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1.
Sci Rep ; 13(1): 15043, 2023 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-37700036

RESUMO

Posttraumatic syringomyelia (PTS) is an enigmatic condition characterized by the development of fluid-filled cysts (syrinxes) within the spinal cord. Perivascular spaces (PVS) are a critical component of fluid transport within the central nervous system (CNS), with dilated PVSs variably implicated in the pathogenesis of syringomyelia. The extent and spatial distribution of dilated PVSs in syringomyelia, however, remains unclear. This study aims to develop a method to assess PVS dimensions across multiple spinal cord segments in rats with PTS. Syrinxes were induced in two Sprague-Dawley rats at C6/7 with computer-controlled motorized spinal cord impaction; two control rats underwent sham laminectomies. Spinal cord segments were obtained at C4, C6 and C8, cleared via tissue clearing protocols, stained with immunofluorescent antibodies and imaged under confocal microscopy. Qualitative and quantitative analyses of PVS size were performed. Arteriolar PVSs were enlarged in the perisyringeal region of the spinal cord, compared to the control cord. No PVS enlargement was observed above or below the syrinx. These results confirm previous incidental findings of enlarged PVSs in the perisyringeal region, providing new insights into PVS dimensions across multiple spinal segments, and providing a novel method for quantifying spinal cord perivascular space size distributions.


Assuntos
Siringomielia , Ratos , Animais , Ratos Sprague-Dawley , Siringomielia/diagnóstico por imagem , Siringomielia/etiologia , Roedores , Sistema Nervoso Central , Hipertrofia
2.
Atherosclerosis ; 284: 153-159, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30913515

RESUMO

BACKGROUND AND AIMS: Atherosclerosis is characterized by lipid deposition, monocyte infiltration and foam cell formation in the artery wall. Translocator protein (TSPO) is abundantly expressed in lipid rich tissues. Recently, TSPO has been identified as a potential diagnostic tool in cardiovascular disease. The purpose of this study was to determine if the TSPO ligand, 18F-PBR111, can identify early atherosclerotic lesions and if TSPO expression can be used to identify distinct macrophage populations during lesion progression. METHODS: ApoE-/- mice were maintained on a high-fat diet for 3 or 12 weeks. C57BL/6J mice maintained on chow diet served as controls. Mice were administered 18F-PBR111 intravenously and PET/CT imaged. After euthanasia, aortas were isolated, fixed and optically cleared. Cleared aortas were immunostained with DAPI, and fluorescently labelled with antibodies to TSPO, the tissue resident macrophage marker F4/80 and the monocyte-derived macrophage marker CD11b. TSPO expression and the macrophage markers were visualised in fatty streaks and established plaques by light sheet microscopy. RESULTS: While tissue resident F4/80 + macrophages were evident in the arteries of animals without atherosclerosis, no CD11b + macrophages were observed in these animals. In contrast, established plaques had high CD11b and low F4/80 expression. A ∼3-fold increase in the uptake of 18F-PBR111 was observed in the aortas of atherosclerotic mice relative to controls. CONCLUSIONS: Imaging of TSPO expression is a new approach for studying atherosclerotic lesion progression and inflammatory cell infiltration. The TSPO ligand, 18F-PBR111, is a potential clinical diagnostic tool for the detection and quantification of atherosclerotic lesion progression in humans.


Assuntos
Aterosclerose/sangue , Aterosclerose/diagnóstico , Antígeno CD11b/fisiologia , Macrófagos , Receptores de GABA/fisiologia , Animais , Antígeno CD11b/biossíntese , Progressão da Doença , Diagnóstico Precoce , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Piridinas/administração & dosagem , Receptores de GABA/biossíntese
3.
Chem Commun (Camb) ; 54(89): 12618-12621, 2018 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-30349928

RESUMO

Polyelectrolyte-protein complexes are widely used to deliver therapeutic proteins. Here, we present a method for imaging the release of drugs from polyion complex (PIC) micelles in 3D tumour spheroids using light-sheet microscopy. A negatively charged block copolymer was condensed with a positively charged model drug, hen egg white lysozyme (HEWL) by electrostatic interaction. We were able to observe the distribution of polymer and protein within the entire tumour spheroid, showing that the protein was released from the polyelectrolyte complex upon cell internalization at the peripheral cell layer of the spheroid.


