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1.
Cell Stem Cell ; 29(4): 528-544.e9, 2022 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-35276096

RESUMO

The autonomic nervous system is a master regulator of homeostatic processes and stress responses. Sympathetic noradrenergic nerve fibers decrease bone mass, but the role of cholinergic signaling in bone has remained largely unknown. Here, we describe that early postnatally, a subset of sympathetic nerve fibers undergoes an interleukin-6 (IL-6)-induced cholinergic switch upon contacting the bone. A neurotrophic dependency mediated through GDNF-family receptor-α2 (GFRα2) and its ligand, neurturin (NRTN), is established between sympathetic cholinergic fibers and bone-embedded osteocytes, which require cholinergic innervation for their survival and connectivity. Bone-lining osteoprogenitors amplify and propagate cholinergic signals in the bone marrow (BM). Moderate exercise augments trabecular bone partly through an IL-6-dependent expansion of sympathetic cholinergic nerve fibers. Consequently, loss of cholinergic skeletal innervation reduces osteocyte survival and function, causing osteopenia and impaired skeletal adaptation to moderate exercise. These results uncover a cholinergic neuro-osteocyte interface that regulates skeletogenesis and skeletal turnover through bone-anabolic effects.


Assuntos
Interleucina-6 , Osteogênese , Colinérgicos , Fibras Colinérgicas , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/fisiologia
2.
Sci Rep ; 10(1): 3397, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32099005

RESUMO

Collagen fibrils are central to the molecular organization of the extracellular matrix (ECM) and to defining the cellular microenvironment. Glycation of collagen fibrils is known to impact on cell adhesion and migration in the context of cancer and in model studies, glycation of collagen molecules has been shown to affect the binding of other ECM components to collagen. Here we use TEM to show that ribose-5-phosphate (R5P) glycation of collagen fibrils - potentially important in the microenvironment of actively dividing cells, such as cancer cells - disrupts the longitudinal ordering of the molecules in collagen fibrils and, using KFM and FLiM, that R5P-glycated collagen fibrils have a more negative surface charge than unglycated fibrils. Altered molecular arrangement can be expected to impact on the accessibility of cell adhesion sites and altered fibril surface charge on the integrity of the extracellular matrix structure surrounding glycated collagen fibrils. Both effects are highly relevant for cell adhesion and migration within the tumour microenvironment.


Assuntos
Colágeno Tipo I/química , Matriz Extracelular/química , Ribosemonofosfatos/química , Animais , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Glicosilação , Humanos , Ribosemonofosfatos/metabolismo
3.
Cancer Discov ; 9(9): 1268-1287, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31263025

RESUMO

Activating KRAS mutations are found in nearly all cases of pancreatic ductal adenocarcinoma (PDAC), yet effective clinical targeting of oncogenic KRAS remains elusive. Understanding of KRAS-dependent PDAC-promoting pathways could lead to the identification of vulnerabilities and the development of new treatments. We show that oncogenic KRAS induces BNIP3L/NIX expression and a selective mitophagy program that restricts glucose flux to the mitochondria and enhances redox capacity. Loss of Nix restores functional mitochondria to cells, increasing demands for NADPH reducing power and decreasing proliferation in glucose-limited conditions. Nix deletion markedly delays progression of pancreatic cancer and improves survival in a murine (KPC) model of PDAC. Although conditional Nix ablation in vivo initially results in the accumulation of mitochondria, mitochondrial content eventually normalizes via increased mitochondrial clearance programs, and pancreatic intraepithelial neoplasia (PanIN) lesions progress to PDAC. We identify the KRAS-NIX mitophagy program as a novel driver of glycolysis, redox robustness, and disease progression in PDAC. SIGNIFICANCE: NIX-mediated mitophagy is a new oncogenic KRAS effector pathway that suppresses functional mitochondrial content to stimulate cell proliferation and augment redox homeostasis. This pathway promotes the progression of PanIN to PDAC and represents a new dependency in pancreatic cancer.This article is highlighted in the In This Issue feature, p. 1143.


