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1.
Methods Mol Biol ; 2273: 189-200, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33604854

RESUMO

Extracellular vesicles (EVs), are membrane-bound nanoparticles of biological origin. These signature molecules of health and disease have raised remarkable attention of the biomedical arena due to its potential diagnostic and therapeutic applicability. Among the many different techniques available for EV isolation, size-exclusion chromatography (SEC) is widely accepted.In this chapter, we present a protocol of size-exclusion high-performance liquid chromatography (SE-HPLC) as a method of EV isolation. This method can be adapted as a low cost but a reliable and scalable method of EV isolation in those laboratories having access to the HPLC systems.


Assuntos
Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Vesículas Extracelulares/química , Animais , Técnicas de Cultura de Células , Cromatografia em Gel/instrumentação , Cromatografia Líquida de Alta Pressão/instrumentação , Meios de Cultivo Condicionados/química , Desenho de Equipamento , Humanos
2.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540846

RESUMO

The adherence and shear-resistance of human umbilical venous endothelial cells (HUVEC) on polymers is determined in vitro in order to qualify cardiovascular implant materials. In these tests, variable fractions of HUVEC do not adhere to the material but remain suspended in the culture medium. Nonadherent HUVEC usually stop growing, rapidly lose their viability and can release mediators able to influence the growth and function of the adherent HUVEC. The aim of this study was the investigation of the time dependent behaviour of HUVEC under controlled nonadherent conditions, in order to gain insights into potential influences of these cells on their surrounding environment in particular adherent HUVEC in the context of in vitro biofunctionality assessment of cardiovascular implant materials. Data from adherent or nonadherent HUVEC growing on polystyrene-based cell adhesive tissue culture plates (TCP) or nonadhesive low attachment plates (LAP) allow to calculate the number of mediators released into the culture medium either from adherent or nonadherent cells. Thus, the source of the inflammatory mediators can be identified. For nonadherent HUVEC, a time-dependent aggregation without further proliferation was observed. The rate of apoptotic/dead HUVEC progressively increased over 90% within two days. Concomitant with distinct blebbing and loss of membrane integrity over time, augmented releases of prostacyclin (PGI2, up to 2.91 ± 0.62 fg/cell) and platelet-derived growth factor BB (PDGF-BB, up to 1.46 ± 0.42 fg/cell) were detected. The study revealed that nonadherent, dying HUVEC released mediators, which can influence the surrounding microenvironment and thereby the results of in vitro biofunctionality assessment of cardiovascular implant materials. Neglecting nonadherent HUVEC bears the risk for under- or overestimation of the materials endothelialization potential, which could lead to the loss of relevant candidates or to uncertainty with regard to their suitability for cardiac applications. One approach to minimize the influence from nonadherent endothelial cells could be their removal shortly after observing initial cell adhesion. However, this would require an individual adaptation of the study design, depending on the properties of the biomaterial used.


Assuntos
Adesão Celular/fisiologia , Técnicas de Cultura de Células , Células Endoteliais da Veia Umbilical Humana/citologia , Apoptose , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Morte Celular , Divisão Celular , Meios de Cultivo Condicionados/química , Citocinas/análise , Epoprostenol/análise , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Mediadores da Inflamação/análise , Peptídeos e Proteínas de Sinalização Intercelular/análise , L-Lactato Desidrogenase/análise , Poliestirenos , Proteínas Recombinantes/farmacologia , Propriedades de Superfície , Tromboxano A2/análise , Fator de Necrose Tumoral alfa/farmacologia
3.
Int J Mol Sci ; 22(3)2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33572938

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global public health emergency. Periodontitis, the most prevalent disease that leads to tooth loss, is caused by infection by periodontopathic bacteria. Periodontitis is also a risk factor for pneumonia and the exacerbation of chronic obstructive pulmonary disease, presumably because of the aspiration of saliva contaminated with periodontopathic bacteria into the lower respiratory tract. Patients with these diseases have increased rates of COVID-19 aggravation and mortality. Because periodontopathic bacteria have been isolated from the bronchoalveolar lavage fluid of patients with COVID-19, periodontitis may be a risk factor for COVID-19 aggravation. However, the molecular links between periodontitis and COVID-19 have not been clarified. In this study, we found that the culture supernatant of the periodontopathic bacterium Fusobacterium nucleatum (CSF) upregulated the SARS-CoV-2 receptor angiotensin-converting enzyme 2 in A549 alveolar epithelial cells. In addition, CSF induced interleukin (IL)-6 and IL-8 production by both A549 and primary alveolar epithelial cells. CSF also strongly induced IL-6 and IL-8 expression by BEAS-2B bronchial epithelial cells and Detroit 562 pharyngeal epithelial cells. These results suggest that when patients with mild COVID-19 frequently aspirate periodontopathic bacteria, SARS-CoV-2 infection is promoted, and inflammation in the lower respiratory tract may become severe in the presence of viral pneumonia.


