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1.
Transl Res ; 215: 57-74, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31541616

RESUMO

During acute myocardial infarction (AMI), Ischemia/Reperfusion (I/R) injury causes cardiomyocyte (CM) death and loss of tissue function, making AMI one of the major causes of death worldwide. Cell-based in vitro models of I/R injury have been increasingly used as a complementary approach to preclinical research. However, most approaches use murine cells in 2D culture setups, which are not able to recapitulate human cellular physiology, as well as nutrient and gas gradients occurring in the myocardium. In this work we established a novel human in vitro model of myocardial I/R injury using CMs derived from human induced pluripotent stem cells (hiPSC-CMs), which were cultured as 3D aggregates in stirred tank bioreactors. We were able to recapitulate important hallmarks of AMI, including loss of CM viability with disruption of cellular ultrastructure, increased angiogenic potential, and secretion of key proangiogenic and proinflammatory cytokines. Conditioned medium was further used to probe human cardiac progenitor cells (hCPCs) response to paracrine cues from injured hiPSC-CMs through quantitative whole proteome analysis (SWATH-MS). I/R injury hiPSC-CM conditioned media incubation caused upregulation of hCPC proteins associated with migration, proliferation, paracrine signaling, and stress response-related pathways, when compared to the control media incubation. Our results indicate that the model developed herein can serve as a novel tool to interrogate mechanisms of action of human cardiac populations upon AMI.


Assuntos
Reatores Biológicos , Modelos Biológicos , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/patologia , Comunicação Parácrina , Agregação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Oxigênio/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Pressão Parcial
2.
Int J Mol Sci ; 20(24)2019 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-31847310

RESUMO

Metastatic renal cell carcinoma (RCC) remains an important clinical issue; the 5-year survival rate of patients with metastasis is approximately 12%, while it is 93% in those with localized disease. There is evidence that blood cadmium and lead levels are elevated in RCC. The current studies were designed to assess the impact of cadmium and lead on the progression of RCC. The disruption of homotypic cell-cell adhesion is an essential step in epithelial-to-mesenchymal transition and tumor metastasis. Therefore, we examined the impact of cadmium and lead on the cadherin/catenin complex in Renca cells-a mouse RCC cell line. Lead, but not cadmium, induced a concentration-dependent loss of E-cadherin, while cadmium, but not lead, increased p120-catenin expression, specifically isoform 1 expression. Lead also induced a substantial increase in matrix metalloproteinase-9 levels. Both cadmium and lead significantly decreased the number of Renca cell aggregates, consistent with the disruption of the cadherin/catenin complex. Both metals enhanced wound healing in a scratch assay, and increased cell migration and invasion. These data suggest that cadmium and lead promote RCC progression.


Assuntos
Cádmio/efeitos adversos , Carcinoma de Células Renais/patologia , Agregação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Neoplasias Renais/patologia , Chumbo/efeitos adversos , Invasividade Neoplásica/patologia , Animais , Caderinas/metabolismo , Carcinoma de Células Renais/metabolismo , Cateninas/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Renais/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos
3.
Sci Rep ; 9(1): 19375, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852975

RESUMO

Rhubarb is commonly used to treat constipation in China for its function of promoting intestinal movement and optimum water content in feces. However, its mechanism of mucus secretion is vague. The aim of the study is to investigate the role of mast cells and enteric neurons in rhubarb extract (RE)-induced mucus secretion in the rat colon. Immunofluorescence was used to detect histamine receptors. Western blotting and 3,3'-diaminobenzidine (DAB) were applied to explore the content changes of mast cells activation. The changes in colonic goblet cells (GCs) were determined by means of PAS/AB staining. An intestinal perfusion system with a Bradford protein assay kit was directly to estimate in vitro secretion. And the cytokines were investigated with ELISA. The longitudinal aspect of this study indicate that the number and water content of faecal pellets were enhanced after the administration of different doses of RE accompanied by mast cells accumulated and increased the content of interferon (IFN) -γ or decreased the levels of interleukin (IL) -10 at doses of 3 and 6 g/kg. Pretreatment with ketotifen, mast cell stabilizer, had partially inhibited on RE-induced mucus secretion. Furthermore, RE induced the release of acetylcholine and mucin-2 in the colonic tissue and the histamine levels from the faeces. The results suggest that RE induced colonic mucus secretion involves mast cell activation and some cytokine.


