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In vitro cell models have undergone a shift from 2D models on glass slides to 3D models that better reflect the native 3D microenvironment. 3D bioprinting promises to progress the field by allowing the high-throughput production of reproducible cell-laden structures with high fidelity. The current stiffness range of printable matrices surrounding the cells that mimic the extracellular matrix environment remains limited. The work presented herein aims to expand the range of stiffnesses by utilizing a four-armed polyethylene glycol with maleimide-functionalized arms. The complementary cross-linkers comprised a matrix metalloprotease-degradable peptide and a four-armed thiolated polymer which were adjusted in ratio to tune the stiffness. The modularity of this system allows for a simple method of controlling stiffness and the addition of biological motifs. The application of this system in drop-on-demand printing is validated using MCF-7 cells, which were monitored for viability and proliferation. This study shows the potential of this system for the high-throughput investigation of the effects of stiffness and biological motif compositions in relation to cell behaviors.
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Bioimpressão , Hidrogéis , Humanos , Matriz Extracelular , Vidro , Células MCF-7RESUMO
Growth factors are key molecules involved in angiogenesis, a process critical for tissue repair and regeneration. Despite the potential of growth factor delivery to stimulate angiogenesis, limited clinical success has been achieved with this approach. Growth factors interact with the extracellular matrix (ECM), and particularly heparan sulphate (HS), to bind and potentiate their signalling. Here we show that engineered short forms of perlecan, the major HS proteoglycan of the vascular ECM, bind and signal angiogenic growth factors, including fibroblast growth factor 2 and vascular endothelial growth factor-A. We also show that engineered short forms of perlecan delivered in porous chitosan biomaterial scaffolds promote angiogenesis in a rat full thickness dermal wound model, with the fusion of perlecan domains I and V leading to superior vascularisation compared to native endothelial perlecan or chitosan scaffolds alone. Together, this study demonstrates the potential of engineered short forms of perlecan delivered in chitosan scaffolds as next generation angiogenic therapies which exert biological activity via the potentiation of growth factors.
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Quitosana , Fator A de Crescimento do Endotélio Vascular , Ratos , Animais , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Proteínas da Matriz ExtracelularRESUMO
Understanding the underlying mechanisms of migration and metastasis is a key focus of cancer research. There is an urgent need to develop in vitro 3D tumor models that can mimic physiological cell-cell and cell-extracellular matrix interactions, with high reproducibility and that are suitable for high throughput (HTP) drug screening. Here, we developed a HTP 3D bioprinted migration model using a bespoke drop-on-demand bioprinting platform. This HTP platform coupled with tunable hydrogel systems enables (i) the rapid encapsulation of cancer cells within in vivo tumor mimicking matrices, (ii) in situ and real-time measurement of cell movement, (iii) detailed molecular analysis for the study of mechanisms underlying cell migration and invasion, and (iv) the identification of novel therapeutic options. This work demonstrates that this HTP 3D bioprinted cell migration platform has broad applications across quantitative cell and cancer biology as well as drug screening.
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Bioimpressão , Neoplasias , Movimento Celular , Humanos , Hidrogéis , Impressão Tridimensional , Reprodutibilidade dos TestesRESUMO
The tumor microenvironment is highly complex owing to its heterogeneous composition and dynamic nature. This makes tumors difficult to replicate using traditional 2D cell culture models that are frequently used for studying tumor biology and drug screening. This often leads to poor translation of results between in vitro and in vivo and is reflected in the extremely low success rates of new candidate drugs delivered to the clinic. Therefore, there has been intense interest in developing 3D tumor models in the laboratory that are representative of the in vivo tumor microenvironment and patient samples. 3D bioprinting is an emerging technology that enables the biofabrication of structures with the virtue of providing accurate control over distribution of cells, biological molecules, and matrix scaffolding. This technology has the potential to bridge the gap between in vitro and in vivo by closely recapitulating the tumor microenvironment. Here, a brief overview of the tumor microenvironment is provided and key considerations in biofabrication of tumor models are discussed. Bioprinting techniques and choice of bioinks for both natural and synthetic polymers are also outlined. Lastly, current bioprinted tumor models are reviewed and the perspectives of how clinical applications can greatly benefit from 3D bioprinting technologies are offered.
