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1.
Methods Mol Biol ; 2206: 151-178, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754817

RESUMO

This protocol focuses on the quantitative description of the angioarchitecture of experimental tumor xenografts. This semiautomatic analysis is carried out on functional vessels and microvessels acquired by confocal imaging and processed into progressively reconstructed angioarchitectures following a caliber-classification step. The protocol can be applied also to the quantification of pathological angioarchitectures other than tumor grafts as well as to the microvasculature of physiological tissue samples.

2.
Int J Mol Sci ; 20(10)2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091708

RESUMO

We performed a three-dimensional (3D) analysis of the microvascular network of the cerebral cortex of twitcher mice (an authentic model of Krabbe disease) using a restricted set of indexes that are able to describe the arrangement of the microvascular tree in CD31-stained sections. We obtained a near-linear graphical "fingerprint" of the microangioarchitecture of wild-type and twitcher animals that describes the amounts, spatial dispersion, and spatial relationships of adjacent classes of caliber-filtered microvessels. We observed significant alterations of the microangioarchitecture of the cerebral cortex of twitcher mice, whereas no alterations occur in renal microvessels, which is keeping with the observation that kidney is an organ that is not affected by the disease. This approach may represent an important starting point for the study of the microvascular changes that occur in the central nervous system (CNS) under different physiopathological conditions.


Assuntos
Córtex Cerebral/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Leucodistrofia de Células Globoides/diagnóstico por imagem , Microvasos/diagnóstico por imagem , Animais , Córtex Cerebral/irrigação sanguínea , Camundongos , Microscopia Confocal/métodos
3.
Int J Mol Sci ; 21(1)2019 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-31905906

RESUMO

Krabbe disease (KD) is an autosomal recessive sphingolipidosis caused by the deficiency of the lysosomal hydrolase ß-galactosylceramidase (GALC). Oligodendroglia degeneration and demyelination of the nervous system lead to neurological dysfunctions which are usually lethal by two years of age. At present, the only clinical treatment with any proven efficacy is hematopoietic stem-cell transplantation, which is more effective when administered in the neonatal period to presymptomatic recipients. Bone marrow (BM) sinusoidal endothelial cells (SECs) play a pivotal role in stem cell engraftment and reconstitution of hematopoiesis. Previous observations had shown significant alterations of microvascular endothelial cells in the brain of KD patients and in Galc mutant twitcher mice, an authentic model of the disease. In the present study, we investigated the vascular component of the BM in the femurs of symptomatic homozygous twitcher mice at postnatal day P36. Histological, immunohistochemical, and two-photon microscopy imaging analyses revealed the presence of significant alterations of the diaphyseal BM vasculature, characterized by enlarged, discontinuous, and hemorrhagic SECs that express the endothelial marker vascular endothelial growth factor receptor-2 (VEGFR2) but lack platelet/endothelial cell adhesion molecule-1 (CD31) expression. In addition, computer-aided image analysis indicates that twitcher CD31-/VEGFR2+ SECs show a significant increase in lumen size and in the number and size of endothelial gaps compared to BM SECs of wild type littermates. These results suggest that morphofunctional defects in the BM vascular niche may contribute to the limited therapeutic efficacy of hematopoietic stem-cell transplantation in KD patients at symptomatic stages of the disease.


Assuntos
Medula Óssea/metabolismo , Galactosilceramidase/metabolismo , Leucodistrofia de Células Globoides/metabolismo , Animais , Medula Óssea/patologia , Encéfalo/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Galactosilceramidase/genética , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patologia , Camundongos , Camundongos Endogâmicos C57BL , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
4.
Sci Rep ; 8(1): 17520, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30504794

