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Mesenchymal stem cells (MSCs) are known to be able to modulate immune responses, possess tissue-protective properties, and exhibit healing capacities with therapeutic potential for various diseases. The ability of MSCs to secrete various cytokines and growth factors provides new insights into autoimmune-diseases such as rheumatoid arthritis (RA). RA is a systemic autoimmune disease that affects the lining of synovial joints, causing stiffness, pain, inflammation, and joint erosion. In recent years, MSCs-based therapies have been widely proposed as promising therapies in the treatment of RA. However, the mechanism involved in disease-specific therapeutic effects of MSCs on RA remains unclear. To clarify the mechanism involved in effects of MSCs on RA, proteomic profiling was performed using an RA mouse model before and after treatment with MSCs. In this study, treatment efficacy of human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) was confirmed using a type II collagen-induced arthritis (CIA) mouse model. Results of measuring incidence rates of arthritis and clinical arthritis index (CAI) revealed that mice administrated with hUCB-MSCs had a significant reduction in arthritis severity. Proteins that might affect disease progression and therapeutic efficacy of hUCB-MSC were identified through LC-MS/MS analysis using serum samples. In addition, L-1000 analysis was performed for hUCB-MSC culture medium. To analysis data obtained from LC-MS/MS and L-1000, tools such as ExDEGA, MEV, and DAVID GO were used. Results showed that various factors secreted from hUCB-MSCs might play roles in therapeutic effects of MSCs on RA, with platelet activation possibly playing a pivotal role. Results of this study also suggest that SERPINE1 and THBS1 among substances secreted by hUCB-MSC might be key factors that can inhibit platelet activation. This paper is expected to improve our understanding of mechanisms involved in treatment effects of stem cells on rheumatoid arthritis.
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Artrite Reumatoide , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Camundongos , Humanos , Animais , Proteômica , Cromatografia Líquida , Espectrometria de Massas em Tandem , Modelos Animais de DoençasRESUMO
Background and Objectives: Human mesenchymal stem cells (MSCs) are emerging as a treatment for atopic dermatitis (AD), a chronic inflammatory skin disorder that affects a large number of people across the world. Treatment of AD using human umbilical cord blood-derived MSCs (hUCB-MSCs) has recently been studied. However, the mechanism underlying their effect needs to be studied continuously. Thus, the objective of this study was to investigate the immunomodulatory effect of epidermal growth factor (EGF) secreted by hUCB-MSCs on AD. Methods and Results: To explore the mechanism involved in the therapeutic effect of MSCs for AD, a secretome array was performed using culture medium of hUCB-MSCs. Among the list of genes common for epithelium development and skin diseases, we focused on the function of EGF. To elucidate the effect of EGF secreted by hUCB-MSCs, EGF was downregulated in hUCB-MSCs using EGF-targeting small interfering RNA. These cells were then co-cultured with keratinocytes, Th2 cells, and mast cells. Depletion of EGF disrupted immunomodulatory effects of hUCB-MSCs on these AD-related inflammatory cells. In a Dermatophagoides farinae-induced AD mouse model, subcutaneous injection of hUCB-MSCs ameliorated gross scoring, histopathologic damage, and mast cell infiltration. It also significantly reduced levels of inflammatory cytokines including interleukin (IL)-4, tumor necrosis factor (TNF)-α, thymus and activation-regulated chemokine (TARC), and IL-22, as well as IgE levels. These therapeutic effects were significantly attenuated at all evaluation points in mice injected with EGF-depleted hUCB-MSCs. Conclusions: EGF secreted by hUCB-MSCs can improve AD by regulating inflammatory responses of keratinocytes, Th2 cells, and mast cells.
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BACKGROUND: Human mesenchymal stem cells (hMSCs) therapy has recently been considered a promising treatment for atopic dermatitis (AD) due to their immunomodulation and tissue regeneration ability. In our previous studies, we demonstrated that hMSCs alleviate allergic inflammation in murine AD model by inhibiting the activation of mast cells and B cells. Also our phase I/IIa clinical trial showed clinical efficacy and safety of hMSCs in moderate-to-severe adult AD patients. However, hMSCs therapy against atopic dermatitis have had poor results in clinical field. Therefore, we investigated the reason behind this result. We hypothesized that drug-cell interaction could interfere with the therapeutic efficacy of stem cells, and investigated whether coadministration with pimecrolimus, one of the topical calcineurin inhibitors, could influence the therapeutic potential of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) in AD. METHODS: hUCB-MSCs were subcutaneously injected to AD-induced mice with or without pimecrolimus topical application. To examine whether pimecrolimus influenced the immunomodulatory activity of hUCB-MSCs, hUCB-MSCs were treated with pimecrolimus. RESULTS: Pimecrolimus disturbed the therapeutic effect of hUCB-MSCs when they were co-administered in murine AD model. Moreover, the inhibitory functions of hUCB-MSCs against type 2 helper T (Th2) cell differentiation and mast cell activation were also deteriorated by pimecrolimus treatment. Interestingly, we found that pimecrolimus decreased the production of PGE2, one of the most critical immunomodulatory factors in hUCB-MSCs. And we demonstrated that pimecrolimus downregulated COX2-PGE2 axis by inhibiting nuclear translocation of NFAT3. CONCLUSIONS: Coadministration of pimecrolimus with hMSCs could interfere with the therapeutic efficacy of hMSCs in atopic dermatitis, and this is the first study that figured out the interaction of hMSCs with other drugs in cell therapy of atopic dermatitis. Therefore, this study might give rise to improvement of the clinical application of hMSCs therapy and facilitate the widespread application of hMSCs in clinical field.