Assuntos
Colo/metabolismo , Sistema Digestório/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Mastócitos/metabolismo , Rheum/química , Animais , Agregação Celular/efeitos dos fármacos , Degranulação Celular/efeitos dos fármacos , Colo/efeitos dos fármacos , Colo/patologia , Citocinas/genética , Sistema Digestório/metabolismo , Sistema Digestório/patologia , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/química , Exocitose/efeitos dos fármacos , Humanos , Mastócitos/efeitos dos fármacos , Mastócitos/patologia , Muco/efeitos dos fármacos , Muco/metabolismo , Ratos
4.
ACS Appl Mater Interfaces ; 11(38): 34560-34574, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31502820

RESUMO

Surface-functionalized microparticles are relevant to fields spanning engineering and biomedicine, with uses ranging from cell culture to advanced cell delivery. Varying topographies of biomaterial surfaces are also being investigated as mediators of cell-material interactions and subsequent cell fate. To investigate competing or synergistic effects of chemistry and topography in three-dimensional cell cultures, methods are required to introduce these onto microparticles without modification of their underlying morphology or bulk properties. In this study, a new approach for surface functionalization of poly(lactic acid) (PLA) microparticles is reported that allows decoration of the outer shell of the polyesters with additional polymers via aqueous atom transfer radical polymerization routes. PLA microparticles with smooth or dimpled surfaces were functionalized with poly(poly(ethylene glycol) methacrylate) and poly[N-(3-aminopropyl)methacrylamide] brushes, chosen for their potential abilities to mediate cell adhesion. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry analysis indicated homogeneous coverage of the microparticles with polymer brushes while maintaining the original topographies. These materials were used to investigate the relative importance of surface chemistry and topography both on the formation of human immortalized mesenchymal stem cell (hiMSCs) particle-cell aggregates and on the enhanced contractility of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs). The influence of surface chemistry was found to be more important on the size of particle-cell aggregates than topographies. In addition, surface chemistries that best promoted hiMSC attachment also improved hiPSC-CM attachment and contractility. These studies demonstrated a new route to obtain topo-chemical combinations on polyester-based biomaterials and provided clear evidence for the predominant effect of surface functionality over micron-scale dimpled topography in cell-microparticle interactions. These findings, thus, provide new guiding principles for the design of biomaterial interfaces to direct cell function.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Microplásticos , Miócitos Cardíacos/metabolismo , Poliésteres , Agregação Celular/efeitos dos fármacos , Linhagem Celular Transformada , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Microplásticos/química , Microplásticos/farmacologia , Miócitos Cardíacos/citologia , Poliésteres/química , Poliésteres/farmacologia
5.
PLoS Genet ; 15(6): e1008188, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31237867

RESUMO

Telomerase, particularly its main subunit, the reverse transcriptase, TERT, prevents DNA erosion during eukaryotic chromosomal replication, but also has poorly understood non-canonical functions. Here, in the model social amoeba Dictyostelium discoideum, we show that the protein encoded by tert has telomerase-like motifs, and regulates, non-canonically, important developmental processes. Expression levels of wild-type (WT) tert were biphasic, peaking at 8 and 12 h post-starvation, aligning with developmental events, such as the initiation of streaming (~7 h) and mound formation (~10 h). In tert KO mutants, however, aggregation was delayed until 16 h. Large, irregular streams formed, then broke up, forming small mounds. The mound-size defect was not induced when a KO mutant of countin (a master size-regulating gene) was treated with TERT inhibitors, but anti-countin antibodies did rescue size in the tert KO. Although, conditioned medium (CM) from countin mutants failed to rescue size in the tert KO, tert KO CM rescued the countin KO phenotype. These and additional observations indicate that TERT acts upstream of smlA/countin: (i) the observed expression levels of smlA and countin, being respectively lower and higher (than WT) in the tert KO; (ii) the levels of known size-regulation intermediates, glucose (low) and adenosine (high), in the tert mutant, and the size defect's rescue by supplemented glucose or the adenosine-antagonist, caffeine; (iii) the induction of the size defect in the WT by tert KO CM and TERT inhibitors. The tert KO's other defects (delayed aggregation, irregular streaming) were associated with changes to cAMP-regulated processes (e.g. chemotaxis, cAMP pulsing) and their regulatory factors (e.g. cAMP; acaA, carA expression). Overexpression of WT tert in the tert KO rescued these defects (and size), and restored a single cAMP signaling centre. Our results indicate that TERT acts in novel, non-canonical and upstream ways, regulating key developmental events in Dictyostelium.


