RESUMEN
Akkermansia muciniphila and Faecalibacterium prausnitzii are next-generation probiotics, which has been reported to protect disease and effectively utilize various carbohydrates (starch and pectin) as nutrients for growth. Atemoya exhibiting fruity flavor, which is suitable for enhancing aroma and attenuating unpleasant taste caused by the koji metabolites. Results indicated that malic acid was increased (from 42.4 to 70.1 mg/100 g) in fermented Atemoya-Amazake. In addition, fermented Atemoya-Amazake elevated growthes in A. muciniphila and F. prausnitzii. Similarly, the populations of Parabacteroides (5.7 fold) and Akkermansia (1.66 fold) were elevated by fermented Atemoya-Amazake treatment in an in vitro simulated gastrointestinal system compared to the control group. Results revealed that fermented Atemoya-Amazake modulated the intestinal microbiota through increasing the production of short-chain fatty acids (exhibiting anti-pathogenic activity) for 2.1, 2.5, 2.6, and 2.1 folds in acetic acid, propionic acid, isobutyric acid, and butyric acid, respectively; suggesting this fermented Atemoya-Amazake could be applied in intestinal protection.
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Annona , Fermentación , Microbioma Gastrointestinal , Probióticos , Probióticos/farmacología , Probióticos/metabolismo , Annona/química , Annona/metabolismo , Annona/microbiología , Ácidos Grasos Volátiles/metabolismo , Bacterias/metabolismo , Bacterias/aislamiento & purificación , Bacterias/clasificación , HumanosRESUMEN
Rice straw is not easy to decompose, it takes a long time to compost, and the anaerobic bacteria involved in the decomposition process produce a large amount of carbon dioxide (CO2), indicating that applications for rice straw need to be developed. Recycling rice straw in agricultural crops is an opportunity to increase the sustainability of grain production. Several studies have shown that the probiotic population gradually decreases in the soil, leading to an increased risk of plant diseases and decreased biomass yield. Because the microorganisms in the soil are related to the growth of plants, when the soil microbial community is imbalanced it seriously affects plant growth. We investigated the feasibility of using composted rice stalks to artificially cultivate microorganisms obtained from the Oryza sativa-planted environment for analyzing the mycobiota and evaluating applications for sustainable agriculture. Microbes obtained from the water-submerged part (group-A) and soil part (group-B) of O. sativa were cultured in an artificial medium, and the microbial diversity was analyzed with internal transcribed spacer sequencing. Paddy field soil was mixed with fermented paddy straw compost, and the microbes obtained from the soil used for O. sativa planting were designated as group-C. The paddy fields transplanted with artificially cultured microbes from group-A were designated as group-D and those from group-B were designated as group-E. We found that fungi and yeasts can be cultured in groups-A and -B. These microbes altered the soil mycobiota in the paddy fields after transplantation in groups-D and -E compared to groups-A and -B. Development in O. sativa post treatment with microbial transplantation was observed in the groups-D and -E compared to group-C. These results showed that artificially cultured microorganisms could be efficiently transplanted into the soil and improve the mycobiota. Phytohormones were involved in improving O. sativa growth and rice yield via the submerged part-derived microbial medium (group-D) or the soil part-derived microbial medium (group-E) treatments. Collectively, these fungi and yeasts may be applied in microbial transplantation via rice straw fermentation to repair soil mycobiota imbalances, facilitating plant growth and sustainable agriculture. These fungi and yeasts may be applied in microbial transplantation to repair soil mycobiota imbalances and sustainable agriculture.
RESUMEN
The titer of recombinant proteins is one of the key parameters in biopharmaceutical manufacturing processes. The fluorescence polarization (FP)-based assay, a homogeneous, high-throughput and real-time analytical method, had emerged as a powerful tool for biochemical analysis and environmental monitoring. In this study, an FP-based bioassay was utilized to quantify antibody fragment crystallizable (Fc)-containing proteins, such as recombinant monoclonal antibodies (mAbs) and mAb derivatives, in the cell culture supernatant, and the impacts of tracer molecular weight and FITC-coupling conditions on fluorescence polarization were methodically examined. Distinct from the fluorescence polarization potency calculated by classical formula, we for the first time proposed a new concept and calculation of fluorescence polarization intensity, based on which an analytical method with broader detection range and analysis window was established for quantifying Fc-containing proteins. This provided new ideas for the practical application of fluorescence polarization theory. The established method could detect 96 samples within 30 minutes, with dynamic titer range of 2.5-400 mg L-1, and a linear fitting R2 between the measured and actual concentration reaching 0.99. The method had great application prospects in determining the titer of recombinant proteins with Fc fragments, especially when applied to large-scale screening of high-yield and stable expression CHO cell lines commonly used in biopharmaceutical industry.
