retains its activity while being vehiculated through keratin. Moreover, PTX and Ce6 act in an additive manner, demonstrating that the combination of the cytostatic blockage of PTX and the oxidative damage of ROS upon light irradiation have a far superior effect compared to singularly administered PTX or Ce6. Our findings provide the proof of principle for the development of a novel, nanotechnology-based drug delivery system for the treatment of osteosarcoma.


Subject(s)
Drug Delivery Systems , Keratins/chemistry , Nanotechnology , Osteosarcoma/drug therapy , Cell Line, Tumor , Cell Survival , Drug Resistance, Neoplasm/drug effects , Humans , Nanoparticles/chemistry , Nanoparticles/toxicity , Nanoparticles/ultrastructure , Osteosarcoma/pathology , Paclitaxel/pharmacology
9.
PLoS Genet ; 10(9): e1004605, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25210889

ABSTRACT

Proteins of the nuclear envelope (NE) are associated with a range of inherited disorders, most commonly involving muscular dystrophy and cardiomyopathy, as exemplified by Emery-Dreifuss muscular dystrophy (EDMD). EDMD is both genetically and phenotypically variable, and some evidence of modifier genes has been reported. Six genes have so far been linked to EDMD, four encoding proteins associated with the LINC complex that connects the nucleus to the cytoskeleton. However, 50% of patients have no identifiable mutations in these genes. Using a candidate approach, we have identified putative disease-causing variants in the SUN1 and SUN2 genes, also encoding LINC complex components, in patients with EDMD and related myopathies. Our data also suggest that SUN1 and SUN2 can act as disease modifier genes in individuals with co-segregating mutations in other EDMD genes. Five SUN1/SUN2 variants examined impaired rearward nuclear repositioning in fibroblasts, confirming defective LINC complex function in nuclear-cytoskeletal coupling. Furthermore, myotubes from a patient carrying compound heterozygous SUN1 mutations displayed gross defects in myonuclear organization. This was accompanied by loss of recruitment of centrosomal marker, pericentrin, to the NE and impaired microtubule nucleation at the NE, events that are required for correct myonuclear arrangement. These defects were recapitulated in C2C12 myotubes expressing exogenous SUN1 variants, demonstrating a direct link between SUN1 mutation and impairment of nuclear-microtubule coupling and myonuclear positioning. Our findings strongly support an important role for SUN1 and SUN2 in muscle disease pathogenesis and support the hypothesis that defects in the LINC complex contribute to disease pathology through disruption of nuclear-microtubule association, resulting in defective myonuclear positioning.


Subject(s)
Cell Nucleus/metabolism , Cytoskeleton/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Microtubule-Associated Proteins/genetics , Muscular Dystrophies/genetics , Muscular Dystrophies/pathology , Nuclear Proteins/genetics , Animals , Cell Nucleus/genetics , Cells, Cultured , Cytoskeleton/genetics , Cytoskeleton/pathology , Female , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Male , Membrane Proteins/metabolism , Mice , Microtubule-Associated Proteins/metabolism , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Muscular Dystrophies/metabolism , Mutation/genetics , Myoblasts/metabolism , Myoblasts/pathology , NIH 3T3 Cells , Nuclear Envelope/genetics , Nuclear Envelope/metabolism , Nuclear Envelope/pathology , Nuclear Proteins/metabolism
10.
J Cell Sci ; 127(Pt 1): 147-57, 2014 Jan 01.
Article in English | MEDLINE | ID: mdl-24155329

ABSTRACT

The dynamic organisation of the cell nucleus is profoundly modified during growth, development and senescence as a result of changes in chromatin arrangement and gene transcription. A plethora of data suggests that the nuclear lamina is a key player in chromatin dynamics and argues in favour of a major involvement of prelamin A in fundamental mechanisms regulating cellular senescence and organism ageing. As the best model to analyse the role of prelamin A in normal ageing, we used cells from centenarian subjects. We show that prelamin A is accumulated in fibroblasts from centenarians owing to downregulation of its specific endoprotease ZMPSTE24, whereas other nuclear envelope constituents are mostly unaffected and cells do not enter senescence. Accumulation of prelamin A in nuclei of cells from centenarians elicits loss of heterochromatin, as well as recruitment of the inactive form of 53BP1, associated with rapid response to oxidative stress. These effects, including the prelamin-A-mediated increase of nuclear 53BP1, can be reproduced by rapamycin treatment of cells from younger individuals. These data identify prelamin A and 53BP1 as new targets of rapamycin that are associated with human longevity. We propose that the reported mechanisms safeguard healthy ageing in humans through adaptation of the nuclear environment to stress stimuli.


