RESUMO
Onychomatricoma (OM) is a rare nail unit tumour with a characteristic presentation of finger-like projections arising from the nail matrix. Due to the lack of transcriptome information, the mechanisms underlying its development are largely unknown. To characterize molecular features involved in the disease pathogenesis, we used digital spatial profiling (DSP) in 2 cases of OM and normal control nail units. Based on the histological evaluation, we selectively profiled 69 regions of interest covering epithelial and stromal compartments of each tissue section. Dermoscopic and histopathologic findings were reviewed in 6 cases. Single-cell RNA sequencing of nail units and DSP were combined to define cell type contributions of OM. We identified 173 genes upregulated in stromal compartments of OM compared to onychodermis, specialized nail mesenchyme. Gene ontology analysis of the upregulated genes suggested the role of Wnt pathway activation in OM pathogenesis. We also found PLA2G2A, a known modulator of Wnt signalling, is strongly and specifically expressed in the OM stroma. The potential role of Wnt pathway was further supported by strong nuclear localization of ß-catenin in OM. Compared to the nail matrix epithelium, only a few genes were increased in OM epithelium. Deconvolution of nail unit cell types showed that onychofibroblasts are the dominant cell type in OM stroma. Altogether, integrated spatial and single-cell multi-omics concluded that OM is a tumour that derives a significant proportion of its origin from onychofibroblasts and is associated with upregulation of Wnt signals, which play a key role in the disease pathogenesis.
Assuntos
Doenças da Unha , Unhas Malformadas , Neoplasias Cutâneas , Humanos , Imuno-Histoquímica , Unhas , Neoplasias Cutâneas/patologia , Unhas Malformadas/metabolismoRESUMO
BACKGROUND: Cutaneous metastasis (CM) refers to the spread of malignancy to the skin. CM is perceived as an advanced stage. It might be the first sign of a primary cancer or an indicator of recurrence. OBJECTIVES: To identify primary cancers associated with CMs and perform a survival analysis according to advanced stage of cutaneous metastasis at a single tertiary centre in Korea. METHODS: A total of 219 patients from Samsung Medical Center from January 2009 to April 2020 were retrospectively analysed to identify cases with biopsy-proven CMs. According to advanced stage of metastasis, patients were divided into three stages, CM only (CMO), CM with lymph node metastasis (CM/LM) and CM with distant metastasis (CM/DM), to analyse clinical characteristics and survival rate. RESULTS: The most common CM from primary cancer was breast cancer, followed by lung cancer, stomach cancer, colorectal cancer and others. When all primary cancers were included, the median survival period was 4.82 years for the CMO stage, 2.15 years for the CM/LM stage and 0.80 years for the CM/DM stage, with a tendency to deteriorate with advancing stage. At 1- and 3-year after occurrence of CM, the CM/DM stage showed a significantly poorer survival rate than the other two stages. CONCLUSIONS: This study showed a median survival period of 22 months for CM patients overall. Breast cancer has greater accessibility to the skin than other cancers. Therefore, breast cancer can metastasize to the skin without involving lymph nodes or other sites.
Assuntos
Neoplasias da Mama , Neoplasias Cutâneas , Humanos , Feminino , Estudos Retrospectivos , Neoplasias Cutâneas/patologia , Linfonodos/patologia , Neoplasias da Mama/patologia , Análise de Sobrevida , República da Coreia/epidemiologia , Estadiamento de NeoplasiasRESUMO
Brain ischemia leads to overstimulation of N-methyl-D-aspartate (NMDA) receptors, referred as excitotoxicity, which mediates neuronal cell death. However, less attention has been paid to changes in synaptic activity and morphology that could have an important impact on cell function and survival following ischemic insult. In this study, we investigated the effects of reperfusion after oxygen/glucose deprivation (OGD) not only upon neuronal cell death, but also on ultrastructural and biochemical characteristics of postsynaptic density (PSD) protein, in the stratum lucidum of the CA3 area in organotypic hippocampal slice cultures. After OGD/reperfusion, neurons were found to be damaged; the organelles such as mitochondria, endoplasmic reticulum, dendrites, and synaptic terminals were swollen; and the PSD became thicker and irregular. Ethanolic phosphotungstic acid staining showed that the density of PSD was significantly decreased, and the thickness and length of the PSD were significantly increased in the OGD/reperfusion group compared to the control. The levels of PSD proteins, including PSD-95, NMDA receptor 1, NMDA receptor 2B, and calcium/calmodulin-dependent protein kinase II, were significantly decreased following OGD/reperfusion. These results suggest that OGD/reperfusion induces significant modifications to PSDs in the CA3 area of organotypic hippocampal slice cultures, both morphologically and biochemically, and this may contribute to neuronal cell death and synaptic dysfunction after OGD/reperfusion.