RESUMO

Blood vessel micro-angioarchitecture plays a pivotal role in tumor progression, metastatic dissemination and response to therapy. Thus, methods able to quantify microvascular trees and their anomalies may allow a better comprehension of the neovascularization process and evaluation of vascular-targeted therapies in cancer. To this aim, the development of a restricted set of indexes able to describe the arrangement of a microvascular tree is eagerly required. We addressed this goal through 3D analysis of the functional microvascular network in sulfo-biotin-stained human multiple myeloma KMS-11 xenografts in NOD/SCID mice. Using image analysis, we show that amounts, spatial dispersion and spatial relationships of adjacent classes of caliber-filtered microvessels provide a near-linear graphical "fingerprint" of tumor micro-angioarchitecture. Position, slope and axial projections of this graphical outcome reflect biological features and summarize the properties of tumor micro-angioarchitecture. Notably, treatment of KMS-11 xenografts with anti-angiogenic drugs affected position and slope of the specific curves without degrading their near-linear properties. The possibility offered by this procedure to describe and quantify the 3D features of the tumor micro-angioarchitecture paves the way to the analysis of the microvascular tree in human tumor specimens at different stages of tumor progression and after pharmacologic interventions, with possible diagnostic and prognostic implications.


Assuntos
Microvasos/patologia , Mieloma Múltiplo/diagnóstico por imagem , Mieloma Múltiplo/patologia , Neovascularização Patológica , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Angiofluoresceinografia , Humanos , Camundongos , Microvasos/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Neovascularização Patológica/diagnóstico por imagem , Neovascularização Patológica/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto
6.
FASEB J ; 32(8): 4190-4202, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29505300

RESUMO

The α3ß4 subtype is the predominant neuronal nicotinic acetylcholine receptor present in the sensory and autonomic ganglia and in a subpopulation of brain neurons. This subtype can form pentameric receptors with either 2 or 3 ß4 subunits that have different pharmacologic and functional properties. To further investigate the role of the fifth subunit, we coexpressed a dimeric construct coding for a single polypeptide containing the ß4 and α3 subunit sequences, with different monomeric subunits. With this strategy, which allowed the formation of single populations of receptors with unique stoichiometry, we demonstrated with immunofluorescence and biochemical and functional assays that only the receptors with 3 ß4 subunits are efficiently expressed at the plasma membrane. Moreover, the LFM export motif of ß4 subunit in the fifth position exerts a unique function in the regulation of the intracellular trafficking of the receptors, their exposure at the cell surface, and consequently, their function, whereas the same export motif present in the ß4 subunits forming the acetylcholine binding site is dispensable.-Crespi, A., Plutino, S., Sciaccaluga, M., Righi, M., Borgese, N., Fucile, S., Gotti, C., Colombo, S. F. The fifth subunit in α3ß4 nicotinic receptor is more than an accessory subunit.


Assuntos
Subunidades Proteicas/metabolismo , Receptores Nicotínicos/metabolismo , Sítios de Ligação/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , Humanos
7.
Angiogenesis ; 18(4): 499-510, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26310512

RESUMO

Defects of the angiogenic process occur in the brain of twitcher mouse, an authentic model of human Krabbe disease caused by genetic deficiency of lysosomal ß-galactosylceramidase (GALC), leading to lethal neurological dysfunctions and accumulation of neurotoxic psychosine in the central nervous system. Here, quantitative computational analysis was used to explore the alterations of brain angioarchitecture in twitcher mice. To this aim, customized ImageJ routines were used to assess calibers, amounts, lengths and spatial dispersion of CD31(+) vessels in 3D volumes from the postnatal frontal cortex of twitcher animals. The results showed a decrease in CD31 immunoreactivity in twitcher brain with a marked reduction in total vessel lengths coupled with increased vessel fragmentation. No significant changes were instead observed for the spatial dispersion of brain vessels throughout volumes or in vascular calibers. Notably, no CD31(+) vessel changes were detected in twitcher kidneys in which psychosine accumulates at very low levels, thus confirming the specificity of the effect. Microvascular corrosion casting followed by scanning electron microscopy morphometry confirmed the presence of significant alterations of the functional angioarchitecture of the brain cortex of twitcher mice with reduction in microvascular density, vascular branch remodeling and intussusceptive angiogenesis. Intussusceptive microvascular growth, confirmed by histological analysis, was paralleled by alterations of the expression of intussusception-related genes in twitcher brain. Our data support the hypothesis that a marked decrease in vascular development concurs to the onset of neuropathological lesions in twitcher brain and suggest that neuroinflammation-driven intussusceptive responses may represent an attempt to compensate impaired sprouting angiogenesis.