Assuntos
Bioimpressão , Neoplasias , Humanos , Bioimpressão/métodos , Medicina de Precisão , Impressão Tridimensional , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Biologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Microambiente TumoralRESUMO
BACKGROUND: Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy with over 80% of cases already disseminated at diagnosis and facing a dismal five-year survival rate of 35%. EOC cells often spread to the greater omentum where they take-up cholesterol. Excessive amounts of cholesterol can be cytocidal, suggesting that cholesterol efflux through transporters may be important to maintain homeostasis, and this may explain the observation that high expression of the ATP-binding cassette A1 (ABCA1) cholesterol transporter has been associated with poor outcome in EOC patients. METHODS: ABCA1 expression was silenced in EOC cells to investigate the effect of inhibiting cholesterol efflux on EOC biology through growth and migration assays, three-dimensional spheroid culture and cholesterol quantification. RESULTS: ABCA1 suppression significantly reduced the growth, motility and colony formation of EOC cell lines as well as the size of EOC spheroids, whilst stimulating expression of ABCA1 reversed these effects. In serous EOC cells, ABCA1 suppression induced accumulation of cholesterol. Lowering cholesterol levels using methyl-B-cyclodextrin rescued the effect of ABCA1 suppression, restoring EOC growth. Furthermore, we identified FDA-approved agents that induced cholesterol accumulation and elicited cytocidal effects in EOC cells. CONCLUSIONS: Our data demonstrate the importance of ABCA1 in maintaining cholesterol balance and malignant properties in EOC cells, highlighting its potential as a therapeutic target for this disease.
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PURPOSE: To investigate the relationship between preoperative keratometry (K) and postoperative refraction and compare the visual outcomes after small-incision lenticule extraction (SMILE) between preoperative flat and steep corneas. METHODS: This study involved 814 consecutive eyes of 409 patients who underwent SMILE. A month later, a linear regression analysis of the relationship between preoperative K and the residual spherical equivalent (SE) along with eyes divided by a single standard deviation between flat and steep corneas (< 41.85 D, > 44.57 D, respectively) was conducted. Eyes were distinguished based on the degree of myopia. RESULTS: One month after surgery, no significant correlation existed between mean preoperative K and residual SE (P = 0.459). Linear regression analysis showed a weak negative correlation between flat corneas (r2 = 0.042, P = 0.025) rather than steep corneas (P = 0.908). Eyes with preoperative low myopia (< 3.00 D) (r2 = 0.233, P = 0.001) had a weak correlation compared with moderate and high myopia (P = 0.272, P = 0.257, respectively). Twelve months later, the predictability, safety, and efficacy did not vary between preoperative flat and steep corneas (P > 0.05). CONCLUSIONS: One month after SMILE for myopia, the corneas were flatter in the preoperative flat corneas or all the low myopic corneas, and they were more overcorrected. However, preoperative corneal curvature does not influence visual outcomes at 1 year after SMILE.
Assuntos
Doenças da Córnea , Cirurgia da Córnea a Laser , Miopia , Córnea/cirurgia , Doenças da Córnea/cirurgia , Substância Própria/cirurgia , Humanos , Lasers de Excimer/uso terapêutico , Microcirurgia , Miopia/cirurgia , Refração Ocular , Resultado do TratamentoRESUMO
Moldable, transparent polydimethylsiloxane (PDMS) elastomer microdevices enable a broad range of complex studies of three-dimensional cellular networks in their microenvironment in vitro. However, the uneven distribution of refractive index change, external to PDMS devices and internally in the sample chamber, creates a significant optical path difference (OPD) that distorts the light sheet beam and so restricts diffraction limited performance. We experimentally showed that an OPD of 120 µm results in the broadening of the lateral point spread function by over 4-fold. In this paper, we demonstrate steps to adapt a commercial inverted selective plane illumination microscope (iSPIM) and remove the OPD so as to achieve sub-micrometer imaging ranging from 0.6 ± 0.04 µm to 0.91 ± 0.03 µm of a fluorescence biological sample suspended in regular saline (RI ≈1.34) enclosed in 1.2 to 2 mm thick micromolded PDMS microdevices. We have proven that the removal of the OPD from the external PDMS layer by refractive index (RI) matching with a readily accessible, inexpensive sucrose solution is critical to achieve a >3-fold imaging resolution improvement. To monitor the RI matching process, a single-mode fiber (SMF) illuminator was integrated into the iSPIM. To remove the OPD inside the PDMS channel, we used an electrically tunable lens (ETL) that par-focuses the light sheet beam with the detection objective lens and so minimised axial distortions to attain sub-micrometer imaging resolution. We termed this new light sheet imaging protocol as modified inverted selective plane illumination microscopy (m-iSPIM). Using the high spatial-temporal 3D imaging of m-iSPIM, we experimentally captured single platelet (≈2 µm) recruitment to a platelet aggregate (22.5 µm × 22.5 µm × 6 µm) under flow at a 150 µm depth within a microfluidic channel. m-iSPIM paves the way for the application of light sheet imaging to a wide range of 3D biological models in microfluidic devices which recapitulate features of the physiological microenvironment and elucidate subcellular responses.
