RESUMEN
In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor κB signal transduction could silence the proinflammatory activation status of endothelial cells. For this, an adenovirus encoding dominant-negative IκB (dnIκB) as a therapeutic transgene was employed. Selectivity for the endothelial cells was achieved by introduction of antibodies specific for inflammatory endothelial adhesion molecules E-selectin or VCAM-1 chemically linked to the virus via polyethylene glycol. In vitro, the retargeted adenoviruses selectively infected cytokine-activated endothelial cells to express functional transgene. The comparison of transductional capacity of both retargeted viruses revealed that E-selectin based transgene delivery exerted superior pharmacological effects. Targeted delivery mediated dnIκB transgene expression in endothelial cells inhibited the induced expression of several inflammatory genes, including adhesion molecules, cytokines, and chemokines. In vivo, in mice suffering from glomerulonephritis, E-selectin-retargeted adenovirus selectively homed in the kidney to microvascular glomerular endothelium. Subsequent downregulation of endothelial adhesion molecule expression 2 days after induction of inflammation demonstrated the pharmacological potential of this gene therapy approach. The data justify further studies towards therapeutic virus design and optimization of treatment schedules to investigate their capacity to interfere with inflammatory disease progression.
Asunto(s)
Adenoviridae/genética , Expresión Génica , Glomerulonefritis , Proteínas I-kappa B/genética , FN-kappa B/antagonistas & inhibidores , Transgenes , Animales , Unión Competitiva , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Selectina E/genética , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/inmunología , Femenino , Glomerulonefritis/genética , Glomerulonefritis/terapia , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Transducción de Señal/genética , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
Endothelial cells in different microvascular segments of the kidney have diverse functions and exhibit differential responsiveness to disease stimuli. The responsible molecular mechanisms are largely unknown. We previously showed that during hemorrhagic shock, VCAM-1 protein was expressed primarily in extraglomerular compartments of the kidney, while E-selectin protein was highly induced in glomeruli only (van Meurs M, Wulfert FM, Knol AJ, de Haes A, Houwertjes M, Aarts LPHJ, Molema G. Shock 29: 291-299, 2008). Here, we investigated the molecular control of expression of these endothelial cell adhesion molecules in mouse models of renal inflammation. Microvascular segment-specific responses to the induction of anti-glomerular basement membrane (anti-GBM), glomerulonephritis and systemic TNF-α treatment showed that E-selectin expression was transcriptionally regulated, with high E-selectin mRNA and protein levels preferentially expressed in the glomerular compartment. In contrast, VCAM-1 mRNA expression was increased in both arterioles and glomeruli, while VCAM-1 protein expression was limited in the glomeruli. These high VCAM-1 mRNA/low VCAM-1 protein levels were accompanied by high local microRNA (miR)-126 and Egfl7 levels, as well as higher Ets1 levels compared with arteriolar expression levels. Using miR-reporter constructs, the functional activity of miR-126 in glomerular endothelial cells could be demonstrated. Moreover, in vivo knockdown of miR-126 function unleashed VCAM-1 protein expression in the glomeruli upon inflammatory challenge. These data imply that miR-126 has a major role in the segmental, heterogenic response of renal microvascular endothelial cells to systemic inflammatory stimuli.
Asunto(s)
Glomerulonefritis/metabolismo , Inflamación/metabolismo , Riñón/metabolismo , MicroARNs/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Línea Celular , Células Cultivadas , Modelos Animales de Enfermedad , Selectina E/genética , Selectina E/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Glomerulonefritis/genética , Humanos , Inflamación/genética , Glomérulos Renales/metabolismo , Ratones , MicroARNs/genética , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/genéticaAsunto(s)
Albúminas/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Oligopéptidos/administración & dosificación , Ftalazinas/administración & dosificación , Piridinas/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Albúminas/química , Albúminas/farmacocinética , Unión Competitiva , Células Cultivadas , Portadores de Fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Oligopéptidos/química , Oligopéptidos/farmacocinética , Ftalazinas/química , Ftalazinas/farmacocinética , Piridinas/química , Piridinas/farmacocinéticaRESUMEN
BACKGROUND: Drug targeting to activated endothelial cells via E-selectin is currently being explored as a new approach to treat chronic inflammatory disorders. This approach uses E-selectin directed antibodies as carrier molecules to selectively deliver anti-inflammatory drugs into activated endothelial cells, thereby theoretically decreasing drug-associated side-effects. Therapeutic effects of developed drug targeting constructs will have to be tested in animal models of inflammation, in which E-selectin is expressed during the course of the disease. In this study several murine models of inflammation were investigated regarding expression of E-selectin. METHODS: E-selectin expression was determined both at the mRNA level using RT-PCR and at the protein level by immunohistochemistry using two monoclonal antibodies (10E9.6 and MES-1). The models studied included delayed type hypersensitivity induced skin inflammation, dextran sodium sulphate induced colitis, kidney ischemia/reperfusion injury, atherosclerosis in ApoE knockout mice, and collagen induced arthritis. RESULTS: In all animal models E-selectin mRNA expression was detected, although to a different extent. In contrast, only the delayed type hypersensitivity model and, to a minor extent, the collagen induced arthritis model showed E-selectin protein expression. CONCLUSION: These results stress the need to determine E-selectin protein expression and not only mRNA expression, when choosing an animal model for testing E-selectin directed drug targeting preparations. In addition, in the arthritis model, E-selectin protein detection was dependent on the particular anti-E-selectin antibody used. This finding may not only have implications for the development and/or choice of homing devices to be used in E-selectin directed drug targeting preparations, but also for inflammation research in general.
