RESUMO
Changes in the homeostasis of tumor necrosis factor α (TNFα) have been demonstrated in patients and experimental models of amyotrophic lateral sclerosis (ALS). However, the contribution of TNFα to the development of ALS is still debated. TNFα is expressed by glia and neurons and acts through the membrane receptors TNFR1 and TNFR2, which may have opposite effects in neurodegeneration. We investigated the role of TNFα and its receptors in the selective motor neuron death in ALS in vitro and in vivo. TNFR2 expressed by astrocytes and neurons, but not TNFR1, was implicated in motor neuron loss in primary SOD1-G93A co-cultures. Deleting TNFR2 from SOD1-G93A mice, there was partial but significant protection of spinal motor neurons, sciatic nerves, and tibialis muscles. However, no improvement of motor impairment or survival was observed. Since the sciatic nerves of SOD1-G93A/TNFR2-/- mice showed high phospho-TAR DNA-binding protein 43 (TDP-43) accumulation and low levels of acetyl-tubulin, two indices of axonal dysfunction, the lack of symptom improvement in these mice might be due to impaired function of rescued motor neurons. These results indicate the interaction between TNFR2 and membrane-bound TNFα as an innovative pathway involved in motor neuron death. Nevertheless, its inhibition is not sufficient to stop disease progression in ALS mice, underlining the complexity of this pathology. We show evidence of the involvement of neuronal and astroglial TNFR2 in the motor neuron degeneration in ALS. Both concur to cause motor neuron death in primary astrocyte/spinal neuron co-cultures. TNFR2 deletion partially protects motor neurons and sciatic nerves in SOD1-G93A mice but does not improve their symptoms and survival. However, TNFR2 could be a new target for multi-intervention therapies.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Neurônios Motores/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Astrócitos/metabolismo , Axônios/metabolismo , Morte Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Camundongos , Neuroglia/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/deficiênciaRESUMO
BACKGROUND AIMS: The Pall Celeris system is a filtration-based point-of-care device designed to obtain a high concentrate of peripheral blood total nucleated cells (PB-TNCs). We have characterized the Pall Celeris-derived TNCs for their in vitro and in vivo angiogenic potency. METHODS: PB-TNCs isolated from healthy donors were characterized through the use of flow cytometry and functional assays, aiming to assess migratory capacity, ability to form capillary-like structures, endothelial trans-differentiation and paracrine factor secretion. In a hind limb ischemia mouse model, we evaluated perfusion immediately and 7 days after surgery, along with capillary, arteriole and regenerative fiber density and local bio-distribution. RESULTS: Human PB-TNCs isolated by use of the Pall Celeris filtration system were shown to secrete a panel of angiogenic factors and migrate in response to vascular endothelial growth factor and stromal-derived factor-1 stimuli. Moreover, after injection in a mouse model of hind limb ischemia, PB-TNCs induced neovascularization by increasing capillary, arteriole and regenerative fiber numbers, with human cells detected in murine tissue up to 7 days after ischemia. CONCLUSIONS: The Pall Celeris system may represent a novel, effective and reliable point-of-care device to obtain a PB-derived cell product with adequate potency for therapeutic angiogenesis.
Assuntos
Isquemia/terapia , Neovascularização Fisiológica , Doença Arterial Periférica/terapia , Sistemas Automatizados de Assistência Junto ao Leito , Animais , Remoção de Componentes Sanguíneos , Diferenciação Celular , Movimento Celular , Separação Celular/métodos , Quimiocina CXCL12/metabolismo , Modelos Animais de Doenças , Células Endoteliais/citologia , Filtração , Citometria de Fluxo , Membro Posterior/irrigação sanguínea , Humanos , Leucócitos/imunologia , Camundongos , Reperfusão , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread.
Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Astrócitos/enzimologia , Exossomos/enzimologia , Neurônios Motores/enzimologia , Proteínas do Tecido Nervoso/metabolismo , Superóxido Dismutase/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Astrócitos/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Morte Celular/genética , Exossomos/genética , Exossomos/patologia , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Mutação , Proteínas do Tecido Nervoso/genética , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Proteína com ValosinaRESUMO
Atrial fibrillation (AF) is characterized by electrical, contractile, and structural remodeling mediated by interstitial fibrosis. It has been shown that human cardiac mesenchymal progenitor cells (CMPCs) can be differentiated into endothelial, smooth muscle, and fibroblast cells. Here, we have investigated, for the first time, the contribution of CMPCs in the fibrotic process occurring in AF. As expected, right auricolae samples displayed significantly higher fibrosis in AF vs control (CTR) patients. In tissue samples of AF patients only, double staining for c-kit and the myofibroblast marker α-smooth muscle actin (α-SMA) was detected. The number of c-kit-positive CMPC was higher in atrial subepicardial regions of CTR than AF cells. AF-derived CMPC (AF-CMPC) and CTR-derived CMPC (Ctr-CMPC) were phenotypically similar, except for CD90 and c-kit, which were significantly more present in AF and CTR cells, respectively. Moreover, AF showed a lower rate of population doubling and fold enrichment vs Ctr-CMPC. When exogenously challenged with the profibrotic transforming growth factor-ß1 (TGF-ß1), AF-CMPC showed a significantly higher nuclear translocation of SMAD2 than Ctr-CMPC. In addition, TGF-ß1 treatment induced the upregulation of COL1A1 and COL1A2 in AF-CMPC only. Further, both a marked production of soluble collagen and α-SMA upregulation have been observed in AF-CMPC only. Finally, electrophysiological studies showed that the inwardly rectifying potassium current (IK1) was evenly present in AF- and Ctr-CMPC in basal conditions and similarly disappeared after TGF-ß1 exposure. All together, these data suggest that AF steers the resident atrial CMPC compartment toward an electrically inert profibrotic phenotype.