Assuntos
/metabolismo , Meios de Cultivo Condicionados/química , Citocinas/metabolismo , Fusobacterium nucleatum/metabolismo , /genética , /virologia , Linhagem Celular , Meios de Cultivo Condicionados/farmacologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Regulação para Cima/efeitos dos fármacos
4.
Biochem Biophys Res Commun ; 534: 14-20, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33310182

RESUMO

Bone represents the most common site for breast cancer metastasis. Bone is a highly dynamic organ that is constantly adapting to its biophysical environment, orchestrated largely by the resident osteocyte network. Osteocytes subjected to physiologically relevant biophysical conditions may therefore represent a source of key factors mediating breast cancer cell metastasis to bone. Therefore, we investigated the potential proliferative and migratory capacity of soluble factors released by mechanically stimulated osteocytes on breast cancer cell behaviour. Interestingly the secretome of mechanically stimulated osteocytes enhanced both the proliferation and migration of cancer cells when compared to the secretome of statically cultured osteocytes, demonstrating that mechanical stimuli is an important physiological stimulus that should be considered when identifying potential targets. Using a cytokine array, we further identified a group of mechanically activated cytokines in the osteocyte secretome, which potentially drive breast cancer metastasis. In particular, CXCL1 and CXCL2 cytokines are highly expressed, mechanically regulated, and are known to interact with one another. Lastly, we demonstrate that these specific factors enhance breast cancer cell migration independently and in a synergistic manner, identifying potential osteocyte derived factors mediating breast cancer metastasis to bone.


Assuntos
Neoplasias da Mama/patologia , Quimiocina CXCL1/farmacologia , Quimiocina CXCL2/farmacologia , Osteócitos/citologia , Animais , Fenômenos Biomecânicos , Neoplasias da Mama/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/metabolismo , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Feminino , Humanos , Células MCF-7 , Camundongos , Osteócitos/fisiologia
5.
J Vis Exp ; (166)2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33369607

RESUMO

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.


Assuntos
Elementos de DNA Transponíveis/genética , Estresse Oxidativo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Transfecção , Antioxidantes/farmacologia , Biomarcadores/metabolismo , Contagem de Células , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/química , Células Epiteliais/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/isolamento & purificação , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/isolamento & purificação , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/isolamento & purificação , Fatores de Crescimento Neural/metabolismo , Neuroproteção/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serpinas/genética , Serpinas/isolamento & purificação , Serpinas/metabolismo , Doadores de Tecidos , Proteína Desacopladora 2/genética , Proteína Desacopladora 2/metabolismo
6.
PLoS One ; 15(7): e0235705, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32649682

RESUMO

Mutations of the SWI/SNF chromatin remodeling complex occur in 20% of all human cancers, including ovarian cancer. Approximately half of ovarian clear cell carcinomas (OCCC) carry mutations in the SWI/SNF subunit ARID1A, while small cell carcinoma of the ovary hypercalcemic type (SCCOHT) presents with inactivating mutations of the SWI/SNF ATPase SMARCA4 alongside epigenetic silencing of the ATPase SMARCA2. Loss of these ATPases disrupts SWI/SNF chromatin remodeling activity and may also interfere with the function of other histone-modifying enzymes that associate with or are dependent on SWI/SNF activity. One such enzyme is lysine-specific histone demethylase 1 (LSD1/KDM1A), which regulates the chromatin landscape and gene expression by demethylating proteins such as histone H3. Cross-cancer analysis of the TCGA database shows that LSD1 is highly expressed in SWI/SNF-mutated tumors. SCCOHT and OCCC cell lines have shown sensitivity to the reversible LSD1 inhibitor SP-2577 (Seclidemstat), suggesting that SWI/SNF-deficient ovarian cancers are dependent on LSD1 activity. Moreover, it has been shown that inhibition of LSD1 stimulates interferon (IFN)-dependent anti-tumor immunity through induction of endogenous retroviral elements and may thereby overcome resistance to checkpoint blockade. In this study, we investigated the ability of SP-2577 to promote anti-tumor immunity and T-cell infiltration in SCCOHT and OCCC cell lines. We found that SP-2577 stimulated IFN-dependent anti-tumor immunity in SCCOHT and promoted the expression of PD-L1 in both SCCOHT and OCCC. Together, these findings suggest that the combination therapy of SP-2577 with checkpoint inhibitors may induce or augment immunogenic responses of SWI/SNF-mutated ovarian cancers and warrants further investigation.


