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BACKGROUND: A reliable preclinical model of patient-derived organoids (PDOs) was developed in a case study of a 69-year-old woman diagnosed with breast cancer (BC) to investigate the tumour evolution before and after neoadjuvant chemotherapy and surgery. The results were achieved due to the development of PDOs from tissues collected before (O-PRE) and after (O-POST) treatment. METHODS: PDO cultures were characterized by histology, immunohistochemistry (IHC), transmission electron microscopy (TEM), scanning electron microscopy (SEM), confocal microscopy, flow cytometry, real-time PCR, bulk RNA-seq, single-cell RNA sequencing (scRNA-seq) and drug screening. RESULTS: Both PDO cultures recapitulated the histological and molecular profiles of the original tissues, and they showed typical mammary gland organization, confirming their reliability as a personalized in vitro model. Compared with O-PRE, O-POST had a greater proliferation rate with a significant increase in the Ki67 proliferation index. Moreover O-POST exhibited a more stem-like and aggressive phenotype, with increases in the CD24low/CD44low and EPCAMlow/CD49fhigh cell populations characterized by increased tumour initiation potential and multipotency and metastatic potential in invasive lobular carcinoma. Analysis of ErbB receptor expression indicated a decrease in HER-2 expression coupled with an increase in EGFR expression in O-POST. In this context, deregulation of the PI3K/Akt signalling pathway was assessed by transcriptomic analysis, confirming the altered transcriptional profile. Finally, transcriptomic single-cell analysis identified 11 cell type clusters, highlighting the selection of the luminal component and the decrease in the number of Epithelial-mesenchymal transition cell types in O-POST. CONCLUSION: Neoadjuvant treatment contributed to the enrichment of cell populations with luminal phenotypes that were more resistant to chemotherapy in O-POST. PDOs represent an excellent 3D cell model for assessing disease evolution.
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BACKGROUND: Deregulation of transcription in the pathogenesis of sporadic Amyotrophic Lateral Sclerosis (sALS) is taking central stage with RNA-sequencing analyses from sALS patients tissues highlighting numerous deregulated long non-coding RNAs (lncRNAs). The oncogenic lncRNA ZEB1-AS1 is strongly downregulated in peripheral blood mononuclear cells of sALS patients. In addition, in cancer-derived cell lines, ZEB1-AS1 belongs to a negative feedback loop regulation with hsa-miR-200c, acting as a molecular sponge for this miRNA. The role of the lncRNA ZEB1-AS1 in sALS pathogenesis has not been characterized yet, and its study could help identifying a possible disease-modifying target. METHODS: the implication of the ZEB1-AS1/ZEB1/hsa-miR-200c/BMI1 pathway was investigated in multiple patients-derived cellular models (patients-derived peripheral blood mononuclear cells and induced pluripotent stem cells-derived neural stem cells) and in the neuroblastoma cell line SH-SY5Y, where its function was inhibited via RNA interference. Molecular techniques such as Real Time PCR, Western Blot and Immunofluorescence were used to assess the pathway dysregulation. RESULTS: Our results show a dysregulation of a signaling pathway involving ZEB1-AS1/hsa-miR-200c/ß-Catenin in peripheral blood mononuclear cells and in induced pluripotent stem cells-derived neural stem cells from sALS patients. These results were validated in vitro on the cell line SH-SY5Y with silenced expression of ZEB1-AS1. Moreover, we found an increase for ZEB1-AS1 during neural differentiation with an aberrant expression of ß-Catenin, highlighting also its aggregation and possible impact on neurite length. CONCLUSIONS: Our results support and describe the role of ZEB1-AS1 pathway in sALS and specifically in neuronal differentiation, suggesting that an impairment of ß-Catenin signaling and an alteration of the neuronal phenotype are taking place.