Subject(s)
Aging/genetics , Antibiotics, Antineoplastic/pharmacology , Fibroblasts/drug effects , Longevity/genetics , Nuclear Proteins/genetics , Protein Precursors/genetics , Sirolimus/pharmacology , Aged, 80 and over , Aging/metabolism , Cell Nucleus/drug effects , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cellular Senescence/drug effects , Cellular Senescence/genetics , Chromatin/drug effects , Chromatin/genetics , Chromatin/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Regulation, Developmental , Humans , Intracellular Signaling Peptides and Proteins/agonists , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lamin Type A , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Membrane Proteins/metabolism , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Nuclear Proteins/agonists , Nuclear Proteins/metabolism , Oxidative Stress , Protein Precursors/agonists , Protein Precursors/metabolism , Signal Transduction , Tumor Suppressor p53-Binding Protein 1
11.
J Cell Physiol ; 230(3): 603-9, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25161103

ABSTRACT

Bone marrow (BM) failure, increased risk of myelodysplastic syndrome, acute leukaemia and solid tumors, endocrinopathies and congenital abnormalities are the major clinical problems in Fanconi anemia patients (FA). Chromosome instability and DNA repair defects are the cellular characteristics used for the clinical diagnosis. However, these biological defects are not sufficient to explain all the clinical phenotype of FA patients. The known defects are structural alteration in cell cytoskeleton, altered structural organization for intermediate filaments, nuclear lamina, and mitochondria. These are associated with different expression and/or maturation of the structural proteins vimentin, mitofilin, and lamin A/C suggesting the involvement of metalloproteinases (MPs). Matrix metalloproteinases (MMP) are involved in normal physiological processes such as human skeletal tissue development, maturation, and hematopoietic reconstitution after bone marrow suppression. Current observations upon the eventual role of MPs in FA cells are largely inconclusive. We evaluated the overall MPs activity in FA complementation group A (FANCA) cells by exposing them to the antioxidants N-acetyl cysteine (NAC) and resveratrol (RV). This work supports the hypothesis that treatment of Fanconi patients with antioxidants may be important in FA therapy.


Subject(s)
Fanconi Anemia/metabolism , Metalloproteases/metabolism , Muscle, Skeletal/growth & development , Oxygen/metabolism , Antioxidants/administration & dosage , Bone Marrow/metabolism , Bone Marrow/pathology , Chromosomal Instability/genetics , DNA Repair/genetics , DNA-Binding Proteins , Fanconi Anemia/drug therapy , Fanconi Anemia/pathology , Female , Humans , Metalloproteases/antagonists & inhibitors , Mitochondrial Proteins/metabolism , Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Vimentin/metabolism
12.
Biochim Biophys Acta ; 1832(3): 411-20, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23261988

ABSTRACT

Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterised by pyramidal, cerebellar, and autonomic disturbances. Duplication of the LMNB1 gene is the genetic cause of ADLD, yet the pathogenetic mechanism is not defined. In this study, we analysed cells and muscle tissue from three patients affected by ADLD, carrying an extra copy of the LMNB1 gene. Lamin B1 levels were dramatically increased in ADLD nuclei, both in skin fibroblasts and skeletal muscle fibres. Since lamin B1 is known to bind Oct-1, a transcription factor involved in the oxidative stress pathway, we investigated Oct-1 fate in ADLD. Oct-1 recruitment to the nuclear periphery was increased in ADLD cells, while nucleoplasmic localisation of the transcription factor under oxidative stress conditions was reduced. Importantly, lamin B1 degradation occurring in some, but not all ADLD cell lines, slowed down lamin B1 and Oct-1 accumulation. In skeletal muscle, focal disorganisation of sarcomeres was observed, while IIB-myosin heavy chain, an Oct-1 target gene, was under-expressed and rod-containing fibres were formed. These data show that a high degree of regulation of lamin B1 expression is implicated in the different clinical phenotypes observed in ADLD and show that altered Oct-1 nuclear localisation contributes to the disease phenotype.