Assuntos
Carcinoma Ductal Pancreático/patologia , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glicólise , Humanos , Proteínas de Membrana/genética , Camundongos , Mitofagia , Mutação , NADP/metabolismo , Transplante de Neoplasias , Oxirredução , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Supressoras de Tumor/genética
4.
Cell Rep ; 27(11): 3124-3138.e13, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31189100

RESUMO

Biomineralization of the extracellular matrix is an essential, regulated process. Inappropriate mineralization of bone and the vasculature has devastating effects on patient health, yet an integrated understanding of the chemical and cell biological processes that lead to mineral nucleation remains elusive. Here, we report that biomineralization of bone and the vasculature is associated with extracellular poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerases in response to oxidative and/or DNA damage. We use ultrastructural methods to show poly(ADP-ribose) can form both calcified spherical particles, reminiscent of those found in vascular calcification, and biomimetically calcified collagen fibrils similar to bone. Importantly, inhibition of poly(ADP-ribose) biosynthesis in vitro and in vivo inhibits biomineralization, suggesting a therapeutic route for the treatment of vascular calcifications. We conclude that poly(ADP-ribose) plays a central chemical role in both pathological and physiological extracellular matrix calcification.


Assuntos
Biomineralização , Dano ao DNA , Poli Adenosina Difosfato Ribose/metabolismo , Calcificação Vascular/metabolismo , Adolescente , Adulto , Idoso , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Bovinos , Linhagem Celular , Células Cultivadas , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Osteoblastos/metabolismo , Osteoblastos/patologia , Estresse Oxidativo , Ratos , Ratos Wistar , Ovinos
5.
J Pathol ; 243(3): 390-400, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28815607

RESUMO

Glomerular scarring, known as glomerulosclerosis, occurs in many chronic kidney diseases and involves interaction between glomerular endothelial cells (GECs), podocytes, and mesangial cells (MCs), leading to signals that promote extracellular matrix deposition and endothelial cell dysfunction and loss. We describe a 3D tri-culture system to model human glomerulosclerosis. In 3D monoculture, each cell type alters its phenotype in response to TGFß, which has been implicated as an important mediator of glomerulosclerosis. GECs form a lumenized vascular network, which regresses in response to TGFß. MCs respond to TGFß by forming glomerulosclerotic-like nodules with matrix deposition. TGFß treatment of podocytes does not alter cell morphology but increases connective tissue growth factor (CTGF) expression. BMP7 prevents TGFß-induced GEC network regression, whereas TGFß-induced MC nodule formation is prevented by SMAD3 siRNA knockdown or ALK5 inhibitors but not BMP7, and increased phospho-SMAD3 was observed in human glomerulosclerosis. In 3D tri-culture, GECs, podocytes, and MCs form a vascular network in which GECs and podocytes interact intimately within a matrix containing MCs. TGFß treatment induces formation of nodules, but combined inhibition of ALK5 and CTGF is required to prevent TGFß-induced nodule formation in tri-cellular cultures. Identification of therapeutic targets for glomerulosclerosis depends on the 3D culture of all three glomerular cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Glomérulos Renais/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Matriz Extracelular/metabolismo , Humanos , Nefropatias/patologia , Glomérulos Renais/metabolismo , Células Mesangiais/citologia , Receptor do Fator de Crescimento Transformador beta Tipo I
6.
Sci Rep ; 7(1): 1373, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28465577

RESUMO

Structural colours in living organisms have been observed and analysed in a large number of species, however the study of how the micro- and nano-scopic natural structures responsible of such colourations develop has been largely ignored. Understanding the interplay between chemical composition, structural morphology on multiple length scales, and mechanical constraints requires a range of investigation tools able to capture the different aspects of natural hierarchical architectures. Here, we report a developmental study of the most widespread strategy for structural colouration in nature: the cuticular multilayer. In particular, we focus on the exoskeletal growth of the dock leaf beetle Gastrophysa viridula, capturing all aspects of its formation: the macroscopic growth is tracked via synchrotron microtomography, while the submicron features are revealed by electron microscopy and light spectroscopy combined with numerical modelling. In particular, we observe that the two main factors driving the formation of the colour-producing multilayers are the polymerization of melanin during the ecdysis and the change in the layer spacing during the sclerotisation of the cuticle. Our understanding of the exoskeleton formation provides a unique insight into the different processes involved during metamorphosis.