RESUMO
The aging brain exhibits a region-specific reduction in synapse number and plasticity. Although astrocytes play central roles in regulating synapses, it is unclear how changes in astrocytes contribute to age-dependent cognitive decline and vulnerability to neurodegenerative diseases. Here, we identified a unique astrocyte subtype that exhibits dysregulated autophagy and morphology in aging hippocampus. In these autophagy-dysregulated astrocytes (APDAs), autophagosomes abnormally accumulate in swollen processes, impairing protein trafficking and secretion. We found that reduced mammalian target of rapamycin (mTOR) and proteasome activities with lysosomal dysfunction generate APDAs in an age-dependent manner. Secretion of synaptogenic molecules and astrocytic synapse elimination were significantly impaired in APDAs, suggesting that APDAs have lost their ability to control synapse number and homeostasis. Indeed, excitatory synapses and dendritic spines associated with APDAs were significantly reduced. Finally, we found that mouse brains with Alzheimer's disease showed a significantly accelerated increase in APDAs, suggesting potential roles for APDAs in age- and Alzheimer's disease-related cognitive decline and synaptic pathology.
Assuntos
Doença de Alzheimer , Camundongos , Animais , Doença de Alzheimer/genética , Astrócitos/metabolismo , Proteostase , Encéfalo/patologia , Envelhecimento , MamíferosRESUMO
Stress is a key risk factor for dystonia, a debilitating motor disorder characterized by cocontractions of muscles leading to abnormal body posture. While the serotonin (5HT) system is known to control emotional responses to stress, its role in dystonia remains unclear. Here, we reveal that 5HT neurons in the dorsal raphe nuclei (DRN) send projections to the fastigial deep cerebellar nuclei (fDCN) and that photostimulation of 5HT-fDCN induces dystonia in wild-type mice. Moreover, we report that photoinhibition of 5HT-fDCN reduces dystonia in a1A tot/tot mice, a genetic model of stress-induced dystonia, and administration of a 5HT-2A receptor inverse agonist (MDL100907; 0.1 to 1 mg/kg) or shRNA-mediated knockdown of the ht2ar gene in fDCN can notably reduce the onset of dystonia in a1A tot/tot mice. These results support the serotonin theory of dystonia and suggest strategies for alleviating symptoms in human patients by blocking 5HT-2A receptors.
RESUMO
Preadipocyte Factor 1 (Pref-1), also known as Delta-like Protein 1 (DLK-1) is an epidermal growth factor-like domain-containing trans-membrane protein that is involved in adipogenesis and cell fate decision. Its function in adipogenesis is reported inconsistently based on different cellular model systems. Here, by using human mesenchymal stem cells (MSCs), we show that Pref-1 is modulated by both dexamethasone and 3-isobutyl-1methylxanthine (IBMX), two components of the adipogenic induction mixture during the adipogenesis in vitro. IBMX induces the expression of Pref-1 in a time- and dose-dependent manner through cyclic AMP and cyclic GMP independent pathway and attenuates adipocyte differentiation by down-regulating PPARgamma (peroxisome proliferator activated receptor gamma) expression. Dexamethasone, on the other hand, is capable of subduing the inhibitory effect of IBMX-induced Pref-1 and initiating the adipogenesis by up-regulating PPARgamma expression. Moreover, the treatment of IBMX or dexamethasone alone fails to develop MSCs into mature adipocytes, however, treating cells with both IBMX and dexamethasone leads to a complete adipocyte differentiation as evaluated by lipid-droplet formation. Taken together, our study demonstrates that IBMX accelerates accumulation of lipid in MSCs only under the circumstance that the negative effect of Pref-1 induced by IBMX on the adipogenesis is overcome by dexamethasone.