Assuntos
Antineoplásicos/farmacologia , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Linfócitos T/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/genética , Histonas/metabolismo , Humanos , Interferons/farmacologia , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Linfócitos T/citologia , Linfócitos T/imunologia , Fatores de Transcrição/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(22): 12071-12079, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32430324

RESUMO

Lesch-Nyhan disease (LND), caused by a deficient salvage purine pathway, is characterized by severe neurological manifestations and uric acid overproduction. However, uric acid is not responsible for brain dysfunction, and it has been suggested that purine nucleotide depletion, or accumulation of other toxic purine intermediates, could be more relevant. Here we show that purine alterations in LND fibroblasts depend on the level of folic acid in the culture media. Thus, physiological levels of folic acid induce accumulation of 5-aminoimidazole-4-carboxamide riboside 5'-monophosphate (ZMP), an intermediary of de novo purine biosynthetic pathway, and depletion of ATP. Additionally, Z-nucleotide derivatives (AICAr, AICA) are detected at high levels in the urine of patients with LND and its variants (hypoxanthine-guanine phosphoribosyltransferase [HGprt]-related neurological dysfunction and HGprt-related hyperuricemia), and the ratio of AICAr/AICA is significantly increased in patients with neurological problems (LND and HGprt-related neurological dysfunction). Moreover, AICAr is present in the cerebrospinal fluid of patients with LND, but not in control individuals. We hypothesize that purine alterations detected in LND fibroblasts may also occur in the brain of patients with LND.


Assuntos
Ácido Fólico/análise , Síndrome de Lesch-Nyhan/etiologia , Purinas/metabolismo , Trifosfato de Adenosina/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Técnicas de Cultura de Células , Meios de Cultivo Condicionados/química , Fibroblastos/metabolismo , Humanos , Hipoxantina Fosforribosiltransferase/metabolismo , Síndrome de Lesch-Nyhan/metabolismo , Ribonucleotídeos/metabolismo
8.
Iran J Immunol ; 17(1): 41-51, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32224540

RESUMO

BACKGROUND: Emerging evidence suggests that secretome of mesenchymal stem cells has many anti-inflammatory and regenerative properties, which makes it a suitable candidate for the treatment of autoimmune and degenerative diseases. Dipeptidyl Peptidase-IV (DPP-IV)/CD26 and Aminopeptidase N (APN)/CD13 are ubiquitous ecto-enzymes which can digest various substrates including some chemokines and neuropeptides that are involved in inflammatory conditions. OBJECTIVE: To evaluate the enzymatic activity of DPP-IV/CD26 and APN/CD13 in MSC conditioned media (MSC-CM). METHODS: The MSCs were isolated from the mouse's abdominal adipose tissues and were cultured with or without preconditioning by adding phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS). The levels of interleukin-10 (IL-10), nitric oxide (NO), as well as the enzymatic activities of DPP-IV/CD26 and APN/CD13 were measured in MSC-CM. RESULTS: The level of IL-10 and the enzyme activity of APN/CD13 did not show any changes in the MSC-CM of stimulated and non-stimulated cells. However, NO production was increased after treatment by LPS or PMA; nevertheless, the DPP-IV/CD26 activity was decreased in MSC-CM merely following the stimulation of cells with LPS. CONCLUSION: Our results indicated that MSC-secretome had DPP-IV/CD26 and APN/CD13 activity. The DPP-IV/CD26 activity was decreased following stimulation of MSCs by toll-like receptor 4 agonist. Further studies are needed to reveal the possible contribution of DPP-IV/CD26 and APN/CD13 in the anti-inflammatory functions of MSC-CM.


