RESUMEN
Unlike Quality by Testing approach, where products were tested only after drug manufacturing, Quality by Design (QbD) is a proactive control quality paradigm, which handles risks from the early development steps. In QbD, regression models built from experimental data are used to predict a risk mapping called Design Space in which the developers can identify values of critical input factors leading to acceptable probabilities to meet the efficacy and safety specifications for the expected product. These empirical models are often limited to quantitative responses. Moreover, in practice the smallness and incompleteness of datasets degrade the quality of predictions. In this study, a Bayesian approach including variable selection, parameter estimation and model quality assessment is proposed and assessed using a real case study devoted to the development of a Cationic Nano-Lipid Structures for siRNA Transfection. Two original model structures are also included to describe both binary and percentage response variables. The results confirm the practical relevance and applicability of the Bayesian implementation of the QbD analysis.
Asunto(s)
Teorema de Bayes , ARN Interferente Pequeño/genética , Control de CalidadRESUMEN
BACKGROUND AND AIMS: Inflammatory bowel diseases are highly debilitating conditions that require constant monitoring and life-long medication. Current treatments are focused on systemic administration of immunomodulatory drugs, but they have a broad range of undesirable side-effects. RNA interference is a highly specific endogenous mechanism that regulates the expression of the gene at the transcript level, which can be repurposed using exogenous short interfering RNA [siRNA] to repress expression of the target gene. While siRNA therapeutics can offer an alternative to existing therapies, with a high specificity critical for chronically administrated drugs, evidence of their potency compared to chemical kinase inhibitors used in clinics is still lacking in alleviating an adverse inflammatory response. METHODS: We provide a framework to select highly specific siRNA, with a focus on two kinases strongly involved in pro-inflammatory diseases, namely JAK1 and JAK3. Using western-blot, real-time quantitative PCR and large-scale analysis, we assessed the specificity profile of these siRNA drugs and compared their efficacy to the most recent and promising kinase inhibitors for Janus kinases [Jakinibs], tofacitinib and filgotinib. RESULTS: siRNA drugs can reach higher efficiency and selectivity at lower doses [5 pM vs 1 µM] than Jakinibs. Moreover, JAK silencing lasted up to 11 days, even with 6 h pulse transfection. CONCLUSIONS: The siRNA-based drugs developed hold the potential to develop more potent therapeutics for chronic inflammatory diseases.
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Enfermedades Inflamatorias del Intestino , Quinasas Janus , Humanos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/genética , Quinasas Janus/genética , Quinasas Janus/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Transducción de SeñalRESUMEN
Nonviral systems, such as lipid nanoparticles, have emerged as reliable methods to enable nucleic acid intracellular delivery. The use of cationic lipids in various formulations of lipid nanoparticles enables the formation of complexes with nucleic acid cargo and facilitates their uptake by target cells. However, due to their small size and highly charged nature, these nanocarrier systems can interact in vivo with antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages. As this might prove to be a safety concern for developing therapies based on lipid nanocarriers, we sought to understand how they could affect the physiology of APCs. In the present study, we investigate the cellular and metabolic response of primary macrophages or DCs exposed to the neutral or cationic variant of the same lipid nanoparticle formulation. We demonstrate that macrophages are the cells affected most significantly and that the cationic nanocarrier has a substantial impact on their physiology, depending on the positive surface charge. Our study provides a first model explaining the impact of charged lipid materials on immune cells and demonstrates that the primary adverse effects observed can be prevented by fine-tuning the load of nucleic acid cargo. Finally, we bring rationale to calibrate the nucleic acid load of cationic lipid nanocarriers depending on whether immunostimulation is desirable with the intended therapeutic application, for instance, gene delivery or messenger RNA vaccines.