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Esclerose Lateral Amiotrófica , MicroRNAs , Neuroblastoma , RNA Longo não Codificante , Humanos , Esclerose Lateral Amiotrófica/genética , beta Catenina/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Leucócitos Mononucleares/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
Brain metastasis (BM) represents a clinical challenge for patients with advanced HER2 + breast cancer (BC). The monoclonal anti-HER2 antibody trastuzumab (TZ) improves survival of BC patients, but it has low central nervous system penetrance, being ineffective in treating BM. Previous studies showed that ferritin nanoparticles (HFn) may cross the blood brain barrier (BBB) through binding to the transferrin receptor 1 (TfR1). However, whether this has efficacy in promoting the trans-BBB delivery of TZ and combating BC BM was not studied yet. Here, we investigated the potential of HFn to drive TZ brain delivery and promote a targeted antitumor response in a murine model of BC BM established by stereotaxic injection of engineered BC cells overexpressing human HER2. HFn were covalently conjugated with TZ to obtain a nanoconjugate endowed with HER2 and TfR1 targeting specificity (H-TZ). H-TZ efficiently achieved TZ brain delivery upon intraperitoneal injection and triggered stable targeting of cancer cells. Treatment with H-TZ plus docetaxel significantly reduced tumor growth and shaped a protective brain microenvironment by engaging macrophage activation toward cancer cells. H-TZ-based treatment also avoided TZ-associated cardiotoxicity by preventing drug accumulation in the heart and did not induce any other major side effects when combined with docetaxel. These results provided in vivo demonstration of the pharmacological potential of H-TZ, able to tackle BC BM in combination with docetaxel. Indeed, upon systemic administration, the nanoconjugate guides TZ brain accumulation, reduces BM growth and limits side effects in off-target organs, thus showing promise for the management of HER2 + BC metastatic to the brain.
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Obesity is a complex disease with multifactorial causes, and its prevalence is becoming a serious health crisis. For this reason, there is a crucial need to identify novel targets and players. With this aim in mind, we analyzed via RNA-sequencing the subcutaneous adipose tissue of normal weight and obesity-affected women, highlighting the differential expression in the two tissues. We specifically focused on long non-coding RNAs, as 6 of these emerged as dysregulated in the diseased-tissue (COL4A2-AS2, RPS21-AS, PELATON, ITGB2-AS1, ACER2-AS and CTEPHA1). For each of them, we performed both a thorough in silico dissection and in vitro validation, to predict their function during adipogenesis. We report the lncRNAs expression during adipose derived stem cells differentiation to adipocytes as model of adipogenesis and their potential modulation by adipogenesis-related transcription factors (C/EBPs and PPARγ). Moreover, inhibiting CTEPHA1 expression we investigated its impact on adipogenesis-related transcription factors, showing its significative dysregulation of C/EBPα expression. Lastly, we dissected the subcellular localization, pathway involvement and disease-correlation for coding differentially expressed genes. Together, these findings highlight a transcriptional deregulation at the basis of obesity, impacted by both coding and long non-coding RNAs.
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RNA Longo não Codificante , Adipócitos/metabolismo , Adipogenia/genética , Tecido Adiposo/metabolismo , Feminino , Humanos , Obesidade/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Gordura Subcutânea/metabolismoRESUMO
The multitasking nature of lncRNAs allows them to play a central role in both physiological and pathological conditions. Often the same lncRNA can participate in different diseases. Specifically, the MYC-induced Long non-Coding RNA MINCR is upregulated in various cancer types, while downregulated in Amyotrophic Lateral Sclerosis patients. Therefore, this work aims to investigate MINCR potential mechanisms of action and its implications in cancer and neurodegeneration in relation to its expression levels in SH-SY5Y cells through RNA-sequencing approach. Our results show that MINCR overexpression causes massive alterations in cancer-related genes, leading to disruption in many fundamental processes, such as cell cycle and growth factor signaling. On the contrary, MINCR downregulation influences a small number of genes involved in different neurodegenerative disorders, mostly concerning RNA metabolism and inflammation. Thus, understanding the cause and functional consequences of MINCR deregulation gives important insights on potential pathogenetic mechanisms both in cancer and in neurodegeneration.
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Neoplasias , RNA Longo não Codificante , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Oncogenes , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transdução de SinaisRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease (NDD) that affects motor neurons, causing weakness, muscle atrophy and spasticity. Unfortunately, there are only symptomatic treatments available. Two important innovations in recent years are three-dimensional (3D) bioprinting and induced pluripotent stem cells (iPSCs). The aim of this work was to demonstrate the robustness of 3D cultures for the differentiation of stem cells for the study of ALS. We reprogrammed healthy and sALS peripheral blood mononuclear cells (PBMCs) in iPSCs and differentiated them in neural stem cells (NSCs) in 2D. NSCs were printed in 3D hydrogel-based constructs and subsequently differentiated first in motor neuron progenitors and finally in motor neurons. Every step of differentiation was tested for cell viability and characterized by confocal microscopy and RT-qPCR. Finally, we tested the electrophysiological characteristics of included NSC34. We found that NSCs maintained good viability during the 3D differentiation. Our results suggest that the hydrogel does not interfere with the correct differentiation process or with the electrophysiological features of the included cells. Such evidence confirmed that 3D bioprinting can be considered a good model for the study of ALS pathogenesis.