Assuntos
Encéfalo/irrigação sanguínea , Circulação Cerebrovascular , Intussuscepção/fisiopatologia , Leucodistrofia de Células Globoides/fisiopatologia , Microcirculação , Microvasos/fisiopatologia , Animais , Modelos Animais de Doenças , Humanos , Intussuscepção/genética , Intussuscepção/patologia , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patologia , Camundongos
8.
Stud Health Technol Inform ; 207: 390-9, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25488245

RESUMO

This paper discusses the problem of fostering lifestyle changes towards healthier habits via tailored user guidance. We present a novel multisensory device, the Wize Mirror, which will be able to detect semeiotic face signs related to cardio-metabolic risk, and encourage users to reduce their risk by improving their lifestyle. Offering a proper user guidance requires solving three main issues: user profiling, definition of a wellness index based on biophysical data, and personalized guidance by means of coaching and supportive messages. For each of these issues, the solutions proposed in the EU FP7 Project SEMEOTICONS are presented, highlighting their advantages with respect to the state-of-the-art.


Assuntos
Doenças Cardiovasculares/prevenção & controle , Expressão Facial , Promoção da Saúde/métodos , Estilo de Vida Saudável , Doenças Metabólicas/prevenção & controle , Pigmentação da Pele , Humanos
9.
PLoS One ; 9(11): e106923, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25379707

RESUMO

3-Iodothyronamine (T1AM) is an endogenous biogenic amine, structurally related to thyroid hormone, which is regarded as a novel chemical messenger. The molecular mechanisms underlying T1AM effects are not known, but it is possible to envisage changes in gene expression, since delayed and long-lasting phenotypic effects have been reported, particularly with regard to the modulation of lipid metabolism and body weight. To test this hypothesis we analysed gene expression profiles in adipose tissue and liver of eight rats chronically treated with T1AM (10 mg/Kg twice a day for five days) as compared with eight untreated rats. In vivo T1AM administration produced significant transcriptional effects, since 378 genes were differentially expressed in adipose tissue, and 114 in liver. The reported changes in gene expression are expected to stimulate lipolysis and beta-oxidation, while inhibiting adipogenesis. T1AM also influenced the expression of several genes linked to lipoprotein metabolism suggesting that it may play an important role in the regulation of cholesterol homeostasis. No effect on the expression of genes linked to toxicity was observed. The assay of tissue T1AM showed that in treated animals its endogenous concentration increased by about one order of magnitude, without significant changes in tissue thyroid hormone concentration. Therefore, the effects that we observed might have physiological or pathophysiological importance. Our results provide the basis for the reported effectiveness of T1AM as a lipolytic agent and gain importance in view of a possible clinical use of T1AM in obesity and/or dyslipidaemia.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Lipólise/efeitos dos fármacos , Tironinas/farmacologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Lipólise/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Ratos , Ratos Wistar , Transcrição Genética/efeitos dos fármacos
10.
J Neuroimmunol ; 271(1-2): 18-29, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24746422