Asunto(s)
Modelos Animales de Enfermedad , Selectina E/genética , Selectina E/metabolismo , Inflamación/genética , Inflamación/metabolismo , Animales , Selectina E/química , Inmunohistoquímica , Inflamación/inducido químicamente , Inflamación/patología , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Piel/metabolismo , Piel/patologíaRESUMEN
Laser dissection microscopy was applied to isolate endothelial cells from tumors obtained from mice treated with TNF-alpha. RNA integrity was demonstrated from whole sections and dissected cells after acetone fixation and hematoxylin staining. RT-PCR for GAPDH, CD31, VCAM-1, ICAM-1, and E-selectin was successfully performed on these samples. These data demonstrate the feasibility of analyzing local endothelial cell responses in diseased tissues at the level of gene expression.
Asunto(s)
Endotelio Vascular/fisiopatología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Animales , Selectina E/genética , Endotelio Vascular/patología , Molécula 1 de Adhesión Intercelular/genética , Rayos Láser , Ratones , Microscopía , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
Aspergillus niger is known for its efficient excretion machinery. However, problems have often arisen in obtaining high amounts of heterologous proteins in the culture medium. Here we present a quick method using sandwiched colonies to evaluate transgenic strains for secretion of heterologous proteins. Expressing the ABH1 hydrophobin of Agaricus bisporus in A. niger, we showed that low production levels of the heterologous protein are probably due to extracellular proteolytic degradation of the protein.
Asunto(s)
Aspergillus niger/genética , Aspergillus niger/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Agaricus/genética , Secuencia de Aminoácidos , Biotecnología , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Expresión Génica , Genes Fúngicos , Micología/métodos , Ingeniería de Proteínas , Procesamiento Proteico-PostraduccionalRESUMEN
Hydrophobins are small (length, about 100 +/- 25 amino acids), cysteine-rich, hydrophobic proteins that are present in large amounts in fungal cell walls, where they form part of the outermost layer (rodlet layer); sometimes, they can also be secreted into the medium. Different hydrophobins are associated with different developmental stages of a fungus, and their biological functions include protection of the hyphae against desiccation and attack by either bacterial or fungal parasites, hyphal adherence, and the lowering of surface tension of the culture medium to permit aerial growth of the hyphae. We identified and isolated a hydrophobin (fruit body hydrophobin 1 [Fbh1]) present in fruit bodies but absent in both monokaryotic and dikaryotic mycelia of the edible mushroom Pleurotus ostreatus. In order to study the temporal and spatial expression of the fbh1 gene, we determined the N-terminal amino acid sequence of Fbh1. We also synthesized and cloned the double-stranded cDNA corresponding to the full-length mRNA of Fbh1 to use it as a probe in both Northern blot and in situ hybridization experiments. Fbh1 mRNA is detectable in specific parts of the fruit body, and it is absent in other developmental stages.
Asunto(s)
Proteínas Fúngicas/biosíntesis , Pleurotus/fisiología , Agaricales , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Membrana Celular/ultraestructura , Clonación Molecular , ADN Complementario , Escherichia coli , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Genes Fúngicos , Hibridación in Situ , Datos de Secuencia Molecular , Pleurotus/genética , ARN Mensajero/análisis , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Transcripción GenéticaRESUMEN
A protein with characteristic properties of a fungal hydrophobin (CoH1) was isolated from the monokaryotic stage of the basidiomycete Coprinus cinereus. A cosmid clone containing the corresponding gene (coH1) was identified using a cDNA probe derived by RT-PCR. Hybridization and sequence analysis identified a second gene, coH2, just 4.1 kb downstream of coH1 encoding a hydrophobin (CoH2) with 64% sequence identity. Both coH1 and coH2 are subject to developmental regulation. They are expressed in vegetative monokaryotic cells but not in the asexual oidia produced on the surface of monokaryons. Transcripts of the genes were barely detected in dikaryotic mycelium and were absent from fruit bodies. Loss of aerial growth due to a mutation known as oid-1 was correlated with lack of both hydrophobins.