Assuntos
Adipogenia , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Células 3T3-L1/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Animais , Proteínas de Ligação ao Cálcio , AMP Cíclico/farmacologia , GMP Cíclico/farmacologia , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Indometacina/farmacologia , Insulina/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Modelos Biológicos , Fatores de TempoRESUMO
The senescence of human mesenchymal stem cells (hMSCs) causes disruption of tissue and organ maintenance, and is thus an obstacle to stem cell-based therapies for disease. Although some researchers have studied changes in the characteristics of hMSCs (decreases in differentiation ability and self-renewal), comparing young and old ages, the mechanisms of stem cell senescence have not yet been defined. In this study, we developed a growth curve for human bone marrow derived MSCs (hBMSCs) which changes into a hyperbolic state after passage number 7. Senescence associated beta-galactosidase (SA beta-gal) staining of hBMSCs showed 10% in passage 9 and 45% in passage 11. We detected an increase in endogenous superoxide levels during senescence that correlated with senescence markers (SA beta-gal, hyperbolic growth curve). Interestingly, even though endogenous superoxide increased in a replicative senescence model, the expression of APE1/Ref-1, which is sensitive to intracellular redox state, decreased. These effects were confirmed in a stress-induced senescence model by exogenous treatment with H(2)O(2). This change is related to the p53 activity that negatively regulates APE1/Ref-1. p21 expression levels, which represent p53 activity, were transiently increased in passage 9, meaning that they correlated with the expression of APE1/Ref-1. Overexpression of APE1/Ref-1 suppressed superoxide production and decreased SA beta-gal in hBMSCs. In conclusion, intracellular superoxide accumulation appears to be the main cause of the senescence of hBMSCs, and overexpression of APE1/Ref-1 can rescue cells from the senescence phenotype. Maintaining characteristics of hBMSCs by regulating intracellular reactive oxygen species production can contribute to tissue regeneration and to improved cell therapy.
Assuntos
Senescência Celular/fisiologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/biossíntese , Células-Tronco Mesenquimais/metabolismo , Estresse Oxidativo/fisiologia , Western Blotting , Linhagem Celular , Regulação para Baixo , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Células-Tronco Mesenquimais/citologia , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxidos/metabolismoRESUMO
Both the accumulation of Amyloid-ß (Aß) in plaques and phosphorylation of Tau protein (p-Tau) in neurofibrillary tangles have been identified as two major symptomatic features of Alzheimer's disease (AD). Despite of critical role of Aß and p-Tau in AD progress, the interconnection of signalling pathways that Aß induces p-Tau remains elusive. Herein, we observed that a popular AD model mouse (APP/PS1) and Aß-injected mouse showed an increase in p-Tyr42 Rho in hippocampus of brain. Low concentrations of Aß (1 µM) induced RhoA-mediated Ser422 phosphorylation of Tau protein (p-Ser422 Tau), but reduced the expression of ATP citrate lyase (ACL) in the HT22 hippocampal neuronal cell line. In contrast, high concentrations of Aß (10 µM) along with high levels of superoxide production remarkably attenuated accumulation of p-Ser422 Tau, but augmented ACL expression and activated sterol regulatory element-binding protein 1 (SREBP1), leading to cellular senescence. Notably, a high concentration of Aß (10 µM) induced nuclear localization of p-Tyr42 Rho, which positively regulated NAD kinase (NADK) expression by binding to the NADK promoter. Furthermore, severe AD patient brain showed high p-Tyr42 Rho levels. Collectively, our findings indicate that both high and low concentrations of Aß are detrimental to neurons via distinct two p-Tyr42 RhoA-mediated signalling pathways in Ser422 phosphorylation of Tau and ACL expression.
