RESUMEN
Understanding the molecular basis of adipogenesis is vital to identify new therapeutic targets to improve anti-obesity drugs. The adipogenic process could be a new target in the management of this disease. Our aim was to evaluate the effect of GMG-43AC, a selective peroxisome proliferator-activated receptor γ (PPARγ) modulator, during adipose differentiation of murine pre-adipocytes and human Adipose Derived Stem Cells (hADSCs). We differentiated 3T3-L1 cells and primary hADSCs in the presence of various doses of GMG-43AC and evaluated the differentiation efficiency measuring lipid accumulation, the expression of specific differentiation markers and the quantification of accumulated triglycerides. The treatment with GMG-43AC is not toxic as shown by cell viability assessments after the treatments. Our findings demonstrate the inhibition of lipid accumulation and the significant decrease in the expression of adipocyte-specific genes, such as PPARγ, FABP-4, and leptin. This effect was long lasting, as the removal of GMG-43AC from culture medium did not allow the restoration of adipogenic process. The above actions were confirmed in hADSCs exposed to adipogenic stimuli. Together, these results indicate that GMG-43AC efficiently inhibits adipocytes differentiation in murine and human cells, suggesting its possible function in the reversal of adipogenesis and modulation of lipolysis.
Asunto(s)
Acetanilidas/farmacología , Adipogénesis/efectos de los fármacos , Lipogénesis/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , PPAR gamma/metabolismo , Fenilpropionatos/farmacología , Triglicéridos/metabolismo , Células 3T3-L1 , Animales , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , RatonesRESUMEN
The adipose tissue is a source of inflammatory proteins, such as TNF, IL-6, and CXCL8. Most of their production occurs in macrophages that act as scavengers of dying adipocytes. The application of an orbital mechanical force for 6-10 min at 97 g to the adipose tissue, lipoaspirated and treated according to Coleman procedures, abolishes the expression of TNF-α and stimulates the expression of the anti-inflammatory protein TNF-stimulated gene-6 (TSG-6). This protein had protective and anti-inflammatory effects when applied to animal models of rheumatic diseases. We examined biopsy, lipoaspirate, and mechanically activated fat and observed that in addition to the increased TSG-6, Sox2, Nanog, and Oct4 were also strongly augmented by mechanical activation, suggesting an effect on stromal cell stemness. Human adipose tissue-derived mesenchymal stem cells (hADSCs), produced from activated fat, grow and differentiate normally with proper cell surface markers and chromosomal integrity, but their anti-inflammatory action is far superior compared to those mesenchymal stem cells (MSCs) obtained from lipoaspirate. The expression and release of inflammatory cytokines from THP-1 cells was totally abolished in mechanically activated adipose tissue-derived hADSCs. In conclusion, we report that the orbital shaking of adipose tissue enhances its anti-inflammatory properties, and derived MSCs maintain such enhanced activity.
Asunto(s)
Tejido Adiposo/citología , Antiinflamatorios/metabolismo , Células Madre Mesenquimatosas/citología , Estrés Mecánico , Adipogénesis , Separación Celular , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Regulación de la Expresión Génica , Humanos , Lipectomía , Células Madre Mesenquimatosas/metabolismo , Osteogénesis , Células Madre Pluripotentes/metabolismoRESUMEN
Since 2005, sex determining region y-box 2 (SOX2) has drawn the attention of the scientific community for being one of the key transcription factors responsible for pluripotency induction in somatic stem cells. Our research investigated the turnover regulation of SOX2 mRNA in human adipose-derived stem cells, considered one of the most valuable sources of somatic stem cells in regenerative medicine. Mitoxantrone is a drug that acts on nucleic acids primarily used to treat certain types of cancer and was recently shown to ameliorate the outcome of autoimmune diseases such as multiple sclerosis. In addition, mitoxantrone has been shown to inhibit the binding of human antigen R (HuR) RNA-binding protein to tumor necrosis factor-α mRNA. Our results show that HuR binds to the 3'-untranslated region of SOX2 mRNA together with the RNA-induced silencing complex miR145. The HuR binding works by stabilizing the interaction between the 3'-untranslated region and the RNA-induced silencing complex. Cell exposure to mitoxantrone leads to HuR detachment and the subsequent prolongation of the SOX2 mRNA half-life. The prolonged SOX2 half-life allows improvement of the spheroid-forming capability of the adipose-derived stem cells. The silencing of HuR confirmed the above observations and illustrates how the RNA-binding protein HuR may be a required molecule for regulation of SOX2 mRNA decay.