Asunto(s)
Coprinus/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Familia de Multigenes , Mutación , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Coprinus/metabolismo , Proteínas Fúngicas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ligamiento Genético , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Propiedades de SuperficieRESUMEN
The Schizophyllum commune hydrophobin Sc3p is a small, hydrophobic, cysteine-rich protein involved in the formation of aerial hyphae. Using an antibody against purified Sc3p we found that the hydrophobin is secreted into the medium at the apices of growing submerged hyphae but in emerging aerial hyphae it accumulates at the hyphal surface. Here, the hydrophobin self-assembles at the wall/air interface into an SDS-insoluble protein membrane, at the aerial site very hydrophobic and with the appearance of a mosaic of 10 nm spaced parallel rodlets. Interfacial self-assembly of the hydrophobin also occurs in vitro. When solutions containing various concentrations of purified Sc3p were dried down onto a glass surface, the amount of assembled Sc3p depended on the area of the interface. Surplus of Sc3p remained in the monomeric form, apparently because formation of a monolayer of assembled Sc3p abolishes the hydrophilic/hydrophobic interface. The 10 nm thick layer of assembled Sc3p at the surface of aerial hyphae thus probably represents a monolayer of the protein.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Schizophyllum/ultraestructura , Animales , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Proteínas Fúngicas/análisis , Inmunohistoquímica , Proteínas de la Membrana/análisis , Microscopía Electrónica , Schizophyllum/metabolismoRESUMEN
The Sc7 and Sc14 genes are specifically expressed in the dikaryon of the basidiomycete fungus Schizophyllum commune during fruiting. These genes are closely linked (within 6 kb) and highly similar in gene structure and nucleotide sequence (70% identical nucleotides in their coding regions). The encoded proteins (204 and 214 amino acids, respectively) have 87% similarity in amino acids (56% of the amino acids are identical). They contain putative signal sequences for secretion, are rich in aromatic amino acids which are generally located at similar positions, and they are generally hydrophilic. Inspection of databanks showed similarities with pathogenesis-related proteins (PR1) from plants, testis-specific proteins from mammals and venom allergen proteins from insects. An antibody raised against a Sc7 fusion protein showed the presence of the Sc7 protein in the culture medium and in the fruit bodies where it is apparently loosely associated with hyphal walls.
Asunto(s)
Proteínas Fúngicas/genética , Genes Fúngicos , Schizophyllum/genética , Secuencia de Aminoácidos , Secuencia de Bases , Espacio Extracelular/metabolismo , Proteínas Fúngicas/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Schizophyllum/crecimiento & desarrollo , Schizophyllum/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The spontaneous and recessive mutation thn in the basidiomycete Schizophyllum commune suppresses the formation of aerial hyphae in the monokaryon and, if present as a double dose, the formation of both aerial hyphae and fruit-bodies in the dikaryon. In the monokaryon, the mutation prevents accumulation of mRNA of the Sc3 gene, and in the dikaryon it also prevents the accumulation of fruiting-specific mRNAs, including mRNAs of the Sc1 and Sc4 genes, which are homologous to the Sc3 gene. These three genes code for hydrophobins, a family of small hydrophobic cysteine-rich proteins. In the thn monokaryon, the only detectable change in synthesized proteins is the disappearance of an abundant protein of apparent Mr = 28 K from the culture medium and from the cell walls. Protein sequencing shows that this is the product of the Sc3 gene. The Sc3 hydrophobin is present in the walls of aerial hyphae as a hot-SDS-insoluble complex. Submerged hyphae excrete large amounts of the hydrophobin into the medium.
Asunto(s)
Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Genes Reguladores , Schizophyllum/genética , Secuencia de Aminoácidos , Proteínas Fúngicas/biosíntesis , Genes Recesivos , Datos de Secuencia Molecular , Fenotipo , ARN de Hongos/biosíntesis , ARN Mensajero/biosíntesis , Schizophyllum/crecimiento & desarrolloRESUMEN
Fungi typically grow by apical extension of hyphae that penetrate moist substrates. After establishing a branched feeding mycelium, the hyphae differentiate and grow away from the substrate into the air where they form various structures such as aerial hyphae and mushrooms. In the basidiomycete species Schizophyllum commune, we previously identified a family of homologous genes that code for small cysteine-rich hydrophobic proteins. We now report that the encoded hydrophobins are excreted in abundance into the culture medium by submerged feeding hyphae but form highly insoluble complexes in the walls of emerging hyphae. The Sc3 gene encodes a hydrophobin present in walls of aerial hyphae. The homologous Sc1 and Sc4 genes, which are regulated by the mating-type genes, encode hydrophobins present in walls of fruit body hyphae. The hydrophobins are probably instrumental in the emergence of these aerial structures.