Assuntos
Doença de Alzheimer , Proteínas tau , ATP Citrato (pro-S)-Liase , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Humanos , Camundongos , Camundongos Transgênicos , Fosforilação , Proteína rhoA de Ligação ao GTP/genética , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Memory loss in Alzheimer's disease (AD) may be related to synaptic defects in damaged hippocampal neurons. We investigated the relationship between amyloid peptide A beta 25-35-induced neuronal death pattern and presynaptic changes in organotypic hippocampal slice cultures. In propidium iodide (PI) uptake and annexin V labeling, A beta 25-35-induced neuronal damage dramatically increased in a concentration dependent manner, indicating both types of cell death. In ultrastructural analysis, apoptotic features in CA1 and CA3 area and synaptic disruption in stratum lucidum were detected in A beta 25-35-treated slices. Immunofluorescence and Western blot analysis for caspase-3 showed A beta 25-35 concentration dependently induced caspase-3 activation. Immunofluorescence and Western blot analysis to determine changes in presynaptic marker proteins demonstrated that expression of synaptosomal-associated protein-25 (SNAP-25) and synaptophysin were reduced by A beta 25-35 in CA1, CA3 and DG area at concentrations >2.5 microM. In conclusion, A beta 25-35-induced apoptotic cell death and caspase-3 activation at relatively low concentration, and induced synaptic disruption and loss of synaptic marker protein at concentrations >2.5 microM in organotypic hippocampal slice cultures. These suggest that A beta 25-35-induced apoptosis via triggering caspase-3 activation and lead to synaptic dysfunction in organotypic hippocampal slice cultures.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Neurônios/citologia , Fragmentos de Peptídeos/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Análise de Variância , Animais , Animais Recém-Nascidos , Anexina A5/metabolismo , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Microscopia Eletrônica de Transmissão/métodos , Neurônios/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Terminações Pré-Sinápticas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Sinaptofisina/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
Brain ischemia activates Ca(2+)-dependent synaptic vesicle exocytosis. The synaptosomal-associated protein 25 (SNAP-25) and syntaxin proteins, located on presynaptic terminals, are components of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex and play a key role in regulating exocytosis. Changes in the expression of SNAREs could affect SNARE complex formation, fusion of vesicles with the presynaptic membrane, and release of neurotransmitters through exocytosis. To investigate the relationship of glucose/oxygen deprivation (GOD)/reperfusion-induced neuronal damage and alteration of presynaptic function, we examined the expression of SNAREs and complexin during GOD and reperfusion using organotypic hippocampal slice cultures. Microtubule-associated protein 2 (MAP-2) staining and transmission electron microscopy showed that neuronal damage increased in a time-dependent manner and both types of neuronal death can occur at different times during GOD and reperfusion. The immunoreactivity of SNAREs such as SNAP-25, vesicle-associated membrane protein and syntaxin and complexin increased in pyramidal cell bodies in the CA1 and CA3 areas in a time-dependent manner following reperfusion. Our data suggest that alteration of presynaptic function may play a partial role in delayed neuronal death during GOD and reperfusion in organotypic hippocampal slice cultures.