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Dermatite Atópica , Células-Tronco Mesenquimais , Animais , Ciclo-Oxigenase 2 , Dermatite Atópica/tratamento farmacológico , Humanos , Camundongos , Tacrolimo/análogos & derivados , Tacrolimo/farmacologiaRESUMO
Human mesenchymal stem cells (MSCs) are promising therapeutics for autoimmune diseases due to their immunomodulatory effects. In particular, human umbilical cord blood-derived MSCs (hUCB-MSCs) have a prominent therapeutic effect on atopic dermatitis (AD). However, the underlying mechanism is unclear. This study investigated the role of transforming growth factor-beta (TGF-ß) in the therapeutic effect of hUCB-MSCs on AD. Small interfering RNA (siRNA)-mediated depletion of TGF-ß disrupted the therapeutic effect of hUCB-MSCs in a mouse model of AD by attenuating the beneficial changes in histopathology, mast cell infiltration, tumor necrosis factor-alpha (TNF-α) expression, and the serum IgE level. To confirm that hUCB-MSCs regulate secretion of TNF-α, we investigated whether they inhibit TNF-α secretion by activated LAD2 cells. Coculture with hUCB-MSCs significantly inhibited secretion of TNF-α by LAD2 cells. However, this effect was abolished by siRNA-mediated depletion of TGF-ß in hUCB-MSCs. TNF-α expression in activated LAD2 cells was regulated by the extracellular signal-related kinase signaling pathway and was suppressed by TGF-ß secreted from hUCB-MSCs. In addition, TGF-ß secreted by hUCB-MSCs inhibited maturation of B cells. Taken together, our findings suggest that TGF-ß plays a key role in the therapeutic effect of hUCB-MSCs on AD by regulating TNF-α in mast cells and maturation of B cells.
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Dermatite Atópica , Imunoglobulina E , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Animais , Dermatite Atópica/terapia , Sangue Fetal , Humanos , Imunoglobulina E/metabolismo , Imunoglobulina E/farmacologia , Mastócitos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Cordão UmbilicalRESUMO
Human tonsil-derived mesenchymal stem cells (T-MSCs) are newly identified MSCs and present typical features of MSCs, including having the differentiation capacity into the three germ layers and excellent proliferation capacity. They are easily sourced and are useful for stem cell therapy in various disease states. We previously reported that T-MSCs could be differentiated into skeletal myocytes and Schwann-like cells; therefore, they are a promising candidate for cell therapies for neuromuscular disease. Motor neurons (MNs), which regulate spontaneous behavior, are affected by a wide range of MN diseases (MNDs) for which there are no effective remedies. We investigated the differentiation potential of MN-like cells derived from T-MSCs (T-MSC-MNCs) for application to therapy of MNDs. After the process of MN differentiation, the expression of MN-related markers, including Islet 1, HB9/HLXB9 (HB9), and choline acetyltransferase (ChAT), was increased when compared with undifferentiated T-MSCs. The secretion of acetylcholine to the conditioned medium was significantly increased after MN differentiation. We cocultured T-MSC-MNCs and human skeletal muscle cells, and confirmed the presence of the acetylcholine receptor clusters, which demonstrated the formation of neuromuscular junctions. The potential functional improvements afforded by these T-MSC-MNCs could be useful in the treatment of MNDs caused by genetic mutation, viral infection, or environmental problems.