Subject(s)
Lamin Type B/metabolism , Nuclear Envelope/metabolism , Octamer Transcription Factor-1/metabolism , Pelizaeus-Merzbacher Disease/metabolism , Blotting, Western , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Cells, Cultured , Female , Fibroblasts/metabolism , Gene Duplication , Humans , Lamin Type B/genetics , Male , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Middle Aged , Muscle, Skeletal/metabolism , Muscle, Skeletal/ultrastructure , Nuclear Envelope/ultrastructure , Pelizaeus-Merzbacher Disease/genetics
14.
ACS Nano ; 18(3): 2047-2065, 2024 Jan 23.
Article in English | MEDLINE | ID: mdl-38166155

ABSTRACT

The use of piezoelectric nanomaterials combined with ultrasound stimulation is emerging as a promising approach for wirelessly triggering the regeneration of different tissue types. However, it has never been explored for boosting chondrogenesis. Furthermore, the ultrasound stimulation parameters used are often not adequately controlled. In this study, we show that adipose-tissue-derived mesenchymal stromal cells embedded in a nanocomposite hydrogel containing piezoelectric barium titanate nanoparticles and graphene oxide nanoflakes and stimulated with ultrasound waves with precisely controlled parameters (1 MHz and 250 mW/cm2, for 5 min once every 2 days for 10 days) dramatically boost chondrogenic cell commitment in vitro. Moreover, fibrotic and catabolic factors are strongly down-modulated: proteomic analyses reveal that such stimulation influences biological processes involved in cytoskeleton and extracellular matrix organization, collagen fibril organization, and metabolic processes. The optimal stimulation regimen also has a considerable anti-inflammatory effect and keeps its ability to boost chondrogenesis in vitro, even in an inflammatory milieu. An analytical model to predict the voltage generated by piezoelectric nanoparticles invested by ultrasound waves is proposed, together with a computational tool that takes into consideration nanoparticle clustering within the cell vacuoles and predicts the electric field streamline distribution in the cell cytoplasm. The proposed nanocomposite hydrogel shows good injectability and adhesion to the cartilage tissue ex vivo, as well as excellent biocompatibility in vivo, according to ISO 10993. Future perspectives will involve preclinical testing of this paradigm for cartilage regeneration.


Subject(s)
Chondrogenesis , Proteomics , Nanogels , Hydrogels/pharmacology , Cell Differentiation , Tissue Engineering
15.
Nat Genet ; 35(4): 367-71, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14625552

ABSTRACT

Collagen VI is an extracellular matrix protein that forms a microfilamentous network in skeletal muscles and other organs. Inherited mutations in genes encoding collagen VI in humans cause two muscle diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy. We previously generated collagen VI-deficient (Col6a1-/-) mice and showed that they have a muscle phenotype that strongly resembles Bethlem myopathy. The pathophysiological defects and mechanisms leading to the myopathic disorder were not known. Here we show that Col6a1-/- muscles have a loss of contractile strength associated with ultrastructural alterations of sarcoplasmic reticulum (SR) and mitochondria and spontaneous apoptosis. We found a latent mitochondrial dysfunction in myofibers of Col6a1-/- mice on incubation with the selective F1F(O)-ATPase inhibitor oligomycin, which caused mitochondrial depolarization, Ca2+ deregulation and increased apoptosis. These defects were reversible, as they could be normalized by plating Col6a1-/- myofibers on collagen VI or by addition of cyclosporin A (CsA), the inhibitor of mitochondrial permeability transition pore (PTP). Treatment of Col6a1-/- mice with CsA rescued the muscle ultrastructural defects and markedly decreased the number of apoptotic nuclei in vivo. These findings indicate that collagen VI myopathies have an unexpected mitochondrial pathogenesis that could be exploited for therapeutic intervention.