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Cationes/química , Técnicas de Transferencia de Gen , Lípidos/química , Liposomas/química , Nanopartículas/química , Ácidos Nucleicos/administración & dosificación , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Línea Celular , Supervivencia Celular , Fenómenos Químicos , Citocinas/química , Portadores de Fármacos , Lipopolisacáridos/química , Ratones , Mitocondrias/metabolismo , Especies Reactivas de OxígenoRESUMEN
Immunoassays have been used for decades in clinical laboratories to quantify proteins in serum and plasma samples. However, their limitations make them inappropriate in some cases. Recently, mass spectrometry (MS) based proteomics analysis has emerged as a promising alternative method when seeking to assess panels of protein biomarkers with a view to providing protein profiles to monitor health status. Up to now, however, translation of MS-based proteomics to the clinic has been hampered by its complexity and the substantial time and human resources necessary for sample preparation. Plasma matrix is particularly tricky to process as it contains more than 3000 proteins with concentrations spanning an extreme dynamic range (1010). To address this preanalytical challenge, we designed a microfluidic device (PepS) automating and accelerating blood sample preparation for bottom-up MS-based proteomics analysis. The microfluidic cartridge is operated through a dedicated compact instrument providing fully automated fluid processing and thermal control. In less than 2 h, the PepS device allows bedside plasma separation from whole blood, volume metering, depletion of albumin, protein digestion with trypsin, and stabilization of tryptic peptides on solid-phase extraction sorbent. For this first presentation, the performance of the PepS device was assessed using discovery proteomics and targeted proteomics, detecting a panel of three protein biomarkers routinely assayed in clinical laboratories (alanine aminotransferase 1, C-reactive protein, and myoglobin). This innovative microfluidic device and its associated instrumentation should help to streamline and simplify clinical proteomics studies.
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Proteínas Sanguíneas/química , Proteómica/métodos , Biomarcadores , Humanos , Dispositivos Laboratorio en un Chip , Sistemas de Atención de Punto , Manejo de EspecímenesRESUMEN
HIV is one of the deadliest pandemics of modern times, having already caused 35 million deaths around the world. Despite the huge efforts spent to develop treatments, the virus cannot yet be eradicated and continues to infect new people. Spread of the virus remains uncontrolled, thus exposing the worldwide population to HIV danger, due to the lack of efficient vaccines. The latest clinical trials describe the challenges associated with developing an effective prophylactic HIV vaccine. These immunological obstacles will only be overcome by smart and innovative solutions applied to the design of vaccine formulations. Here, we describe the use of nanostructured lipid carriers (NLC) for the delivery of p24 protein as a model HIV antigen, with the aim of increasing its immunogenicity. We have designed vaccine formulations comprising NLC grafted with p24 antigen, together with cationic NLC optimized for the delivery of immunostimulant CpG. This tailored system significantly enhanced immune responses against p24, in terms of specific antibody production and T-cell activation in mice. More importantly, the capacity of NLC to induce specific immune responses against this troublesome HIV antigen was further supported by a 7-month study on non-human primates (NHP). This work paves the way toward the development of a future HIV vaccine, which will also require the use of envelope antigens.
RESUMEN
Nucleic acid amplification testing is a very powerful method to perform efficient and early diagnostics. However, the integration of a DNA amplification reaction with its associated detection in a low-cost, portable, and autonomous device remains challenging. Addressing this challenge, the use of screen-printed electrochemical sensor is reported. To achieve the detection of the DNA amplification reaction, a real-time monitoring of the hydronium ions concentration, a byproduct of this reaction, is performed. Such measurements are done by potentiometry using polyaniline (PAni)-based working electrodes and silver/silver chloride reference electrodes. The developed potentiometric sensor is shown to enable the real-time monitoring of a loop-mediated isothermal amplification (LAMP) reaction with an initial number of DNA strands as low as 10 copies. In addition, the performance of this PAni-based sensor is compared to fluorescence measurements, and it is shown that similar results are obtained for both methods.
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Compuestos de Anilina/química , ADN/análisis , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas Electroquímicas , Electrodos , Concentración de Iones de Hidrógeno , MicrofluídicaRESUMEN
New vaccine formulations are still highly anticipated in the near-future to face incoming health challenges, such as emergence or reemergence of severe infectious diseases, immunosenescence associated with elderly or the spread of pathogens resistant to antibiotics. In particular, new nanoparticle-based adjuvants are promising for sub-unit vaccines in order to elicit potent and long lasting immune responses with a better control on their safety. In this context, an innovative delivery system of protein antigens has been designed based on the chemical grafting of the antigen onto the shell of Nanostructured Lipid Carriers (NLC). By using the well-known ovalbumin (OVA) as model of protein antigen, we have compared the immunogenicity properties in mice of different formulations of NLC grafted with OVA, by studying the influence of two main parameters: the size (80 nm versus 120 nm) and the surface charge (anionic versus cationic). We have shown that all mice immunized with OVA delivered through NLC produced much higher antibody titers for all tested formulations as compared to that immunized with OVA or OVA formulated in Complete Freund Adjuvant (CFA, positive control). More interestingly, the 80 nm anionic lipid particles were the most efficient antigen carrier for eliciting higher humoral immune response, as well as cellular immune response characterized by a strong secretion of gamma interferon (IFN-γ). These results associated with the demonstrated non-immunogenicity of the NLC carrier by itself open new avenues for the design of smart sub-unit vaccines containing properly engineered lipid nanoparticles which could stimulate or orient the immune system in a specific way.