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Anticuerpos Monoclonales , Cricetulus , Polarización de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Fragmentos Fc de Inmunoglobulinas , Proteínas Recombinantes , Proteínas Recombinantes/química , Proteínas Recombinantes/análisis , Células CHO , Polarización de Fluorescencia/métodos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/química , Ensayos Analíticos de Alto Rendimiento/métodos , Fragmentos Fc de Inmunoglobulinas/química , Bioensayo/métodos , AnimalesRESUMEN
Clinical application of PD-1 and PD-L1 monoclonal antibodies (mAbs) is hindered by their relatively low response rates and the occurrence of drug resistance. Co-expression of B7-H3 with PD-L1 has been found in various solid tumors, and combination therapies that target both PD-1/PD-L1 and B7-H3 pathways may provide additional therapeutic benefits. Up to today, however, no bispecific antibodies targeting both PD-1 and B7-H3 have reached the clinical development stage. In this study, we generated a stable B7-H3×PD-L1 bispecific antibody (BsAb) in IgG1-VHH format by coupling a humanized IgG1 mAb against PD-L1 with a humanized camelus variable domain of the heavy-chain of heavy-chain antibody (VHH) against human B7-H3. The BsAb exhibited favorable thermostability, efficient T cell activation, IFN-γ production, and antibody-dependent cell-mediated cytotoxicity (ADCC). In a PBMC humanized A375 xenogeneic tumor model, treatment with BsAb (10 mg/kg, i.p., twice a week for 6 weeks) showed enhanced antitumor activities compared to monotherapies and, to some degree, combination therapies. Our results suggest that targeting both PD-1 and B7-H3 with BsAbs increases their specificities to B7-H3 and PD-L1 double-positive tumors and induces a synergetic effect. We conclude that B7-H3×PD-L1 BsAb is favored over mAbs and possibly combination therapies in treating B7-H3 and PD-L1 double-positive tumors.
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Antígeno B7-H1 , Receptor de Muerte Celular Programada 1 , Humanos , Antígeno B7-H1/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Leucocitos Mononucleares/metabolismo , Anticuerpos Monoclonales , Inmunoglobulina G/metabolismoRESUMEN
Vascular development is a complex multistep process involving the coordination of cellular functions such as migration, proliferation, and differentiation. How mechanical forces generated by cells and transmission of these physical forces control vascular development is poorly understood. Using an endothelial-specific genetic model in mice, we show that deletion of the scaffold protein Angiomotin (Amot) inhibits migration and expansion of the physiological and pathological vascular network. We further show that Amot is required for tip cell migration and the extension of cellular filopodia. Exploiting in vivo and in vitro molecular approaches, we show that Amot binds Talin and is essential for relaying forces between fibronectin and the cytoskeleton. Finally, we provide evidence that Amot is an important component of the endothelial integrin adhesome and propose that Amot integrates spatial cues from the extracellular matrix to form a functional vascular network.
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Citoesqueleto/metabolismo , Fibronectinas/metabolismo , Integrinas/metabolismo , Neovascularización Fisiológica/fisiología , Angiomotinas/metabolismo , Animales , Membrana Celular/metabolismo , Movimiento Celular/fisiología , Endotelio/metabolismo , Ratones Transgénicos , Sustitutos del Plasma/farmacología , Seudópodos/metabolismoRESUMEN
OBJECTIVE: The meta-analysis was conducted to assess the effectiveness and safety of intravenous administration of dexmedetomidine for cesarean section under general anesthesia, as well as neonatal outcomes. MATERIALS AND METHODS: We searched PubMed, Embase, Cochrane Central Register of Controlled Trials and the China National Knowledge Infrastructure database for relevant randomized controlled trials (RCTs) about the application of intravenous dexmedetomidine under general anesthesia for cesarean section. RevMan 5.3 was used to conduct the meta-analysis of the outcomes of interest. RESULTS: Eight RCTs involved 376 participants were included in this study. The meta-analysis showed that the mean blood pressure at the time of intubation (weighted mean difference [WMD]: -15.67, 95% CI: -21.21, -10.13, P<0.00001), skin incision (WMD: -12.83, 95% CI -20.53, -5.14, P=0.001), and delivery (WMD: -11.65, 95% CI -17.18, -6.13, P<0.0001) in dexmedetomidine group were significantly lower than that in the control group. The heart rate (HR) at the time of intubation (WMD: -31.41, 95% CI -35.01, -27.81, P<0.00001), skin incision (WMD: -22.32, 95% CI -34.55, -10.10, P=0.0003), and delivery (WMD: -19.07, 95% CI -22.09, -16.04, P<0.00001) were also lower than that in control group. For neonatal parameters, no differences existed in umbilical blood gases at delivery, and Apgar scores at 1 minute (WMD: -0.12, 95% CI -0.37, 0.12, P=0.33) and 5 minutes (WMD: -0.17, 95% CI -0.13, 0.46, P=0.27) among two groups. CONCLUSION: Intravenous administration of dexmedetomidine could efficiently attenuate the maternal cardiovascular response during cesarean section, without affecting Apgar score of the neonate.