Assuntos
Agregação Celular/genética , Dictyostelium/genética , Morfogênese/genética , Telomerase/genética , Adenosina/genética , Animais , Agregação Celular/efeitos dos fármacos , Quimiotaxia/genética , AMP Cíclico/genética , Dictyostelium/crescimento & desenvolvimento , Inibidores Enzimáticos/farmacologia , Técnicas de Inativação de Genes , Glucose/genética , Transdução de Sinais/genética , Telomerase/antagonistas & inibidores
6.
Biofabrication ; 11(4): 045003, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31091518

RESUMO

The elasticity of the cell and that of the supporting extracellular matrices (ECMs) in tissue are correlated. In some cases, the modulus of the ECM varies with a high spatial gradient. To study the effect of such a modulus gradient on the cell culture behavior, we proposed a novel yet straightforward method to fabricate elastomeric micropillar substrates with different height gradients, which could provide a large range of elasticity gradient from 2.4 kPa to 60 kPa. The micropillars were integrated into a microfluidic chip to demonstrate the elasticity variation, with the theoretical results proving that the elasticity of the two micropillar substrates was in the same range but with distinguished gradient strengths. Fibroblast seeded on the micropillar substrates showed migration toward the stiffer area but their elongation highly depended on the strength of the elasticity gradient. In the case of high gradient strength, cells could easily migrate to the stiffer area and then elongated perpendicularly to their migration direction. Otherwise, cells were mostly elongated in the direction of the gradient. Our results also showed that when the cell density was sufficiently high, cells tended to be oriented in the same direction locally, which was affected by both underneath pillars and cell-cell contact. The elasticity gradients could also be generated in a ripple shape, and the cell behavior showed the feasibility of using the micropillars for cell patterning applications. Moreover, the gradient pillar substrates were further used for the aggregate formation of induced pluripotent stem cells, thus providing an alternative substrate to study the effect of substrate elasticity on stem cell behavior and differentiation.


Assuntos
Movimento Celular/efeitos dos fármacos , Elasticidade , Elastômeros/farmacologia , Animais , Agregação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Dispositivos Lab-On-A-Chip , Camundongos , Células NIH 3T3 , Imagem com Lapso de Tempo
7.
PLoS One ; 14(5): e0217517, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31145754

RESUMO

Staphylococcus aureus formed bacterial aggregates in the plasma fraction of the hemolymph of silkworm, the larva of Bombyx mori, in a growth-dependent manner. The addition of arabinose or galactose inhibited the formation of S. aureus aggregates in the silkworm plasma. Formation of the bacterial aggregates depended on S. aureus genes required for the synthesis of bacterial surface polysaccharides-ypfP and ltaA, which are involved in lipoteichoic acid synthesis, and the tagO gene, which is involved in wall teichoic acid synthesis. These findings suggest that S. aureus forms bacterial aggregates in the silkworm plasma via bacterial surface teichoic acids.


Assuntos
Bombyx/genética , Agregação Celular/efeitos dos fármacos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Animais , Arabinose/farmacologia , Bombyx/metabolismo , Bombyx/microbiologia , Agregação Celular/genética , Galactose/farmacologia , Glicosiltransferases/genética , Hemolinfa/metabolismo , Hemolinfa/microbiologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/genética , Lipopolissacarídeos/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Ácidos Teicoicos/biossíntese
8.
Gynecol Endocrinol ; 35(11): 985-990, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31124382

RESUMO

Oocytes are extremely sensitive to radiation and chemotherapy, and premature ovarian failure (POF) is one of the side effects of anti-tumor therapy. The pathogenesis of POF is very complex and still not fully elucidated. A mouse POF model was established after 14 days of cyclophosphamide injection. POF mice presented ovarian atrophy, destroyed follicular structure, a reduction in the number of primordial and mature follicles, and an decrease in the number of corpora luteal along with increased level of follicle-stimulating hormone (FSH), decreased levels of estradiol (E2), and anti-Mullerian hormone (AMH). Additionally, the proportion of bone marrow myeloid-derived suppressor cells (MDSCs) in peripheral blood, spleen, and ovarian tissue increased. MDSCs were mainly distributed around follicles and corpora luteal. Levels of mTOR and p-mTOR increased in ovarian tissue and inhibition of mTOR with rapamycin reduced the aggregation of MDSCs in peripheral blood, spleen, and ovarian tissue. This investigation sheds new light on the modulatory role of mTOR and demonstrates that an increase in MDSC number may play a key role in the pathological reaction during POF. Inhibition of mTOR and reduction of MDSCs in the ovary may represent a novel strategy for the treatment of POF.