Assuntos
Exoesqueleto/crescimento & desenvolvimento , Besouros/anatomia & histologia , Besouros/crescimento & desenvolvimento , Exoesqueleto/ultraestrutura , Animais , Besouros/ultraestrutura , Cor
7.
ACS Nano ; 11(3): 2652-2664, 2017 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-28221763

RESUMO

There is a potential for silver nanowires (AgNWs) to be inhaled, but there is little information on their health effects and their chemical transformation inside the lungs in vivo. We studied the effects of short (S-AgNWs; 1.5 µm) and long (L-AgNWs; 10 µm) nanowires instilled into the lungs of Sprague-Dawley rats. S- and L-AgNWs were phagocytosed and degraded by macrophages; there was no frustrated phagocytosis. Interestingly, both AgNWs were internalized in alveolar epithelial cells, with precipitation of Ag2S on their surface as secondary Ag2S nanoparticles. Quantitative serial block face three-dimensional scanning electron microscopy showed a small, but significant, reduction of NW lengths inside alveolar epithelial cells. AgNWs were also present in the lung subpleural space where L-AgNWs exposure resulted in more Ag+ve macrophages situated within the pleura and subpleural alveoli, compared with the S-AgNWs exposure. For both AgNWs, there was lung inflammation at day 1, disappearing by day 21, but in bronchoalveolar lavage fluid (BALF), L-AgNWs caused a delayed neutrophilic and macrophagic inflammation, while S-AgNWs caused only acute transient neutrophilia. Surfactant protein D (SP-D) levels in BALF increased after S- and L-AgNWs exposure at day 7. L-AgNWs induced MIP-1α and S-AgNWs induced IL-18 at day 1. Large airway bronchial responsiveness to acetylcholine increased following L-AgNWs, but not S-AgNWs, exposure. The attenuated response to AgNW instillation may be due to silver inactivation after precipitation of Ag2S with limited dissolution. Our findings have important consequences for the safety of silver-based technologies to human health.


Assuntos
Pulmão/efeitos dos fármacos , Nanofios/química , Prata/química , Animais , Instilação de Medicamentos , Pulmão/metabolismo , Pulmão/patologia , Nanofios/administração & dosagem , Ratos , Ratos Sprague-Dawley , Prata/administração & dosagem
8.
Exp Neurol ; 285(Pt A): 72-81, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27632900

RESUMO

Recently it has been shown that there is impaired cerebral endothelial function in many chronic neurodegenerative disorders including Alzheimer's and Huntington's disease. Such problems have also been reported in Parkinson's disease, in which α-synuclein aggregation is the pathological hallmark. However, little is known about the relationship between misfolded α-synuclein and endothelial function. In the present study, we therefore examined whether α-synuclein preformed fibrils affect endothelial function in vitro. Using a well-established endothelial cell model, we found that the expression of tight junction proteins, in particular zona occludens-1 and occludin, was significantly perturbed in the presence of fibril-seeded neurotoxicity. Disrupted expression of these proteins was also found in the postmortem brains of patients dying with Parkinson's disease. There was though little evidence in vitro of functional impairments in endothelial cell function in terms of transendothelial electrical resistance and permeability. This study therefore shows for the first time that misfolded α-synuclein can interact and affect the cerebral endothelial system, although its relevance to the pathogenesis of Parkinson's disease remains to be elucidated.