RESUMO

Calcitonin Gene-Related Peptide (CGRP) inhibits microglia inflammatory activation in vitro. We here analyzed the involvement of CGRP and Receptor Component Protein (RCP) in experimental autoimmune encephalomyelitis (EAE). Alpha-CGRP deficiency increased EAE scores which followed the scale alpha-CGRP null>heterozygote>wild type. In wild type mice, CGRP delivery into the cerebrospinal fluid (CSF) 1) reduced chronic EAE (C-EAE) signs, 2) inhibited microglia activation (revealed by quantitative shape analysis), and 3) did not alter GFAP expression, cell density, lymphocyte infiltration, and peripheral lymphocyte production of IFN-gamma, TNF-alpha, IL-17, IL-2, and IL-4. RCP (probe for receptor involvement) was expressed in white matter microglia, astrocytes, oligodendrocytes, and vascular-endothelial cells: in EAE, also in infiltrating lymphocytes. In relapsing-remitting EAE (R-EAE) RCP increased during relapse, without correlation with lymphocyte density. RCP nuclear localization (stimulated by CGRP in vitro) was I) increased in microglia and decreased in astrocytes (R-EAE), and II) increased in microglia by CGRP CSF delivery (C-EAE). Calcitonin like receptor was rarely localized in nuclei of control and relapse mice. CGRP increased in motoneurons. In conclusion, CGRP can inhibit microglia activation in vivo in EAE. CGRP and its receptor may represent novel protective factors in EAE, apparently acting through the differential cell-specific intracellular translocation of RCP.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Regulação da Expressão Gênica/fisiologia , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Adrenomedulina/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/genética , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Peptídeo Relacionado com Gene de Calcitonina/uso terapêutico , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/genética , Inibidores Enzimáticos/farmacologia , Adjuvante de Freund/imunologia , Adjuvante de Freund/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , Glicoproteína Mielina-Oligodendrócito/imunologia , Glicoproteína Mielina-Oligodendrócito/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/farmacologia , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/genética
11.
PLoS One ; 8(3): e59691, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23555747

RESUMO

Quantitative characterization of the in vivo effects of vascular-targeted therapies on tumor vessels is hampered by the absence of useful 3D vascular network descriptors aside from microvessel density. In this study, we extended the quantification of planar vessel distribution to the analysis of vascular volumes by studying the effects of antiangiogenic (sorafenib and sunitinib) or antivascular (combretastatin A4 phosphate) treatments on the quantity and spatial distributions of thin microvessels. These observations were restricted to perinecrotic areas of treated human multiple myeloma tumors xenografted in immunodeficient mice and to microvessels with an approximate cross-sectional area lower than 75 µm(2). Finally, vessel skeletonization minimized artifacts due to possible differential wall staining and allowed a comparison of the various treatment effects. Antiangiogenic drug treatment reduced the number of vessels of every caliber (at least 2-fold fewer vessels vs. controls; p<0.001, n = 8) and caused a heterogeneous distribution of the remaining vessels. In contrast, the effects of combretastatin A4 phosphate mainly appeared to be restricted to a homogeneous reduction in the number of thin microvessels (not more than 2-fold less vs. controls; p<0.001, n = 8) with marginal effects on spatial distribution. Unexpectedly, these results also highlighted a strict relationship between microvessel quantity, distribution and cross-sectional area. Treatment-specific changes in the curves describing this relationship were consistent with the effects ascribed to the different drugs. This finding suggests that our results can highlight differences among vascular-targeted therapies, providing hints on the processes underlying sample vascularization together with the detailed characterization of a pathological vascular tree.


Assuntos
Transformação Celular Neoplásica , Imageamento Tridimensional/métodos , Linfoma/patologia , Terapia de Alvo Molecular , Neovascularização Patológica , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Artefatos , Linhagem Celular Tumoral , Feminino , Humanos , Linfoma/tratamento farmacológico , Linfoma/fisiopatologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Neovascularização Patológica/tratamento farmacológico , Estilbenos/farmacologia , Estilbenos/uso terapêutico
12.
Angiogenesis ; 16(3): 707-22, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23605004