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Esclerose Lateral Amiotrófica , Células-Tronco Pluripotentes Induzidas , Doenças Neurodegenerativas , Esclerose Lateral Amiotrófica/patologia , Humanos , Hidrogéis/farmacologia , Leucócitos Mononucleares/patologiaRESUMO
Different mechanisms were proposed as responsible for COVID-19 neurological symptoms but a clear one has not been established yet. In this work we aimed to study SARS-CoV-2 capacity to infect pediatric human cortical neuronal HCN-2 cells, studying the changes in the transcriptomic profile by next generation sequencing. SARS-CoV-2 was able to replicate in HCN-2 cells, that did not express ACE2, confirmed also with Western blot, and TMPRSS2. Looking for pattern recognition receptor expression, we found the deregulation of scavenger receptors, such as SR-B1, and the downregulation of genes encoding for Nod-like receptors. On the other hand, TLR1, TLR4 and TLR6 encoding for Toll-like receptors (TLRs) were upregulated. We also found the upregulation of genes encoding for ERK, JNK, NF-κB and Caspase 8 in our transcriptomic analysis. Regarding the expression of known receptors for viral RNA, only RIG-1 showed an increased expression; downstream RIG-1, the genes encoding for TRAF3, IKKε and IRF3 were downregulated. We also found the upregulation of genes encoding for chemokines and accordingly we found an increase in cytokine/chemokine levels in the medium. According to our results, it is possible to speculate that additionally to ACE2 and TMPRSS2, also other receptors may interact with SARS-CoV-2 proteins and mediate its entry or pathogenesis in pediatric cortical neurons infected with SARS-CoV-2. In particular, TLRs signaling could be crucial for the neurological involvement related to SARS-CoV-2 infection.
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COVID-19/metabolismo , Córtex Cerebral/metabolismo , Neurônios/virologia , SARS-CoV-2/patogenicidade , Receptores Toll-Like/metabolismo , COVID-19/genética , COVID-19/imunologia , Criança , Citocinas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Neurônios/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , Transdução de Sinais/genética , Receptores Toll-Like/genética , Replicação ViralRESUMO
The inflammatory response plays a central role in the complications of congenital pulmonary airway malformations (CPAM) and severe coronavirus disease 2019 (COVID-19). The aim of this study was to evaluate the transcriptional changes induced by SARS-CoV-2 exposure in pediatric MSCs derived from pediatric lung (MSCs-lung) and CPAM tissues (MSCs-CPAM) in order to elucidate potential pathways involved in SARS-CoV-2 infection in a condition of exacerbated inflammatory response. MSCs-lung and MSCs-CPAM do not express angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TRMPSS2). SARS-CoV-2 appears to be unable to replicate in MSCs-CPAM and MSCs-lung. MSCs-lung and MSCs-CPAM maintained the expression of stemness markers MSCs-lung show an inflammatory response (IL6, IL1B, CXCL8, and CXCL10), and the activation of Notch3 non-canonical pathway; this route appears silent in MSCs-CPAM, and cytokine genes expression is reduced. Decreased value of p21 in MSCs-lung suggested no cell cycle block, and cells did not undergo apoptosis. MSCs-lung appears to increase genes associated with immunomodulatory function but could contribute to inflammation, while MSCs-CPAM keeps stable or reduce the immunomodulatory receptors expression, but they also reduce their cytokines expression. These data indicated that, independently from their perilesional or cystic origin, the MSCs populations already present in a patient affected with CPAM are not permissive for SARS-CoV-2 entry, and they will not spread the disease in case of infection. Moreover, these MSCs will not undergo apoptosis when they come in contact with SARS-CoV-2; on the contrary, they maintain their staminality profile.