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Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Junção Neuromuscular/fisiologia , Tonsila Palatina/citologia , Acetilcolina/metabolismo , Biomarcadores , Células Cultivadas , Expressão Gênica , Humanos , Imuno-Histoquímica , Fibras Musculares Esqueléticas/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismoRESUMO
Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited motor and sensory neuropathy, and is caused by duplication of PMP22, alterations of which are a characteristic feature of demyelination. The clinical phenotype of CMT1A is determined by the degree of axonal loss, and patients suffer from progressive muscle weakness and impaired sensation. Therefore, we investigated the potential of Schwann-like cells differentiated from human tonsil-derived stem cells (T-MSCs) for use in neuromuscular regeneration in trembler-J (Tr-J) mice, a model of CMT1A. After differentiation, we confirmed the increased expression of Schwann cell (SC) markers, including glial fibrillary acidic protein (GFAP), nerve growth factor receptor (NGFR), S100 calcium-binding protein B (S100B), glial cell-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF), which suggests the differentiation of T-MSCs into SCs (T-MSC-SCs). To test their functional efficiency, the T-MSC-SCs were transplanted into the caudal thigh muscle of Tr-J mice. Recipients' improved locomotive activity on a rotarod test, and their sciatic function index, which suggests that transplanted T-MSC-SCs ameliorated demyelination and atrophy of nerve and muscle in Tr-J mice. Histological and molecular analyses showed the possibility of in situ remyelination by T-MSC-SCs transplantation. These findings demonstrate that the transplantation of heterologous T-MSC-SCs induced neuromuscular regeneration in mice and suggest they could be useful for the therapeutic treatment of patients with CMT1A disease.
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Diferenciação Celular , Doença de Charcot-Marie-Tooth/terapia , Células-Tronco Mesenquimais/metabolismo , Tonsila Palatina/metabolismo , Recuperação de Função Fisiológica , Células de Schwann/transplante , Animais , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Modelos Animais de Doenças , Xenoenxertos , Humanos , Masculino , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Mutantes , Tonsila Palatina/patologia , Células de Schwann/metabolismo , Células de Schwann/patologiaRESUMO
INTRODUCTION: Mesenchymal stem cells (MSCs) can differentiate into various cell types. METHODS: In this study we investigated the potential of human tonsil-derived MSCs (T-MSCs) for neuromuscular regeneration in trembler-J (Tr-J) mice, a model for Charcot-Marie-Tooth disease type 1A (CMT1A). RESULTS: T-MSCs differentiated toward skeletal myocytes with increased expression of skeletal muscle-related markers (including troponin I type 1, and myogenin), and the formation of myotubes in vitro. In-situ transplantation of T-MSC-derived myocytes (T-MSC myocytes) into the gastrocnemius muscle in Tr-J mice enhanced motor function, with recovery of compound muscle action potential amplitudes. Morphology of the sciatic nerve and skeletal muscle recovered without the formation of teratomas, and the expression levels of nerve growth factor and glial-cell-line-derived neurotrophic factor were increased significantly in T-MSC myocytes compared with T-MSCs in vitro. DISCUSSION: Transplantation of T-MSC myocytes could enable neuromuscular regeneration in patients with CMT1A. Muscle Nerve 57: 478-486, 2018.
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Doença de Charcot-Marie-Tooth/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Músculo Esquelético/fisiopatologia , Tonsila Palatina/citologia , Potenciais de Ação/fisiologia , Animais , Diferenciação Celular/fisiologia , Doença de Charcot-Marie-Tooth/fisiopatologia , Modelos Animais de Doenças , Masculino , CamundongosRESUMO
Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation and are thus a valuable source for the replacement of diseased or damaged organs. Previously, we reported that the tonsils can be an excellent reservoir of MSCs for the regeneration of skeletal muscle (SKM) damage. However, the mechanisms involved in the differentiation from tonsil-derived MSCs (T-MSCs) to myocytes via myoblasts remain unclear. To clarify these mechanisms, we analyzed gene expression profiles of T-MSCs during differentiation into myocytes compared with human skeletal muscle cells (hSKMCs). Total RNA was extracted from T-MSCs, T-MSC-derived myoblasts and myocytes, and hSKMCs and was subjected to analysis using a microarray. Microarray analysis of the three phases of myogenic differentiation identified candidate genes associated with myogenic differentiation. The expression pattern of undifferentiated T-MSCs was distinguishable from the myogenic differentiated T-MSCs and hSKMCs. In particular, we selected FNBP1L, which among the upregulated genes is essential for antibacterial autophagy, since autophagy is related to SKM metabolism and myogenesis. T-MSCs differentiated toward myoblasts and skeletal myocytes sequentially, as evidenced by increased expression of autophagy-related markers (including Beclin-1, LC3B and Atg5) and decreased expression of Bcl-2. Furthermore, we reconfirmed that autophagy has an effect on the mechanism of skeletal myogenic differentiation derived from T-MSCs by treatment with 5-azacytidine and bafilomycin A1. These data suggest that the transcriptome of the T-MSC-derived myocytes is similar to that of hSKMCs, and that autophagy has an important role in the mechanism of myogenic differentiation of T-MSCs.