Subject(s)
Apoptosis , Collagen Type VI/deficiency , Mitochondria, Muscle/pathology , Mitochondrial Diseases/pathology , Muscular Diseases/pathology , Animals , Calcium/metabolism , Cyclosporine/pharmacology , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Female , Fibroblasts/metabolism , Homozygote , Immunosuppressive Agents/pharmacology , In Situ Nick-End Labeling , Male , Membrane Potentials/drug effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Skeletal/drug effects , Oligomycins/pharmacology , Sarcoplasmic Reticulum/ultrastructure
16.
Cancer Gene Ther ; 30(9): 1285-1295, 2023 09.
Article in English | MEDLINE | ID: mdl-37353558

ABSTRACT

Ewing sarcoma (EWS) is a challenging pediatric cancer characterized by vast intra-tumor heterogeneity. We evaluated the RNA-binding protein IGF2BP3, whose high expression correlates with a poor prognosis and an elevated tendency of metastases, as a possible soluble mediator of inter-cellular communication in EWS. Our data demonstrate that (i) IGF2BP3 is detected in cell supernatants, and it is released inside extracellular vesicles (EVs); (ii) EVs from IGF2BP3-positive or IGF2BP3-negative EWS cells reciprocally affect cell migration but not the proliferation of EWS recipient cells; (iii) EVs derived from IGF2BP3-silenced cells have a distinct miRNA cargo profile and inhibit the PI3K/Akt pathway in recipient cells; (iv) the 11 common differentially expressed miRNAs associated with IGF2BP3-positive and IGF2BP3-negative EVs correctly group IGF2BP3-positive and IGF2BP3-negative clinical tissue specimens. Overall, our data suggest that IGF2BP3 can participate in the modulation of phenotypic heterogeneity.


Subject(s)
Extracellular Vesicles , Sarcoma, Ewing , Child , Humans , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Extracellular Vesicles/metabolism , Gene Expression Regulation, Neoplastic , Oncogene Proteins, Fusion/genetics , Phosphatidylinositol 3-Kinases/genetics , Proto-Oncogene Proteins c-akt/metabolism , Sarcoma, Ewing/genetics , Sarcoma, Ewing/metabolism , Sarcoma, Ewing/pathology
17.
Biochim Biophys Acta ; 1812(7): 711-8, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21419220

ABSTRACT

Mandibuloacral dysplasia type A (MADA) is a rare disease caused by mutations in the LMNA gene encoding A type lamins. Patients affected by mandibuloacral dysplasia type A suffer from partial lipodystrophy, skin abnormalities and accelerated aging. Typical of mandibuloacral dysplasia type A is also bone resorption at defined districts including terminal phalanges, mandible and clavicles. Little is known about the biological mechanism underlying osteolysis in mandibuloacral dysplasia type A. In the reported study, we analyzed an osteoblast primary culture derived from the cervical vertebrae of a mandibuloacral dysplasia type A patient bearing the homozygous R527H LMNA mutation. Mandibuloacral dysplasia type A osteoblasts showed nuclear abnormalities typical of laminopathic cells, but they proliferated in culture and underwent differentiation upon stimulation with dexamethasone and beta-glycerophosphate. Differentiated osteoblasts showed proper production of bone mineral matrix until passage 8 in culture, suggesting a good differentiation activity. In order to evaluate whether mandibuloacral dysplasia type A osteoblast-derived factors affected osteoclast differentiation or activity, we used a conditioned medium from mandibuloacral dysplasia type A or control cultures to treat normal human peripheral blood monocytes and investigated whether they were induced to differentiate into osteoclasts. A higher osteoclast differentiation and matrix digestion rate was obtained in the presence of mandibuloacral dysplasia type A osteoblast medium with respect to normal osteoblast medium. Further, TGFbeta 2 and osteoprotegerin expression were enhanced in mandibuloacral dysplasia type A osteoblasts while the RANKL/osteoprotegerin ratio was diminished. Importantly, inhibition of TGFbeta 2 by a neutralizing antibody abolished the effect of mandibuloacral dysplasia type A conditioned medium on osteoclast differentiation. These data argue in favor of an altered bone turnover in mandibuloacral dysplasia type A, caused by upregulation of bone-derived stimulatory cytokines, which activate non-canonical differentiation stimuli. In this context, TGFbeta 2 appears as a major player in the osteolytic process that affects mandibuloacral dysplasia type A patients.