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Células-Tronco Mesenquimais/metabolismo , Anormalidades do Sistema Respiratório , SARS-CoV-2/fisiologia , Transcriptoma , COVID-19/genética , COVID-19/metabolismo , COVID-19/patologia , Estudos de Casos e Controles , Células Cultivadas , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Humanos , Lactente , Pulmão/anormalidades , Pulmão/metabolismo , Pulmão/patologia , Masculino , Células-Tronco Mesenquimais/patologia , Células-Tronco Mesenquimais/virologia , RNA-Seq , Anormalidades do Sistema Respiratório/genética , Anormalidades do Sistema Respiratório/patologia , Anormalidades do Sistema Respiratório/virologiaRESUMO
Obesity is a major risk factor for a large number of secondary diseases, including cancer. Specific insights into the role of gender differences and secondary comorbidities, such as type 2 diabetes (T2D) and cancer risk, are yet to be fully identified. The aim of this study is thus to find a correlation between the transcriptional deregulation present in the subcutaneous adipose tissue of obese patients and the oncogenic signature present in multiple cancers, in the presence of T2D, and considering gender differences. The subcutaneous adipose tissue (SAT) of five healthy, normal-weight women, five obese women, five obese women with T2D and five obese men were subjected to RNA-sequencing, leading to the identification of deregulated coding and non-coding RNAs, classified for their oncogenic score. A panel of DE RNAs was validated via Real-Time PCR and oncogene expression levels correlated the oncogenes with anthropometrical parameters, highlighting significant trends. For each analyzed condition, we identified the deregulated pathways associated with cancer, the prediction of possible prognosis for different cancer types and the lncRNAs involved in oncogenic networks and tissues. Our results provided a comprehensive characterization of oncogenesis correlation in SAT, providing specific insights into the possible molecular targets implicated in this process. Indeed, the identification of deregulated oncogenes also in SAT highlights hypothetical targets implicated in the increased oncogenic risk in highly obese subjects. These results could shed light on new molecular targets to be specifically modulated in obesity and highlight which cancers should receive the most attention in terms of better prevention in obesity-affected patients.
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Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Obesidade/genética , Oncogenes , Fases de Leitura Aberta/genética , RNA Longo não Codificante/genética , Gordura Subcutânea/metabolismo , Gordura Subcutânea/patologia , Adulto , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Feminino , Humanos , Masculino , Neoplasias/genética , Obesidade/complicações , Prognóstico , RNA Longo não Codificante/metabolismo , Caracteres Sexuais , Transdução de Sinais/genética , Transcrição GênicaRESUMO
Neurodegenerative disorders (i.e., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and spinal cord injury) represent a great problem worldwide and are becoming prevalent because of the increasing average age of the population. Despite many studies having focused on their etiopathology, the exact cause of these diseases is still unknown and until now, there are only symptomatic treatments. Biomaterials have become important not only for the study of disease pathogenesis, but also for their application in regenerative medicine. The great advantages provided by biomaterials are their ability to mimic the environment of the extracellular matrix and to allow the growth of different types of cells. Biomaterials can be used as supporting material for cell proliferation to be transplanted and as vectors to deliver many active molecules for the treatments of neurodegenerative disorders. In this review, we aim to report the potentiality of biomaterials (i.e., hydrogels, nanoparticles, self-assembling peptides, nanofibers and carbon-based nanomaterials) by analyzing their use in the regeneration of neural and glial cells their role in axon outgrowth. Although further studies are needed for their use in humans, the promising results obtained by several groups leads us to suppose that biomaterials represent a potential therapeutic approach for the treatments of neurodegenerative disorders.
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Materiais Biocompatíveis , Doenças Neurodegenerativas/terapia , Medicina Regenerativa , Animais , Materiais Biocompatíveis/química , Humanos , Nanoestruturas , Nanotecnologia/métodos , Regeneração Nervosa , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Nanomedicina TeranósticaRESUMO
Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) are neurodegenerative disorders characterized by a progressive degeneration of the central or peripheral nervous systems. A central role of the RNA metabolism has emerged in these diseases, concerning mRNAs processing and non-coding RNAs biogenesis. We aimed to identify possible common grounds or differences in the dysregulated pathways of AD, PD, and ALS. To do so, we performed RNA-seq analysis to investigate the deregulation of both coding and long non-coding RNAs (lncRNAs) in ALS, AD, and PD patients and controls (CTRL) in peripheral blood mononuclear cells (PBMCs). A total of 293 differentially expressed (DE) lncRNAs and 87 mRNAs were found in ALS patients. In AD patients a total of 23 DE genes emerged, 19 protein coding genes and four lncRNAs. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses, we found common affected pathways and biological processes in ALS and AD. In PD patients only five genes were found to be DE. Our data brought to light the importance of lncRNAs and mRNAs regulation in three principal neurodegenerative disorders, offering starting points for new investigations on deregulated pathogenic mechanisms.