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Autofagia , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Tonsila Palatina/metabolismo , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Fibras Musculares Esqueléticas/citologia , Tonsila Palatina/citologiaRESUMO
INTRODUCTION: Recent studies have revealed that vitamin D and its synthetic analogues have a protective effect on experimental ischemia/reperfusion (I/R) models in several organs, but little is known about its effect on the liver. The aim of this study was to evaluate the beneficial effects of vitamin D in a model of liver I/R in rats, focusing on Toll-like receptor (TLR) 4 signaling, which has been shown to be involved in I/R injury. MATERIAL AND METHODS: Twenty-four male Wistar rats were randomized into four groups: Saline + Sham, Saline + I/R, Paricalcitol + Sham, and Paricalcitol + I/R. A synthetic vitamin D2 analogue, paricalcitol, was intraperitoneally injected 24 h prior to surgery. The animals were subjected to 60 min of partial warm ischemia (70%), followed by reperfusion for 6 h on the same day. The ischemic lobe of the liver and blood were collected for molecular biochemical analyses. RESULTS: Liver damage following I/R was diminished by pretreatment with paricalcitol. Pretreatment with paricalcitol decreased the levels of pro-inflammatory mediators, such as interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and macrophage migration inhibitory factor (MIF), in both plasma and liver tissue. In addition, pretreatment with paricalcitol markedly down-regulated the expression of TLR4, HMGB1, TNF-α and NF-κB. CONCLUSIONS: The vitamin D analogue paricalcitol attenuates hepatic I/R injury through down-regulation of the TLR4 signaling pathway and might be considered to be a potential nutritional therapeutic agent against I/R injury in the liver.
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Schwann cells (SCs), which produce neurotropic factors and adhesive molecules, have been reported previously to contribute to structural support and guidance during axonal regeneration; therefore, they are potentially a crucial target in the restoration of injured nervous tissues. Autologous SC transplantation has been performed and has shown promising clinical results for treating nerve injuries and donor site morbidity, and insufficient production of the cells have been considered as a major issue. Here, we performed differentiation of tonsil-derived mesenchymal stem cells (T-MSCs) into SC-like cells (T-MSC-SCs), to evaluate T-MSC-SCs as an alternative to SCs. Using SC markers such as CAD19, GFAP, MBP, NGFR, S100B, and KROX20 during quantitative real-time PCR we detected the upregulation of NGFR, S100B, and KROX20 and the downregulation of CAD19 and MBP at the fully differentiated stage. Furthermore, we found myelination of axons when differentiated SCs were cocultured with mouse dorsal root ganglion neurons. The application of T-MSC-SCs to a mouse model of sciatic nerve injury produced marked improvements in gait and promoted regeneration of damaged nerves. Thus, the transplantation of human T-MSCs might be suitable for assisting in peripheral nerve regeneration.
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Células-Tronco Mesenquimais/citologia , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/reabilitação , Células de Schwann/citologia , Nervo Isquiático/lesões , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Criança , Técnicas de Cocultura , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Expressão Gênica , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Tonsila Palatina/citologia , Tonsila Palatina/metabolismo , Tonsila Palatina/cirurgia , Traumatismos dos Nervos Periféricos/metabolismo , Traumatismos dos Nervos Periféricos/patologia , Traumatismos dos Nervos Periféricos/cirurgia , Recuperação de Função Fisiológica , Células de Schwann/metabolismo , Células de Schwann/transplante , Nervo Isquiático/metabolismo , Tonsilectomia , Transplante HeterólogoRESUMO
Stem cells are regarded as an important source of cells which may be used to promote the regeneration of skeletal muscle (SKM) which has been damaged due to defects in the organization of muscle tissue caused by congenital diseases, trauma or tumor removal. In particular, mesenchymal stem cells (MSCs), which require less invasive harvesting techniques, represent a valuable source of cells for stem cell therapy. In the present study, we demonstrated that human tonsil-derived MSCs (T-MSCs) may differentiate into myogenic cells in vitro and that the transplantation of myoblasts and myocytes generated from human T-MSCs mediates the recovery of muscle function in vivo. In order to induce myogenic differentiation, the T-MSC-derived spheres were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12) supplemented with 1 ng/ml transforming growth factor-ß, non-essential amino acids and insulintransferrin-selenium for 4 days followed by culture in myogenic induction medium [low-glucose DMEM containing 2% fetal bovine serum (FBS) and 10 ng/ml insulinlike growth factor 1 (IGF1)] for 14 days. The T-MSCs sequentially differentiated into myoblasts and skeletal myocytes, as evidenced by the increased expression of skeletal myogenesis-related markers [including α-actinin, troponin I type 1 (TNNI1) and myogenin] and the formation of myotubes in vitro. The in situ transplantation of T-MSCs into mice with a partial myectomy of the right gastrocnemius muscle enhanced muscle function, as demonstrated by gait assessment (footprint analysis), and restored the shape of SKM without forming teratomas. Thus, T-MSCs may differentiate into myogenic cells and effectively regenerate SKM following injury. These results demonstrate the therapeutic potential of T-MSCs to promote SKM regeneration following injury.