Assuntos
Amiloide/metabolismo , Córtex Cerebral/citologia , Células Endoteliais/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Junções Íntimas/metabolismo , alfa-Sinucleína , Amiloide/química , Amiloide/ultraestrutura , Animais , Astrócitos/metabolismo , Astrócitos/ultraestrutura , Linhagem Celular Transformada , Células Cultivadas , Técnicas de Cocultura , Citocinas/metabolismo , Impedância Elétrica , Células Endoteliais/ultraestrutura , Feto , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Ocludina/metabolismo , Estatísticas não Paramétricas , Junções Íntimas/ultraestrutura , Fatores de Tempo , Proteína da Zônula de Oclusão-1/metabolismo
9.
ACS Nano ; 10(5): 5070-85, 2016 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-27035850

RESUMO

Uptake and translocation of short functionalized multi-walled carbon nanotubes (short-fMWCNTs) through the pulmonary respiratory epithelial barrier depend on physicochemical property and cell type. Two monoculture models, immortalized human alveolar epithelial type 1 (TT1) cells and primary human alveolar epithelial type 2 cells (AT2), which constitute the alveolar epithelial barrier, were employed to investigate the uptake and transport of 300 and 700 nm in length, poly(4-vinylpyridine)-functionalized, multi-walled carbon nanotubes (p(4VP)-MWCNTs) using quantitative imaging and spectroscopy techniques. The p(4VP)-MWCNT exhibited no toxicity on TT1 and AT2 cells, but significantly decreased barrier integrity (*p < 0.01). Uptake of p(4VP)-MWCNTs was observed in 70% of TT1 cells, correlating with compromised barrier integrity and basolateral p(4VP)-MWCNT translocation. There was a small but significantly greater uptake of 300 nm p(4VP)-MWCNTs than 700 nm p(4VP)-MWCNTs by TT1 cells. Up to 3% of both the 300 and 700 nm p(4VP)-MWCNTs reach the basal chamber; this relatively low amount arose because the supporting transwell membrane minimized the amount of p(4VP)-MWCNT translocating to the basal chamber, seen trapped between the basolateral cell membrane and the membrane. Only 8% of AT2 cells internalized p(4VP)-MWCNT, accounting for 17% of applied p(4VP)-MWCNT), with transient effects on barrier function, which initially fell then returned to normal; there was no MWCNT basolateral translocation. The transport rate was MWCNT length modulated. The comparatively lower p(4VP)-MWCNT uptake by AT2 cells is proposed to reflect a primary barrier effect of type 2 cell secretions and the functional differences between the type 1 and type 2 alveolar epithelial cells.


Assuntos
Células Epiteliais , Pulmão/citologia , Nanotubos de Carbono , Alvéolos Pulmonares/citologia , Técnicas de Cultura de Células , Humanos , Mucosa Respiratória
10.
ACS Nano ; 10(1): 307-16, 2016 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-26649752

RESUMO

Surface-enhanced Raman spectroscopy (SERS) is an ultrasensitive vibrational fingerprinting technique widely used in analytical and biosensing applications. For intracellular sensing, typically gold nanoparticles (AuNPs) are employed as transducers to enhance the otherwise weak Raman spectroscopy signals. Thus, the signature patterns of the molecular nanoenvironment around intracellular unlabeled AuNPs can be monitored in a reporter-free manner by SERS. The challenge of selectively identifying molecular changes resulting from cellular processes in large and multidimensional data sets and the lack of simple tools for extracting this information has resulted in limited characterization of fundamental cellular processes by SERS. Here, this shortcoming in analysis of SERS data sets is tackled by developing a suitable methodology of reference-based PCA-LDA (principal component analysis-linear discriminant analysis). This method is validated and exemplarily used to extract spectral features characteristic of the endocytic compartment inside cells. The voluntary uptake through vesicular endocytosis is widely used for the internalization of AuNPs into cells, but the characterization of the individual stages of this pathway has not been carried out. Herein, we use reporter-free SERS to identify and visualize the stages of endocytosis of AuNPs in cells and map the molecular changes via the adaptation and advantageous use of chemometric methods in combination with tailored sample preparation. Thus, our study demonstrates the capabilities of reporter-free SERS for intracellular analysis and its ability to provide a way of characterizing intracellular composition. The developed analytical approach is generic and enables the application of reporter-free SERS to identify unknown components in different biological matrices and materials.