Assuntos
Lentes , Microscopia , Dimetilpolisiloxanos , Dispositivos Lab-On-A-Chip , IluminaçãoRESUMO
The ability of the N-MYC transcription factor to drive cancer progression is well demonstrated in neuroblastoma, the most common extracranial pediatric solid tumor, where MYCN amplification heralds a poor prognosis, with only 11% of high-risk patients surviving past 5 years. However, decades of attempts of direct inhibition of N-MYC or its paralogues has led to the conclusion that this protein is "undruggable." Therefore, targeting pathways upregulated by N-MYC signaling presents an alternative therapeutic approach. Here, we show that MYCN-amplified neuroblastomas are characterized by elevated rates of protein synthesis and that high expression of ABCE1, a translation factor directly upregulated by N-MYC, is itself a strong predictor of poor clinical outcome. Despite the potent ability of N-MYC in heightening protein synthesis and malignant characteristics in cancer cells, suppression of ABCE1 alone selectively negated this effect, returning the rate of translation to baseline levels and significantly reducing the growth, motility, and invasiveness of MYCN-amplified neuroblastoma cells and patient-derived xenograft tumors in vivo. The growth of nonmalignant cells or MYCN-nonamplified neuroblastoma cells remained unaffected by reduced ABCE1, supporting a therapeutic window associated with targeting ABCE1. Neuroblastoma cells with c-MYC overexpression also required ABCE1 to maintain cell proliferation and translation. Taken together, ABCE1-mediated translation constitutes a critical process in the progression of N-MYC-driven and c-MYC-driven cancers that warrants investigations into methods of its therapeutic inhibition. SIGNIFICANCE: These findings demonstrate that N-MYC-driven cancers are reliant on elevated rates of protein synthesis driven by heightened expression of ABCE1, a vulnerability that can be exploited through suppression of ABCE1.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Progressão da Doença , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Proteína Proto-Oncogênica N-Myc/metabolismo , Biossíntese de Proteínas , RNA MensageiroRESUMO
Epithelial ovarian cancer (EOC) is a complex disease comprising discrete histological and molecular subtypes, for which survival rates remain unacceptably low. Tailored approaches for this deadly heterogeneous disease are urgently needed. Efflux pumps belonging to the ATP-binding cassette (ABC) family of transporters are known for roles in both drug resistance and cancer biology and are also highly targetable. Here we have investigated the association of ABCC4/MRP4 expression to clinical outcome and its biological function in endometrioid and serous tumors, common histological subtypes of EOC. We found high expression of ABCC4/MRP4, previously shown to be directly regulated by c-Myc/N-Myc, was associated with poor prognosis in endometrioid EOC (P = .001) as well as in a subset of serous EOC with a "high-MYCN" profile (C5/proliferative; P = .019). Transient siRNA-mediated suppression of MRP4 in EOC cells led to reduced growth, migration and invasion, with the effects being most pronounced in endometrioid and C5-like serous cells compared to non-C5 serous EOC cells. Sustained knockdown of MRP4 also sensitized endometrioid cells to MRP4 substrate drugs. Furthermore, suppression of MRP4 decreased the growth of patient-derived EOC cells in vivo. Together, our findings provide the first evidence that MRP4 plays an important role in the biology of Myc-associated ovarian tumors and highlight this transporter as a potential therapeutic target for EOC.