Assuntos
Antígenos CD13/metabolismo , Dipeptidil Peptidase 4/metabolismo , Células-Tronco Mesenquimais/enzimologia , Células-Tronco Mesenquimais/imunologia , Animais , Meios de Cultivo Condicionados/química , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Acetato de Tetradecanoilforbol/farmacologia
9.
Mol Biol (Mosk) ; 54(1): 128-136, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32163396

RESUMO

Neuroinflammation plays a key role in the pathogenesis of neurodegenerative diseases. Microglial cells are the main immune cells of the central nervous system. On exposure to lipopolysaccharides (LPS, components of the cell wall of Gram-negative enterobacteria), microglia is activated to produce reactive oxygen species (ROS), cytokines, and inflammatory mediators, which may cause neuron death. Exogenous recombinant human heat shock protein 70 (HSP70) was tested for effect on the activation of human microglial and neuroblastoma cells in response to LPS from Escherichia coli. Experiments included cell cultivation separately and transferring the conditioned medium from A-172 microglial cells to SK-N-SH neuroblastoma cells to simulate the effect of microglia treated with LPS and/or HSP70. The levels of ROS, TNFα, and apoptosis in LPS-treated cells were estimated in the presence or absence of HSP70. HSP70 was found to reduce the LPS-induced ROS generation, TNFα production, apoptosis, and necrosis, in both separate cell cultures and neuroblastoma cells grown in the conditioned medium from microglial cells. Signaling pathways involving protein kinases p38MAPK, JNK, and PI3K were demonstrated to play an important role in HSP70-mediated protection of microglial and neuroblastoma cells from LPS-induced apoptosis and ROS production.


Assuntos
Meios de Cultivo Condicionados/química , Proteínas de Choque Térmico HSP70/farmacologia , Lipopolissacarídeos/toxicidade , Neuroblastoma/tratamento farmacológico , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Humanos , Lipopolissacarídeos/imunologia , Microglia/efeitos dos fármacos , Microglia/imunologia , Microglia/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Espécies Reativas de Oxigênio/metabolismo
10.
Sci Rep ; 10(1): 2708, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066783

RESUMO

Prostate-specific antigen (PSA) is the most frequently used biomarker for the screening of prostate cancer. Understanding the structure of cancer-specific glycans can help us improve PSA assay. In the present study, we analysed the glycans of PSA obtained from culture medium containing cancer tissue-originated spheroids (CTOS) which have similar characteristics as that of the parent tumour to explore the new candidates for cancer-related glycoforms of PSA. The glycan profile of PSA from CTOS was determined by comparing with PSA from normal seminal plasma and cancer cell lines (LNCaP and 22Rv1) using lectin chromatography and mass spectrometry. PSA from CTOS was mostly sialylated and the content of Wisteria floribunda agglutinin reactive glycan (LacdiNAc) was similar to that of PSA derived from seminal plasma and 22Rv1. Conversely, concanavalin A (Con A)-unbound PSA was definitely detected from the three cancer origins but was almost negligible in seminal PSA. Two novel types of PSA were elucidated in the Con A-unbound fraction: one is a high molecular weight PSA with highly branched N-glycans, and the other is a low molecular weight PSA without N-glycans. Furthermore, the existence of Lewis X antigen group on PSA was indicated. These PSAs will be candidates for new cancer-related markers.


Assuntos
Biomarcadores Tumorais/metabolismo , Polissacarídeos/química , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/diagnóstico , Processamento de Proteína Pós-Traducional , Esferoides Celulares/metabolismo , Biomarcadores Tumorais/química , Sequência de Carboidratos , Linhagem Celular Tumoral , Cromatografia de Afinidade , Concanavalina A/química , Meios de Cultivo Condicionados/química , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Antígenos CD15/química , Antígenos CD15/metabolismo , Masculino , Lectinas de Plantas/química , Polissacarídeos/isolamento & purificação , Polissacarídeos/metabolismo , Próstata/metabolismo , Próstata/patologia , Antígeno Prostático Específico/química , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores de N-Acetilglucosamina/química , Sêmen/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Esferoides Celulares/química , Esferoides Celulares/patologia
11.
Exp Cell Res ; 386(2): 111737, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31759058