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Antígenos/administración & dosificación , Portadores de Fármacos/química , Lípidos/química , Nanoestructuras/química , Ovalbúmina/administración & dosificación , Animales , Antígenos/inmunología , Femenino , Inmunidad Celular , Inmunidad Humoral , Inmunización , Interferón gamma/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células 3T3 NIH , Ovalbúmina/inmunologíaRESUMEN
AIM: We report the physicochemical analysis of nanosystems intended for cardiovascular applications and their toxicological characterization in static and dynamic cell culture conditions. METHODS: Size, polydispersity and ζ-potential were determined in 10 nanoparticle systems including liposomes, lipid nanoparticles, polymeric and iron oxide nanoparticles. Nanoparticle effects on primary human endothelial cell viability were monitored using real-time cell analysis and live-cell microscopy in static conditions, and in a flow model of arterial bifurcations. RESULTS & CONCLUSIONS: The majority of tested nanosystems were well tolerated by endothelial cells up to the concentration of 100 µg/ml in static, and up to 400 µg/ml in dynamic conditions. Pilot experiments in a pig model showed that intravenous administration of liposomal nanoparticles did not evoke the hypersensitivity reaction. These findings are of importance for future clinical use of nanosystems intended for intravascular applications.
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Nanopartículas/química , Nanopartículas/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Compuestos Férricos/química , Compuestos Férricos/toxicidad , Células Endoteliales de la Vena Umbilical Humana , Humanos , Liposomas/química , Liposomas/toxicidad , Masculino , Polímeros/química , Polímeros/toxicidad , PorcinosRESUMEN
Near-infrared (NIR) fluorescence imaging using FDA-approved indocyanine green (ICG) has been the subject of numerous studies during the past few years. It could constitute a potentially exciting new paradigm shift in veterinary oncology, especially to develop in vivo fluorescence imaging diagnostics and surgery guidance methods. The objective of this study was to evaluate the pharmacologic and toxicological characteristics in healthy beagle dogs of LipImage™ 815, a formulation made of NIR-dye-loaded lipid nanoparticles. The initial dosage for the evaluation of biodistribution was extrapolated from data in mice and then adapted to define the more adapted dose (MAD) according to the fluorescence results obtained in 5 dogs using a Fluobeam® 800 imaging device (phase 0 study). A single dose acute toxicity study was then performed (3 dogs, phase I study). Before the systemic administration of LipImage™ 815, the dogs presented a very mild residual fluorescence, particularly in the liver and kidneys. After injection, the plasma fluorescence continuously decreased, and the signal was relatively homogeneously distributed throughout the different organs, though more pronounced in the liver and to a lesser extent in the steroid-rich organs (adrenal, ovaries), intestines, lymph nodes and kidneys. A MAD of 2.0µg/kg was found. No evidence of acute or delayed general, hepatic, renal or hematologic toxicity was observed at 1-fold, 5-fold or 10-fold MAD. The results of this phase-0/phase-I study showed that an optimal dosage of LipImage™ 815 of 2.0µg/kg allowed the achievement of a fluorescence signal suitable for surgery guidance application without any acute side effects.