Assuntos
Células Supressoras Mieloides/efeitos dos fármacos , Ovário/efeitos dos fármacos , Ovário/patologia , Insuficiência Ovariana Primária/induzido quimicamente , Serina-Treonina Quinases TOR/metabolismo , Animais , Agregação Celular/efeitos dos fármacos , Ciclofosfamida , Modelos Animais de Doenças , Feminino , Ovário/metabolismo , Insuficiência Ovariana Primária/metabolismo , Insuficiência Ovariana Primária/patologia , Perda de Peso
9.
Biomater Sci ; 7(4): 1286-1298, 2019 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-30865196

RESUMO

A supramolecular hybrid hydrogel displaying a wide array of dynamic physical properties along with enhanced in vivo stem cell retention has been developed. The key strategy is facilely polymerizing bioactive gelatin methacrylate (GelMA) with 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) and 2-(3-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)ureido)ethyl methacrylate (UPyMA) to generate one hybrid branched copolymer. Rapid gelation occurs upon increasing the temperature above the lower critical solution temperature (LCST) of this supramolecular copolymer, where PMEO2MA segments dehydrate and assemble into clusters, providing a hydrophobic microenvironment facilitating UPy dimerization to connect polymer chains, thus forming quadruple hydrogen bond reinforced crosslinking networks. The biodegradable, self-healing, thermo-reversible and injectable properties of the supramolecular hydrogel are finely tunable by changing the hydrogel formulation. Mesenchymal stem cells encapsulated in the hydrogel show high viability and proliferation. The subcutaneous study shows that the stem cells delivered within the in situ formed hydrogel are well protected from mechanical damage and have significantly enhanced in vivo cell retention for three weeks. These results suggest that the dynamic supramolecular hydrogel can be utilized to regulate stem cells for tissue regeneration applications.


Assuntos
Materiais Biocompatíveis/farmacologia , Hidrogéis/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Temperatura , Animais , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Agregação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Hidrogéis/síntese química , Hidrogéis/química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Imagem Óptica , Tamanho da Partícula , Propriedades de Superfície
10.
Nanomedicine ; 20: 101977, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30878658

RESUMO

Plasma transfusion induces some transfusion related acute lung injury (TRALI) mediated through neutrophil extracellular traps (NETs). We investigated whether extracellular vesicles (EVs) present in plasma or obtained from resting (N-PEVs) or thrombin activated platelets (T-PEVs) can trigger NETs, and whether 75 nm-nanofiltration, to partially remove EVs, prohibits NETs formation. EVs size and concentration were determined by conventional biophysical approaches and by an original NanoBioAnalytical (NBA) platform based on EV immunocapture biochip, combining Surface Plasmon Resonance Imaging (SPRi) and Atomic Force Microscopy (AFM) exploration. EVs effective diameter was in the 25-1000 nm range, with a majority (≈ 90%) ≤ 100 nm. Both T-PEVs in buffer (but not N-PEVs) and non-nanofiltered plasma containing T-PEVs triggered NETs formation. Nanofiltration depleted large EVs (> 70 nm) and decreased NETs formation. The NBA platform was found to be a suitable tool to investigate the safety of plasma for transfusion.


Assuntos
Transfusão de Sangue , Vesículas Extracelulares/metabolismo , Nanotecnologia/métodos , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Agregação Celular/efeitos dos fármacos , Vesículas Extracelulares/efeitos dos fármacos , Filtração , Humanos , Nanopartículas/química , Nanoporos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Trombina/farmacologia
11.
Mol Nutr Food Res ; 63(11): e1801148, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30848861