RESUMO

The presence of elevated T lymphocytic microparticles (TLMPs) during respiratory illness is associated with airway and lung inflammation and epithelial injuries. Although inflammasome and IL-1ß signaling are crucial in airway inflammation, little was known about their regulatory mechanism. We hypothesized that TLMPs trigger inflammasome activation and IL-1ß production in bronchial and alveolar epithelial cells to induce airway and lung inflammation. In this study, TLMPs induced IL-1ß and IL-18 secretion through NLRP3 inflammasome activation and upregulated TLR4 mRNA and protein expression in alveolar (A549) and human airway epithelial (16HBE) cells. Pretreatment with CLI-095, a specific inhibitor of TLR4 signaling, dramatically diminished the TLMP-induced release of IL-1ß and IL-18 by inhibiting the formation of NLRP3/ASC/pro-caspase-1 inflammasome in a dose-dependent manner. The TLMP-induced autophagy inhibition in epithelial cells was dependent on the PI3K/Akt signaling pathway, which significantly increased NLRP3 expression and enhanced TLMP-induced inflammation. TLR4, IL-1ß, and IL-18 proteins harbored in TLMPs were nonessential for the pro-inflammatory effect. In conclusion, TLMPs induce bronchial and alveolar epithelial cell secretion of IL-1ß and IL-18 cytokines by activating the TLR4 and PI3K/Akt signaling pathways and inhibiting autophagy. These effects lead to NLRP3 inflammasome formation and accumulation. TLMPs may be regarded as deleterious markers of airway and lung damage in respiratory diseases.


Assuntos
Micropartículas Derivadas de Células/imunologia , Meios de Cultivo Condicionados/farmacologia , Inflamassomos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Células A549 , Anti-Inflamatórios/farmacologia , Brônquios/citologia , Brônquios/imunologia , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/imunologia , Caspase 1/genética , Caspase 1/imunologia , Linhagem Celular , Micropartículas Derivadas de Células/química , Meios de Cultivo Condicionados/química , Dactinomicina/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Regulação da Expressão Gênica , Humanos , Inflamassomos/genética , Inflamassomos/imunologia , Inflamação , Interleucina-18/genética , Interleucina-18/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais/imunologia , Sulfonamidas/farmacologia , Receptor 4 Toll-Like/imunologia
12.
Mater Sci Eng C Mater Biol Appl ; 106: 110286, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31753397

RESUMO

Autologous chondrocyte implantation (ACI) is a promising approach to repair cartilage defects; however, the cartilage trauma-induced inflammatory environment compromises its clinical outcomes. Cell-derived decellularized extracellular matrix (DECM) has been used as a culture substrate for mesenchymal stem cells (MSCs) to improve the cell proliferation and lineage-specific differentiation. In this study, DECM deposited by synovium-derived MSCs was used as an in vitro expansion system for rabbit articular chondrocytes and the response of DECM-expanded chondrocytes to pro-inflammatory cytokines such as interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) was evaluated. Compared with those grown on tissue culture polystyrene (TCPS), the proliferation rate was significantly improved in DECM-expanded chondrocytes. TCPS- and DECM-expanded chondrocytes were isolated and induced to redifferentiation in a high-density pellet culture. DECM-expanded chondrocytes exerted a stronger resistance to 1 ng/mL of IL-1ß than TCPS-expanded cells, but the production of cartilage matrix in both groups was inhibited by 5 ng/mL of IL-1ß. When exposed to 1 or 5 ng/mL of TNF-α, DECM-expanded chondrocytes showed higher levels of cartilage matrix synthesis than TCPS-expanded cells. In addition, the gene expression of IL-1ß- or TNF-α-induced matrix degrading enzymes (MMP3, MMP9, MMP13, and ADAMTS5) was significantly lower in DECM-expanded chondrocytes than TCPS-expanded cells. Furthermore, we found that SIRT1 inhibition by nicotinamide completely counteracted the protective effect of DECM on chondrocytes in the presence of IL-1ß or TNF-α, indicating that the SIRT1 signaling pathway was involved in the DECM-mediated enhancement of anti-inflammatory properties of chondrocytes. Taken together, this work suggests that stem cell-derived DECM is a superior culture substrate for in vitro chondrocyte expansion by improving proliferation and enhancing the anti-inflammatory properties of chondrocytes. DECM-expanded chondrocytes with enhanced anti-inflammatory properties hold great potential in clinically ACI-based cartilage repair.


Assuntos
Condrócitos/metabolismo , Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Sirtuína 1/metabolismo , Agrecanas/metabolismo , Animais , Cartilagem Articular/citologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Proliferação de Células/efeitos dos fármacos , Condrócitos/citologia , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Regulação para Baixo/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Células-Tronco Mesenquimais/citologia , Coelhos , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/genética , Membrana Sinovial/citologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
13.
Mol Cell Biochem ; 463(1-2): 67-78, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31602539