Assuntos
Endossomos/ultraestrutura , Ouro/química , Lisossomos/ultraestrutura , Nanopartículas Metálicas/química , Neurônios/ultraestrutura , Análise Espectral Raman/métodos , Transporte Biológico/fisiologia , Linhagem Celular Tumoral , DNA/química , Análise Discriminante , Endocitose/fisiologia , Endossomos/metabolismo , Humanos , Hidrólise , Lisossomos/metabolismo , Neurônios/metabolismo , Análise de Componente Principal , RNA/química
11.
ACS Biomater Sci Eng ; 2(8): 1273-1285, 2016 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-33434981

RESUMO

Fe-encapsulated multiwall carbon nanotubes (Fe@MWCNTs) are candidates for magnetically targeted Drug Delivery Systems (mt-DDSs) against breast cancer. However, their full potential as versatile and biosafe vectors has yet to be developed. Key challenges that remain are relating surface functionalization to cytotoxicity and inducing selective cytotoxicity to cancer cells. We have studied quantitative uptake of pristine and functionalized Fe@MWCNTs (f-Fe@MWCNTs) in correlation to their in vitro cytotoxicity. Human monocyte macrophages (HMMs) and T47D breast cancer cells were selected as models to test selective cytotoxicity. [2+1]-Cycloaddition of nitrenes to Fe@MWCNTs yielded both effective functionalization and drug "tethering". Hydrophilization of Fe@MWCNTs was critical for efficient active cell uptake. f-Fe@MWCNTs were considerably more toxic to T47D cells than HMMs, in spite of longer exposure times of the latter. Eventually, Fe@MWCNTs loaded with 5-fluorouracil in a ß-cyclodextrin cage or with covalently linked purpurin emerged as the most cytotoxic and steerable in a magnetic field toward promising mt-DDSs.

12.
Nat Commun ; 6: 7495, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26151378

RESUMO

Vascular calcification is a complex biological process that is a hallmark of atherosclerosis. While macrocalcification confers plaque stability, microcalcification is a key feature of high-risk atheroma and is associated with increased morbidity and mortality. Positron emission tomography and X-ray computed tomography (PET/CT) imaging of atherosclerosis using (18)F-sodium fluoride ((18)F-NaF) has the potential to identify pathologically high-risk nascent microcalcification. However, the precise molecular mechanism of (18)F-NaF vascular uptake is still unknown. Here we use electron microscopy, autoradiography, histology and preclinical and clinical PET/CT to analyse (18)F-NaF binding. We show that (18)F-NaF adsorbs to calcified deposits within plaque with high affinity and is selective and specific. (18)F-NaF PET/CT imaging can distinguish between areas of macro- and microcalcification. This is the only currently available clinical imaging platform that can non-invasively detect microcalcification in active unstable atherosclerosis. The use of (18)F-NaF may foster new approaches to developing treatments for vascular calcification.


Assuntos
Aterosclerose/diagnóstico , Artérias Carótidas/patologia , Tomografia por Emissão de Pósitrons/métodos , Fluoreto de Sódio/química , Calcificação Vascular/diagnóstico , Idoso , Aterosclerose/patologia , Feminino , Radioisótopos de Flúor , Humanos , Masculino
13.
Nat Nanotechnol ; 10(4): 361-9, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25751305

RESUMO

In humans and other mammals it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer's patches, small areas of the intestine concentrated with particle-scavenging immune cells. In wild-type mice, intestinal immune cells containing these naturally formed nanoparticles expressed the immune tolerance-associated molecule 'programmed death-ligand 1', whereas in NOD1/2 double knockout mice, which cannot recognize peptidoglycan, programmed death-ligand 1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and show how this helps to shape intestinal immune homeostasis.