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Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/patologia , Genes myc/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Prognóstico , RNA Interferente Pequeno/genética , Taxa de SobrevidaRESUMO
Survival rates for pediatric patients suffering from mixed lineage leukemia (MLL)-rearranged leukemia remain below 50% and more targeted, less toxic therapies are urgently needed. A screening method optimized to discover cytotoxic compounds selective for MLL-rearranged leukemia identified CCI-006 as a novel inhibitor of MLL-rearranged and CALM-AF10 translocated leukemias that share common leukemogenic pathways. CCI-006 inhibited mitochondrial respiration and induced mitochondrial membrane depolarization and apoptosis in a subset (7/11, 64%) of MLL-rearranged leukemia cell lines within a few hours of treatment. The unresponsive MLL-rearranged leukemia cells did not undergo mitochondrial membrane depolarization or apoptosis despite a similar attenuation of mitochondrial respiration by the compound. In comparison to the sensitive cells, the unresponsive MLL-rearranged leukemia cells were characterized by a more glycolytic metabolic phenotype, exemplified by a more pronounced sensitivity to glycolysis inhibitors and elevated HIF1α expression. Silencing of HIF1α expression sensitized an intrinsically unresponsive MLL-rearranged leukemia cell to CCI-006, indicating that this pathway plays a role in determining sensitivity to the compound. In addition, unresponsive MLL-rearranged leukemia cells expressed increased levels of MEIS1, an important leukemogenic MLL target gene that plays a role in regulating metabolic phenotype through HIF1α. MEIS1 expression was also variable in a pediatric MLL-rearranged ALL patient dataset, highlighting the existence of a previously undescribed metabolic variability in MLL-rearranged leukemia that may contribute to the heterogeneity of the disease. This study thus identified a novel small molecule that rapidly kills MLL-rearranged leukemia cells by targeting a metabolic vulnerability in a subset of low HIF1α/low MEIS1-expressing MLL-rearranged leukemia cells.
Assuntos
Acrilatos/farmacologia , Antineoplásicos/farmacologia , Furanos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Nitrilas/farmacologia , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Rearranjo Gênico , Histona-Lisina N-Metiltransferase/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos Endogâmicos , Mitocôndrias/fisiologia , Proteína Meis1/genética , Proteína de Leucina Linfoide-Mieloide/genética , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Diffuse Intrinsic Pontine Gliomas (DIPG) are the most devastating of all pediatric brain tumors. They mostly affect young children and, as there are no effective treatments, almost all patients with DIPG will die of their tumor within 12 months of diagnosis. A key feature of this devastating tumor is its intrinsic resistance to all clinically available therapies. It has been shown that glioma development is associated with metabolic reprogramming, redox state disruption and resistance to apoptotic pathways. The mitochondrion is an attractive target as a key organelle that facilitates these critical processes. PENAO is a novel anti-cancer compound that targets mitochondrial function by inhibiting adenine nucleotide translocase (ANT). Here we found that DIPG neurosphere cultures express high levels of ANT2 protein and are sensitive to the mitochondrial inhibitor PENAO through oxidative stress, while its apoptotic effects were found to be further enhanced upon co-treatment with mTOR inhibitor temsirolimus. This combination therapy was found to act through inhibition of PI3K/AKT/mTOR pathway, HSP90 and activation of AMPK. In vivo experiments employing an orthotopic model of DIPG showed a marginal anti-tumour effect likely due to poor penetration of the inhibitors into the brain. Further testing of this anti-DIPG strategy with compounds that penetrate the BBB is warranted.