Assuntos
Sistemas de Liberação de Medicamentos , Micelas , Muramidase/química , Polímeros/química , Esferoides Celulares/química , Portadores de Fármacos/química , Humanos , Íons/química , Células MCF-7 , Microscopia de Fluorescência , Estrutura Molecular , Muramidase/metabolismo , Esferoides Celulares/metabolismo , Eletricidade Estática
4.
Nat Commun ; 5: 5452, 2014 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-25406832

RESUMO

The evolutionarily conserved peripheral benzodiazepine receptor (PBR), or 18-kDa translocator protein (TSPO), is thought to be essential for cholesterol transport and steroidogenesis, and thus life. TSPO has been proposed as a biomarker of neuroinflammation and a new drug target in neurological diseases ranging from Alzheimer's disease to anxiety. Here we show that global C57BL/6-Tspo(tm1GuWu(GuwiyangWurra))-knockout mice are viable with normal growth, lifespan, cholesterol transport, blood pregnenolone concentration, protoporphyrin IX metabolism, fertility and behaviour. However, while the activation of microglia after neuronal injury appears to be unimpaired, microglia from (GuwiyangWurra)TSPO knockouts produce significantly less ATP, suggesting reduced metabolic activity. Using the isoquinoline PK11195, the ligand originally used for the pharmacological and structural characterization of the PBR/TSPO, and the imidazopyridines CLINDE and PBR111, we demonstrate the utility of (GuwiyangWurra)TSPO knockouts to provide robust data on drug specificity and selectivity, both in vitro and in vivo, as well as the mechanism of action of putative TSPO-targeting drugs.


Assuntos
Glândulas Suprarrenais/diagnóstico por imagem , Encéfalo/diagnóstico por imagem , Rim/diagnóstico por imagem , Microglia/metabolismo , Receptores de GABA/genética , Trifosfato de Adenosina/metabolismo , Animais , Comportamento Animal , Colesterol/metabolismo , Fertilidade/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tomografia por Emissão de Pósitrons , Pregnenolona/sangue , Protoporfirinas/metabolismo , Baço/diagnóstico por imagem , Testículo/diagnóstico por imagem , Imagem Corporal Total
5.
BMC Cell Biol ; 13: 12, 2012 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-22583596

RESUMO

BACKGROUND: The behaviour of tumour cells depends on factors such as genetics and the tumour microenvironment. The latter plays a crucial role in normal mammary gland development and also in breast cancer initiation and progression. Breast cancer tissues tend to be highly desmoplastic and dense matrix as a pre-existing condition poses one of the highest risk factors for cancer development. However, matrix influence on tumour cell gene expression and behaviour such as cell migration is not fully elucidated. RESULTS: We generated high-density (HD) matrices that mimicked tumour collagen content of 20 mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1 mg/cm3. Live-cell imaging showed breast cancer cells utilizing cytoplasmic streaming and cell body contractility for migration within HD matrix. Cell migration was blocked in the presence of both the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugs individually. This suggests roles for ROCK1 and MMP in cell migration are complicated by compensatory mechanisms. ROCK1 expression and protein activity, were significantly upregulated in HD matrix but these were blocked by treatment with a histone deacetylase (HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirect and relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA or DAPT abrogated the inhibition of ROCK1 by MS-275. CONCLUSION: Increased matrix density elevates ROCK1 activity, which aids in cell migration via cell contractility. The upregulation of ROCK1 is epigenetically regulated in an indirect manner involving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS-275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1 expression.


Assuntos
Receptor Notch1/metabolismo , Quinases Associadas a rho/metabolismo , Amidas/farmacologia , Animais , Benzamidas/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Colágeno/química , Colágeno/metabolismo , Dipeptídeos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz/farmacologia , Piridinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Quinases Associadas a rho/antagonistas & inibidores
6.
Biophys J ; 95(3): 1523-30, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18645198

RESUMO

The extracellular availability of growth factors, hormones, chemokines, and neurotransmitters under gradient conditions is required for directional cellular responses such as migration, axonal pathfinding, and tissue patterning. These responses are, in turn, important in disease and developmental processes. This article addresses critical barriers toward devising a chemotaxis assay that is broadly applicable for different kinds of cancer cells through the design of a microfluidic chamber that produces a steep gradient of chemoattractant. Photolithography was used to create microchannels for chemoattractant delivery, flow diversion barriers/conduits, and small outlets in the form of apertures. The 1-microm apertures were made at the active surface by uncapping a thin (1.5 microm) layer of AZ1518. This process also created a vertical conduit that diverted the flow such that it occurred perpendicularly to the active, experimental surface where the gradients were measured. The other side of the vertical conduit opened to underlying 20-microm deep channels that carried microfluidic flows of tracer dyes/growth factors. Modeled data using computational fluid dynamics produced gradients that were steep along the horizontal, active surface. This simulation mirrors empirically derived gradients obtained from the flow analyses of fluorescent compounds. The open chamber contains a large buffer volume, which prevents chemoattractant saturation and permits easy cell and compound manipulation. The technique obviates the use of membranes or laminar flow that may hinder imaging, rinsing steps, cell seeding, and treatment. The utility of the chamber in the study of cell protrusion, an early step during chemotaxis, was demonstrated by growing cancer cells in the chamber, inducing a chemoattractant gradient using compressed air at 0.7 bar, and performing time-lapse microscopy. Breast cancer cells responded to the rapidly developed and stable gradient of epidermal growth factor by directing centroid positions toward the gradient and by forming a leading edge at a speed of 0.45 microm/min.