RESUMO

The proapoptotic death receptor 5 (DR5) expressed by tumor associated endothelial cells (TECs) mediates vascular disrupting effects of human CD34(+) cells engineered to express membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (CD34-TRAIL (+) cells) in mice. Indeed, lack of DR5 on TECs causes resistance to CD34-TRAIL (+) cells. By xenografting in nonobese diabetic/severe combined immunodeficient mice the TRAIL-resistant lymphoma cell line SU-DHL-4V, which generates tumors lacking endothelial DR5 expression, here we demonstrate for the first time that the Akt inhibitor perifosine induces in vivo DR5 expression on TECs, thereby overcoming tumor resistance to the vascular disruption activity of CD34-TRAIL (+) cells. In fact, CD34-TRAIL (+) cells combined with perifosine, but not CD34-TRAIL (+) cells alone, exerted marked antivascular effects and caused a threefold increase of hemorrhagic necrosis in SU-DHL-4V tumors. Consistent with lack of DR5 expression, CD34-TRAIL (+) cells failed to affect the growth of SU-DHL-4V tumors, but CD34-TRAIL (+) cells plus perifosine reduced tumor volumes by 60 % compared with controls. In view of future clinical studies using membrane-bound TRAIL, our results highlight a strategy to rescue patients with primary or acquired resistance due to the lack of DR5 expression in tumor vasculature.


Assuntos
Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Neovascularização Patológica/fisiopatologia , Fosforilcolina/análogos & derivados , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Análise de Variância , Animais , Antígenos CD34/metabolismo , Western Blotting , Linhagem Celular Tumoral , Primers do DNA/genética , Feminino , Citometria de Fluxo , Imunofluorescência , Transplante de Células-Tronco Hematopoéticas , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microscopia Confocal , Fosforilcolina/farmacologia , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
13.
PLoS One ; 8(4): e61603, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23620775

RESUMO

The anti-lymphoma activity and mechanism(s) of action of the multikinase inhibitor sorafenib were investigated using a panel of lymphoma cell lines, including SU-DHL-4V, Granta-519, HD-MyZ, and KMS-11 cell lines. In vitro, sorafenib significantly decreased cell proliferation and phosphorylation levels of MAPK and PI3K/Akt pathways while increased apoptotic cell death. In vivo, sorafenib treatment resulted in a cytostatic rather than cytotoxic effect on tumor cell growth associated with a limited inhibition of tumor volumes. However, sorafenib induced an average 50% reduction of tumor vessel density and a 2-fold increase of necrotic areas. Upon sorafenib treatment, endothelial and tumor cells from SU-DHL-4V, Granta-519, and KMS-11 nodules showed a potent inhibition of either phospho-ERK or phospho-AKT, whereas a concomitant inhibition of phospho-ERK and phospho-AKT was only observed in HD-MyZ nodules. In conclusion, sorafenib affects the growth of lymphoid cell lines by triggering antiangiogenic mechanism(s) and directly targeting tumor cells.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Linfoma/tratamento farmacológico , Linfoma/enzimologia , Sistema de Sinalização das MAP Quinases , Neovascularização Patológica/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Humanos , Linfoma/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos SCID , Necrose , Neovascularização Patológica/enzimologia , Neovascularização Patológica/patologia , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Pericitos/efeitos dos fármacos , Pericitos/patologia , Compostos de Fenilureia/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Sorafenibe , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Arch Ital Biol ; 151(2): 76-98, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24442985

RESUMO

In recent years many tools have been developed to cope with the interpretation of gene expression results from microarray experiments. The effectiveness of these tools largely depends on their ease of use by biomedical researchers. Tools based on effective computational methods, indeed, cannot be fully exploited by users if they are not supported by an intuitive interface, a large set of utilities and effective outputs. In this paper, 10 tools for the interpretation of gene expression microarray results have been tested on 11 microarray datasets and evaluated according to eight assessment criteria: 1. interface design and usability, 2. easiness of input submission, 3. effectiveness of output representation and 4. of the downloaded outputs, 5. possibility to submit multiple gene IDs, 6. sources of information, 7. provision of different statistical tests and 8. of multiple test correction methods. Strengths and weaknesses of each tool are highlighted to: a. provide useful tips to users dealing with the biological interpretation of microarray results; b. draw the attention of software developers on the usability of their tools.