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Colorantes Fluorescentes/química , Verde de Indocianina/química , Lípidos/química , Nanopartículas/química , Espectroscopía Infrarroja Corta , Animales , Perros , Femenino , Colorantes Fluorescentes/farmacocinética , Verde de Indocianina/farmacocinética , Indoles/química , Indoles/farmacocinética , Lípidos/farmacocinética , Masculino , Espectroscopía Infrarroja Corta/métodos , Distribución Tisular/efectos de los fármacos , Distribución Tisular/fisiologíaRESUMEN
Quantification of cell proliferation and monitoring its kinetics are essential in fields of research such as developmental biology, oncology, etc. Although several proliferation assays exist, monitoring cell proliferation kinetics remains challenging. We present a novel cell proliferation assay based on real-time monitoring of cell culture inside a standard incubator using a lensfree video-microscope, combined with automated detection of single cell divisions over a population of several thousand cells. Since the method is based on direct visualization of dividing cells, it is label-free, continuous, and not sample destructive. Kinetics of cell proliferation can be monitored from a few hours to several days. We compare our method to a standard assay, the EdU proliferation assay, and as proof of principle, we demonstrate concentration-dependent and time-dependent effect of actinomycin D-a cell proliferation inhibitor.
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Proliferación Celular , Técnicas Citológicas/instrumentación , Técnicas Citológicas/métodos , Microscopía por Video/instrumentación , Microscopía por Video/métodos , Animales , Células Cultivadas , Cinética , Ratones , Células 3T3 NIHRESUMEN
Among various attempts to enhance the therapeutic efficacy of photodynamic therapy (PDT), the specific delivery of photosensitizer (PS) in the tumor tissue is expected to improve its clinical applications. The aim of this study was to engineer lipid nanoparticles (LNP) with different sizes and various PS contents, using simple solvent-free and easily scale up manufacturing processes. Meso-(tetrahydroxyphenyl) chlorin (mTHPC) is one of the most potent photoactive compounds for clinical use. We demonstrated that mTHPC was efficiently incorporated into the lipid core of LNP, leading to a large range of stable and reproducible mTHPC-loaded LNP with narrow size distribution. Photophysical and physico-chemical properties of mTHPC-loaded LNP were assessed as well as absorption spectra and singlet oxygen emission, colloidal stability, particle size and zeta potential. The photocytotoxicity of selected mTHPC-loaded solid LNP was demonstrated on MCF-7 cells under irradiation at 652nm with a range of light fluence from 1.0 to 10J/cm(2). All physico-chemical, photophysical and biological results allow us to conclude that solid LNP appear as a very promising nano-mTHPC delivery system for PDT.
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Portadores de Fármacos/química , Lípidos/química , Mesoporfirinas/química , Nanopartículas/química , Fármacos Fotosensibilizantes/química , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/administración & dosificación , Composición de Medicamentos , Humanos , Luz , Células MCF-7 , Mesoporfirinas/administración & dosificación , Nanopartículas/administración & dosificación , Fotoquimioterapia , Fármacos Fotosensibilizantes/administración & dosificación , Oxígeno Singlete/químicaRESUMEN
A new contrast agent, LipImage™ 815, has been designed and compared to previously described indocyanine green (ICG)-loaded lipid nanoparticles (ICG-lipidots®). Both contrast agents display similar size (50-nm diameter), zeta potential, high IC50 in cellular studies, near-infrared absorption and emission wavelengths in the "imaging window," long-term shelf colloidal and optical stabilities with high brightness (>106 L mol-1 cm-1) in ready-to-use storage conditions in aqueous buffer (4°C in dark), therefore being promising fluorescence contrast agents for in vivo imaging. However, while ICG-lipidots® display a relatively short plasma lifetime, LipImage™ 815 circulates in blood for longer times, allowing the efficient uptake of fluorescence signal in human prostate cancer cells implanted in mice. Prolonged tumor labeling is observed for more than 21 days.
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Colorantes Fluorescentes/química , Lípidos/química , Nanopartículas/química , Imagen Óptica/métodos , Espectroscopía Infrarroja Corta/métodos , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Colorantes Fluorescentes/farmacocinética , Colorantes Fluorescentes/toxicidad , Humanos , Lípidos/farmacocinética , Masculino , Ratones , Ratones Desnudos , Tamaño de la Partícula , Distribución TisularRESUMEN
Lymphatic vessels are critical for the maintenance of tissue fluid homeostasis and their dysfunction contributes to several human diseases. The activin receptor-like kinase 1 (ALK1) is a transforming growth factor-ß family type 1 receptor that is expressed on both blood and lymphatic endothelial cells (LECs). Its high-affinity ligand, bone morphogenetic protein 9 (BMP9), has been shown to be critical for retinal angiogenesis. The aim of this work was to investigate whether BMP9 could play a role in lymphatic development. We found that Bmp9 deficiency in mice causes abnormal lymphatic development. Bmp9-knockout (KO) pups presented hyperplastic mesenteric collecting vessels that maintained LYVE-1 expression. In accordance with this result, we found that BMP9 inhibited LYVE-1 expression in LECs in an ALK1-dependent manner. Bmp9-KO pups also presented a significant reduction in the number and in the maturation of mesenteric lymphatic valves at embryonic day 18.5 and at postnatal days 0 and 4. Interestingly, the expression of several genes known to be involved in valve formation (Foxc2, Connexin37, EphrinB2, and Neuropilin1) was upregulated by BMP9 in LECS. Finally, we demonstrated that Bmp9-KO neonates and adult mice had decreased lymphatic draining efficiency. These data identify BMP9 as an important extracellular regulator in the maturation of the lymphatic vascular network affecting valve development and lymphatic vessel function.