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Analgésicos no Narcóticos/efectos adversos , Analgésicos no Narcóticos/farmacología , Anestésicos Intravenosos/efectos adversos , Anestésicos Intravenosos/farmacología , Sistema Cardiovascular/efectos de los fármacos , Cesárea , Dexmedetomidina/efectos adversos , Dexmedetomidina/farmacología , Analgésicos no Narcóticos/administración & dosificación , Anestesia Raquidea , Anestésicos Intravenosos/administración & dosificación , Presión Sanguínea/efectos de los fármacos , Procedimientos Quirúrgicos Cardiovasculares , Dexmedetomidina/administración & dosificación , Humanos , Inyecciones Intravenosas , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
The assembly of individual epithelial or endothelial cells into a tight cellular sheet requires stringent control of cell packing and organization. These processes are dependent on the establishment and further integration of cellular junctions, the cytoskeleton and the formation of apical-basal polarity. However, little is known how these subcellular events are coordinated. The (Angiomotin) Amot protein family consists of scaffold proteins that interact with junctional cadherins, polarity proteins and the cytoskeleton. In this report, we have studied how these protein complexes integrate to control cellular shapes consistent with organ function. Using gene-inactivating studies in zebrafish and cell culture systems in vitro, we show that Par3 to be essential for localization of AmotL2 to cellular junctions to associate with VE/E-cadherin and subsequently the organization of radial actin filaments. Our data provide mechanistic insight in how critical processes such as aortic lumen expansion as well as epithelial packing into hexagonal shapes are controlled.
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Uniones Adherentes/metabolismo , Proteínas Portadoras/genética , Polaridad Celular/genética , Forma de la Célula/genética , Proteínas de la Membrana/genética , Proteínas de Pez Cebra/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Angiomotinas , Animales , Animales Modificados Genéticamente , Cadherinas/genética , Cadherinas/metabolismo , Proteínas Portadoras/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Interferencia de ARN , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
Transmission of mechanical force via cell junctions is an important component that molds cells into shapes consistent with proper organ function. Of particular interest are the cadherin transmembrane proteins, which play an essential role in connecting cell junctions to the intra-cellular cytoskeleton. Understanding how these biomechanical complexes orchestrate intrinsic and extrinsic forces is important for our understanding of the underlying mechanisms driving morphogenesis. We have previously identified the Amot protein family, which are scaffold proteins that integrate polarity, junctional, and cytoskeletal cues to modulate cellular shape in endothelial as well as epithelial cells. In this report, we show that AmotL1 is a novel partner of the N-cadherin protein complex. We studied the role of AmotL1 in normal retinal as well as tumor angiogenesis using inducible endothelial-specific knock-out mice. We show that AmotL1 is essential for normal establishment of vascular networks in the post-natal mouse retina as well as in a transgenic breast cancer model. The observed phenotypes were consistent with a non-autonomous pericyte defect. We show that AmotL1 forms a complex with N-cadherin present on both endothelial cells and pericytes. We propose that AmotL1 is an essential effector of the N-cadherin mediated endothelial/pericyte junctional complex.