Assuntos
Hipocampo/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Piramidais/metabolismo , Traumatismo por Reperfusão/metabolismo , Proteínas SNARE/metabolismo , Regulação para Cima , Animais , Morte Celular/fisiologia , Hipóxia Celular , Imunofluorescência , Hipocampo/ultraestrutura , Hipóxia-Isquemia Encefálica/patologia , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Células Piramidais/ultraestrutura , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/patologia , Proteína 25 Associada a Sinaptossoma/metabolismo , Técnicas de Cultura de TecidosRESUMO
OBJECTIVE: The purpose of this study is to evaluate the effect of dexamethasone on the damaged blood-ocular barrier caused by triolein emulsion, using contrast-enhanced MR imaging. MATERIALS AND METHODS: An emulsion of 0.1-mL triolein in 20 mL of saline was infused into the carotid arteries of 32 cats, 12 cats were placed in the treatment group and 18 cats were placed in the Control group. Thirty minutes after the infusion of triolein emulsion, a set of orbital pre- and post-contrast T1-weighted MR images (T1WIs) were obtained. Infusion of 10 mg/kg dexamethasone into the ipsilateral carotid artery of each of the cats in the treatment group cats and 20 mL saline in each of the cats in the control group was given. A second set of pre- and post-contrast orbital T1WIs were obtained three hours following triolein emulsion infusion. Qualitative analysis was performed for the the anterior chamber (AC), the posterior chamber (PC), and in the vitreous humor of the ipsilateral and contralateral eyes. The signal intensity ratios of the ipsilateral eye over the contralateral eye were quantitatively evaluated in the three ocular chambers on the first and second set of T1WIs, and were then statistically compared. RESULTS: Qualitatively, the AC, the PC or the vitreous did not show immediate contrast enhancement on the first and the second set of post-contrast T1WIs. However, the AC and the PC showed delayed contrast enhancement for both groups of cats on the second pre-contrast T1WIs. No enhancement or minimally delayed enhancement was seen for the vitreous humor. Quantitatively, the signal intensity ratios in the PC of the treatment group of cats were statistically lower than the ratios of the control group of cats for the second set of T1WIs (p = 0.037). The AC and vitreous showed no statistically significant difference between the feline treatment group and control group (p > 0.05). CONCLUSION: Contrast-enhanced MR images revealed increased vascular permeability in the PC of the eye after infusion of triolein emulsion. Dexamethasone seems to decrease the breakdown of the blood-aqueous barrier in the PC.
Assuntos
Barreira Hematoaquosa/efeitos dos fármacos , Barreira Hematorretiniana/efeitos dos fármacos , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Imageamento por Ressonância Magnética/métodos , Trioleína/efeitos adversos , Animais , Permeabilidade Capilar/efeitos dos fármacos , Gatos , Meios de Contraste , Emulsões , Aumento da ImagemRESUMO
Glial cells are receiving much attention since they have been recognized as important regulators of many aspects of brain function and disease. Recent evidence has revealed that two different glial cells, astrocytes and microglia, control synapse elimination under normal and pathological conditions via phagocytosis. Astrocytes use the MEGF10 and MERTK phagocytic pathways, and microglia use the classical complement pathway to recognize and eliminate unwanted synapses. Notably, glial phagocytosis also contributes to the clearance of disease-specific protein aggregates, such as ß-amyloid, huntingtin, and α-synuclein. Here we reivew recent findings showing that glial cells are active regulators in brain functions through phagocytosis and that changes in glial phagocytosis contribute to the pathogenesis of various neurodegenerative diseases. A better understanding of the cellular and molecular mechanisms of glial phagocytosis in healthy and diseased brains will greatly improve our current approach in treating these diseases.
RESUMO
Propofol is the most widely accepted intravenous anesthetic available for clinical use. However, neurotoxicity of propofol in the developing brain has been reported. This study investigated the effects of propofol on cognitive function in normal healthy adult mice. Thirty-three GFP-LC3 adult mice were included. Propofol was injected for anesthesia (nâ¯=â¯22). The sham control (nâ¯=â¯11) received intralipid injections. The mice completed a Y-maze test on 3 and 7â¯days after being anesthetized. Western blotting, immunofluorescence staining, and transmission electron microscopic (TEM) analyses were performed with their hippocampi. In addition, we conducted a separate ex vivo experiment using organotypic hippocampal slice cultures (OHSCs) to investigate the effects of propofol on induced autophagy. There was a significantly lower percentage of alternation in the Y-maze test on day 3 after propofol anesthesia than the control, but no difference was observed on day 7. Western blot analyses and immunofluorescence assays showed that the levels of cognitive function-related proteins significantly decreased in the propofol group compared to the control on day 3 but had recovered by day 7. In terms of autophagy-related proteins, western blot analyses and immunofluorescence assays showed that propofol increased autophagic induction, flux, and degradation of autophagosomes. Ex vivo experiments showed that propofol enhanced autophagic flux of the induced autophagy. In conclusion, although transient cognitive dysfunction occurred, adult mice recovered their cognitive function after the administration of propofol anesthesia. And this finding may be associated with enhanced autophagic flux.