Assuntos
Biologia Computacional , Bases de Dados Genéticas , Expressão Gênica , Análise em Microsséries , Animais , Humanos , Software , Interface Usuário-Computador
15.
Mol Cell Neurosci ; 51(1-2): 12-21, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22800606

RESUMO

Several lines of evidence indicate that neuromuscular junction (NMJ) destruction and disassembly is an early phenomenon in amyotrophic lateral sclerosis (ALS). Here we analyzed by confocal and electron microscopy the NMJ structure in the diaphragm of SOD1G93A mice at symptom onset. In these mice, which provide a model for familial ALS, diaphragm denervation (~50%) as well as gastrocnemius denervation (~40%) was found. In addition, the size of the synaptic vesicle pool was reduced and alterations of mitochondria were observed in approximately 40% of the remaining presynaptic terminals. Chronic treatment of SOD1G93A mice with the anabolic steroid nandrolone during the presymptomatic stage preserved the diaphragm muscle mass and features indicative of synaptic activity. These features were represented by the number of vesicles docked within 200 nm from the presynaptic membrane and area of acetylcholine receptor clusters. Structural preservation of mitochondria was documented in presynaptic terminals. However, innervation of diaphragm muscle fibers was only slightly increased in nandrolone-treated SOD1-mutant mice. Altogether the results point out and define fine structural alterations of diaphragm NMJs in the murine model of familial ALS at symptom onset, and indicate that nandrolone may prevent or delay structural alterations in NMJ mitochondria and stimulate presynaptic activity but does not prevent muscle denervation during the disease.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Anabolizantes/farmacologia , Nandrolona/farmacologia , Junção Neuromuscular/ultraestrutura , Superóxido Dismutase/genética , Esclerose Amiotrófica Lateral/genética , Animais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/ultraestrutura , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/fisiologia , Mutação , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/genética , Junção Neuromuscular/fisiopatologia , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Vesículas Sinápticas/ultraestrutura
16.
Neoplasia ; 14(12): 1236-48, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23308055

RESUMO

Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by two molecularly distinct self-renewing leukemic stem cell (LSC) populations most closely related to normal progenitors and organized as a hierarchy. A requirement for WNT/ß-catenin signaling in the pathogenesis of AML has recently been suggested by a mouse model. However, its relationship to a specific molecular function promoting retention of self-renewing leukemia-initiating cells (LICs) in human remains elusive. To identify transcriptional programs involved in the maintenance of a self-renewing state in LICs, we performed the expression profiling in normal (n = 10) and leukemic (n = 33) human long-term reconstituting AC133(+) cells, which represent an expanded cell population in most AML patients. This study reveals the ligand-dependent WNT pathway activation in AC133(bright) AML cells and shows a diffuse expression and release of WNT10B, a hematopoietic stem cell regenerative-associated molecule. The establishment of a primary AC133(+) AML cell culture (A46) demonstrated that leukemia cells synthesize and secrete WNT ligands, increasing the levels of dephosphorylated ß-catenin in vivo. We tested the LSC functional activity in AC133(+) cells and found significant levels of engraftment upon transplantation of A46 cells into irradiated Rag2(-/-)γc(-/-) mice. Owing to the link between hematopoietic regeneration and developmental signaling, we transplanted A46 cells into developing zebrafish. This system revealed the formation of ectopic structures by activating dorsal organizer markers that act downstream of the WNT pathway. In conclusion, our findings suggest that AC133(bright) LSCs are promoted by misappropriating homeostatic WNT programs that control hematopoietic regeneration.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Regeneração/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/metabolismo , Antígeno AC133 , Animais , Antígenos CD/metabolismo , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Glicoproteínas/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Peptídeos/metabolismo , Fosforilação , Cultura Primária de Células , Proteínas Proto-Oncogênicas/genética , Proteínas Wnt/genética , Peixe-Zebra
17.
J Cell Sci ; 124(Pt 19): 3356-68, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21940798