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Cadherinas/metabolismo , Células Endoteliales/fisiología , Uniones Intercelulares , Proteínas de la Membrana/metabolismo , Neovascularización Patológica , Neovascularización Fisiológica , Pericitos/fisiología , Proteína 1 Similar a la Angiopoyetina , Animales , Neoplasias de la Mama/patología , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Retina/fisiologíaRESUMEN
Protein misfolding is implicated in neurodegenerative diseases such as ALS, where mutations of superoxide dismutase 1 (SOD1) account for about 20% of the inherited mutations. Human SOD1 (hSOD1) contains four cysteines, including Cys(57) and Cys(146), which have been linked to protein stability and folding via forming a disulfide bond, and Cys(6) and Cys(111) as free thiols. But the roles of the cellular oxidation-reduction (redox) environment in SOD1 folding and aggregation are not well understood. Here we explore the effects of cellular redox systems on the aggregation of hSOD1 proteins. We found that the known hSOD1 mutations G93A and A4V increased the capability of the thioredoxin and glutaredoxin systems to reduce hSOD1 compared with wild-type hSOD1. Treatment with inhibitors of these redox systems resulted in an increase of hSOD1 aggregates in the cytoplasm of cells transfected with mutants but not in cells transfected with wild-type hSOD1 or those containing a secondary C111G mutation. This aggregation may be coupled to changes in the redox state of the G93A and A4V mutants upon mild oxidative stress. These results strongly suggest that the thioredoxin and glutaredoxin systems are the key regulators for hSOD1 aggregation and may play critical roles in the pathogenesis of ALS.
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Esclerosis Amiotrófica Lateral , Estrés Oxidativo , Agregación Patológica de Proteínas , Pliegue de Proteína , Superóxido Dismutasa-1 , Sustitución de Aminoácidos , Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/genética , Animales , Línea Celular Tumoral , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Humanos , Mutación Missense , Oxidación-Reducción , Agregación Patológica de Proteínas/enzimología , Agregación Patológica de Proteínas/genética , Ratas , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
The aryl hydrocarbon receptor (AHR) is a transcription factor which activates gene transcription by binding to its corresponding enhancer as the heterodimer, which is consisted of AHR and the aryl hydrocarbon receptor nuclear translocator (ARNT). Human AHR can be rather difficult to study, when compared among the AHR of other species, since it is relatively unstable and less sensitive to some ligands in vitro. Overexpression of human AHR has been limited to the baculovirus expression, which is costly and tedious due to the need of repetitive baculovirus production. Here we explored whether we could generate abundant amounts of human AHR and ARNT in a better overexpression system for functional study. We observed that human AHR and ARNT can be expressed in Pichia pastoris with yields that are comparable to the baculovirus system only if their cDNAs are optimized for Pichia expression. Fusion with a c-myc tag at their C-termini seems to increase the expression yield. These Pichia expressed proteins can effectively heterodimerize and form the ternary AHR/ARNT/enhancer complex in the presence of ß-naphthoflavone or kynurenine. Limited proteolysis using thermolysin can be used to study the heterodimerization of these human AHR and ARNT proteins.
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Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Pichia/genética , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Translocador Nuclear del Receptor de Aril Hidrocarburo/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Codón , ADN Complementario/genética , Expresión Génica , Humanos , Unión Proteica , Mapas de Interacción de Proteínas , Multimerización de Proteína , Proteolisis , Receptores de Hidrocarburo de Aril/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termolisina/metabolismo , Regulación hacia ArribaRESUMEN
Thioredoxin systems, composed of thioredoxin reductase (TrxR), thioredoxin (Trx) and NADPH, play important roles in maintaining cellular redox homeostasis and redox signaling. Recently the cytosolic Trx1 system has been shown to be a cellular target of arsenic containing compounds. To elucidate the relationship of the structure of arsenic compounds with their ability of inhibiting TrxR1 and Trx1, and cytotoxicity, we have investigated the reaction of Trx1 system with seven arsenic trithiolates: As(Cys)3, As(GS)3, As(Penicillamine)3, As(Mercaptoethanesulfonate)3, As(Mercaptopurine)3, As(2-mercaptopyridine)3 and As(2-mercaptopyridine N-oxide)3. The cytotoxicity of these arsenicals was consistent with their ability to inhibit TrxR1 in vitro and in cells. Unlike other arsenicals, As(Mercaptopurine)3 which did not show inhibitory effects on TrxR1 had very weak cytotoxicity, indicating that TrxR1 is a reliable drug target for arsenicals. Moreover, the two aromatic compounds As(2-mercaptopyridine)3 and As(2-mercaptopyridine N-oxide)3 showed stronger cytotoxicity than the others. As(2-mercaptopyridine)3 which selectively oxidized two structural cysteines (Cys62 and Cys69) in Trx1 showed mild improvement in cytotoxicity. As(2-mercaptopyridine N-oxide)3 oxidized all the Cys residues in Trx1, exhibiting the strongest cytotoxicity. Oxidation of Trx1 by As(2-mercaptopyridine)3 and As(2-mercaptopyridine N-oxide)3 affected electron transfer from NADPH and TrxR1 to peroxiredoxin 1 (Prx1), which could result in the reactive oxygen species elevation and trigger cell death process. These results suggest that oxidation of structural cysteine residues in Trx1 by aromatic group in TrxR1-targeting drugs may sensitize tumor cells to cell death, providing a novel approach to regulate cellular redox signaling and also a basis for rational design of new anticancer agents.