Subject(s)
Acro-Osteolysis/pathology , Cell Differentiation , Osteoblasts/pathology , Osteoclasts/pathology , Acro-Osteolysis/blood , Alkaline Phosphatase/metabolism , Base Sequence , Blotting, Western , Cells, Cultured , DNA Primers , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Gene Expression Profiling , Humans , Microscopy, Electron
18.
Histochem Cell Biol ; 138(4): 643-51, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22706480

ABSTRACT

Mandibuloacral dysplasia type A (MADA) is a rare laminopathy characterized by growth retardation, craniofacial anomalies, bone resorption at specific sites including clavicles, phalanges and mandibula, mottled cutaneous pigmentation, skin rigidity, partial lipodystrophy, and insulin resistance. The disorder is caused by recessive mutations of the LMNA gene encoding for A-type lamins. The molecular feature of MADA consists in the accumulation of the unprocessed lamin A precursor, which is detected at the nuclear rim and in intranuclear aggregates. Here, we report the characterization of prelamin A post-translational modifications in MADA cells that induce alterations in the chromatin arrangement and dislocation of nuclear envelope-associated proteins involved in correct nucleo-cytoskeleton relationships. We show that protein post-translational modifications change depending on the passage number, suggesting the onset of a feedback mechanism. Moreover, we show that treatment of MADA cells with the farnesyltransferase inhibitors is effective in the recovery of the chromatin phenotype, altered in MADA, provided that the cells are at low passage number, while at high passage number, the treatment results ineffective. Moreover, the distribution of the lamin A interaction partner SUN2, a constituent of the nuclear envelope, is altered by MADA mutations, as argued by the formation of a highly disorganized lattice. Treatment with statins partially rescues proper SUN2 organization, indicating that its alteration is caused by farnesylated prelamin A accumulation. Given the major role of SUN1 and SUN2 in the nucleo-cytoskeleton interactions and in regulation of nuclear positioning in differentiating cells, we hypothesise that mechanisms regulating nuclear membrane-centrosome interplay and nuclear movement may be affected in MADA fibroblasts.


Subject(s)
Acro-Osteolysis/drug therapy , Acro-Osteolysis/physiopathology , Chromatin Assembly and Disassembly/drug effects , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Intracellular Signaling Peptides and Proteins/genetics , Lipodystrophy/drug therapy , Lipodystrophy/physiopathology , Lovastatin/pharmacology , Membrane Proteins/genetics , Blotting, Western , Cells, Cultured , Chromatin Assembly and Disassembly/genetics , Fibroblasts/drug effects , Fluorescent Antibody Technique , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Lamin Type A , Mandible/abnormalities , Mandible/physiopathology , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Nuclear Envelope/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Protein Precursors/chemistry , Protein Precursors/genetics , Protein Processing, Post-Translational , Skin/cytology
20.
Gels ; 8(12)2022 Nov 24.
Article in English | MEDLINE | ID: mdl-36547290

ABSTRACT

Autophagy is a cellular process that contributes to the maintenance of cell homeostasis through the activation of a specific path, by providing the necessary factors in stressful and physiological situations. Autophagy plays a specific role in chondrocyte differentiation; therefore, we aimed to analyze this process in adipose-derived mesenchymal stromal cells (ASCs) laden in three-dimensional (3D) hydrogel. We analyzed chondrogenic and autophagic markers using molecular biology, immunohistochemistry, and electron microscopy. We demonstrated that ASCs embedded in 3D hydrogel showed an increase expression of typical autophagic markers Beclin 1, LC3, and p62, associated with clear evidence of autophagic vacuoles in the cytoplasm. During ASCs chondrogenic differentiation, we showed that autophagic markers declined their expression and autophagic vesicles were rare, while typical chondrogenic markers collagen type 2, and aggrecan were significantly increased. In line with developmental animal models of cartilage, our data showed that in a 3D hydrogel, ASCs increased their autophagic features. This path is the fundamental prerequisite for the initial phase of differentiation that contributes to fueling the cells with energy and factors necessary for chondrogenic differentiation.

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