RESUMO

POF1B is a candidate gene for premature ovarian failure (POF); it is mainly expressed in polarised epithelial tissues, but its function in these tissues and the relationship with the disorder are unknown. Here we show colocalisation of POF1B with markers of both adherens and tight junctions in human jejunum. The tight junction localisation was maintained by the human POF1B stably expressed in the MDCK polarised epithelial cell line, whereas it was lost by the POF1B R329Q variant associated with POF. Localisation of apico-basal polarity markers and ultrastructure of the tight junctions were maintained in cells expressing the mutant. However, tight junction assembly was altered, cells were dysmorphic and the monolayer organisation was also altered in three-dimensional culture systems. Moreover, cells expressing the POF1B R329Q variant showed defects in ciliogenesis and cystogenesis as a result of misorientation of primary cilia and mitotic division. All of these defects were explained by interference of the mutant with the content and organisation of F-actin at the junctions. A role for POF1B in the regulation of the actin cytoskeleton was further verified by shRNA silencing of the endogenous protein in human intestinal Caco-2 cells. Taken together, these data indicate that localisation of POF1B to tight junctions has a key role in the organisation of epithelial monolayers by regulating the actin cytoskeleton.


Assuntos
Polaridade Celular/genética , Células Epiteliais/fisiologia , Insuficiência Ovariana Primária/genética , Proteínas/genética , Actinas/metabolismo , Substituição de Aminoácidos , Animais , Células CACO-2 , Forma Celular , Cílios/fisiologia , Cães , Células Epiteliais/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Jejuno/citologia , Microscopia de Fluorescência , Transporte Proteico , Proteínas/metabolismo , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Junções Íntimas/metabolismo
18.
Traffic ; 11(7): 877-85, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20406421

RESUMO

C-tail-anchored (TA) proteins constitute a heterogeneous group of membrane proteins that are inserted into membranes by unique post-translational mechanisms and that play key roles within cells. During recent years, bioinformatic screens on eukaryotic genomes have helped to obtain comprehensive pictures of the number, intracellular distribution and functions of TA proteins, but similar screens had not yet been carried out on prokaryotic cells. Here, we report the results of a bioinformatic screen of the genomes of two bacteria and one archeon. We find that all three of these prokaryotes contain TA proteins in proportions approaching those found in eukaryotic cells, indicating that this protein group is present in all three domains of life. Although some of our hits correspond to proteins of unknown function, others are enzymes with hydrophobic substrates or have functions carried out at the inner face of the cytoplasmic membrane. To generate hypotheses on the insertion mechanisms of prokaryotic TA proteins, we compared the sequences of the prokaryotic and eukaryotic versions of Asna1/Trc40/GET3, a cytosolic ATPase that plays a key role in TA protein post-translational delivery to membranes in eukaryotic cells. We found that hydrophobic residues involved in TA binding by the eukaryotic chaperone (Mateja et al., Nature 2009;461:361-366) are generally replaced with equally hydrophobic amino acids in the archeal homologue (ArsA), whereas this is not the case for the bacterial protein. Thus, eukaryotes may have inherited the GET3 targeting pathway from our archeal ancestor, while the bacterial homologue may be exclusively dedicated to heavy metal resistance.