RESUMO

Adipose-derived stem cells (ADSCs) and their derivatives have aroused intense interest in fields of dermatological and aesthetic medicine. As a major component detected in ADSCs secretome, platelet-derived growth factor AA (PDGF-AA) has been reported mediating extracellular matrix deposition and remodeling, thus might contribute to its anti-aging effect. On the basis of establishing an experimental model that simulate actual skin aging by exposing HDFs to both intrinsic and extrinsic aging factors, we pretreated human dermal fibroblasts (HDFs) with ADSC-conditioned medium (ADSC-CM) before being irradiated, aiming at exploring preventive effects of ADSCs secretome against aging damages. 48 h after irradiation, we detected cellular proliferation; ß-galactosidase stain; mRNA expressions of MMP-1, MMP-9, and TIMP-1; and protein expressions of collagen I, collagen III, and elastin. Moreover, we detected related protein expression of PI3K/Akt signal pathway, which can be activated by PDGF-AA and was newly found to promote extracellular matrix protein synthesis. Concentration of PDGF-AA in the prepared ADSC-CM decreased over time and maintained excellent bioactivity at low temperature until the 11th week. ADSC-CM pretreatment can slightly or significantly improve cellular proliferative activity and reduce cellular senescence in irradiated HDFs. Besides, ADSC-CM pretreatment increased collagen I, collagen III, elastin, and TIMP-1 expressions but decreased MMP-1 and MMP-9 expressions both in irradiated and nonirradiated HDFs. ADSC-CM pretreatment significantly increased pAkt protein expression, and ECM protein expression greatly decreased in case of LY294002 application. The results were similar in three generations of HDFs, yet varied with different degrees. Generally, ADSC-CM we prepared demonstrates a certain degree of positive role in preventing HDFs from intrinsic and extrinsic aging damages and that PDGF-AA may contribute to making it become effective with some other components in ADSC-CM.


Assuntos
Tecido Adiposo/metabolismo , Senescência Celular , Fibroblastos/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Raios Ultravioleta , Tecido Adiposo/citologia , Adulto , Senescência Celular/efeitos dos fármacos , Senescência Celular/efeitos da radiação , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Fibroblastos/citologia , Humanos , Masculino , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Células-Tronco/citologia
14.
J Clin Endocrinol Metab ; 105(1)2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31588501

RESUMO

CONTEXT: Human granulosa cells (hGCs) produce and respond to insulin-like growth factor 2 (IGF2) but whether the oocyte participates in IGF2 regulation in humans is unknown. OBJECTIVE: To determine the role of oocyte-secreted factors (OSFs) such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in IGF2 production by hGCs. DESIGN: Primary human cumulus GCs in culture. SETTING: University infertility center. PATIENTS OR OTHER PARTICIPANTS: GCs of women undergoing in vitro fertilization. INTERVENTION(S): Cells treated with GDF9 and BMP15 in the presence of vehicle, follicle-stimulating hormone (FSH), dibutyryl cyclic-AMP (dbcAMP), or mothers against decapentaplegic homolog (SMAD) inhibitors. MAIN OUTCOME MEASURE(S): Quantification of mRNA, protein, promoter activity, and DNA methylation. RESULTS: FSH stimulation of IGF2 (protein and mRNA) was significantly potentiated by the GDF9 and BMP15 (G+B) combination (P < 0.0001) in a concentration-dependent manner showing a maximal effect at 5 ng/mL each. However, GDF9 or BMP15 alone or in combination (G+B) have no effect on IGF2 in the absence of FSH. FSH stimulated IGF2 promoter 3 activity, but G+B had no effect on promoter activity. G+B potentiated IGF2 stimulation by cAMP. SMAD3 inhibitors inhibited G+B enhancement of IGF2 stimulation by FSH (P < 0.05) but had no effect on FSH induction. Moreover, inhibition of insulin-like growth factor receptor partially blocked G+B potentiation of FSH actions (P < 0.009). CONCLUSIONS: For the first time, we show that the oocyte actively participates in the regulation of IGF2 expression in hGCs, an effect that is mediated by the specific combination of G+B via SMAD2/3, which in turn target mechanisms downstream of the FSH receptor.