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Arsenicales/farmacología , Supervivencia Celular/efectos de los fármacos , Cisteína/química , Neuroblastoma/patología , Tiorredoxina Reductasa 1/antagonistas & inhibidores , Tiorredoxinas/metabolismo , Western Blotting , Citosol/metabolismo , Humanos , NADP/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Oxidación-Reducción , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Tiorredoxina Reductasa 1/genética , Tiorredoxina Reductasa 1/metabolismo , Tiorredoxinas/genética , Células Tumorales CultivadasRESUMEN
Maternal immunization is successfully applied against some life-threatening infectious diseases as it can protect the mother and her offspring through the passive transfer of maternal antibodies. Here, we sought to evaluate whether the concept of maternal immunization could also be applied to cancer immune-prevention. We have previously shown that antibodies induced by DNA vaccination against rat Her2 (neu) protect heterozygous neu-transgenic female (BALB-neuT) mice from autochthonous mammary tumor development. We, herein, seek to evaluate whether a similar maternal immunization can confer antitumor protection to BALB-neuT offspring. Significantly extended tumor-free survival was observed in BALB-neuT offspring born and fed by mothers vaccinated against neu, as compared to controls. Maternally derived anti-neu immunoglobulin G (IgG) was successfully transferred from mothers to newborns and was responsible for the protective effect. Vaccinated mothers and offspring also developed active immunity against neu as revealed by the presence of T-cell-mediated cytotoxicity against the neu immunodominant peptide. This active response was due to the milk transfer of immune complexes that were formed between the neu extracellular domain, shed from vaccine-transfected muscle cells, and the anti-neu IgG induced by the vaccine. These findings show that maternal immunization has the potential to hamper mammary cancer in genetically predestinated offspring and to develop into applications against lethal neonatal cancer diseases for which therapeutic options are currently unavailable.
RESUMEN
The establishment and maintenance of apical-basal cell polarity is essential for the functionality of glandular epithelia. Cell polarity is often lost in advanced tumours correlating with acquisition of invasive and malignant properties. Despite extensive knowledge regarding the formation and maintenance of polarity, the mechanisms that deregulate polarity in metastasizing cells remain to be fully characterized. Here we show that AmotL2 expression correlates with loss of tissue architecture in tumours from human breast and colon cancer patients. We further show that hypoxic stress results in activation of c-Fos-dependent expression of AmotL2 leading to loss of polarity. c-Fos/hypoxia-induced p60 AmotL2 interacts with the Crb3 and Par3 polarity complexes retaining them in large vesicles and preventing them from reaching the apical membrane. The resulting loss of polarity potentiates the response to invasive cues in vitro and in vivo in mice. These data provide a molecular mechanism how hypoxic stress deregulates cell polarity during tumour progression.
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Neoplasias de la Mama/genética , Proteínas Portadoras/genética , Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica , Hipoxia/genética , Proteínas Adaptadoras Transductoras de Señales , Angiomotinas , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Células CACO-2 , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Polaridad Celular , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Neoplasias del Colon/cirugía , Femenino , Células HeLa , Humanos , Hipoxia/metabolismo , Hipoxia/patología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Ganglios Linfáticos/cirugía , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Glándulas Mamarias Humanas/cirugía , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones SCID , Invasividad Neoplásica , Estadificación de Neoplasias , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transducción de Señal , Vesículas Transportadoras/metabolismoRESUMEN
The assembly of individual endothelial cells into multicellular tubes is a complex morphogenetic event in vascular development. Extracellular matrix cues and cell-cell junctional communication are fundamental to tube formation. Together they determine the shape of endothelial cells and the tubular structures that they ultimately form. Little is known regarding how mechanical signals are transmitted between cells to control cell shape changes during morphogenesis. Here we provide evidence that the scaffold protein amotL2 is needed for aortic vessel lumen expansion. Using gene inactivation strategies in zebrafish, mouse and endothelial cell culture systems, we show that amotL2 associates to the VE-cadherin adhesion complex where it couples adherens junctions to contractile actin fibres. Inactivation of amotL2 dissociates VE-cadherin from cytoskeletal tensile forces that affect endothelial cell shape. We propose that the VE-cadherin/amotL2 complex is responsible for transmitting mechanical force between endothelial cells for the coordination of cellular morphogenesis consistent with aortic lumen expansion and function.