Assuntos
Anestésicos Intravenosos/efeitos adversos , Autofagia/efeitos dos fármacos , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/fisiopatologia , Propofol/efeitos adversos , Recuperação de Função Fisiológica/fisiologia , Animais , Autofagia/fisiologia , Cognição/efeitos dos fármacos , Cognição/fisiologia , Disfunção Cognitiva/patologia , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Hipocampo/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/fisiologia , Distribuição Aleatória , Técnicas de Cultura de TecidosRESUMO
Gabexate mesilate (GM), a synthetic protease inhibitor, has an antiproteinase activity on various types of plasma serine proteases. However, its role on matrix metalloproteinases (MMPs) has not been identified. In this study, we investigated the effect of GM on MMPs and on the invasion and metastasis of human colon cancer cell lines and neoangiogenesis. The activities of MMPs secreted from these cells were significantly reduced by GM but unaffected by the serine protease inhibitor aprotinin. GM directly inhibited purified progelatinase A derived from T98G human glioblastoma cells. In vitro, GM significantly reduced the invasive ability of colon cancer cells but not cellular motility, whereas aprotinin affected neither. Liver metastatic ability and tumorigenic potential in nude mice were remarkably reduced on treatment with GM. Immunohistochemical analysis of GM-treated tumors in mice showed a marked increase in apoptosis and a significant reduction in tumor angiogenesis. Human umbilical vein endothelial cell proliferation, tube formation, and neoangiogenesis in the rabbit cornea and Matrigel implanted in mice were significantly inhibited by GM. These results suggest that GM is a novel inhibitor of MMPs and that it may inhibit the invasion and metastasis of human colon cancer cells by blocking MMPs and neoangiogenesis.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Gabexato/farmacologia , Neovascularização Patológica , Animais , Capilares/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Células Cultivadas , Colágeno/química , Colágeno/metabolismo , Neoplasias do Colo/patologia , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Endotélio Vascular/citologia , Feminino , Géis , Humanos , Imuno-Histoquímica , Laminina/química , Laminina/metabolismo , Fígado/patologia , Neoplasias Hepáticas/patologia , Masculino , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Proteoglicanas/química , Proteoglicanas/metabolismo , Coelhos , Inibidores de Serina Proteinase/farmacologia , Temperatura , Fatores de Tempo , Veias Umbilicais/citologia , Ativador de Plasminogênio Tipo Uroquinase/biossínteseRESUMO
hBMSCs are multipotent cells that are useful for tissue regeneration to treat degenerative diseases and others for their differentiation ability into chondrocytes, osteoblasts, adipocytes, hepatocytes and neuronal cells. In this study, biodegradable elastic hydrogels consisting of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic poly(ε-caprolactone) (PCL) scaffolds were evaluated for tissue engineering because of its biocompatibility and the ability to control the release of bioactive peptides. The primary cultured cells from human bone marrow are confirmed as hBMSC by immunohistochemical analysis. Mesenchymal stem cell markers (collagen type I, fibronectin, CD54, integrin1ß, and Hu protein) were shown to be positive, while hematopoietic stem cell markers (CD14 and CD45) were shown to be negative. Three different hydrogel scaffolds with different block compositions (PEG:PCL=6:14 and 14:6 by weight) were fabricated using the salt leaching method. The hBMSCs were expanded, seeded on the scaffolds, and cultured up to 8 days under static conditions in Iscove's Modified Dulbecco's Media (IMDM). The growth of MSCs cultured on the hydrogel with PEG/PCL= 6/14 was faster than that of the others. In addition, the morphology of MSCs seemed to be normal and no cytotoxicity was found. The coating of the vascular endothelial growth factor (VEGF) containing scaffold with Matrigel slowed down the release of VEGF in vitro and promoted the angiogenesis when transplanted into BALB/c nude mice. These results suggest that hBMSCs can be supported by a biode gradable hydrogel scaffold for effective cell growth, and enhance the angiogenesis by Matrigel coating.