Asunto(s)
Proteína 1 Similar a ELAV/metabolismo , Células Madre Mesenquimatosas/metabolismo , ARN Mensajero/fisiología , Factores de Transcripción SOXB1/fisiología , Células Cultivadas , Femenino , Humanos , Masculino , Unión Proteica/fisiologíaRESUMEN
Although effective and safe, carotid endarterectomy (CEA) implies a reduced blood flow to the brain and likely an ischemia/reperfusion event. The high rate of uneventful outcomes associated with CEA suggests the activation of brain endogenous protection mechanisms aimed at limiting the possible ischemia/reperfusion damage. This study aims at assessing whether CEA triggers protective mechanisms such as brain release of erythropoietin and nitric oxide. CEA was performed in 12 patients; blood samples were withdrawn simultaneously from the surgically exposed ipsilateral jugular and leg veins before, during (2 and 40 min) and after clamp removal (2 min). Plasma antioxidant capacity, carbonylated proteins, erythropoietin, nitrates and nitrites (NOx) were determined. No changes in intraoperative EEG, peripheral and transcranial blood oxygen saturation were detectable, and no patients showed any neurologic sign after the intervention. Antioxidant capacity and protein carbonylation in plasma were unaffected. Differently, erythropoietin, VEGF, TNF-α and NOx increased during clamping in the jugular blood (2 and 40 min), while no changes were observed in the peripheral circulation. These results show that blood erythropoietin, VEGF, TNF-α, and NOx increased in the brain during uncomplicated CEA. This may represent an endogenous self-activated neuroprotective mechanism aimed at the prevention of ischemia/reperfusion damage.
Asunto(s)
Encéfalo/metabolismo , Endarterectomía Carotidea/efectos adversos , Eritropoyetina/sangre , Óxido Nítrico/sangre , Factor de Necrosis Tumoral alfa/sangre , Anciano , Anciano de 80 o más Años , Animales , Encéfalo/irrigación sanguínea , Citocinas/metabolismo , Femenino , Humanos , Masculino , Ratones , Persona de Mediana EdadRESUMEN
Tuberous Sclerosis Complex (TSC) is characterized by the presence of benign tumors in the brain, kidneys, heart, eyes, lungs, and skin. The typical brain lesions are cortical tubers, subependimal nodules and subependymal giant-cell astrocytomas. The occurrence of malignant astrocytomas such as glioblastoma is rare. We report on a child with a clinical diagnosis of TSC and a rapidly evolving glioblastoma multiforme. Genetic analysis identified a de novo mutation in TSC2. Molecular characterization of the tumor was performed and discussed, as well as a review of the literature where cases of TSC and glioblastoma multiforme are described. Although the co-occurrence of TSC and glioblastoma multiforme seems to be rare, this possible association should be kept in mind, and proper clinical and radiological follow up should be recommended in these patients.
Asunto(s)
Neoplasias Encefálicas/genética , Glioblastoma/genética , Mutación , Esclerosis Tuberosa/genética , Proteínas Supresoras de Tumor/genética , Antineoplásicos/uso terapéutico , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/efectos de la radiación , Neoplasias Encefálicas/complicaciones , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Niño , Resultado Fatal , Rayos gamma/uso terapéutico , Expresión Génica , Glioblastoma/complicaciones , Glioblastoma/patología , Glioblastoma/terapia , Humanos , Masculino , Esclerosis Tuberosa/complicaciones , Esclerosis Tuberosa/patología , Esclerosis Tuberosa/terapia , Proteína 2 del Complejo de la Esclerosis TuberosaRESUMEN
The only available treatment of traumatic spinal cord injury (TSCI) is high-dose methylprednisolone (MP) administered acutely after injury. However, as the efficacy of MP is controversial, we assessed the superiority of erythropoietin (EPO) versus MP in improving clinical outcome of acute TSCI. Patients aged 18 to 65 years after C5-T12 injury, and grade A or B of the ASIA Impairment Scale (AIS), admitted within 8 h, hemodynamically stable, were randomized to MP according to the NASCIS III protocol or EPO iv (500 UI/kg, repeated at 24 and 48 h). Patients were assessed by an investigator blind to treatment assignment at baseline and at day 3, 7, 14, 30, 60 and 90. Primary end point: number of responders (reduction of at least one AIS grade). Secondary end points: treatment safety and the effects of drugs on a number of disability measures. Frequentistic and post hoc Bayesian analyses were performed. Eight patients were randomized to MP and 11 to EPO. Three patients (27.3 %) on EPO and no patients on MP reached the primary end point (p = 0.17). No significant differences were found for the other disability measures. No adverse events or serious adverse events were reported in both groups. The Bayesian analysis detected a 91.8 % chance of achieving higher success rates on the primary end point with EPO in the intention-to-treat population with a 95 % chance the difference between EPO and MP falling in the range (-0.10, 0.51) and a median value of 0.2. The results of Bayesian analysis favored the experimental treatment.