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Doença de Alzheimer/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Doença de Parkinson/metabolismo , Doença de Alzheimer/genética , Esclerose Lateral Amiotrófica/genética , Humanos , Doença de Parkinson/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , RNA-SeqRESUMO
3D cell cultures are becoming more and more important in the field of regenerative medicine due to their ability to mimic the cellular physiological microenvironment. Among the different types of 3D scaffolds, we focus on the Nichoid, a miniaturized scaffold with a structure inspired by the natural staminal niche. The Nichoid can activate cellular responses simply by subjecting the cells to mechanical stimuli. This kind of influence results in different cellular morphology and organization, but the molecular bases of these changes remain largely unknown. Through RNA-Seq approach on murine neural precursors stem cells expanded inside the Nichoid, we investigated the deregulated genes and pathways showing that the Nichoid causes alteration in genes strongly connected to mechanobiological functions. Moreover, we fully dissected this mechanism highlighting how the changes start at a membrane level, with subsequent alterations in the cytoskeleton, signaling pathways, and metabolism, all leading to a final alteration in gene expression. The results shown here demonstrate that the Nichoid influences the biological and genetic response of stem cells thorough specific alterations of cellular signaling. The characterization of these pathways elucidates the role of mechanical manipulation on stem cells, with possible implications in regenerative medicine applications.
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Mecanotransdução Celular/genética , Células-Tronco Neurais/metabolismo , Transcriptoma/genética , Animais , Técnicas de Cultura de Células , Células Cultivadas , Citoesqueleto/genética , Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos C57BL , Medicina Regenerativa/métodos , Transdução de Sinais/genética , Nicho de Células-Tronco/genética , Alicerces Teciduais/químicaRESUMO
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.
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Acetanilidas/farmacologia , Adipogenia/efeitos dos fármacos , Lipogênese/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , PPAR gama/metabolismo , Fenilpropionatos/farmacologia , Triglicerídeos/metabolismo , Células 3T3-L1 , Animais , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , CamundongosRESUMO
LncRNAs play crucial roles in cellular processes and their regulatory effects in the adult brain and neural stem cells (NSCs) remain to be entirely characterized. We report that 10 lncRNAs (LincENC1, FABL, lincp21, HAUNT, PERIL, lincBRN1a, lincBRN1b, HOTTIP, TUG1 and FENDRR) are expressed during murine NSCs differentiation and interact with the RNA-binding protein ELAVL1/HuR. Furthermore, we characterize the function of two of the deregulated lncRNAs, lincBRN1a and lincBRN1b, during NSCs' differentiation. Their inhibition leads to the induction of differentiation, with a concomitant decrease in stemness and an increase in neuronal markers, indicating that they exert key functions in neuronal cells differentiation. Furthermore, we describe here that HuR regulates their half-life, suggesting their synergic role in the differentiation process. We also identify six human homologs (PANTR1, TUG1, HOTTIP, TP53COR, ELDRR and FENDRR) of the mentioned 10 lncRNAs and we report their deregulation during human iPSCs differentiation into neurons. In conclusion, our results strongly indicate a key synergic role for lncRNAs and HuR in neuronal stem cells fate.