Assuntos
Antígenos/imunologia , Intestinos/citologia , Intestinos/imunologia , Peptidoglicano/imunologia , Nódulos Linfáticos Agregados/imunologia , Fosfatos/imunologia , Animais , Cálcio/imunologia , Fosfatos de Cálcio/imunologia , Células Cultivadas , Humanos , Intestinos/química , Camundongos , Camundongos Endogâmicos BALB C , Minerais/imunologia , Chaperonas Moleculares/imunologia , Nanopartículas/química , Nanopartículas/ultraestrutura , Tamanho da Partícula , Nódulos Linfáticos Agregados/citologia
14.
Circ Res ; 116(8): 1312-23, 2015 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-25711438

RESUMO

RATIONALE: Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. OBJECTIVE: The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. METHODS AND RESULTS: Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. CONCLUSIONS: This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.


Assuntos
Cálcio/metabolismo , Exocitose , Exossomos/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Vesículas Secretórias/metabolismo , Calcificação Vascular/fisiopatologia , Adolescente , Adulto , Estudos de Casos e Controles , Células Cultivadas , Citocinas/metabolismo , Exossomos/patologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Transporte Proteico , Proteômica/métodos , Interferência de RNA , Vesículas Secretórias/patologia , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Tetraspaninas/metabolismo , Fatores de Tempo , Transfecção , Calcificação Vascular/genética , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia , Adulto Jovem , alfa-2-Glicoproteína-HS/metabolismo
15.
Plant Physiol ; 167(2): 558-73, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25489023

RESUMO

The accumulation of carbon storage compounds by many unicellular algae after nutrient deprivation occurs despite declines in their photosynthetic apparatus. To understand the regulation and roles of photosynthesis during this potentially bioenergetically valuable process, we analyzed photosynthetic structure and function after nitrogen deprivation in the model alga Chlamydomonas reinhardtii. Transcriptomic, proteomic, metabolite, and lipid profiling and microscopic time course data were combined with multiple measures of photosynthetic function. Levels of transcripts and proteins of photosystems I and II and most antenna genes fell with differing trajectories; thylakoid membrane lipid levels decreased, while their proportions remained similar and thylakoid membrane organization appeared to be preserved. Cellular chlorophyll (Chl) content decreased more than 2-fold within 24 h, and we conclude from transcript protein and (13)C labeling rates that Chl synthesis was down-regulated both pre- and posttranslationally and that Chl levels fell because of a rapid cessation in synthesis and dilution by cellular growth rather than because of degradation. Photosynthetically driven oxygen production and the efficiency of photosystem II as well as P700(+) reduction and electrochromic shift kinetics all decreased over the time course, without evidence of substantial energy overflow. The results also indicate that linear electron flow fell approximately 15% more than cyclic flow over the first 24 h. Comparing Calvin-Benson cycle transcript and enzyme levels with changes in photosynthetic (13)CO2 incorporation rates also pointed to a coordinated multilevel down-regulation of photosynthetic fluxes during starch synthesis before the induction of high triacylglycerol accumulation rates.


Assuntos
Chlamydomonas reinhardtii/fisiologia , Nitrogênio/deficiência , Fotossíntese , Ciclo do Carbono , Isótopos de Carbono , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/ultraestrutura , Clorofila/metabolismo , Regulação para Baixo/genética , Metabolismo Energético , Fluorescência , Regulação da Expressão Gênica de Plantas , Lipídeos/análise , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Força Próton-Motriz , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Amido/biossíntese , Tilacoides/metabolismo , Tilacoides/ultraestrutura
16.
Plant J ; 81(4): 611-24, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25515814