Asunto(s)
Eritropoyetina/uso terapéutico , Metilprednisolona/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Traumatismos de la Médula Espinal/tratamiento farmacológico , Adolescente , Adulto , Anciano , Teorema de Bayes , Vértebras Cervicales , Simulación por Computador , Eritropoyetina/efectos adversos , Femenino , Humanos , Italia , Masculino , Metilprednisolona/efectos adversos , Persona de Mediana Edad , Fármacos Neuroprotectores/efectos adversos , Método Simple Ciego , Vértebras Torácicas , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
Tuberous sclerosis complex (TSC) is caused by mutations in TSC1 or TSC2 genes. Lymphangioleiomyomatosis (LAM) can be sporadic or associated with TSC and is characterized by widespread pulmonary proliferation of abnormal α-smooth muscle (ASM)-like cells. We investigated the features of ASM cells isolated from chylous thorax of a patient affected by LAM associated with TSC, named LAM/TSC cells, bearing a germline TSC2 mutation and an epigenetic defect causing the absence of tuberin. Proliferation of LAM/TSC cells is epidermal growth factor (EGF)-dependent and blockade of EGF receptor causes cell death as we previously showed in cells lacking tuberin. LAM/TSC cells spontaneously detach probably for the inactivation of the focal adhesion kinase (FAK)/Akt/mTOR pathway and display the ability to survive independently from adhesion. Non-adherent LAM/TSC cells show an extremely low proliferation rate consistent with tumour stem-cell characteristics. Moreover, LAM/TSC cells bear characteristics of stemness and secrete high amount of interleukin (IL)-6 and IL-8. Anti-EGF receptor antibodies and rapamycin affect proliferation and viability of non-adherent cells. In conclusion, the understanding of LAM/TSC cell features is important in the assessment of cell invasiveness in LAM and TSC and should provide a useful model to test therapeutic approaches aimed at controlling their migratory ability.
Asunto(s)
Epigénesis Genética , Linfangioleiomiomatosis/genética , Linfangioleiomiomatosis/patología , Proteínas Supresoras de Tumor/genética , Anticuerpos/farmacología , Secuencia de Bases , Adhesión Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Factor de Crecimiento Epidérmico/farmacología , Epigénesis Genética/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Receptores ErbB/inmunología , Femenino , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Datos de Secuencia Molecular , Sirolimus/farmacología , Esclerosis Tuberosa/genética , Esclerosis Tuberosa/patología , Proteína 2 del Complejo de la Esclerosis TuberosaRESUMEN
In this review, we describe a new avenue that involves the therapeutic use of human adipose tissue. In the past two decades, thousands of papers have described the potential clinical use of human fat and adipose tissue. Moreover, mesenchymal stem cells have been a source of great enthusiasm in clinical studies, and these have generated curiosity at academic levels. On the other hand, they have created considerable commercial business opportunities. High expectations have emerged for curing some recalcitrant diseases or reconstructing anatomically defective human body parts, but several concerns have been raised by generating criticism on the clinical practice that have not been substantiated by rigorous scientific evidence. However, in general, the consensus is that human adipose-derived mesenchymal stem cells inhibit the production of inflammatory cytokines and stimulate the production of anti-inflammatory cytokines. Here, we show that the application of a mechanical elliptical force for several minutes to human abdominal fat activates anti-inflammatory properties and gene-related expression. This may pave the way for new unexpected clinical developments.