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Antígenos CD/metabolismo , Aorta/crecimiento & desarrollo , Cadherinas/metabolismo , Proteínas Contráctiles/metabolismo , Proteínas de la Membrana/metabolismo , Neovascularización Fisiológica/fisiología , Proteínas de Pez Cebra/metabolismo , Citoesqueleto de Actina/metabolismo , Uniones Adherentes/metabolismo , Angiomotinas , Animales , Aorta/citología , Comunicación Celular , Forma de la Célula , Células Endoteliales/citología , Endotelio Vascular/crecimiento & desarrollo , Endotelio Vascular/metabolismo , Matriz Extracelular/metabolismo , Técnicas de Silenciamiento del Gen , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfogénesis , Morfolinos/genética , Interferencia de ARN , ARN Interferente Pequeño , Pez CebraRESUMEN
Angiomotin (Amot) is one of several identified angiostatin receptors expressed by the endothelia of angiogenic tissues. We have shown that a DNA vaccine targeting Amot overcome immune tolerance and induce an antibody response that hampers the progression of incipient tumors. Following our observation of increased Amot expression on tumor endothelia concomitant with the progression from pre-neoplastic lesions to full-fledged carcinoma, we evaluated the effect of anti-Amot vaccination on clinically evident tumors. Electroporation of plasmid coding for the human Amot (pAmot) significantly delayed the progression both of autochthonous tumors in cancer prone BALB-neuT and PyMT genetically engineered mice and transplantable TUBO tumor in wild-type BALB/c mice. The intensity of the inhibition directly correlated with the titer of anti-Amot antibodies induced by the vaccine. Tumor inhibition was associated with an increase of vessels diameter with the formation of lacunar spaces, increase in vessel permeability, massive tumor perivascular necrosis and an effective epitope spreading that induces an immune response against other tumor associated antigens. Greater tumor vessel permeability also markedly enhances the antitumor effect of doxorubicin. These data provide a rationale for the development of novel anticancer treatments based on anti-Amot vaccination in conjunction with chemotherapy regimens.
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Vacunas contra el Cáncer/farmacología , Permeabilidad Capilar/inmunología , Tolerancia Inmunológica , Péptidos y Proteínas de Señalización Intercelular/inmunología , Proteínas de Microfilamentos/inmunología , Neoplasias Experimentales/terapia , Neovascularización Patológica/terapia , Vacunas de ADN/farmacología , Angiomotinas , Animales , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Permeabilidad Capilar/genética , Línea Celular Tumoral , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Neovascularización Patológica/genética , Neovascularización Patológica/inmunología , Neovascularización Patológica/patología , Ratas , Vacunas de ADN/genética , Vacunas de ADN/inmunologíaRESUMEN
Alterations in mitochondrial structure and function are a hallmark of cancer cells compared to normal cells and thus targeting mitochondria has emerged as an novel approach to cancer therapy. The mitochondrial thioredoxin 2 (Trx2) system is critical for cell viability, but its role in cancer biology is not well understood. Recently some cationic triphenylmethanes such as brilliant green (BG) and gentian violet were shown to have antitumor and antiangiogenic activity with unknown mechanisms. Here we demonstrate that BG killed cells at nanomolar concentrations and targeted mitochondrial Trx2, which was oxidized and degraded. HeLa cells were more sensitive to BG than fibroblasts. In HeLa cells, Trx2 down-regulation by siRNA resulted in increased sensitivity to BG, whereas for fibroblasts, the same treatments had no effect. BG was observed to accumulate in mitochondria and cause a rapid and dramatic decrease in mitochondrial Trx2 protein. With a redox Western blot method, we found that treatment with BG caused oxidation of both Trx1 and Trx2, followed by release of cytochrome c and apoptosis-inducing factor from the mitochondria into the cytosol. Moreover, this treatment resulted in an elevation of the mRNA level of Lon protease, a protein quality control enzyme in the mitochondrial matrix, suggesting that the oxidized Trx2 may be degraded by Lon protease.