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BACKGROUND: The Myc oncogene family has been implicated in many human malignancies and is often associated with particularly aggressive disease, suggesting Myc as an attractive prognostic marker and therapeutic target. However, for epithelial ovarian cancer (EOC), there is little consensus on the incidence and clinical relevance of Myc aberrations. Here we comprehensively investigated alterations in gene copy number, expression, and activity for Myc and evaluated their clinical significance in EOC. METHODS: To address inconsistencies in the literature regarding the definition of copy number variations, we developed a novel approach using quantitative polymerase chain reaction (qPCR) coupled with a statistical algorithm to estimate objective thresholds for detecting Myc gain/amplification in large cohorts of serous (n = 150) and endometrioid (n = 80) EOC. MYC, MYCN, and MYCL1 mRNA expression and Myc activity score for each case were examined by qPCR. Kaplan-Meier and Cox-regression analyses were conducted to assess clinical significance of Myc aberrations. RESULTS: Using a large panel of cancer cell lines (n = 34), we validated the statistical algorithm for determining clear thresholds for Myc gain/amplification. MYC was the most predominantly amplified of the Myc oncogene family members, and high MYC mRNA expression levels were associated with amplification in EOC. However, there was no association between prognosis and increased copy number or gene expression of MYC/MYCN/MYCL1 or with a pan-Myc transcriptional activity score, in EOC, although MYC amplification was associated with late stage and high grade in endometrioid EOC. CONCLUSION: A systematic and comprehensive analysis of Myc genes, transcripts, and activity levels using qPCR revealed that although such aberrations commonly occur in EOC, overall they have limited impact on outcome, suggesting that the biological relevance of Myc oncogene family members is limited to certain subsets of this disease.
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Heparin has been used clinically as an anti-coagulant for more than 100 y and the major source of this therapeutic is still animal tissues. Contamination issues in some batches of heparin over 10 y ago have highlighted the need to develop alternative methods of production of this essential drug. 1 Bioengineering heparin by expressing serglycin in mammalian cells is a promising approach that was recently reported by the authors. 2 This addendum explores the approaches that the authors are taking to increase the yield of recombinantly expressed serglycin decorated with heparin/heparan sulfate focusing on cell culture and bioreactor conditions and proposes that the cell microenvironment is a key modulator of heparin biosynthesis.
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Melhoramento Genético/métodos , Glucose/metabolismo , Heparina/biossíntese , Heparitina Sulfato/biossíntese , Proteoglicanas/genética , Proteínas de Transporte Vesicular/genética , Células HEK293 , Heparina/genética , Heparina/isolamento & purificação , Heparitina Sulfato/genética , Heparitina Sulfato/isolamento & purificação , Humanos , Proteoglicanas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
Myc transcriptional activity is frequently deregulated in human cancers, but a Myc-driven gene signature with prognostic ability across multiple tumor types remains lacking. Here, we selected 18 Myc-regulated genes from published studies of Myc family targets in epithelial ovarian cancer (EOC) and neuroblastoma. A Myc family activity score derived from the 18 genes was correlated to MYC/MYCN/MYCL1 expression in a panel of 35 cancer cell lines. The prognostic ability of this signature was evaluated in neuroblastoma, medulloblastoma, diffuse large B-cell lymphoma (DLBCL), and EOC microarray gene expression datasets using Kaplan-Meier and multivariate Cox regression analyses and was further validated in 42 primary neuroblastomas using qPCR. Cell lines with high MYC, MYCN, and/or MYCL1 gene expression exhibited elevated expression of the signature genes. Survival analysis showed that the signature was associated with poor outcome independently of well-defined prognostic factors in neuroblastoma, breast cancer, DLBCL, and medulloblastoma. In EOC, the 18-gene Myc activity signature was capable of identifying a group of patients with poor prognosis in a "high-MYCN" molecular subtype but not in the overall cohort. The predictive ability of this signature was reproduced using qPCR analysis of an independent cohort of neuroblastomas, including a subset of tumors without MYCN amplification. These data reveal an 18-gene Myc activity signature that is highly predictive of poor prognosis in diverse Myc-associated malignancies and suggest its potential clinical application in the identification of Myc-driven tumors that might respond to Myc-targeted therapies. Cancer Res; 77(4); 971-81. ©2016 AACR.