Asunto(s)
Tejido Adiposo , Enfermedades del Sistema Nervioso , Humanos , Tejido Adiposo/metabolismo , Antiinflamatorios , Citocinas/metabolismoRESUMEN
Tuberous sclerosis complex (TSC) is a multi-systemic syndrome caused by mutations in TSC1 or TSC2 gene. In TSC2-null cells, Rheb, a member of the Ras family of GTPases, is constitutively activated. Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase and block the synthesis of isoprenoid lipids with inhibition of Rheb farnesylation and RhoA geranylgeranylation. The effects of rosuvastatin on the function of human TSC2(-/-) and TSC2(-/meth) α-actin smooth muscle (ASM) cells have been investigated. The TSC2(-/-) and TSC2(-/meth) ASM cells, previously isolated in our laboratory from the renal angiomyolipoma of two TSC patients, do not express tuberin and bear loss of heterozigosity caused by a double hit on TSC2 and methylation of TSC2 promoter, respectively. Exposure to rosuvastatin affected TSC2(-/meth) ASM cell growth and promoted tuberin expression by acting as a demethylating agent. This occurred without changes in interleukin release. Rosuvastatin also reduced RhoA activation in TSC2(-/meth) ASM cells, and it required coadministration with the specific mTOR (mammalian target of rapamycin) inhibitor rapamycin to be effective in TSC2(-/-) ASM cells. Rapamycin enhanced rosuvastatin effect in inhibiting cell proliferation in TSC2(-/-) and TSC2(-/meth) ASM cells. Rosuvastatin alone did not alter phosphorylation of S6 and extracellular signal-regulated kinase (ERK), and at the higher concentration, rosuvastatin and rapamycin slightly decreased ERK phosphorylation. These results suggest that rosuvastatin may potentially represent a treatment adjunct to the therapy with mTOR inhibitors now in clinical development for TSC. In particular, rosuvastatin appears useful when the disease is originated by epigenetic defects.
Asunto(s)
Antiinflamatorios , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Fluorobencenos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Pirimidinas/farmacología , Sulfonamidas/farmacología , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genética , Actinas/metabolismo , Adulto , Western Blotting , Línea Celular Tumoral , Proliferación Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Guanosina Trifosfato/metabolismo , Humanos , Masculino , Ácido Mevalónico/metabolismo , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Rosuvastatina Cálcica , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Lymphangioleiomyomatosis (LAM) is an interstitial lung disease characterized by invasion and proliferation of abnormal smooth muscle (ASM) cells in lung parenchyma and axial lymphatics. LAM cells bear mutations in tuberous sclerosis (TSC) genes. TSC2(-/-) ASM cells, derived from a human renal angiomyolipoma, require epidermal growth factor (EGF) for proliferation. Blockade of EGF receptors (EGFR) causes cell death. TSC2(-/-) ASM cells, previously labeled with PKH26-GL dye, were endonasally administered to 5-week-old immunodeficient female nude mice, and 4 or 26 weeks later anti-EGFR antibody or rapamycin was administered twice a week for 4 consecutive weeks. TSC2(-/-) ASM cells infiltrated lymph nodes and alveolar lung walls, causing progressive destruction of parenchyma. Parenchymal destruction was efficiently reversed by anti-EGFR treatment and partially by rapamycin treatment. Following TSC2(-/-) ASM cell administration, lymphangiogenesis increased in lungs as indicated by more diffuse LYVE1 expression and high murine VEGF levels. Anti-EGFR antibody and rapamycin blocked the increase in lymphatic vessels. This study shows that TSC2(-/-) ASM cells can migrate and invade lungs and lymph nodes, and anti-EGFR antibody is more effective than rapamycin in promoting lung repair and reducing lymphangiogenesis. The development of a model to study metastasis by TSC cells will also help to explain how they invade different tissues and metastasize to the lung.