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Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/fisiologia , Tonsila Palatina/citologia , Regeneração , Adipogenia , Animais , Biomarcadores , Diferenciação Celular/genética , Regulação da Expressão Gênica , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais , Camundongos , Músculo Esquelético/citologia , OsteogêneseRESUMO
BACKGROUND: Mutations in MPV17 cause the autosomal recessive disorder mitochondrial DNA depletion syndrome 6 (MTDPS6), also called Navajo neurohepatopathy (NNH). Clinical features of MTDPS6 is infantile onset of progressive liver failure with seldom development of progressive neurologic involvement. METHODS: Whole exome sequencing (WES) was performed to isolate the causative gene of two unrelated neuropathy patients (9 and 13 years of age) with onset of the syndrome. Clinical assessments and biochemical analysis were performed. RESULTS: A novel homozygous mutation (p.R41Q) in MPV17 was found by WES in both patients. Both showed axonal sensorimotor polyneuropathy without liver and brain involvement, which is neurophysiologically similar to axonal Charcot-Marie-Tooth disease (CMT). A distal sural nerve biopsy showed an almost complete loss of the large and medium-sized myelinated fibers compatible with axonal neuropathy. An in vitro assay using mouse motor neuronal cells demonstrated that the abrogation of MPV17 significantly affected cell integrity. In addition, the expression of the mutant protein affected cell proliferation. These results imply that both the loss of normal function of MPV17 and the gain of detrimental effects of the mutant protein might affect neuronal function. CONCLUSION: We report a novel homozygous mutation in MPV17 from two unrelated patients harboring axonal sensorimotor polyneuropathy without hepatoencephalopathy. This report expands the clinical spectrum of diseases caused by mutations of MPV17, and we recommend MPV17 gene screening for axonal peripheral neuropathies.
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Homozigoto , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Mutação , Polineuropatias/genética , Adulto , Povo Asiático/genética , Feminino , Humanos , Masculino , Linhagem , República da CoreiaRESUMO
BACKGROUND: Mutations in heat shock 27 kDa protein 1 (HSP27 or HSPB1) cause distal hereditary motor neuropathy (dHMN) or Charcot-Marie-Tooth disease type 2 F (CMT2F) according to unknown factors. Mutant HSP27 proteins affect axonal transport by reducing acetylated tubulin. RESULTS: We generated a transgenic mouse model overexpressing HSP27-S135F mutant protein driven by Cytomegalovirus (CMV) immediate early promoter. The mouse phenotype was similar to dHMN patients in that they exhibit motor neuropathy. To determine the phenotypic aberration of transgenic mice, behavior test, magnetic resonance imaging (MRI), electrophysiological study, and pathology were performed. Rotarod test showed that founder mice exhibited lowered motor performance. MRI also revealed marked fatty infiltration in the anterior and posterior compartments at calf level. Electrophysiologically, compound muscle action potential (CMAP) but not motor nerve conduction velocity (MNCV) was reduced in the transgenic mice. Toluidine staining with semi-thin section of sciatic nerve showed the ratio of large myelinated axon fiber was reduced, which might cause reduced locomotion in the transgenic mice. Electron microscopy also revealed abundant aberrant myelination. Immunohistochemically, neuronal dysfunctions included elevated level of phosphorylated neurofilament and reduced level of acetylated tubulin in the sural nerve of transgenic mice. There was no additional phenotype besides motor neuronal defects. CONCLUSIONS: Overexpression of HSP27-S135F protein causes peripheral neuropathy. The mouse model can be applied to future development of therapeutic strategies for dHMN or CMT2F.