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Neoplasias/mortalidade , Proteínas Proto-Oncogênicas c-myc/fisiologia , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Humanos , Meduloblastoma/mortalidade , Proteína Proto-Oncogênica N-Myc/genética , Neoplasias/terapia , Neoplasias Epiteliais e Glandulares/mortalidade , Neuroblastoma/mortalidade , Neoplasias Ovarianas/mortalidade , Reação em Cadeia da Polimerase , Prognóstico , Modelos de Riscos ProporcionaisRESUMO
The mammalian HSPG2 gene encodes the proteoglycan protein core perlecan, which has important functions in biology including cell adhesion via integrins, binding to the extracellular matrix via various protein-protein interactions and binding of growth factors via the heparan sulfate chains decorating the N-terminal domain I. Here we show that, in the human mast cell line HMC-1, the transcription of this gene results in a population of mRNA that is processed in such a way to provide a relative increase of transcripts corresponding to domain V or the C-terminus compared to transcripts from either domain III or the N-terminal domain I. This paper also presents evidence of splicing of the HSPG2 gene in HMC-1 cells at exons 2/3 and after comparing this sequence with those published in various databases, a model is postulated to explain what might be happening in these cells with regard to the transcription of the HSPG2 gene. As domain V of perlecan contains the α2ß1 integrin binding site that modulates angiogenesis, we hypothesize that the transcriptional control of the HSPG2 gene in mast cells to synthesize these transcripts supports their stimulatory and specific role in wound healing and tissue regeneration.
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Processamento Alternativo/fisiologia , Células Endoteliais/metabolismo , Proteoglicanas de Heparan Sulfato/genética , Mastócitos/metabolismo , Transcrição Gênica , Processamento Alternativo/genética , Sequência de Aminoácidos , Análise de Variância , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Glicosaminoglicanos/metabolismo , Humanos , Integrina alfa2beta1/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Implantation of a foreign material almost certainly results in the formation of a fibrous capsule around the implant however, mechanistic events leading to its formation are largely unexplored. Mast cells are an inflammatory cell type known to play a role in the response to material implants, through the release of pro-inflammatory proteases and cytokines from their α-granules following activation. This study examined the in vivo and in vitro response of mast cells to chitosan, through detection of markers known to be produced by mast cells or involved with the inflammatory response. Mast cells, identified as Leder stained positive cells, were shown to be present in response to material implants. Additionally, the mast cell receptor, c-kit, along with collagen, serglycin, perlecan and chondroitin sulphate were detected within the fibrous capsules, where distribution varied between material implants. In conjunction, rat mast cells (RBL-2H3) were shown to be activated following exposure to chitosan as indicated by the release of ß-hexosaminidase. Proteoglycan and glycosaminoglycans produced by the cells showed similar expression and localisation when in contact with chitosan to when chemically activated. These data support the role that mast cells play in the inflammatory host response to chitosan implants, where mediators released from their α-granules impact on the formation of a fibrous capsule by supporting the production and organisation of collagen fibres.
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Quitosana/administração & dosagem , Mastócitos/citologia , Proteoglicanas/metabolismo , Animais , Linhagem Celular , Quitosana/farmacologia , Feminino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Mast cells are derived from hematopoietic progenitors that are known to migrate to and reside within connective and mucosal tissues, where they differentiate and respond to various stimuli by releasing pro-inflammatory mediators, including histamine, growth factors, and proteases. This study demonstrated that primary human mast cells as well as the rat and human mast cell lines, RBL-2H3 and HMC-1, produce the heparan sulfate proteoglycan, perlecan, with a molecular mass of 640 kDa as well as smaller molecular mass species of 300 and 130 kDa. Utilizing domain-specific antibodies coupled with N-terminal sequencing, it was confirmed that both forms contained the C-terminal module of the protein core known as endorepellin, which were generated by mast cell-derived proteases. Domain-specific RT-PCR experiments demonstrated that transcripts corresponding to domains I and V, including endorepellin, were present; however, mRNA transcripts corresponding to regions of domain III were not present, suggesting that these cells were capable of producing spliced forms of the protein core. Fractions from mast cell cultures that were enriched for these fragments were shown to bind endothelial cells via the α(2)ß(1) integrin and stimulate the migration of cells in "scratch assays," both activities of which were inhibited by incubation with either anti-endorepellin or anti-perlecan antibodies. This study shows for the first time that mast cells secrete and process the extracellular proteoglycan perlecan into fragments containing the endorepellin C-terminal region that regulate angiogenesis and matrix turnover, which are both key events in wound healing.