RESUMO

SCOPE: Obese adipose tissue (AT) is infiltrated by inflammatory immune cells including IL-17A-producing-T (Th17) cells. It has been previously demonstrated that adipose-derived stem cells from obese (ob-ASCs), but not lean AT promote Th17 cells. Because n-3 PUFAs are known to inhibit obese AT inflammation, it is tested here whether they could inhibit ob-ASC-mediated IL-17A secretion. METHODS AND RESULTS: The n-3 PUFA precursor, alpha-linolenic acid (ALA), or its derivatives, eicosapentaenoic, or docosahexaenoic acid, is added to co-cultures of human ob-ASCs and mononuclear cells (MNCs). All three inhibited IL-17A, but not IL-1ß, IL-6, nor TNFα  secretion. As a control, palmitic acid (PA), a saturated fatty acid, did not inhibit IL-17A secretion. ALA also inhibited IL-17A secretion mediated by adipocytes differentiated from ob-ASCs. Toll-like-receptor 4 is shown to be involved in ob-ASC-mediated-IL-17A secretion, and to be inhibited by ALA, together with Cyclo-Oxygenase-2 and Signal-Transducer-and-Activator-of-transcription-3. In addition, ALA down-regulated Intercellular-Adhesion-Molecule-1 (ICAM-1) expression in both monocytes and ASCs, which resulted in decreased interactions between ob-ASCs and MNCs, and inhibition of IL-17A secretion. CONCLUSION: It is demonstrated herein that ALA inhibits Th17 cell promotion, through decreased ICAM-1expression in both ob-ASCs and monocytes. This novel mechanism may contribute to explain the beneficial effects of n-3 PUFA in IL-17A-related inflammatory pathologies.


Assuntos
Tecido Adiposo/citologia , Ácidos Graxos Ômega-3/farmacologia , Molécula 1 de Adesão Intercelular/genética , Interleucina-17/antagonistas & inibidores , Obesidade/metabolismo , Células-Tronco/fisiologia , Células Th17/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Técnicas de Cocultura , Humanos , Interleucina-17/biossíntese , Fator de Transcrição STAT3/antagonistas & inibidores , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologia , Células Th17/imunologia , Receptor 4 Toll-Like/antagonistas & inibidores , Ácido alfa-Linoleico/farmacologia
12.
Sci Rep ; 9(1): 183, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30655573

RESUMO

The discovery of giant viruses in the last years has fascinated the scientific community due to virus particles size and genome complexity. Among such fantastic discoveries, we have recently described tupanviruses, which particles present a long tail, and has a genome that contains the most complete set of translation-related genes ever reported in the known virosphere. Here we describe a new kind of virus-host interaction involving tupanvirus. We observed that tupanvirus-infected amoebas were induced to aggregate with uninfected cells, promoting viral dissemination and forming giant host cell bunches. Even after mechanical breakdown of bunches, amoebas reaggregated within a few minutes. This remarkable interaction between infected and uninfected cells seems to be promoted by the expression of a mannose receptor gene. Our investigations demonstrate that the pre-treatment of amoebas with free mannose inhibits the formation of bunches, in a concentration-dependent manner, suggesting that amoebal-bunch formation correlates with mannose receptor gene expression. Finally, our data suggest that bunch-forming cells are able to interact with uninfected cells promoting the dissemination and increase of tupanvirus progeny.


Assuntos
Amoeba/virologia , Agregação Celular/efeitos dos fármacos , Vírus Gigantes/patogenicidade , Interações Hospedeiro-Patógeno , Viroses/transmissão , Amoeba/citologia , Vírus Gigantes/genética , Lectinas Tipo C/metabolismo , Manose/farmacologia , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo
13.
ACS Chem Neurosci ; 10(3): 1603-1614, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30452227

RESUMO

Detailed study of the molecular mechanism behind the pathogenesis of Huntington's disease (HD) suggests that polyglutamine aggregation is one of the fundamental reasons for HD. Despite the discovery of many potential molecules, HD therapy is still limited to symptomatic relief. Among these molecules, few mechanism based peptide inhibitors of polyglutamine aggregation (QBP1, NT17 and PGQ9P2) have shown promising activity; however, poor blood-brain barrier (BBB) penetration, low bioavailability, and low half-life may hinder their therapeutic potential. Hence, to deliver them to the brain for assessing their efficacy, we have designed and synthesized peptide loaded poly-d,l-lactide- co-glycolide (PLGA) nanoparticles of less than 200 nm in size by carbodiimide chemistry and nanoprecipitation protocols. For brain delivery, PLGA nanoparticles were coated with polysorbate 80 which aids receptor mediated internalization. Using the in vitro BBB model of Madin-Darby canine kidney cells and healthy mice, the translocation of polysorbate 80 coated fluorescent nanoparticles was confirmed. Moreover, QBP1, NT17, and PGQ9P2 loaded PLGA nanoparticles showed dose dependent inhibition of polyglutamine aggregation in cell models of HD (Neuro 2A and PC12 cells) and improved motor performance in Drosophila model of HD. Additionally, no toxicity in cells and animals confirmed biocompatibility of the nanoparticulate formulations. Based on this work, future studies can be designed in higher animal models to test peptide loaded nanoparticles in HD and other polyglutamine expansion related diseases.