Assuntos
Fibrilação Atrial/patologia , Células-Tronco Mesenquimais/patologia , Miocárdio/patologia , Miofibroblastos/patologia , Idoso , Fibrilação Atrial/fisiopatologia , Diferenciação Celular , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Becker muscular dystrophy (BMD) is a dystrophinopathy caused by mutations in the dystrophin gene on chromosome Xp21. BMD mutations result in truncated semi-functional dystrophin isoforms. Consequently, less severe clinical symptoms become apparent later in life compared to Duchenne muscular dystrophy. Dermal fibroblasts from a BMD patient were electroporated with episomal plasmids containing reprogramming factors to create the induced pluripotent stem cell line: CCMi002BMD-A-9 that showed pluripotent markers, were karyotypically normal and capable of trilineage differentiation. MLPA analyses performed on DNA extracted from CCMi002BMD-A-9 showed an in-frame deletion of exons 45 to 55 (CCMi002BMD-A-9 Δ45-55).
Assuntos
Técnicas de Cultura de Células/métodos , Distrofina/genética , Éxons/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Deleção de Sequência/genética , Adulto , Humanos , MasculinoRESUMO
Duchenne muscular dystrophy (DMD) is caused by abnormalities in the dystrophin gene and is clinically characterised by childhood muscle degeneration and cardiomyopathy. We produced an induced pluripotent stem cell line from a DMD patient's dermal fibroblasts by electroporation with episomal vectors containing: hL-MYC, hLIN28, hSOX2, hKLF4, hOCT3/4. The resultant DMD iPSC line (CCMi001DMD-A-3) displayed iPSC morphology, expressed pluripotency markers, possessed trilineage differentiation potential and was karyotypically normal. MLPA analyses performed on DNA extracted from CCMi001DMD-A-3 showed a deletion of exons 49 and 50 (CCMi001DMD-A-3, ∆49, ∆50).
Assuntos
Éxons/genética , Células-Tronco Pluripotentes Induzidas/citologia , Distrofia Muscular de Duchenne/enzimologia , Adulto , Células Cultivadas , Reprogramação Celular/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , MasculinoRESUMO
Doxorubicin (DOXO) treatment is limited by its cardiotoxicity, since it causes cardiac-progenitor-cell depletion. Although the cardioprotective role of the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 (SDF1/CXCR4) axis is well established, its involvement during DOXO-induced cardiotoxicity has never been investigated. We showed that in a mouse model of DOXO-induced cardiomyopathy, CXCR4+ cells were increased in response to DOXO, mainly in human cardiac mesenchymal progenitor cells (CmPC), a subpopulation with regenerative potential. Our in vitro results showed a CXCR4 induction after 24 h of DOXO exposure in CmPC. SDF1 administration protected from DOXO-induced cell death and promoted CmPC migration. CXCR4 promoter analysis revealed zinc finger E-box binding homeobox 1 (ZEB1) binding sites. Upon DOXO treatment, ZEB1 binding decreased and RNA-polymerase-II increased, suggesting a DOXO-mediated transcriptional increase in CXCR4. Indeed, DOXO induced the upregulation of miR-200c, that directly targets ZEB1. SDF1 administration in DOXO-treated mice partially reverted the adverse remodeling, decreasing left ventricular (LV) end diastolic volume, LV ejection fraction and LV anterior wall thickness in diastole, recovering LV end systolic pressure and reducing±dP/dt. Moreover, in vivo administration of SDF1 partially reverted DOXO-induced miR-200c and p53 protein upregulation in mouse hearts. In addition, downmodulation of ZEB1 mRNA and protein by DOXO was significantly increased by SDF1. In keeping, p21 mRNA, that is induced by p53 and inhibited by ZEB1, is induced by DOXO treatment and is decreased by SDF1 administration. This study showed new players of the DOXO-induced cardiotoxicity, that can be exploited to ameliorate DOXO-associated cardiomyopathy.