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Apoptosis/efectos de los fármacos , Violeta de Genciana/farmacología , Mitocondrias/metabolismo , Compuestos de Amonio Cuaternario/farmacología , Tiorredoxinas/antagonistas & inhibidores , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Factor Inductor de la Apoptosis/análisis , Factor Inductor de la Apoptosis/metabolismo , Cationes/química , Supervivencia Celular/efectos de los fármacos , Citocromos c/análisis , Citocromos c/metabolismo , Fibroblastos , Violeta de Genciana/química , Violeta de Genciana/uso terapéutico , Células HeLa , Humanos , Neoplasias/tratamiento farmacológico , Oxidación-Reducción , Proteasa La/metabolismo , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/uso terapéutico , ARN Interferente Pequeño/farmacología , Especies Reactivas de Oxígeno/metabolismo , Tiorredoxinas/biosíntesis , Compuestos de Tritilo/química , Compuestos de Tritilo/farmacología , Compuestos de Tritilo/uso terapéutico , Regulación hacia ArribaRESUMEN
RATIONALE: We have previously shown that angiomotin (Amot) is essential for endothelial cell migration during mouse embryogenesis. However, approximately 5% of Amot knockout mice survived without any detectable vascular defects. Angiomotin-like protein 1 (AmotL1) potentially compensates for the absence of Amot as it is 62% homologous to Amot and exhibits similar expression pattern in endothelial cells. OBJECTIVE: Here, we report the identification of a novel isoform of AmotL1 that controls endothelial cell polarization and directional migration. METHODS AND RESULTS: Small interfering RNA-mediated silencing of AmotL1 in mouse aortic endothelial cells caused a significant reduction in migration. In confluent mouse pancreatic islet endothelial cells (MS-1), AmotL1 colocalized with Amot to tight junctions. Small interfering RNA knockdown of both Amot and AmotL1 in MS-1 cells exhibited an additive effect on increasing paracellular permeability compared to that of knocking down either Amot or AmotL1, indicating both proteins were required for proper tight junction activity. Moreover, as visualized using high-resolution 2-photon microscopy, the morpholino-mediated knockdown of amotl1 during zebrafish embryogenesis resulted in vascular migratory defect of intersegmental vessels with strikingly decreased junction stability between the stalk cells and the aorta. However, the phenotype was quite distinct from that of amot knockdown which affected polarization of the tip cells of intersegmental vessels. Double knockdown resulted in an additive phenotype of depolarized tip cells with no or decreased connection of the stalk cells to the dorsal aorta. CONCLUSIONS: These results cumulatively validate that Amot and AmotL1 have similar effects on endothelial migration and tight junction formation in vitro. However, in vivo Amot appears to control the polarity of vascular tip cells whereas AmotL1 mainly affects the stability of cell-cell junctions of the stalk cells.
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Polaridad Celular/fisiología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Uniones Intercelulares/metabolismo , Proteínas de la Membrana/metabolismo , Neovascularización Fisiológica/fisiología , Proteínas de Pez Cebra/metabolismo , Secuencia de Aminoácidos , Angiomotinas , Proteína 1 Similar a la Angiopoyetina , Animales , Animales Modificados Genéticamente , Bovinos , Adhesión Celular/fisiología , Línea Celular , Movimiento Celular/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/genética , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Dominios PDZ/genética , Isoformas de Proteínas/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
OBJECTIVE: Angiogenesis and lymphangiogenesis are complex phenomena that involve the interplay of several growth factors and receptors. Recently, we have demonstrated that in Keratin-14 (K14) promoter-driven Vegf-A transgenic (Tg) mice, not only angiogenesis but also lymphangiogenesis is stimulated. However, the mechanism by which VEGFR1 is involved in lymphangiogenesis remains unclear. METHODS AND RESULTS: To examine how important the tyrosine kinase (TK) of VEGFR1 is in lymphangiogenesis in K14 Vegf-A Tg mice, we crossed the K14 Vegf-A Tg mice with VEGFR1-TK-deficient mice to generate double mutant K14 Vegf-A Tg Vegfr1 tk(-/-) mice. K14 Vegf-A Tg Vegfr1 tk(-/-) mice exhibit a remarkable decrease in lymphangiogensis as well as angiogenesis in subcutaneous tissues. To address the mechanism underlying the decrease in lymphangiogensis, we investigated the recruitment of monocyte-macrophage-lineage cells into the skin. The recruitment of VEGFR1-expressing macrophages driven by VEGF-A was reduced in K14 Vegf-A Tg Vegfr1 tk(-/-) mice. Vegf-A Tg mice that received VEGFR1-TK-deficient bone marrow showed a reduction of macrophage recruitment, lymphangiogenesis and angiogenesis compared with those in K14 Vegf-A Tg mice. CONCLUSIONS: VEGFR1 signaling promotes lymphangiogenesis as well as angiogenesis mainly by increasing bone marrow-derived macrophage recruitment.