Asunto(s)
Modelos Animales de Enfermedad , Linfangioleiomiomatosis/patología , Miocitos del Músculo Liso/trasplante , Proteínas Supresoras de Tumor/deficiencia , Administración Intranasal , Animales , Anticuerpos/farmacología , Recuento de Células , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Femenino , Humanos , Pulmón/efectos de los fármacos , Pulmón/patología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/patología , Ratones , Ratones Desnudos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/enzimología , Fosforilación/efectos de los fármacos , Condicionamiento Físico Animal , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Sirolimus/farmacología , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
A novel type of adult neural precursor cells (NPCs) has been isolated from the subventricular zone of the mouse 6 hr after animal death (T6-NPCs). This condition is supposed to select hypoxia-resistant cells of scientific and clinical interest. Ionic channels are ultimately the expression of the functional maturation of neurons, so the aim of this research was to characterize the pattern of the main voltage-dependent ionic channels in T6-NPCs differentiating to a neuronal phenotype, comparing it with NPCs isolated soon after death (T0-NPCs). T6- and T0-NPCs grow in medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Differentiation was performed in small wells without the addition of growth factors, in the presence of adhesion molecules, fetal bovine serum, and leukemia inhibitory factor. Ionic currents, recorded by means of whole-cell patch-clamp, namely, I(Ca2+) HVA, both L- and non-L-type, I(K+) delayed rectifying, I(K+) inward rectifier, transient I(K+A) , and TTX-sensitive I(Na+) have been found, although Na(+) currents were found in only a small percentage of cells and after the fifth week of differentiation. No significant differences in current types, density, orcell capacitance were observed between T6-NPCs and T0-NPCs. The sequence in which the markers appear in new neural cells is not necessarily a fixed program, but the discrepancies in morphological, biochemical, and electrophysiological maturation of mouse NPCs to neurons, possibly different in vivo, suggest that the various steps of the differentiation are independently regulated. Therefore, in addition to morphological and biochemical data, functional tests should be considered for characterizing the maturation of neurons.
Asunto(s)
Células Madre Adultas/fisiología , Encéfalo/citología , Diferenciación Celular/fisiología , Canales Iónicos/metabolismo , Neuronas/fisiología , Cambios Post Mortem , Células Madre Adultas/efectos de los fármacos , Animales , Biofisica , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio , Diferenciación Celular/efectos de los fármacos , Estimulación Eléctrica , Factor de Crecimiento Epidérmico , Factor 2 de Crecimiento de Fibroblastos , Proteína Ácida Fibrilar de la Glía/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/fisiología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio , Tetraetilamonio/farmacología , Tetrodotoxina/farmacologíaRESUMEN
Hair follicle (HF) regression is characterized by the activation of apoptosis in HF cells. Dermal papilla cells play a leading role in the regulation of HF development and cycling. Human follicular dermal papilla cells (HFDPC) were used to investigate the protective activities of rutin, sperimidine and zeaxanthine. HFDP cell incubation with staurosporine caused apoptosis, which was completely inhibited by exposure to rutin (2.2 µM), spermidine (1 µM) and zeaxanthin (80 µM). These agents were much less effective when applied as single compounds. Moreover, treatment preserved the expression of anti-apoptotic molecules such as Bcl-2, MAP-kinases and their phosphorylated forms. In conclusion, the investigated agents may represent an effective treatment for the prevention of apoptosis, one of the leading events involved in hair bulb regression.
Asunto(s)
Apoptosis/efectos de los fármacos , Folículo Piloso/efectos de los fármacos , Rutina/farmacología , Espermidina/farmacología , Xantófilas/farmacología , Células Cultivadas , Dermis/citología , Dermis/efectos de los fármacos , Folículo Piloso/citología , Humanos , ZeaxantinasRESUMEN
This study was aimed at the isolation of neural precursor cells (NPCs) capable of resisting to a prolonged ischemic insult as this may occur at the site of traumatic and ischemic CNS injuries. Adult mice were anesthetized and then killed by cervical dislocation. The cadavers were maintained at room temperature or at 4°C for different time periods. Post mortem neural precursors (PM-NPCs) were isolated, grown in vitro and their differentiation capability was investigated by evaluating the expression of different neuronal markers. PM-NPCs differentiate mostly in neurons, show activation of hypoxia-inducible factor-1 and MAPK, and express both erythropoietin (EPO) and its receptor (EPO-R). The exposure of PM-NPCs to neutralizing antibodies to EPO or EPO-R dramatically reduced the extent of neuronal differentiation to about 11% of total PM-NPCs. The functionality of mTOR and MAPK is also required for the expression of the neuronal phenotype by PM-NPCs. These results suggest that PM-NPCs can be isolated from animal cadaver even several hours after death and their self-renewable capability is comparable to normal neural precursors. Differently, their ability to achieve a neural phenotype is superior to that of NPCs, and this is mediated by the activation of hypoxia-induced factor 1 and EPO signaling. PM-NPCs may represent good candidates for transplantation studies in animal models of neurodegenerative diseases.