Assuntos
Doxorrubicina/farmacologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Receptores CXCR4/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Feminino , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Receptores CXCR4/genética , Transdução de Sinais , Regulação para Cima/efeitos dos fármacos , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Stem cells are being used in the treatment of cardivovascular diseases. Here, we review the physiologic and pathologic conditions that impact the regenerative potential of stem cells in the treatment of cardiovascular diseases which include the influence of donor age and the presence of metabolic syndromes. We will also discuss strategies such as pretreatment of the recipient tissue or autologous or allogeneic stem cells by growth factors or drugs and by providing a synthetic scaffold and genetic modifications that impact the regenerative potential of stem cells. Finally, we will evaluate the current state of treatment of acute or chronic cardiovascular diseases with allogeneic stem cells.
Assuntos
Regeneração/fisiologia , Células-Tronco/fisiologia , Envelhecimento/patologia , Envelhecimento/fisiologia , Aloenxertos , Animais , Autoenxertos , Doenças Cardiovasculares/patologia , Doenças Cardiovasculares/fisiopatologia , Doenças Cardiovasculares/terapia , Coração/fisiopatologia , Humanos , Síndrome Metabólica/patologia , Síndrome Metabólica/fisiopatologia , Miocárdio/patologia , Transplante de Células-Tronco/métodosRESUMO
Epigenetics harbours all regulatory information that, beyond nucleotide sequences, allows cells to "make decisions" throughout their lifetime in response to the external environment. The information can be transitory or relatively stable, and is even transmittable either to daughter cells or to the next generations through the germ line. Recent discoveries shed light on numerous connections between metabolites and epigenetic chromatin-modifying enzymes, providing a link between the metabolic state of the cell and epigenetics, and ultimately between metabolism, gene expression and cell fate. In this review, we discuss the possible connections between metabolism and epigenetic regulation of stem cell differentiation and self-renewal. Moreover, we describe pertinent literature that could explain how altered metabolic state and nutrition can contribute to disease development through epigenetic modifications. A special section is dedicated to the emerging link between the circadian clock, metabolic transcriptional regulation by epigenetic mechanisms and their implication in stem cell homeostasis.
Assuntos
Diferenciação Celular/genética , Epigênese Genética , Metabolômica , Células-Tronco/metabolismo , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , HumanosRESUMO
According to the European Medicine Agency (EMA) regulatory frameworks, Advanced Therapy Medicinal Products (ATMP) represent a new category of drugs in which the active ingredient consists of cells, genes, or tissues. ATMP-CD133 has been widely investigated in controlled clinical trials for cardiovascular diseases, making CD133(+) cells one of the most well characterized cell-derived drugs in this field. To ensure high quality and safety standards for clinical use, the manufacturing process must be accomplished in certified facilities following standard operative procedures (SOPs). In the present work, we report the fully compliant GMP-grade production of ATMP-CD133 which aims to address the treatment of chronic refractory ischemic heart failure. Starting from bone marrow (BM), ATMP-CD133 manufacturing output yielded a median of 6.66 × 10(6) of CD133(+) cells (range 2.85 × 10(6)-30.84 × 10(6)), with a viability ranged between 96,03% and 99,97% (median 99,87%) and a median purity of CD133(+) cells of 90,60% (range 81,40%-96,20%). Based on these results we defined our final release criteria for ATMP-CD133: purity ≥ 70%, viability ≥ 80%, cellularity between 1 and 12 × 10(6) cells, sterile, and endotoxin-free. The abovementioned criteria are currently applied in our Phase I clinical trial (RECARDIO Trial).
Assuntos
Antígenos CD/metabolismo , Células da Medula Óssea/citologia , Transplante de Medula Óssea/normas , Cardiomiopatias/terapia , Glicoproteínas/metabolismo , Isquemia Miocárdica/terapia , Peptídeos/metabolismo , Transplante de Células-Tronco/normas , Antígeno AC133 , Animais , Cardiomiopatias/etiologia , Cardiomiopatias/patologia , Aprovação de Equipamentos/normas , Europa (Continente) , Fidelidade a Diretrizes , Humanos , Isquemia Miocárdica/complicações , Isquemia Miocárdica/patologia , Guias de Prática Clínica como Assunto , Células-TroncoRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder involving the selective degeneration of motor neurons. In a small proportion of patients, ALS is caused by mutations in copper/zinc superoxide dismutase (SOD1), and mice overexpressing SOD1(G93A) mutant develop a syndrome that closely resembles the human disease. Excitotoxicity mediated by glutamate AMPA receptors has been suggested to be implicated in the selective susceptibility of motor neurons occurring in ALS. In SOD1(G93A) mice, we found that levels of GluR2 AMPA subunit, which plays a pivotal role in the maintenance of calcium impermeability of AMPA receptors, are decreased in spinal motor neurons before symptom onset in concomitance with a modest increase of GluR3 expression, a calcium-permeable AMPA subunit. This effect can result in a higher number of calcium-permeable AMPA receptors on motor neurons of SOD1(G93A) mice, predisposing these cells to be injured by AMPA-mediated glutamate firing. In support of this, we showed that treatment with a new noncompetitive AMPA antagonist, ZK 187638, partially protected motor neurons, improved motor function, and prolonged the survival of SOD1(G93A) mice.