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Doença de Charcot-Marie-Tooth/genética , Proteínas de Choque Térmico/biossíntese , Atrofia Muscular Espinal/genética , Proteínas de Neoplasias/biossíntese , Doenças do Sistema Nervoso Periférico/genética , Animais , Doença de Charcot-Marie-Tooth/fisiopatologia , Modelos Animais de Doenças , Proteínas de Choque Térmico/genética , Humanos , Camundongos , Camundongos Transgênicos , Chaperonas Moleculares , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/patologia , Mutação , Proteínas de Neoplasias/genética , Doenças do Sistema Nervoso Periférico/fisiopatologiaRESUMO
Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the gene encoding the α-galactosidase A (α-Gal A) lysosomal enzyme, which results in globotriaosylceramide (Gb3) storage in vascular endothelial cells and different cell types throughout the body. Involvement of the kidney and heart is life threatening, and fibrosis of these organs is considered to be involved in the pathogenesis of Fabry disease. An increased concentration of deacylated Gb3 (lysoGb3) in the plasma of symptomatic patients has also been suggested as a causative molecular event. To elucidate the molecular mechanisms involved in renal fibrosis in Fabry disease, the present analyzed the changes in global gene expression prior to and following Gb3 or lysoGb3 treatment in two types of kidney cell lines, human proximal renal tubular epithelial (HK2) and mouse renal glomerular mesangial (SV40 MES 13) cells. Gb3 and lysoGb3 treatment regulated the expression of 199 and 328 genes in each cell type, demonstrating a >2.0fold change. The majority of the biological functions of the regulated genes were associated with fibrogenesis or epithelialmesenchymal transition (EMT). The gene expression patterns of sphingolipidtreated HK2 cells were distinguishable from the patterns in the SV40 MES 13 cells. Several genes associated with the EMT were selected and evaluated further in kidney cells and in Fabry mouse kidney tissues. In the SV40 MES 13 cells, the DLL1, F8, and HOXA11 genes were downregulated, and FOXP2 was upregulated by treatment with Gb3 or lysoGb3. In the HK2 cells, the ADAMTS6, BEST1, IL4, and MYH11 genes were upregulated. Upregulation of the FOXP2, COL15A1, IL4, and MYH11 genes was also observed in the Fabry mouse kidney tissues. The gene expression profiles in kidney cells following the addition of Gb3 or lysoGb3 revealed substratespecific and cellspecific patterns. These findings suggested that Gb3 and lysoGb3 lead to renal fibrosis in Fabry disease through different biochemical modulations.
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Células Epiteliais/metabolismo , Doença de Fabry/genética , Regulação da Expressão Gênica , Túbulos Renais/metabolismo , Células Mesangiais/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteínas ADAMTS , Animais , Bestrofinas , Proteínas de Ligação ao Cálcio , Linhagem Celular Transformada , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Doença de Fabry/metabolismo , Doença de Fabry/patologia , Perfilação da Expressão Gênica , Glicolipídeos/farmacologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Masculino , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/patologia , Camundongos , Camundongos Transgênicos , Anotação de Sequência Molecular , Especificidade de Órgãos , Transdução de Sinais , Esfingolipídeos/farmacologia , Transcriptoma , Triexosilceramidas/farmacologiaRESUMO
The changes in DNA methylation status in cancer cells are characterized by hypermethylation of promoter CpG islands and diffuse genomic hypomethylation. Alu and long interspersed nucleotide element-1 (LINE-1) are non-coding genomic repetitive sequences and methylation of these elements can be used as a surrogate marker for genome-wide methylation status. This study was designed to evaluate the changes of Alu and LINE-1 hypomethylation during breast cancer progression from normal to pre-invasive lesions and invasive breast cancer (IBC), and their relationship with characteristics of IBC. We analyzed the methylation status of Alu and LINE-1 in 145 cases of breast samples including normal breast tissue, atypical ductal hyperplasia/flat epithelial atypia (ADH/FEA), ductal carcinoma in situ (DCIS) and IBC, and another set of 129 cases of IBC by pyrosequencing. Alu methylation showed no significant changes during multistep progression of breast cancer, although it tended to decrease during the transition from DCIS to IBC. In contrast, LINE-1 methylation significantly decreased from normal to ADH/FEA, while it was similar in ADH/FEA, DCIS and IBC. In IBC, Alu hypomethylation correlated with negative estrogen receptor (ER) status, and LINE-1 hypomethylation was associated with negative ER status, ERBB2 (HER2) amplification and p53 overexpression. Alu and LINE-1 methylation status was significantly different between breast cancer subtypes, and the HER2 enriched subtype had lowest methylation levels. In survival analyses, low Alu methylation status tended to be associated with poor disease-free survival of the patients. Our findings suggest that LINE-1 hypomethylation is an early event and Alu hypomethylation is probably a late event during breast cancer progression, and prominent hypomethylation of Alu and LINE-1 in HER2 enriched subtype may be related to chromosomal instability of this specific subtype.