Asunto(s)
Células Madre Adultas/citología , Senescencia Celular/fisiología , Eritropoyetina/fisiología , Células-Madre Neurales/citología , Neuronas/citología , Cambios Post Mortem , Células Madre Adultas/metabolismo , Animales , Diferenciación Celular/fisiología , Hipoxia de la Célula/fisiología , Células Cultivadas , Eritropoyetina/biosíntesis , Eritropoyetina/metabolismo , Ratones , Ratones Endogámicos , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , Receptores de Eritropoyetina/biosíntesis , Receptores de Eritropoyetina/fisiología , Transducción de Señal/fisiologíaRESUMEN
Spinal cord injury (SCI) triggers a complex ischemic and inflammatory reaction, involving activation of neurotransmitter systems, in particular glutamate, culminating in cell death. We hypothesized that SCI might lead to alteration in the RNA editing of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors that govern critical determinants of neuronal survival. To this end, we examined the molecular changes set in motion by SCI that affect the channel properties of AMPA receptors. SCI strongly reduced the level of AMPA receptor R/G editing, involving not only the site of the lesion but also adjacent areas of the cord spared by the lesion. The effects, with changes for some subunits and loci, were observed as long as 30 days after lesioning and may correlate with a partial decrease in enzymatic activity of adenosine deaminase acting on RNA 2 (ADAR2), as deduced from the analysis of ADAR2 self-editing. The reduced editing at the R/G site of glutamate receptor subunits (GluRs) is likely to reduce post-synaptic excitatory responses to glutamate, thus limiting the progression of cell death; however, prolonged suppression of GluR function in later stages may hinder synaptic plasticity. These observations provide the first direct evidence of the potential contribution of RNA editing to excitatory neural injury and recovery after SCI.
Asunto(s)
Edición de ARN , Receptores AMPA/genética , Traumatismos de la Médula Espinal/metabolismo , Adenosina Desaminasa/biosíntesis , Adenosina Desaminasa/genética , Empalme Alternativo , Animales , Actividad Motora , ARN Mensajero/biosíntesis , Proteínas de Unión al ARN , Ratas , Ratas Sprague-Dawley , Receptores AMPA/biosíntesis , Médula Espinal/metabolismo , Médula Espinal/patología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
Tuberous sclerosis complex (TSC) is an autosomal-dominant disease that is caused by mutations in either the TSC1 or TSC2 gene. Smooth muscle-like cells (ASMs) were isolated from an angiomyolipoma of a patient with TSC. These cells lacked tuberin, were labeled by both HMB45 and CD44v6 antibodies, and had constitutive S6 phosphorylation. The cells bear a germline TSC2 intron 8-exon 9 junction mutation, but DNA analysis and polymerase chain reaction amplification failed to demonstrate loss of heterozygosity. Testing for an epigenetic alteration, we detected methylation of the TSC2 promoter. Its biological relevance was confirmed by tuberin expression and a reduction in HMB45 labeling and S6 constitutive phosphorylation after exposure to the chromatin-remodeling agents, trichostatin A and 5-azacytidine. These cells were named TSC2(-/meth) ASMs. Their proliferation required epidermal growth factor in the medium as previously described for TSC2(-/-) ASMs. Blockade of epidermal growth factor with monoclonal antibodies caused the death of TSC2(-/meth) ASMs. In addition, rapamycin effectively blocked the proliferation of these cells. Our data show for the first time that methylation of the TSC2 promoter might cause a complete loss of tuberin in TSC2 cells, and that the pathogenesis of angiomyolipomas might also originate from epigenetic defects in smooth muscle cells. Additionally, the effect of chromatin-remodeling agents in these cells suggests a further avenue for the treatment of TSC as well as lymphangioleiomyomatosis.