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Factor 2 de Diferenciación de Crecimiento/fisiología , Linfangiogénesis/genética , Vasos Linfáticos/fisiología , Mesenterio/embriología , Animales , Animales Recién Nacidos , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Factor 2 de Diferenciación de Crecimiento/genética , Factor 2 de Diferenciación de Crecimiento/metabolismo , Humanos , Linfangiogénesis/fisiología , Vasos Linfáticos/metabolismo , Proteínas de Transporte de Membrana , Mesenterio/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Considering toxicity requirements for clinical translation of fluorescence imaging applications, the use of biocompatible carriers for designing near infrared emitting contrast agents appears as an attractive alternative to semiconductor nanocrystals. Lipid nanoparticles (LNP) have been designed to serve as carriers for indocyanine green (ICG), the presently only human-use approved near infrared dye. The cytotoxicity and hemocompatibility of these nanoparticle-based probes are determined in vitro, respectively in mouse 3T3 fibroblasts and human blood samples. Comparative biodistribution of free ICG and ICG-LNP in mice is monitored, and an ex vivo fluorescence organ quantification is performed considering large animal cohorts. Good tolerability and very low hemolytic activity are demonstrated for naked and ICG-loaded LNP. Interestingly, ICG-LNP lead to long-term plasma fluorescence (> 24 hours) but also a partial intestinal reabsorption of ICG between 5 and 24 hours after injection. This novel ICG nanoformulation is foreseen to expand rapidly the field of clinical fluorescence imaging applications.
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Verde de Indocianina/farmacocinética , Verde de Indocianina/toxicidad , Lípidos/farmacocinética , Lípidos/toxicidad , Nanopartículas/química , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Eritrocitos/efectos de los fármacos , Femenino , Fluorescencia , Hemólisis/efectos de los fármacos , Humanos , Verde de Indocianina/química , Lípidos/química , Ratones , Ratones Desnudos , Células 3T3 NIH , Nanopartículas/toxicidad , Distribución TisularRESUMEN
Fluorescence imaging is opening a new era in image-guided surgery and other medical applications. The only FDA approved contrast agent in the near infrared is IndoCyanine Green (ICG), which despites its low toxicity, displays poor chemical and optical properties for long-term and sensitive imaging applications in human. Lipid nanoparticles are investigated for improving ICG optical properties and in vivo fluorescence imaging sensitivity. 30 nm diameter lipid nanoparticles (LNP) are loaded with ICG. Their characterization and use for tumor and lymph node imaging are described. Nano-formulation benefits dye optical properties (6 times improved brightness) and chemical stability (>6 months at 4 degrees C in aqueous buffer). More importantly, LNP vectorization allows never reported sensitive and prolonged (>1 day) labeling of tumors and lymph nodes. Composed of human-use approved ingredients, this novel ICG nanometric formulation is foreseen to expand rapidly the field of clinical fluorescence imaging applications.