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Diferenciação Celular/genética , Proteína Semelhante a ELAV 1/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , RNA Longo não Codificante/genética , Animais , Biomarcadores , Autorrenovação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Humanos , Imuno-Histoquímica , Masculino , CamundongosRESUMO
BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease, presenting with midbrain dopaminergic neurons degeneration. A number of studies suggest that microglial activation may have a role in PD. It has emerged that inflammation-derived oxidative stress and cytokine-dependent toxicity may contribute to nigrostriatal pathway degeneration and exacerbate the progression of the disease in patients with idiopathic PD. Cell therapies have long been considered a feasible regenerative approach to compensate for the loss of specific cell populations such as the one that occurs in PD. We recently demonstrated that erythropoietin-releasing neural precursors cells (Er-NPCs) administered to MPTP-intoxicated animals survive after transplantation in the recipient's damaged brain, differentiate, and rescue degenerating striatal dopaminergic neurons. Here, we aimed to investigate the potential anti-inflammatory actions of Er-NPCs infused in an MPTP experimental model of PD. METHODS: The degeneration of dopaminergic neurons was caused by MPTP administration in C57BL/6 male mice. 2.5 × 105 GFP-labeled Er-NPCs were administered by stereotaxic injection unilaterally in the left striatum. Functional recovery was assessed by two independent behavioral tests. Neuroinflammation was investigated measuring the mRNAs levels of pro-inflammatory and anti-inflammatory cytokines, and immunohistochemistry studies were performed to evaluate markers of inflammation and the potential rescue of tyrosine hydroxylase (TH) projections in the striatum of recipient mice. RESULTS: Er-NPC administration promoted a rapid anti-inflammatory effect that was already evident 24 h after transplant with a decrease of pro-inflammatory and increase of anti-inflammatory cytokines mRNA expression levels. This effect was maintained until the end of the observational period, 2 weeks post-transplant. Here, we show that Er-NPCs transplant reduces macrophage infiltration, directly counteracting the M1-like pro-inflammatory response of murine-activated microglia, which corresponds to the decrease of CD68 and CD86 markers, and induces M2-like pro-regeneration traits, as indicated by the increase of CD206 and IL-10 expression. Moreover, we also show that this activity is mediated by Er-NPCs-derived erythropoietin (EPO) since the co-injection of cells with anti-EPO antibodies neutralizes the anti-inflammatory effect of the Er-NPCs treatment. CONCLUSION: This study shows the anti-inflammatory actions exerted by Er-NPCs, and we suggest that these cells may represent good candidates for cellular therapy to counteract neuroinflammation in neurodegenerative disorders.
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Encefalite/etiologia , Encefalite/cirurgia , Eritropoetina/uso terapêutico , Células-Tronco Neurais/transplante , Transtornos Parkinsonianos/complicações , Recuperação de Função Fisiológica/fisiologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Técnicas de Cocultura , Corpo Estriado/metabolismo , Corpo Estriado/cirurgia , Citocinas/metabolismo , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Eritropoetina/genética , Eritropoetina/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Força Muscular/efeitos dos fármacos , Força Muscular/fisiologia , Células-Tronco Neurais/fisiologia , Transtornos Parkinsonianos/etiologia , Transtornos Parkinsonianos/patologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/genética , Olfato/efeitos dos fármacos , Olfato/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
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.
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Tecido Adiposo/citologia , Anti-Inflamatórios/metabolismo , Células-Tronco Mesenquimais/citologia , Estresse Mecânico , Adipogenia , Separação Celular , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Lipectomia , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Células-Tronco Pluripotentes/metabolismoRESUMO
In the last decade, the advances made into the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) led to great improvements towards their use as models of diseases. In particular, in the field of neurodegenerative diseases, iPSCs technology allowed to culture in vitro all types of patient-specific neural cells, facilitating not only the investigation of diseases' etiopathology, but also the testing of new drugs and cell therapies, leading to the innovative concept of personalized medicine. Moreover, iPSCs can be differentiated and organized into 3D organoids, providing a tool which mimics the complexity of the brain's architecture. Furthermore, recent developments in 3D bioprinting allowed the study of physiological cell-to-cell interactions, given by a combination of several biomaterials, scaffolds, and cells. This technology combines bio-plotter and biomaterials in which several types of cells, such as iPSCs or differentiated neurons, can be encapsulated in order to develop an innovative cellular model. IPSCs and 3D cell cultures technologies represent the first step towards the obtainment of a more reliable model, such as organoids, to facilitate neurodegenerative diseases' investigation. The combination of iPSCs, 3D organoids and bioprinting will also allow the development of new therapeutic approaches. Indeed, on the one hand they will lead to the development of safer and patient-specific drugs testing but, also, they could be developed as cell-therapy for curing neurodegenerative diseases with a regenerative medicine approach.
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Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Neurológicos , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/terapia , Neurônios/citologia , Organoides/citologia , Animais , HumanosRESUMO
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.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Células-Tronco Mesenquimais/metabolismo , RNA Mensageiro/fisiologia , Fatores de Transcrição SOXB1/fisiologia , Células Cultivadas , Feminino , Humanos , Masculino , Ligação Proteica/fisiologiaRESUMO
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.