Assuntos
Elementos Alu , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Metilação de DNA , Elementos Nucleotídeos Longos e Dispersos , Receptor ErbB-2/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Progressão da Doença , Epigênese Genética , Feminino , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Avaliação de Resultados da Assistência ao Paciente , Fenótipo , Receptor ErbB-2/metabolismo , Adulto JovemRESUMO
DNA methylation is one of the main epigenetic mechanisms and hypermethylation of CpG islands at tumor suppressor genes switches off these genes. To find novel DNA methylation markers in hepatocellular carcinoma (HCC), we performed pharmacological unmasking (treatment with 5-aza-2'-deoxycytidine or trichostatin A) followed by microarray analysis in HCC cell lines. Of the 239 promoter CpG island loci hypermethylated in HCC cell lines (as revealed by methylation-specific PCR), 221 loci were found to be hypermethylated in HCC or nonneoplastic liver tissues. Thirty-three loci showed a 20% higher methylation frequency in tumors than in adjacent nonneoplastic tissues. Correlation of individual cancer-related methylation markers with clinicopathological features of HCC patients (n = 95) revealed that the number of hypermethylated genes in HCC tumors was higher in older than in younger patients. Univariate and multivariate survival analysis revealed that the HIST1H2AE methylation status is closely correlated with the patient's overall survival (P = 0.022 and P = 0.010, respectively). In conclusion, we identified 221 novel DNA methylation markers for HCC. One promising prognostic marker, HIST1H2AE, should be further validated in the prognostication of HCC patients.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Metilação de DNA/efeitos dos fármacos , Neoplasias Hepáticas/genética , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Carcinoma Hepatocelular/mortalidade , Linhagem Celular Tumoral , Ilhas de CpG , Decitabina , Regulação para Baixo , Feminino , Células Hep G2 , Humanos , Ácidos Hidroxâmicos/farmacologia , Fígado/metabolismo , Neoplasias Hepáticas/mortalidade , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Análise de SobrevidaRESUMO
Although DNA methylation profiles in breast cancer have been connected to breast cancer molecular subtype, there have been no studies of the association of DNA methylation with stem cell phenotype. This study was designed to evaluate the promoter CpG island methylation of 15 genes in relation to breast cancer subtype, and to investigate whether the patterns of CpG island methylation in each subtype are associated with their cancer stem cell phenotype represented by CD44+/CD24- and ALDH1 expression. We performed MethyLight analysis of the methylation status of 15 promoter CpG island loci involved in breast cancer progression (APC, DLEC1, GRIN2B, GSTP1, HOXA1, HOXA10, IGF2, MT1G, RARB, RASSF1A, RUNX3, SCGB3A1, SFRP1, SFRP4, and TMEFF2) and determined cancer stem cell phenotype by CD44/CD24 and ALDH1 immunohistochemistry in 36 luminal A, 33 luminal B, 30 luminal-HER2, 40 HER2 enriched, and 40 basal-like subtypes of breast cancer. The number of CpG island loci methylated differed significantly between subtypes, and was highest in the luminal-HER2 subtype and lowest in the basal-like subtype. Methylation frequencies and levels in 12 of the 15 genes differed significantly between subtypes, and the basal-like subtype had significantly lower methylation frequencies and levels in nine of the genes than the other subtypes. CD44+/CD24- and ALDH1+ putative stem cell populations were most enriched in the basal-like subtype. Methylation of promoter CpG islands was significantly lower in CD44+/CD24-cell (+) tumors than in CD44+/CD24-cell (-) tumors, even within the basal-like subtype. ALDH1 (+) tumors were also less methylated than ALDH1 (-) tumors. Our findings showed that promoter CpG island methylation was different in relation to breast cancer subtype and stem cell phenotype of tumor, suggesting that breast cancers have distinct patterns of CpG island methylation according to molecular subtypes and these are associated with different stem cell phenotypes of the tumor.
Assuntos
Neoplasias da Mama/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Células-Tronco Neoplásicas/patologia , Regiões Promotoras Genéticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Família Aldeído Desidrogenase 1 , Neoplasias da Mama/classificação , Neoplasias da Mama/patologia , Antígeno CD24/análise , Antígeno CD24/biossíntese , Feminino , Perfilação da Expressão Gênica , Humanos , Receptores de Hialuronatos/análise , Receptores de Hialuronatos/biossíntese , Imuno-Histoquímica , Isoenzimas/análise , Isoenzimas/biossíntese , Pessoa de Meia-Idade , Fenótipo , Retinal Desidrogenase/análise , Retinal Desidrogenase/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