Assuntos
Meios de Cultivo Condicionados/farmacologia , Células da Granulosa/citologia , Fator de Crescimento Insulin-Like II/genética , Oócitos/metabolismo , Proteína Morfogenética Óssea 15/farmacologia , Células Cultivadas , Meios de Cultivo Condicionados/química , AMP Cíclico/metabolismo , Combinação de Medicamentos , Feminino , Hormônio Foliculoestimulante/genética , Hormônio Foliculoestimulante/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/metabolismo , Fator 9 de Diferenciação de Crescimento/farmacologia , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Oócitos/citologia , Cultura Primária de Células/métodos
15.
Methods Mol Biol ; 2043: 265-273, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31463919

RESUMO

Cell surface proteolysis controls numerous biological processes including cell-cell attachment and the communication between cells. The membrane-tethered families of matrix metalloproteinases (MT-MMPs) and disintegrin metalloproteinases (ADAMs) are major enzymes involved in the cleavage of molecules at the cell surface, and their activity is finely regulated by their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). The biological function of a metalloproteinase closely depends on the subset of substrates that it cleaves. Similarly, molecular processes that are regulated by a specific TIMP strictly depend on its unique inhibitory profile.Herein, we describe a mass spectrometry-based method for the quantitative analysis of protein abundance in conditioned media of cultured cells that is particularly suited for substrate identification of membrane-tethered metalloproteinases and for the identification of membrane proteins whose cleavage is regulated by TIMPs. This unbiased proteomic method represents a valuable tool to investigate biological functions of metalloproteinases and TIMPs at the "omic" level.


Assuntos
Metaloproteinases da Matriz/metabolismo , Proteômica/métodos , Inibidores Teciduais de Metaloproteinases/metabolismo , Animais , Técnicas de Cultura de Células , Células Cultivadas , Meios de Cultivo Condicionados/química , Humanos , Espectrometria de Massas
16.
Methods Mol Biol ; 2043: 275-284, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31463920

RESUMO

Here we describe the use of a decellularized ECM produced in vitro by BALB/c 3T3 fibroblasts for the identification of ADAMTS substrates. Seeding of ADAMTS protease-producing HEK cells on top of the cell-free ECM followed by analysis of the conditioned medium by liquid chromatography tandem mass spectrometry (LC-MS/MS), allows for screening of ADAMTS substrates without prior purification of full-length protease.


Assuntos
Proteínas ADAMTS/metabolismo , Meios de Cultivo Condicionados/química , Matriz Extracelular/metabolismo , Animais , Células 3T3 BALB , Sistema Livre de Células , Cromatografia Líquida , Células HEK293 , Humanos , Camundongos , Espectrometria de Massas em Tandem
17.
Tissue Eng Regen Med ; 16(6): 615-630, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31824824

RESUMO

Background: Mesenchymal Stem/Stromal Cells (MSCs) from the decidua parietalis (DPMSCs) of human term placenta express several molecules with important biological and immunological properties. DPMSCs induce natural killer cell expression of inflammatory receptors and their cytotoxic activity against cancer cells. These properties make DPMSCs promising therapeutical agent for cancer. The successful development of MSCs as an anti-cancer therapeutic cells rely on their ability to function in a hostile inflammatory and oxidative stress cancer environment. Here, we studied the effects of conditioned medium obtained from the culture of breast cancer cells (CMMDA-231) on the functional and phenotypic properties of DPMSCs. Methods: DPMSCs were cultured with CMMDA-231 and important functions of DPMSCs were measured. The effect of CMMDA-231 on DPMSC expression of several genes with different functions was also evaluated. Results: DPMSCs were able to function in response to CMMDA-231, but with reduced proliferative and adhesive potentials. Preconditioning of DPMSCs with CMMDA-231 enhanced their adhesion while reducing their invasion. In addition, CMMDA-231 modulated DPMSC expression of many genes with various functional (i.e., proliferation, adhesion, and invasion) properties. DPMSCs also showed increased expression of genes with anti-cancer property. Conclusion: These data show the ability of DPMSCs to survive and function in cancer environment. In addition, preconditioning of DPMSCs with CMMDA-231 enhanced their anti-cancer properties and thus demonstrating their potential as an anti-cancer therapeutic agent. However, future studies are essential to reveal the mechanism underlying the effects of MDA-231 on DPMSC functional activities and also to confirm the anti-cancer therapeutic potential of DPMSCs.