Assuntos
Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Animais , Archaea/genética , Proteínas Arqueais/química , Proteínas de Bactérias/química , Membrana Celular/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Chaperonas Moleculares/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico/fisiologia , Proteoma/análise
19.
Blood ; 115(11): 2231-40, 2010 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-20075160

RESUMO

Adenovirus-transduced CD34+ cells expressing membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (CD34-TRAIL+ cells) exert potent antitumor activity. To further investigate the mechanism(s) of action of CD34-TRAIL+ cells, we analyzed their homing properties as well as antitumor and antivascular effects using a subcutaneous myeloma model in immunodeficient mice. After intravenous injection, transduced cells homed in the tumor peaking at 48 hours when 188 plus or minus 25 CD45+ cells per 10(5) tumor cells were detected. Inhibition experiments showed that tumor homing of CD34-TRAIL+ cells was largely mediated by vascular cell adhesion molecule-1 and stromal cell-derived factor-1. Both CD34-TRAIL+ cells and soluble (s)TRAIL significantly reduced tumor volume by 40% and 29%, respectively. Computer-aided analysis of TdT-mediated dUTP nick end-labeling-stained tumor sections demonstrated significantly greater effectiveness for CD34-TRAIL+ cells in increasing tumor cell apoptosis and necrosis over sTRAIL. Proteome array analysis indicated that CD34-TRAIL+ cells and sTRAIL activate similar apoptotic machinery. In vivo staining of tumor vasculature with sulfosuccinimidyl-6-(biotinamido) hexanoate-biotin revealed that CD34-TRAIL+ cells but not sTRAIL significantly damaged tumor vasculature, as shown by TdT-mediated dUTP nick end-labeling+ endothelial cells, appearance of hemorrhagic areas, and marked reduction of endothelial area. These results demonstrate that tumor homing of CD34-TRAIL+ cells induces early vascular disruption, resulting in hemorrhagic necrosis and tumor destruction.


Assuntos
Antígenos CD34/metabolismo , Membrana Celular/metabolismo , Engenharia Genética , Neoplasias/irrigação sanguínea , Neoplasias/terapia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Apoptose , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Linhagem Celular Tumoral , Transplante de Células , Quimiocina CXCL12/metabolismo , Hemorragia/patologia , Humanos , Camundongos , Necrose , Neoplasias/genética , Neoplasias/patologia , Neovascularização Patológica/patologia , Neovascularização Patológica/terapia , Ligação Proteica , Distribuição Tecidual , Molécula 1 de Adesão de Célula Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Lab Invest ; 89(9): 1063-70, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19652644

RESUMO

Experimental approaches currently used to quantify the activity of antiangiogenic treatments in cancer therapy do not generally address the importance of spatial distribution of microvessels in target tissues. We report a new computerized method to assess tumor vascularization by quantifying the distribution of functional microvessels as revealed by in vivo staining with sulfosuccinimidyl-6-(biotinamido) hexanoate. Our approach was based on pixel dilation of digital images of blood vessels and addressed the space-filling property of the vessel layouts. This was practically achieved computing the number of dilation cycles (Halo index) needed to permeate a pre-defined amount of each image. Our approach was validated on human tumor xenografts in nonobese diabetic/severe combined immunodeficient mice treated with the antiangiogenic drug sorafenib. For each experimental model, area normalization allowed the unbiased comparison of several hundreds of images showing different amounts of vascular tissue. In two different tumor types, comparison of Halo values showed statistically significant differences between control and sorafenib-treated samples. Conversely, this effect was not observed in samples from an additional xenograft known to resist the antiangiogenic treatment. By separating the analysis of vessel area from the quantification of vessel distributions, our approach can potentially contribute to a better evaluation of the antiangiogenic or vascular-disrupting activity of new drugs or treatments.


Assuntos
Antineoplásicos/farmacologia , Biologia Computacional , Interpretação de Imagem Assistida por Computador/métodos , Microvasos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Inibidores da Angiogênese/farmacologia , Animais , Benzenossulfonatos/farmacologia , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos SCID , Microvasos/patologia , Transplante de Neoplasias , Neoplasias/irrigação sanguínea , Neovascularização Patológica/patologia , Niacinamida/análogos & derivados , Compostos de Fenilureia , Piridinas/farmacologia , Sorafenibe , Ensaios Antitumorais Modelo de Xenoenxerto
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