RESUMO

Drastic alteration in macronutrients causes large changes in gene expression in the photosynthetic unicellular alga Chlamydomonas reinhardtii. Preliminary data suggested that cells follow a biphasic response to this change hinging on the initiation of lipid accumulation, and we hypothesized that drastic repatterning of metabolism also followed this biphasic modality. To test this hypothesis, transcriptomic, proteomic, and metabolite changes that occur under nitrogen (N) deprivation were analyzed. Eight sampling times were selected covering the progressive slowing of growth and induction of oil synthesis between 4 and 6 h after N deprivation. Results of the combined, systems-level investigation indicated that C. reinhardtii cells sense and respond on a large scale within 30 min to a switch to N-deprived conditions turning on a largely gluconeogenic metabolic state, which then transitions to a glycolytic stage between 4 and 6 h after N depletion. This nitrogen-sensing system is transduced to carbon- and nitrogen-responsive pathways, leading to down-regulation of carbon assimilation and chlorophyll biosynthesis, and an increase in nitrogen metabolism and lipid biosynthesis. For example, the expression of nearly all the enzymes for assimilating nitrogen from ammonium, nitrate, nitrite, urea, formamide/acetamide, purines, pyrimidines, polyamines, amino acids and proteins increased significantly. Although arginine biosynthesis enzymes were also rapidly up-regulated, arginine pool size changes and isotopic labeling results indicated no increased flux through this pathway.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Nitrogênio/metabolismo , Triglicerídeos/biossíntese , Adaptação Fisiológica , Arginina/biossíntese , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Chlamydomonas reinhardtii/ultraestrutura , Perfilação da Expressão Gênica , Poliaminas/metabolismo , Proteínas/metabolismo , Biologia de Sistemas , Regulação para Cima
17.
PLoS One ; 9(5): e97565, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24849210

RESUMO

Calcification is a detrimental process in vascular ageing and in diseases such as atherosclerosis and arthritis. In particular, small calcium phosphate (CaP) crystal deposits are associated with inflammation and atherosclerotic plaque de-stabilisation. We previously reported that CaP particles caused human vascular smooth muscle cell (VSMC) death and that serum reduced the toxic effects of the particles. Here, we found that the serum proteins fetuin-A and albumin (≥ 1 µM) reduced intracellular Ca2+ elevations and cell death in VSMCs in response to CaP particles. In addition, CaP particles functionalised with fetuin-A, but not albumin, were less toxic than naked CaP particles. Electron microscopic studies revealed that CaP particles were internalised in different ways; via macropinocytosis, membrane invagination or plasma membrane damage, which occurred within 10 minutes of exposure to particles. However, cell death did not occur until approximately 30 minutes, suggesting that plasma membrane repair and survival mechanisms were activated. In the presence of fetuin-A, CaP particle-induced damage was inhibited and CaP/plasma membrane interactions and particle uptake were delayed. Fetuin-A also reduced dissolution of CaP particles under acidic conditions, which may contribute to its cytoprotective effects after CaP particle exposure to VSMCs. These studies are particularly relevant to the calcification observed in blood vessels in patients with kidney disease, where circulating levels of fetuin-A and albumin are low, and in pathological situations where CaP crystal formation outweighs calcification-inhibitory mechanisms.


Assuntos
Albuminas/farmacologia , Fosfatos de Cálcio/química , Fosfatos de Cálcio/toxicidade , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Nanopartículas/toxicidade , alfa-2-Glicoproteína-HS/farmacologia , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citotoxinas/química , Citotoxinas/toxicidade , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo
18.
Science ; 344(6185): 742-6, 2014 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-24833391

RESUMO

Nuclear magnetic resonance (NMR) spectroscopy is useful to determine molecular structure in tissues grown in vitro only if their fidelity, relative to native tissue, can be established. Here, we use multidimensional NMR spectra of animal and in vitro model tissues as fingerprints of their respective molecular structures, allowing us to compare the intact tissues at atomic length scales. To obtain spectra from animal tissues, we developed a heavy mouse enriched by about 20% in the NMR-active isotopes carbon-13 and nitrogen-15. The resulting spectra allowed us to refine an in vitro model of developing bone and to probe its detailed structure. The identification of an unexpected molecule, poly(adenosine diphosphate ribose), that may be implicated in calcification of the bone matrix, illustrates the analytical power of this approach.