Assuntos
Agregação Celular/efeitos dos fármacos , Doença de Huntington/tratamento farmacológico , Nanopartículas/química , Peptídeos/uso terapêutico , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Encéfalo/patologia , Modelos Animais de Doenças , Drosophila , Doença de Huntington/metabolismo , Ácido Láctico/química
14.
Biochem Biophys Res Commun ; 508(3): 715-721, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30528229

RESUMO

EphA3, a member of the Eph family of receptor tyrosine kinases, has been reported to be overexpressed in some human cancers including glioblastoma. Here, we found that expression of EphA3 is up-regulated in response to epidermal growth factor (EGF) stimulation and promotes formation of cell aggregates in suspension culture of glioblastoma cells. Suppression of EphA3 expression by short hairpin RNA-mediated knockdown or CRISPR/Cas9-mediated gene deletion inhibited EGF-induced promotion of cell aggregate formation, whereas overexpression of EphA3 promoted formation of cell aggregates in suspension culture. EGF-induced EphA3 expression and promotion of cell aggregate formation required Akt activity. Furthermore, N-cadherin, whose expression was regulated by EGF and EphA3, contributed to the formation of cell aggregates in suspension culture. These results suggest that the regulation of EphA3 expression plays a critical role in glioblastoma cell growth in non-adherent conditions.


Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Fator de Crescimento Epidérmico/farmacologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Receptores Proteína Tirosina Quinases/genética , Regulação para Cima/genética , Caderinas/metabolismo , Agregação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Suspensões , Regulação para Cima/efeitos dos fármacos
15.
Tissue Eng Part A ; 25(5-6): 446-456, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30343640

RESUMO

IMPACT STATEMENT: The interactions of hypoxia and TGF-ß3 in aggregates of human meniscus fibrochondrocytes are synergistic in nature, suggesting combinatorial strategies using these factors are promising for tissue engineering the inner meniscus regions. Hypoxia alone in the absence of TGF-ß supplementation may be insufficient to initiate an inner meniscus-like extracellular matrix-forming response in this model.


Assuntos
Condrócitos/citologia , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Menisco/crescimento & desenvolvimento , Fator de Crescimento Transformador beta3/farmacologia , Adulto , Agregação Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Matriz Extracelular/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Pessoa de Meia-Idade , Análise de Componente Principal , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adulto Jovem
16.
Cell Mol Neurobiol ; 39(1): 61-71, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30415355

RESUMO

Axons of a peripheral nerve grow faster after an axotomy if it attains a prior injury a few days earlier. This is called conditioning lesion effect (CLE) and very much valued since it may provide new insights into neuron biology and axonal regeneration. There are established in vivo experimental paradigms to study CLE, however, there is a need to have an in vitro conditioning technique where CLE occurs in a maximally controlled environment. Mouse primary sensory neurons were isolated from lumbar 4-5 dorsal root ganglia and incubated at 37 °C on a silicon-coated watch glass that prevents cell attachment. After this conditioning period they were transferred to laminin coated culture dishes. Similar cultures were set up with freshly isolated neurons from control animals and from the animals that received a sciatic nerve cut 3 days earlier. All preparations were placed on a live cell imaging microscopy providing physiological conditions and photographed for 48 h. Axonal regeneration and neuronal survival was assessed. During the conditioning incubation period neurons remained in suspended aggregates and did not grow axons. The regeneration rate of the in vitro conditioned neurons was much higher than the in vivo conditioned and control preparations during the first day of normal incubation. However, higher regeneration rates were compromised by progressive substantial neuronal death in both types of conditioned cultures but not in the control preparations. By using neutralizing antibodies, we demonstrated that activity of endogenous leukemia inhibitory factor is essential for induction of CLE in this model.