Assuntos
Técnicas de Cultura de Células/instrumentação , Fenômenos Fisiológicos Celulares/efeitos dos fármacos , Fatores Quimiotáticos/administração & dosagem , Fatores Quimiotáticos/química , Análise de Injeção de Fluxo/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Cultura de Células/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Análise de Injeção de Fluxo/métodos , Técnicas Analíticas Microfluídicas/métodos
7.
Cancer Res ; 68(12): 4525-30, 2008 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-18559496

RESUMO

The transcriptional repressor Snail2 is overexpressed in head and neck squamous cell carcinomas (HNSCC) relative to nonmalignant head and neck mucosal epithelium, and in locally recurrent relative to nonrecurrent HNSCCs. We investigated the mechanisms by which Snails might contribute to the pathogenesis of HNSCCs using cell biological and molecular analyses. Oral keratinocytes that expressed Snails acquired an enhanced ability to attract monocytes and to invade a dense interstitial collagen matrix. They were also found to up-regulate production of proinflammatory cytokines and cyclooxygenase-2 (COX2), which have previously been shown to correlate with malignancy. Induction of nuclear factor-kappaB transcriptional activity by Snails was weak and not sufficient to account for the elevated levels of COX2, interleukin (IL)-6, IL8, or CXCL1. In addition, expression of Snails in oral keratinocytes impaired desquamation in vitro and strongly repressed expression of both ELF3 and matriptase-1, which play important roles in the terminal differentiation of keratinocytes. Reexpression of matriptase-1 in Snail-expressing cells partially rescued desquamation. This implicates Snails as contributing to malignancy both at the early stages, by impeding terminal differentiation, and at later stages, when invasion and inflammation are important.


Assuntos
Diferenciação Celular , Quimiocina CXCL1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Queratinócitos/metabolismo , Mucosa Bucal/metabolismo , Fatores de Transcrição/fisiologia , Animais , Caderinas/genética , Linhagem Celular , Movimento Celular , Proteínas de Ligação a DNA/metabolismo , Cães , Ensaio de Imunoadsorção Enzimática , Humanos , Queratinócitos/citologia , Rim/citologia , Rim/metabolismo , Luciferases/metabolismo , Mucosa Bucal/citologia , NF-kappa B/genética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets , Serina Endopeptidases/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Regulação para Cima
8.
BMC Cancer ; 6: 151, 2006 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-16756685

RESUMO

BACKGROUND: Non-small cell lung cancer is the most common cause of early casualty from malignant disease in western countries. The heterogeneous nature of these cells has been identified by histochemical and microarray biomarker analyses. Unfortunately, the morphological, molecular and biological variation within cell lines used as models for invasion and metastasis are not well understood. In this study, we test the hypothesis that heterogeneous cancer cells exhibit variable motility responses such as chemokinesis and chemotaxis that can be characterized molecularly. METHODS: A subpopulation of H460 lung cancer cells called KINE that migrated under chemokinetic (no gradient) conditions was harvested from Boyden chambers and cultured. Time-lapsed microscopy, immunofluorescence microscopy and microarray analyses were then carried out comparing chemokinetic KINE cells with the unselected CON cell population. RESULTS: Time-lapsed microscopy and analysis showed that KINE cells moved faster but less directionally than the unselected control population (CON), confirming their chemokinetic character. Of note was that chemokinetic KINE cells also chemotaxed efficiently. KINE cells were less adhesive to substrate than CON cells and demonstrated loss of mature focal adhesions at the leading edge and the presence of non-focalized cortical actin. These characteristics are common in highly motile amoeboid cells that may favour faster motility speeds. KINE cells were also significantly more invasive compared to CON. Gene array studies and real-time PCR showed the downregulation of a gene called, ROM, in highly chemokinetic KINE compared to mainly chemotactic CON cells. ROM was also reduced in expression in a panel of lung cancer cell lines compared to normal lung cells. CONCLUSION: This study shows that cancer cells that are efficient in both chemokinesis and chemotaxis demonstrate high invasion levels. These cells possess different morphological, cytoskeletal and adhesive properties from another population that are only efficient at chemotaxis, indicating a loss in polarity. Understanding the regulation of polarity in the context of cell motility is important in order to improve control and inhibition of invasion and metastasis.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Linhagem Celular Tumoral , Movimento Celular , Quimiotaxia , Genes Neoplásicos , Humanos , Cinética , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos
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