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Linfangiogénesis/fisiología , Macrófagos/fisiología , Neovascularización Fisiológica , Receptor 1 de Factores de Crecimiento Endotelial Vascular/fisiología , Animales , Trasplante de Médula Ósea , Permeabilidad Capilar/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/fisiología , Factor C de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/deficiencia , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
OBJECTIVE: VEGF-E(NZ7)/PlGF molecules composed of Orf virus-derived VEGF-E(NZ7) and human PlGF1 were previously proven to be potent angiogenic factors stimulating angiogenesis without significant enhancement of vascular leakage and inflammation in vivo. For its future clinical application, there is a pressing need to better understand the beneficial effects of VEGF-E(NZ7)/PlGF during wound healing in adulthood. METHODS AND RESULTS: In this study, several angiogenic factors were administrated to skin punched wounds of both wild-type and diabetic mice. The treatment with VEGF-E(NZ7)/PlGF accelerated wound closure accompanied with enhanced angiogenesis, the process was occurring slightly faster than that in VEGF-A164 group. Moreover, the macrophage infiltration and lymphangiogenesis level in healed wounds were strikingly lower in VEGF-E(NZ7)/PlGF group than VEGF-A164 group, suggesting that the increased inflammation was the key issue preventing speedy wound healing of VEGF-A164-treated skin. Considering clinical safety, we further examined the antigenicity of chimeric VEGF-E(NZ7)/PlGF. Compared with the original VEGF-E(NZ7), the immunogenicity of VEGF-E(NZ7)/PlGF molecules was markedly decreased in mice and squirrel monkeys with the increase of PlGF1 humanized ratio. CONCLUSION: These results indicate that VEGF-E(NZ7)/PlGF molecules are superior to VEGF-A for the acceleration of either normal or delayed skin wound healing and might be regarded as potential drugs in therapeutic angiogenesis.
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Neovascularización Fisiológica/efectos de los fármacos , Proteínas/farmacología , Piel/lesiones , Factor A de Crecimiento Endotelial Vascular/farmacología , Cicatrización de Heridas/efectos de los fármacos , Animales , Western Blotting , Diabetes Mellitus/fisiopatología , Inmunohistoquímica , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neovascularización Fisiológica/fisiología , Proteínas de Fusión Oncogénica/farmacología , Probabilidad , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Valores de Referencia , Sensibilidad y Especificidad , Piel/patología , Cicatrización de Heridas/fisiologíaRESUMEN
OBJECTIVE: Vascular endothelial growth factor (VEGF) plays critical roles in the regulation of angiogenesis and lymphangiogenesis. However, tissue edema, hemorrhage, and inflammation occur when VEGF-A is used for angiogenic therapy. To design a novel angiogenic factor without severe side effects, we examined the biological function of chimeric VEGF-E(NZ7)/placental growth factor (PlGF), which is composed of Orf-Virus(NZ7)-derived VEGF-E(NZ7) and human PlGF1, in a transgenic (Tg) mouse model. METHODS AND RESULTS: A strong angiogenic response was observed in both VEGF-E(NZ7)/PlGF and VEGF-A165 Tg mice. Notably, the vascular leakage of VEGF-E(NZ7)/PlGF-induced blood vessels was 4-fold lower than that of VEGF-A165-induced blood vessels. Furthermore, the monocyte/macrophage recruitment in the skin of VEGF-E(NZ7)/PlGF Tg mice was approximately 8-fold decreased compared with that of VEGF-A165 Tg mice. In addition, the lymphatic vessels in VEGF-E(NZ7)/PlGF Tg mice were structurally normal, whereas they were markedly dilated in VEGF-A165 Tg mice, possibly because of the high vascular leakage. Receptor binding assay demonstrated that VEGF-E(NZ7)/PlGF was the ligand only activating VEGF receptor (VEGFR)-2. CONCLUSIONS: These results indicated that neither the hyperpermeability in response to simultaneous stimulation of VEGFR-1 and VEGFR-2 nor VEGFR-1-mediated severe inflammation was associated with VEGF-E(NZ7)/PlGF-induced angiogenesis. The unique receptor binding property may shed light on VEGF-E(NZ7)/PlGF as a novel candidate for therapeutic angiogenesis.