Assuntos
Colágeno/metabolismo , Laminina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica , Poliésteres/metabolismo , Polietilenoglicóis/metabolismo , Proteoglicanas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Medula Óssea/metabolismo , Transplante de Células , Células Cultivadas , Colágeno/toxicidade , Combinação de Medicamentos , Humanos , Hidrogéis/metabolismo , Hidrogéis/toxicidade , Laminina/toxicidade , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos BALB C , Poliésteres/toxicidade , Polietilenoglicóis/toxicidade , Proteoglicanas/toxicidadeRESUMO
Synapsin I is believed to be involved in regulating neurotransmitter release and in synapse formation. Its interactions with the actin filaments and synaptic vesicles are regulated by phosphorylation. Because exocytosis and synapsin I phosphorylation are a Ca(2+)-dependent process, it is possible that an ischemic insult modifies the presynaptic proteins. However, the neuronal damage and the changes in synapsin I as well as its phosphorylation level as a result of glucose/oxygen deprivation (GOD) and reperfusion in organotypic hippocampal slice cultures have not been established. In this study, the level of synapsin I and phosphosynapsin was measured in organotypic hippocampal slice cultures in order to determine the role of synapsin I in the presynaptic nerve terminals during GOD/reperfusion. Propidium iodide fluorescence was observed in the CA1 area after GOD for 30 min, which could be detected in the whole pyramidal cell layer during reperfusion for 24 h. The immunofluorescence of the neuron specific nuclear protein, NeuN, showed a negative correlation with the PI fluorescence. During GOD/reperfusion, the immunofluorescence of synapsin I increased in the stratum radiatum and the stratum oriens of the CA1 area and the stratum lucidum and the stratum oriens of the CA3 area. The phosphosynapsin level evidently increased in the stratum lucidum of the CA3 area after GOD for 30 min, which was reduced to the control level after reperfusion. These results suggested that the neuronal damage and degenerations were observed as a result of GOD/reperfusion and the increase in synapsin I and its phosphorylation might play a role in modulating the release of neurotransmitters via exocytosis and in the formation of new synapses after brain ischemia.
Assuntos
Glucose/metabolismo , Hipocampo/metabolismo , Oxigênio/metabolismo , Sinapsinas/metabolismo , Animais , Animais Recém-Nascidos , Morte Celular , Hipóxia Celular/fisiologia , Imunofluorescência/métodos , Técnicas In Vitro , Fosfopiruvato Hidratase/metabolismo , Fosforilação , Propídio/metabolismo , Ratos , Ratos Sprague-Dawley , Reperfusão/métodos , Fatores de TempoRESUMO
Toll-like receptors (TLRs), which have been implicated in various neuroinflammatory responses, are thought to act in defense mechanisms by inhibiting neuronal cell death in Alzheimer's disease. In this study, we evaluated the effects of TLR2 on amyloid beta peptide 25-35 (Aß25-35)-induced neuronal cell death, synaptic dysfunction, and microglial activation in organotypic hippocampal slice cultures (OHSCs) from wild-type (WT) C57BL/6 mice and TLR2-knockout (KO) mice. In WT mice, Aß25-35 induced ß-amyloid aggregation and surrounding TLR2 expression. And, propidium iodide (PI) uptake, which is a measure of cell death, increased in a dose-dependent manner in slices with Aß25-35 treatment. In the Aß25-35-treated TLR2-KO OHSCs, the PI uptake was significantly attenuated to the control level, indicating that the cells were less susceptible to Aß25-35-induced neuronal toxicity. In the ultrastructural analysis, nuclear shrinkage, slightly swollen mitochondria, and degraded organelles were detected in the Aß25-35-treated slices from WT mice but not in the Aß25-35-treated slices from TLR2-KO, suggesting the resistance of TLR2-KO to Aß25-35-induced neurotoxicity. In Aß25-35-treated OHSCs of WT mice, the levels of phosphorylated tau were increased and the levels of synaptophysin were decreased in a dose-dependent manner, but they were not changed in OHSCs of TLR2-KO mice. In WT mice, Aß25-35 increased total protein level and immunoreactivity of Iba-1, which was colocalized with TLR2. However, there were no significant changes in the slices of Aß25-35-treated TLR2-KO mice. These results suggested that TLR2 may play a role in Aß25-35-induced neuronal cell loss and synaptic dysfunction through the activation of microglia in OHSCs.