Assuntos
Axônios/fisiologia , Modelos Biológicos , Animais , Axônios/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Células Cultivadas , Fator Inibidor de Leucemia/farmacologia , Camundongos Endogâmicos BALB C , Regeneração Nervosa/efeitos dos fármacos
17.
Int J Biol Macromol ; 123: 600-608, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30414418

RESUMO

In this work, we evaluated the ability of Punica granatum sarcotesta lectin (PgTeL) to impair the growth and viability of the Staphylococcus aureus clinical isolates 8325-4 (non-resistant) and LAC USA300 (MRSA strain). The effects of this lectin on aggregating, hemolytic activity, biofilm-forming ability, and expression of virulence genes (hla, rnaIII, and spa) were also investigated. PgTeL showed antibacterial activity against 8325-4 and LAC USA300 strains by interfering with both the growth (MIC50 of 6.25 and 12.5 µg/mL, respectively) and survival (MBC values of 25.0 and 50.0 µg/mL, respectively). Culture growth started only at the ninth (8325-4) and tenth (LAC USA300) hour in the presence of PgTeL at MIC50, while growth was detected since the first hour in the control. The lectin caused markedly altered cell morphology in both the strains. Although, at the MIC50, PgTeL caused structural alterations, most cells were still viable, while at the MBC it promoted cell injury and death. PgTeL showed anti-aggregation effect and exhibited antibiofilm activity against both the isolates. However, the lectin did not interfere with the hemolytic activity of LAC USA300 and with the expression of hla, rnaIII, and spa genes. In conclusion, PgTeL is a lectin with multiple inhibitory effects on S. aureus clinical isolates.


Assuntos
Biofilmes/efeitos dos fármacos , Lectinas/química , Lythraceae/química , Staphylococcus aureus/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Agregação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Lectinas/farmacologia , Staphylococcus aureus/patogenicidade
18.
Folia Biol (Praha) ; 65(5-6): 246-255, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-32362308

RESUMO

In vitro produced ß-like cells can provide promising cell therapy for curing the epidemic of diabetes. In this context, we aimed to investigate the effects of different concentrations of γ-aminobutyric acid (GABA) on the differentiation of rat pancreatic ductal epithelial-like stem cells (PDESCs) into ß-like cells. The PDESC line cells were cultured in the basal media (DMEM/F12 + 10% FBS + 1% penicillinstreptomycin) supplemented with 0 µM, 5 µM, 50 µM, 500 µM, and 5 mM of GABA for 28 days to induce their differentiation. The differentiated cells were detected by cell morphology, dithizone (DTZ) staining, immunofluorescence staining, real-time polymerase chain reaction (qPCR), and glucose-stimulated insulin secretion (GSIS) assay to validate their identity. At the end of 28 days, compared with the control group, enrichment of induced cells was high among the 5 µM, 50 µM, 500 µM, and 5 mM GABA induction groups. The formation of islet-like cell clusters (ICCs) began at 14 days, and the cell clusters showed a growth trend with the culture time. The induced ICCs were positive for DTZ staining, while the control group showed negative results for DTZ staining and the differentiated cells were also positive for ß-cell-specific markers (Ins1 and Pdx1). GSIS assay of 50 µM induction group cells at 28 days showed significantly higher levels of C-peptide and insulin secretion than the control, 5 µM, 500 µM, and 5 mM GABA-treated groups (P < 0.01). At the same time, the 50 µM induction group cells also showed significantly higher levels of Ins1, Pdx1 and Nkx6.1 mRNA as compared to the 5 µM, 500 µM and 5 mM GABA groups (P < 0.01). Thus, the addition of GABA to the basal medium effectively induced differentiation of adult rat PDESCs into insulin-secreting ß-like cells, and 50 µM was the most effective concentration for the induction.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Ductos Pancreáticos/citologia , Células-Tronco/citologia , Ácido gama-Aminobutírico/farmacologia , Animais , Peptídeo C/metabolismo , Agregação Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ratos Sprague-Dawley , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Transativadores/genética , Transativadores/metabolismo
19.
Acta Cir Bras ; 33(11): 954-963, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30517322