Asunto(s)
Angiomiolipoma/genética , Angiomiolipoma/patología , Metilación de ADN/genética , Miocitos del Músculo Liso/patología , Proteínas Supresoras de Tumor/genética , Adulto , Angiomiolipoma/etiología , Antibióticos Antineoplásicos/farmacología , Antígenos de Neoplasias , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Análisis Mutacional de ADN , Mutación de Línea Germinal , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Masculino , Antígenos Específicos del Melanoma , Microscopía Fluorescente , Miocitos del Músculo Liso/efectos de los fármacos , Proteínas de Neoplasias , Regiones Promotoras Genéticas/genética , Sirolimus/farmacología , Esclerosis Tuberosa/complicaciones , Esclerosis Tuberosa/genética , Esclerosis Tuberosa/patología , Proteína 2 del Complejo de la Esclerosis TuberosaRESUMEN
Spinal cord injury (SCI) is a devastating disease, which leads to paralysis and is associated to substantially high costs for the individual and society. At present, no effective therapies are available. Here, the use of mechanically-activated lipoaspirate adipose tissue (MALS) in a murine experimental model of SCI is presented. Our results show that, following acute intraspinal MALS transplantation, there is an engraftment at injury site with the acute powerful inhibition of the posttraumatic inflammatory response, followed by a significant progressive improvement in recovery of function. This is accompanied by spinal cord tissue preservation at the lesion site with the promotion of endogenous neurogenesis as indicated by the significant increase of Nestin-positive cells in perilesional areas. Cells originated from MALS infiltrate profoundly the recipient cord, while the extra-dural fat transplant is gradually impoverished in stromal cells. Altogether, these novel results suggest the potential of MALS application in the promotion of recovery in SCI.
Asunto(s)
Tejido Adiposo/metabolismo , Nestina/metabolismo , Neurogénesis , Neuroprotección , Recuperación de la Función , Traumatismos de la Médula Espinal/terapia , Tejido Adiposo/citología , Tejido Adiposo/trasplante , Animales , Masculino , RatonesRESUMEN
Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into multiple cell types, including adipocytes, osteoblasts, and chondrocytes. The role of adipose-derived stem cells (ADSCs) in cancers is significantly relevant. They seem to be involved in the promotion of tumour development and progression and relapse processes. For this reason, investigating the effects of breast cancer microenvironment on ADSCs is of high importance in order to understand the relationship between tumour cells and the surrounding stromal cells. With the current study, we aimed to investigate the specific characteristics of human ADSCs isolated from the adipose tissue of breast tumour patients. We compared ADSCs obtained from periumbilical fat (PF) of controls with ADSCs obtained from adipose tissue of breast cancer- (BC-) bearing patients. We analysed the surface antigens and the adipogenic differentiation ability of both ADSC populations. C/EBPδ expression was increased in PF and BC ADSCs induced to differentiate compared to the control while PPARγ and FABP4 expressions were enhanced only in PF ADSCs. Conversely, adiponectin expression was reduced in PF-differentiated ADSCs while it was slightly increased in differentiated BC ADSCs. By means of Oil Red O staining, we further observed an impaired differentiation capability of BC ADSCs. To investigate this aspect more in depth, we evaluated the effect of selective PPARγ activation and nutritional supplementation on the differentiation efficiency of BC ADSCs, noting that it was only with a strong differentiation stimuli that the process took place. Furthermore, we observed no response in BC ADSCs to the PPARγ inhibitor T0070907, showing an impaired activation of this receptor in adipose cells surrounding the breast cancer microenvironment. In conclusion, our study shows an impaired adipogenic differentiation capability in BC ADSCs. This suggests that the tumour microenvironment plays a key role in the modulation of the adipose microenvironment located in the surrounding tissue.
RESUMEN
The aim of the study was the assessment of the effects of adult neural stem cell (NSC) transplantation in a mouse model of spinal cord injury (SCI). The contusion injury was performed by means of the Infinite Horizon Device to allow the generation of reproducible traumatic lesion to the cord. We administered green fluorescent-labeled (GFP-)NSCs either by intravenous (i.v.) injection or by direct transplantation into the spinal cord (intraspinal route). We report that NSCs significantly improved recovery of hind limb function and greatly attenuated secondary degeneration. The i.v. route of NSC administration yielded better recovery than the intraspinal route of administration. About 2% of total i.v.-administered NSCs homed to the spinal cord injury site, and survived almost undifferentiated; thus the positive effect of NSC treatment cannot be ascribed to damaged tissue substitution. The NSCs homing to the injury site triggered, within 48 h, a large increase of the expression of neurotrophic factors and chemokines. One wk after transplantation, exogenous GFP-NSCs still retained their proliferation potential and produced neurospheres when recovered from the lesion site and cultured in vitro. At a later time, GFP-NSC were phagocytated by macrophages. We suggest that the process of triggering the recovery of function might be strongly related to the viability of GFP-NSC, still capable ex vivo of producing neurospheres, and their ability to modify the lesion environment in a positive fashion.