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Hipocampo/metabolismo , Fragmentos de Peptídeos/toxicidade , Receptor 2 Toll-Like/fisiologia , Animais , Western Blotting , Imunofluorescência , Hipocampo/patologia , Hipocampo/ultraestrutura , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Fosforilação , Sinaptofisina/metabolismo , Receptor 2 Toll-Like/genética , Proteínas tau/metabolismoRESUMO
The development of epilepsy is often associated with marked changes in central nervous system cell structure and function. Along these lines, reactive gliosis and granule cell axonal sprouting within the dentate gyrus of the hippocampus are commonly observed in individuals with temporal lobe epilepsy (TLE). Here we used the pilocarpine model of TLE in mice to screen the proteome and phosphoproteome of the dentate gyrus to identify molecular events that are altered as part of the pathogenic process. Using a two-dimensional gel electrophoresis-based approach, followed by liquid chromatography-tandem mass spectrometry, 24 differentially expressed proteins, including 9 phosphoproteins, were identified. Functionally, these proteins were organized into several classes, including synaptic physiology, cell structure, cell stress, metabolism and energetics. The altered expression of three proteins involved in synaptic physiology, actin, profilin 1 and α-synuclein was validated by secondary methods. Interestingly, marked changes in protein expression were detected in the supragranular cell region, an area where robust mossy fibers sprouting occurs. Together, these data provide new molecular insights into the altered protein profile of the epileptogenic dentate gyrus and point to potential pathophysiologic mechanisms underlying epileptogenesis.
Assuntos
Giro Denteado/metabolismo , Epilepsia/patologia , Proteoma/metabolismo , Proteômica/métodos , Actinas/metabolismo , Animais , Giro Denteado/patologia , Modelos Animais de Doenças , Eletroencefalografia/métodos , Eletroforese em Gel Bidimensional/métodos , Epilepsia/induzido quimicamente , Fluoresceínas , Masculino , Camundongos , Neurônios/efeitos dos fármacos , Compostos Orgânicos , Fosfoproteínas/metabolismo , Pilocarpina , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos , Fatores de TempoRESUMO
Rottlerin is widely used as a protein kinase C-delta inhibitor. Recently, several reports have shown the possible apoptosis-inducing effect of rottlerin in some cancer cell lines. Here we report that rottlerin induces not only apoptosis but also autophagy via a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells. Rottlerin treatment induced a dose- and time-dependent inhibition of cell growth, and cytoplasmic vacuolations were markedly shown. These vacuoles were identified as acidic autolysosomes by electron microscopy, acidic vesicular organelle (AVO) staining and transfection of green fluorescent protein-LC3. The LC3-II protein level also increased after treatment with rottlerin. Prolonged exposure to rottlerin eventually caused apoptosis via loss of mitochondrial membrane potential and translocation of AIF from mitochondria to the nucleus. However, the activities of caspase-3, -8 and -9 were not changed, and PARP did not show signs of cleavage. Interestingly, the pretreatment of cells with a specific inhibitor of autophagy (3-methyladenine) accelerated rottlerin-induced apoptosis as revealed by an analysis of the subdiploid fraction and TUNEL assay. Nevertheless, the knockdown of PKC-delta by RNA interference neither affected cell growth nor acidic vacuole formation. Similarly, rottlerin-induced cell death was not prevented by PKC-delta overexpression. Taken together, these findings suggest that rottlerin induces early autophagy and late apoptosis in a PKC-delta-independent manner, and the rottlerin-induced early autophagy may act as a survival mechanism against late apoptosis in HT1080 human fibrosarcoma cells.