RESUMO

PURPOSE: The effect of a prophylactic oleuropein-rich diet before anesthesia accompanied by the widely-used steroid-based neuromuscular drug rocuronium on mast cell activation was investigated in the study. METHODS: 14 rabbits used in the study. The rabbits in the oleuropein group were given oleuropein-rich extract added to the animals' water at doses of 20 mg/kg oleuropein for 15 days orally. After 15 days, all rabbits in the two groups were given general anesthesia with rocuronium of 1 mg/kg. After 1 day, animals were sacrificed and the liver tissue sections stained with H&E, toluidine blue and tryptase for immunohistochemical study. RESULTS: There was no statistically significant difference between ALT, AST and albumin averages of the oleuropein and control groups (p> 0.05). The tryptase average of the control group was higher than the tryptase average of the oleuropein group and this difference was statistically significant (p=0.003). The T. blue average in the oleuropein group was higher than the control group. However, there was no statistically significant difference between groups (p=0.482). CONCLUSIONS: Rocuronium adverse effects, like hypersensitivity and anaphylaxis, may limit routine use of this substance. The use of oleuropein reduced the number of inflammatory cells and prevented degranulation.


Assuntos
Anestesia Geral/efeitos adversos , Anti-Inflamatórios/administração & dosagem , Iridoides/administração & dosagem , Mastócitos/efeitos dos fármacos , Fármacos Neuromusculares não Despolarizantes/efeitos adversos , Rocurônio/efeitos adversos , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Agregação Celular/efeitos dos fármacos , Degranulação Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Dietoterapia/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Imuno-Histoquímica , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Mastócitos/patologia , Profilaxia Pré-Exposição/métodos , Coelhos , Distribuição Aleatória , Reprodutibilidade dos Testes , Albumina Sérica/análise
20.
Stem Cell Res Ther ; 9(1): 342, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30526677

RESUMO

BACKGROUND: Three-dimensional (3D) floating culture clumps of mesenchymal stem cell (MSC)/extracellular matrix (ECM) complexes (C-MSCs) consist of cells and self-produced ECM. Previous studies have demonstrated that C-MSCs can be transplanted into bony lesions without an artificial scaffold to induce bone regeneration. Moreover, osteoinductive medium (OIM)-treated C-MSCs (OIM-C-MSCs) have shown rapid and increased new bone formation in vivo. To apply OIM-C-MSCs for novel bone regenerative cell therapy, their cellular properties at the molecular level must be elucidated. The transcriptional co-activators yes-associated protein/transcriptional co-activator with PDZ-binding motif (YAP/TAZ) have been recognized as key players in the mechanotransduction cascade, controlling cell lineage commitment in MSCs. It is plausible that 3D C-MSCs/OIM-C-MSCs cultured in floating conditions could provide distinct microenvironments compared to conventional 2D culture systems and thereby induce unique mechanotransduction cascades. Therefore, this study investigated the YAP/TAZ activity in 3D-cultured C-MSCs/OIM-C-MSCs in floating conditions. METHODS: Human bone marrow-derived MSCs were cultured in growth medium supplemented with ascorbic acid. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and were then torn off. The sheet was rolled to make round clumps of cells. Then, YAP/TAZ activity, filamentous actin (F-actin) integrity, collagen type I (COL1) production, and the differentiation potency in 3D floating culture C-MSCs/OIM-C-MSCs were analyzed. RESULTS: C-MSCs cultured in floating conditions lost their actin cytoskeleton to downregulate YAP/TAZ activity, which directed cells to undergo adipogenesis/chondrogenesis. OIM treatment induced abundant COL1 deposition, which facilitated Intß1-dependent actin fiber formation and YAP/TAZ activity to elevate the expression levels of osteogenic master transcriptional factor runt-related transcription factor 2 (RUNX2) mRNA in C-MSCs. Importantly, elevation of YAP/TAZ activity via OIM was associated with COL1 deposition and F-actin integrity, suggesting a positive feedback loop in OIM-C-MSCs. CONCLUSION: These findings suggest that OIM-C-MSCs, which form a unique microenvironment that maintains high YAP/TAZ activity, can serve as better candidates for bone regenerative cell therapy than C-MSCs.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Técnicas de Cultura de Células/métodos , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osseointegração , Fosfoproteínas/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Adipogenia/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Células Cultivadas , Condrogênese/efeitos dos fármacos , Meios de Cultura/farmacologia , Regulação para Baixo/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Retroalimentação Fisiológica , Humanos , Integrina beta1/metabolismo , Mecanotransdução Celular , Células-Tronco Mesenquimais/efeitos dos fármacos , Modelos Biológicos , Osseointegração/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Transdução de Sinais , Fatores de Transcrição , Quinases Associadas a rho/metabolismo
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