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Proteoglicanas de Heparan Sulfato/metabolismo , Mastócitos/metabolismo , Neovascularização Fisiológica , Fragmentos de Peptídeos/metabolismo , Cicatrização , Sequência de Aminoácidos , Animais , Adesão Celular , Linhagem Celular , Movimento Celular , Vasos Coronários/citologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Glicosaminoglicanos/biossíntese , Proteoglicanas de Heparan Sulfato/química , Proteoglicanas de Heparan Sulfato/genética , Proteoglicanas de Heparan Sulfato/isolamento & purificação , Humanos , Integrina alfa2beta1/metabolismo , Pulmão/citologia , Mastócitos/citologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteoglicanas/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas de Transporte Vesicular/biossínteseRESUMO
Cerium oxide nanoparticles (nanoceria) are promising materials for intracellular oxygen free radical scavenging providing a potential therapy for reactive oxygen species (ROS)-mediated inflammatory processes. In this study rhombohedral-shaped nanoceria were synthesized by flame spray pyrolysis with tuneable particle diameters between 3 and 94 nm by changing the liquid precursor flow rate. Monocytes and macrophages are major players in inflammatory processes as their production of ROS species has important downstream effects on cell signalling. Therefore, this study examined the ability of the nanoceria to be internalised by the human monocytic cell line, U937, and scavenge intracellular ROS. U937 cells activated in the presence of phorbol 12-myristate 13-acetate (PMA) were found to be more responsive to the nanoceria than U937 cells, which may not be surprising given the role of monocyte/macrophages in phagocytosing foreign material. The smaller particles were found to contain more crystal lattice defects with which to scavenge ROS, however a greater proportion of both the U937 and activated U937 cell populations responded to the larger particles. Hence all nanoceria particle sizes examined in this study were equally effective in scavenging intracellular ROS.
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Cério/química , Endocitose , Monócitos/citologia , Monócitos/metabolismo , Nanopartículas/química , Espécies Reativas de Oxigênio/metabolismo , Sobrevivência Celular , Citometria de Fluxo , Fluorescência , Humanos , Espaço Intracelular/metabolismo , Nanopartículas/ultraestrutura , Análise Espectral Raman , Temperatura , Células U937RESUMO
Chitosan has been shown to promote initial wound closure events to prevent blood loss. Platelet adhesion and activation are crucial early events in these processes after traumatic bleeding leading to thrombus formation. Platelet adhesion to chitosan was found to be enhanced in the presence of adsorbed plasma and extracellular matrix proteins and was found to be primarily mediated by α(IIb)ß(3) integrins, while α(2)ß(1) integrins were found to be involved in platelet adhesion to collagen and perlecan. Platelets were found to be activated by chitosan, as shown by an increase in the expression of α(IIb)ß(3) integrins and P-selectin, while the extent of activation was modulated by the presence of proteins including perlecan and fibrinogen. Collagen-coated chitosan was found to activate platelets to the same extent as either chitosan or collagen alone. These data support the role of plasma and extracellular matrix proteins in promoting chitosan mediated platelet adhesion and activation supporting the hypothesis that chitosan promotes wound healing via these interactions.
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Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Proteínas Sanguíneas/metabolismo , Quitosana/farmacologia , Proteínas da Matriz Extracelular/metabolismo , Adesividade Plaquetária/efeitos dos fármacos , Adsorção , Humanos , Cicatrização/efeitos dos fármacosRESUMO
The purpose of this study was to determine the incidence and risk factors of hypotony and the effectiveness of medical and surgical treatment. A total 117 eyes of 103 patients that underwent trabeculectomy with mitimycin C (MMC) between 1993 and 2000 were reviewed. Hypotony was treated with the following methods in a stepwise manner: medical treatment, intrableb autologous blood injection, additional sutures to the scleral flap, necrotic bleb excision and advancement of the forniceal conjunctival flap. Hypotony developed in 30 eyes (25.6%) of 26 patients, among, which hypotonic maculopathy developed in 11 eyes (9.4%). The risk factors of hypotony were young age and primary open angle glaucoma. Because of no light sense, 6 of the 30 hypotonic eyes were not treated. Nineteen (79.2%) of the 24 treated hypotonic eyes were successfully managed. Five eyes, 3 with hypotony but maintaining visual acuity and 2 with follow-up loss, were not included in the success group. The mean intraocular pressure (IOP) before treatment, 2.5 +/- 1.2 mmHg, increased to 8.3 +/- 4.0 mmHg at 18.5 months follow-up. The stepwise treatment seems to be a useful method to manage hypotony after trabeculectomy with MMC.