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Neoplasias de la Mama/patología , Verde de Indocianina , Lípidos/química , Ganglios Linfáticos/patología , Microscopía Fluorescente/métodos , Nanocápsulas/química , Animales , Línea Celular Tumoral , Aumento de la Imagen/métodos , Verde de Indocianina/química , Ganglios Linfáticos/cirugía , Ratones , Ratones DesnudosRESUMEN
The use of fluorescent nanostructures can bring several benefits on the signal to background ratio for in vitro microscopy, in vivo small animal imaging, and image-guided surgery. Fluorescent quantum dots (QDs) display outstanding optical properties, with high brightness and low photobleaching rate. However, because of their toxic element core composition and their potential long term retention in reticulo-endothelial organs such as liver, their in vivo human applications seem compromised. The development of new dye-loaded (DiO, DiI, DiD, DiR, and Indocyanine Green (ICG)) lipid nanoparticles for fluorescence imaging (lipidots) is described here. Lipidot optical properties quantitatively compete with those of commercial QDs (QTracker(®)705). Multichannel in vivo imaging of lymph nodes in mice is demonstrated for doses as low as 2 pmols of particles. Along with their optical properties, fluorescent lipidots display very low cytotoxicity (IC(50) > 75 nM), which make them suitable tools for in vitro, and especially in vivo, fluorescence imaging applications.
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Diagnóstico por Imagen/métodos , Colorantes Fluorescentes/química , Nanopartículas/química , Aceite de Soja/química , Espectrometría de Fluorescencia/métodos , Análisis de Varianza , Animales , Supervivencia Celular , Medios de Contraste/química , Ganglios Linfáticos/química , Ganglios Linfáticos/metabolismo , Ratones , Ratones Desnudos , Células 3T3 NIH , Tamaño de la Partícula , Fosfolípidos/química , Puntos Cuánticos , Tensoactivos/química , Distribución TisularRESUMEN
Brain effects of erythropoietin (Epo) are proposed to involve a heteromeric receptor comprising the classical Epo receptor (Epo-R) and the common beta chain (betac). However, data documenting the pattern of betac gene expression in the healthy brain, in comparison with that of the Epo-R gene, are still lacking. The present study is the first to investigate at the same time betac, Epo-R, and Epo gene expression within different rat brain areas throughout the life span, from neonatal to elderly stages, using quantitative RT-PCR for transcripts. Corresponding proteins were localized by using immunohistochemistry. We demonstrate that the betac transcript level does not correlate with that of Epo-R or Epo, whereas the Epo-R transcript level strongly correlates with that of Epo throughout the life span in all brain structures analyzed. Both Epo and Epo-R were detected primarily in neurons. In the hippocampus, the greatest Epo-R mRNA levels were measured during the early postnatal period and in middle-aged rats, associated with an intense neuronal immunolabeling. Conversely, betac protein was barely detectable in the brain at all ages, even in neurons expressing high levels of Epo-R. Finally, betac transcript could not be detected in PC12 cells, even after nerve growth factor-induced neuritogenesis, which is a condition that dramatically enhances Epo-R transcript level. Altogether, our data suggest that most neurons are likely to express high levels of Epo-R but low, if not null, levels of betac. Given that Epo protects extended populations of neurons after injury, a yet-to-be-identified receptor heterocomplex including Epo-R may exist in the large population of brain neurons that does not express betac.
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Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Eritropoyetina/metabolismo , Receptores de Eritropoyetina/metabolismo , Envejecimiento/metabolismo , Análisis de Varianza , Animales , Astrocitos/metabolismo , Expresión Génica , Inmunohistoquímica , Masculino , Microglía/metabolismo , Neuritas/metabolismo , Neuronas/metabolismo , Células PC12 , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Heparanase is an endo-beta-d-glucuronidase which specifically cleaves extracellular and cell surface heparan sulphates at intra-chain sites. Its enzymatic activity is strongly implicated in cell dissemination associated with tumor metastasis and inflammation. Indeed, heparanase gene is expressed in various tumors and its over-expression is correlated with increased tumor vascularity and metastatic potential of tumor cells. However, heparanase expression in non-invasive and non-immune tissue, including brain, has received less attention. Using RT-qPCR, western blot and histological analysis, we demonstrate in the adult rat that heparanase transcript is differentially expressed according to brain area, and that heparanase protein is mainly detected in neurons. Furthermore, we provide evidence that heparanase transcript and protein reach their greatest levels at early postnatal stages, in particular within the neocortex characterized by intensive structural plasticity. Using the in vitro model of PC12-induced neuronal differentiation, we suggest that developmental regulation of heparanase may coincide with axonal and dendritic pathfinding. At adulthood, we demonstrate that the increased heparanase transcript level correlates in the hippocampus with enhanced angiogenesis following repeated hypoxia exposures. Taken together, our results emphasize the potential importance of heparanase in brain homeostasis, both during development and adaptative responses to severe environmental challenges.