Assuntos
Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/química , Decídua/citologia , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Gravidez
18.
Reprod Biol Endocrinol ; 17(1): 113, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31883523

RESUMO

BACKGROUND: The Hippo pathway plays critical roles in regulating cell proliferation, differentiation and survival among species. Hippo pathway proteins are expressed in the ovary and are involved in ovarian function. Deletion of Lats1 causes germ cell loss, ovarian stromal tumors and reduced fertility. Ovarian fragmentation induces nuclear YAP1 accumulation and increased follicular development. At ovulation, follicular cells stop proliferating and terminally differentiate, but the mechanisms controlling this transition are not completely known. Here we explore the role of Hippo signaling in mouse granulosa cells before and during ovulation. METHODS: To assess the effect of oocytes on Hippo transcripts in cumulus cells, cumulus granulosa cells were cultured with oocytes and cumulus oocyte complexes (COCs) were cultured with a pSMAD2/3 inhibitor. Secondly, to evaluate the criticality of YAP1 on granulosa cell proliferation, mural granulosa cells were cultured with oocytes, YAP1-TEAD inhibitor verteporfin or both, followed by cell viability assay. Next, COCs were cultured with verteporfin to reveal its role during cumulus expansion. Media progesterone levels were measured using ELISA assay and Hippo transcripts and expansion signatures from COCs were assessed. Lastly, the effects of ovulatory signals (EGF in vitro and hCG in vivo) on Hippo protein levels and phosphorylation were examined. Throughout, transcripts were quantified by qRT-PCR and proteins were quantified by immunoblotting. Data were analyzed by student's t-test or one-way ANOVA followed by Tukey's post-hoc test or Dunnett's post-hoc test. RESULTS: Our data show that before ovulation oocytes inhibit expression of Hippo transcripts and promote granulosa cell survival likely through YAP1. Moreover, the YAP1 inhibitor verteporfin, triggers premature differentiation as indicated by upregulation of expansion transcripts and increased progesterone production from COCs in vitro. In vivo, ovulatory signals cause an increase in abundance of Hippo transcripts and stimulate Hippo pathway activity as indicated by increased phosphorylation of the Hippo targets YAP1 and WWTR1 in the ovary. In vitro, EGF causes a transient increase in YAP1 phosphorylation followed by decreased YAP1 protein with only modest effects on WWTR1 in COCs. CONCLUSIONS: Our results support a YAP1-mediated mechanism that controls cell survival and differentiation of granulosa cells during ovulation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Ciclo Celular/fisiologia , Células da Granulosa/fisiologia , Ovulação/fisiologia , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Proliferação de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados/química , Células do Cúmulo/fisiologia , Fator de Crescimento Epidérmico/farmacologia , Feminino , Camundongos , Oócitos/fisiologia , Progesterona/análise , RNA Mensageiro/análise , Transdução de Sinais/genética , Verteporfina
19.
Biomed Res Int ; 2019: 5768285, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31886229

RESUMO

We investigated the effects of a human umbilical cord mesenchymal stem cell-conditioned medium (MSC-CM)/chitosan/collagen/ß-glycerophosphate (ß-GP) thermosensitive hydrogel (MSC-CM/hydrogel) on mice with third-degree burns. MSC-CM was collected and mixed with chitosan, collagen, and ß-GP to generate the thermosensitive MSC-CM/hydrogel, which was stored in the liquid phase at 4°C. The wounds of established third-degree burned mice were then externally covered with the MSC-CM/hydrogel, which formed a gel when placed on the wounds at physiological temperature. Injured mice in three additional groups were treated with unconditioned MSC medium (UM), MSC-CM, or UM/chitosan/collagen/ß-GP thermosensitive hydrogels. Skin wound samples were obtained 4, 14, and 28 days after burning for further analysis by hematoxylin and eosin and Ki-67 staining. Wound healing rates and times, in addition to immunohistochemical results, were then compared and analyzed among the four groups. Application of the MSC-CM/hydrogel shortened healing time, limited the area of inflammation, enhanced reepithelialization, promoted the formation of high-quality, well-vascularized granulation tissue, and attenuated the formation of fibrotic and hypertrophic scar tissue. In summary, MSC-CM/hydrogel effectively promotes wound healing in third-degree burned mice.


Assuntos
Queimaduras/patologia , Meios de Cultivo Condicionados/farmacologia , Hidrogéis/farmacologia , Células-Tronco Mesenquimais , Cordão Umbilical , Cicatrização/efeitos dos fármacos , Animais , Células Cultivadas , Quitosana/química , Colágeno/química , Meios de Cultivo Condicionados/química , Glicerofosfatos/química , Humanos , Hidrogéis/química , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Temperatura , Cordão Umbilical/citologia , Cordão Umbilical/metabolismo
20.
Sci Rep ; 9(1): 17574, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31772251

RESUMO

RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles.


Assuntos
Vesículas Extracelulares/genética , Plasma/química , Plasma Rico em Plaquetas/química , Análise de Sequência de RNA , Urina/química , Meios de Cultivo Condicionados/química , Humanos , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Sequência de RNA/normas
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