Assuntos
Desenvolvimento Ósseo , Calcificação Fisiológica , Ressonância Magnética Nuclear Biomolecular/métodos , Poli Adenosina Difosfato Ribose/análise , Animais , Isótopos de Carbono , Matriz Extracelular/química , Lâmina de Crescimento/crescimento & desenvolvimento , Camundongos , Modelos Biológicos , Isótopos de Nitrogênio , Ovinos
19.
Ann Neurol ; 76(1): 31-42, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24798518

RESUMO

OBJECTIVE: Huntington disease (HD) is caused by a genetically encoded pathological protein (mutant huntingtin [mHtt]), which is thought to exert its effects in a cell-autonomous manner. Here, we tested the hypothesis that mHtt is capable of spreading within cerebral tissue by examining genetically unrelated fetal neural allografts within the brains of patients with advancing HD. METHODS: The presence of mHtt aggregates within the grafted tissue was confirmed using 3 different types of microscopy (bright-field, fluorescence, and electron), 2 additional techniques consisting of Western immunoblotting and infrared spectroscopy, and 4 distinct antibodies targeting different epitopes of mHtt aggregates. RESULTS: We describe the presence of mHtt aggregates within intracerebral allografts of striatal tissue in 3 HD patients who received their transplants approximately 1 decade earlier and then died secondary to the progression of their disease. The mHtt(+) aggregates were observed in the extracellular matrix of the transplanted tissue, whereas in the host brain they were seen in neurons, neuropil, extracellular matrix, and blood vessels. INTERPRETATION: This is the first demonstration of the presence of mHtt in genetically normal and unrelated allografted neural tissue transplanted into the brain of affected HD patients. These observations raise questions on protein spread in monogenic neurodegenerative disorders of the central nervous system characterized by the formation of mutant protein oligomers/aggregates.


Assuntos
Aloenxertos/metabolismo , Transplante de Tecido Encefálico , Doença de Huntington/terapia , Mutação/genética , Proteínas do Tecido Nervoso/genética , Adulto , Ensaios Clínicos como Assunto/tendências , Transplante de Tecido Fetal , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/patologia , Pessoa de Meia-Idade , Neostriado/embriologia , Neostriado/transplante
20.
Proc Natl Acad Sci U S A ; 111(14): E1354-63, 2014 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-24706850

RESUMO

We provide evidence that citrate anions bridge between mineral platelets in bone and hypothesize that their presence acts to maintain separate platelets with disordered regions between them rather than gradual transformations into larger, more ordered blocks of mineral. To assess this hypothesis, we take as a model for a citrate bridging between layers of calcium phosphate mineral a double salt octacalcium phosphate citrate (OCP-citrate). We use a combination of multinuclear solid-state NMR spectroscopy, powder X-ray diffraction, and first principles electronic structure calculations to propose a quantitative structure for this material, in which citrate anions reside in a hydrated layer, bridging between apatitic layers. To assess the relevance of such a structure in native bone mineral, we present for the first time, to our knowledge, (17)O NMR data on bone and compare them with (17)O NMR data for OCP-citrate and other calcium phosphate minerals relevant to bone. The proposed structural model that we deduce from this work for bone mineral is a layered structure with thin apatitic platelets sandwiched between OCP-citrate-like hydrated layers. Such a structure can explain a number of known structural features of bone mineral: the thin, plate-like morphology of mature bone mineral crystals, the presence of significant quantities of strongly bound water molecules, and the relatively high concentration of hydrogen phosphate as well as the maintenance of a disordered region between mineral platelets.


Assuntos
Osso e Ossos/metabolismo , Ácido Cítrico/metabolismo , Minerais/metabolismo , Animais , Fosfatos de Cálcio/metabolismo , Cavalos , Espectroscopia de Ressonância Magnética , Difração de Pó , Coelhos
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