Asunto(s)
Neuronas/trasplante , Traumatismos de la Médula Espinal/cirugía , Médula Espinal/cirugía , Trasplante de Células Madre/métodos , Células Madre/citología , Animales , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Actividad Motora , Neuronas/citología , Recuperación de la Función , Médula Espinal/fisiología , Médula Espinal/trasplante , Células Madre/fisiologíaRESUMEN
Acute lesions of the spinal cord lead to dramatic changes in neuronal function. In the present study, we examined the possible involvement of neurotrophic factors in the action of the drug of choice for the treatment of such an emergency, i.e. the glucocorticoid methylprednisolone is compared to erythropoietin, a cytokine recently shown to markedly shorten the time necessary for motor recovery following injury [Gorio, A., Gokmen, N., Erbayraktar, S., Yilmaz, O., Madaschi, L., Cichetti, C., Di Giulio, A.M., Vardar, E., Cerami, A., Brines, M., 2002. Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma. Proc. Natl. Acad. Sci. 99, 9450-9455]. We found that methylprednisolone reduces the lesion-enhanced Nerve Growth Factor (NGF) mRNA levels 3 h after injury in the trauma epicenter and caudal section of the cord whereas erythropoietin reinforced the NGF gene expression. Three days after the occurrence of the lesion, erythropoietin, but not methylprednisolone, significantly up-regulated the NGF gene expression both caudally and rostrally to the lesion site, an effect that, based on the chemo-attractant properties of neurotrophin, might facilitate the growth of injured axons toward NGF-rich sites and contribute to the enhancement of the regenerative process. The differences between the effects of methylprednisolone and erythropoietin dissipate 7 days after the lesion when they both enhance NGF mRNA levels at the epicenter. These data show that methylprednisolone and erythropoietin display a different pattern of activation of the neurotrophin NGF which is strictly dependent on the portion of the cord examined and the time elapsed from the injury. Based on our results, we suggest that the higher increase of NGF expression mediated by erythropoietin soon after the injury might explain, at least in part, the improved recovery of motor functions produced by erythropoietin compared to methylprednisolone and saline.
Asunto(s)
Eritropoyetina/farmacología , Glucocorticoides/farmacología , Metilprednisolona/farmacología , Factor de Crecimiento Nervioso/efectos de los fármacos , Traumatismos de la Médula Espinal/tratamiento farmacológico , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Factor de Crecimiento Nervioso/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patología , Traumatismos de la Médula Espinal/patología , Factores de TiempoRESUMEN
Polymeric materials based on epsilon-caprolactone (CL), 1,5-dioxepan-2-one (DXO), and trimethylene carbonate (TMC) were prepared and evaluated as possible candidates for polymer-on-multielectrode (PoM) applications. CL was copolymerized with either DXO or TMC in the presence of the diol initiator 1,4-benzenedimethanol (BDM). The ring-opening polymerization experiments, carried out in bulk and using tin(II) catalysis, yielded the desired low molecular weight random copolymer diols, as evidenced by NMR, IR, MALDI-ToF MS, and DSC techniques. Upon reaction with acryloyl chloride, the corresponding diacrylate end-capped copolymers were obtained. The latter were characterized by NMR and IR spectroscopy, and their photocross-linking (in the presence of a UV initiator) was followed by ATR-FTIR spectroscopy. Transparent and soft thin films of the copoly(ether-ester) and copoly(ester-carbonate) diacrylates were prepared and cured under UV irradiation. The resulting polymeric films showed good biocompatibility properties as far as in vitro neural stem cells proliferation and differentiation to neurons and astrocytes are concerned. Noteworthy are the beneficial effects obtained upon preconditioning the copolymers by means of the cell-culture medium and the excellent properties shown particularly by the CL-TMC copolymer. Moreover, preliminary results show that microchannel formation by photocuring is possible with the synthesized polymers.