This study was designed to evaluate the changes in promoter CpG islands hypermethylation during breast cancer progression from pre-invasive lesions [flat epithelial atypia (FEA), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS)] to invasive ductal carcinoma (IDC). We performed MethyLight analysis for the methylation status of 57 promoter CpG island loci in 20 IDCs and their paired normal breast tissues. After selecting 15 CpG island loci showing breast cancer-specific DNA methylation, another set of normal breast tissue (n = 10), ADH/FEA (n = 30), DCIS (n = 35), and IDC (n = 30) of the breast were analyzed for these loci. We found six new methylation markers of breast cancer, namely DLEC1, GRIN2B, HOXA1, MT1G, SFRP4, and TMEFF2, in addition to APC, GSTP1, HOXA10, IGF2, RARB, RASSF1A, RUNX3, SCGB3A1 (HIN-1), and SFRP1. The number of methylated genes increased stepwise from normal breast to ADH/FEA and DCIS, while IDC did not differ from DCIS. Methylation levels and frequencies of APC, DLEC1, HOXA1, and RASSF1A promoter CpG islands were significantly higher in ADH/FEA than in normal breast tissue. GRIN2B, GSTP1, HOXA1, RARB, RUNX3, SFRP1, and TMEFF2 showed higher methylation levels and frequencies in DCIS than in ADH/FEA. DICS and IDC did not differ in the methylation levels or frequencies for most CpG island loci except SFRP1 and HOXA10. Our findings showed that promoter CpG island methylation changed significantly in pre-invasive lesions, and was similar in IDC and DCIS, suggesting that CpG island methylation of tumor-related genes is an early event in breast cancer progression.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ilhas de CpG/genética , Metilação de DNA/fisiologia , DNA de Neoplasias/metabolismo , Progressão da Doença , Regiões Promotoras Genéticas/genética , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/metabolismo , Carcinoma Intraductal não Infiltrante/patologia , Metilação de DNA/genética , DNA de Neoplasias/genética , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Hiperplasia/genética , Hiperplasia/metabolismo , Hiperplasia/patologia , Proteínas de Membrana/metabolismo , Metalotioneína/metabolismo , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Estudos Retrospectivos , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Gastric carcinoma (GC) is one of the human cancers in which promoter CpG island hypermethylation is frequently found. CpG island methylator phenotype (CIMP) refers to a subset of GCs which harbor concordant methylation of multiple promoter CpG island loci. However, little is known regarding clinicopathological features of CIMP-positive (CIMP-high) GC. Our study aimed to characterize clinicopathological features of CIMP-high GC. We analyzed 196 cases of GCs for their methylation status in 16 cancer-specific CpG island loci using MethyLight assay and arbitrarily defined CIMP-high GC as those with methylation at 13 or more CpG island loci. With exclusion of microsatellite instability-positive GC and EBV-positive GC from the analysis, CIMP-high GC (n = 10, 6.7%) demonstrated tendency toward higher cancer stage, infiltrative growth type, poor differentiation, and diffuse or mixed type of Lauren classification. CIMP-high GC showed significantly shortened survival compared with that of CIMP-negative GC. When CIMP-negative GC (methylation at 12 or less) was divided into CIMP-intermediate and CIMP-low (methylation at one or none), CIMP-low exhibited better clinical outcome than CIMP-intermediate. Hypermethylation at 14 CpG island loci or more was closely associated with poor clinical outcome and found to be an independent prognostic factor. Our findings that CIMP-high GCs were featured with characteristic clinicopathological parameters, including poor prognosis are distinct from previous studies. More extensive, large-scaled study is necessary to validate the findings of the present study.
Assuntos
Ilhas de CpG , Metilação de DNA , Neoplasias Gástricas/genética , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologiaRESUMO
There is a controversy about the capacity of the mesenchymal stem cells (MSCs) from aged individuals to proliferate and differentiate into cartilage. The purpose of this study was to investigate the optimal condition to culture human MSCs from the aged individuals (>50 years) for cartilage tissue engineering. We tested the hypothesis that effective proliferation and chondrogenesis can be achieved with human MSCs from aged individuals under appropriate conditions. To investigate the best condition for proliferation, MSCs were cultured in medium containing four concentrations subsets (0, 0.05, 0.5, 5 ng/mL) of recombinant human TGF-beta2 and FGF-2, either with or without fetal calf serum. The cell numbers were counted 0, 1, 3, and 7 days after growth factors were given. For the induction of chondrogenesis in 3-dimensional (3-D) culture, cells were cultured in pellets with chondrogenic medium containing combinations of various growth factors. After 4 weeks of culture, the pellets were fixed and evaluated with Safranin-O staining for proteoglycan and immunohistochemical staining for type II collagen. RT-PCR was also performed for the mRNAs of type I collagen, type II collagen, and cartilage oligomeric protein (COMP). In a monolayer culture, TGF-beta2 in concentrations of 0.5 and 5 ng/mL caused significant reduction in cell number irrespective of the presence of serum. FGF-2 of 5 ng/mL most effectively increased cell number even in the absence of serum. In a pellet culture, remarkable chondrocyte-like differentiation of cells was induced around the peripheral areas of a pellet with 5 ng/mL of TGF-beta2, accompanied by increased proteoglycan and type II collagen production. The addition of 100 ng/mL of IGF-I induced notable increase in proteoglycan contents. The results of RT-PCR mirrored those of histological studies. This study shows that an effective proliferation and chondrogenesis may be obtained with proper combinations of growth factors and mesenchymal stem cells from aged individuals.