Name: vasodilation of isolated vessels and the isolation of the extracellular matrix of tight-skin mice
Uploaded: 2017-03-24T13:00:00
Description: Watch this Scientific Journal Video about Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice at JoVE.com

This work was supported by NIH grants HL-089779 (DW), HL-112270 (KAP) and HL-102836 (KAP) and Cimphoni Life Sciences (part of DW salary). The authors thank Meghann Sytsma for editing the manuscript.

Este estudio se llevó a cabo en estricta conformidad con las recomendaciones de la Guía para el Cuidado y Uso de Animales de Laboratorio de los Institutos Nacionales de Salud. El protocolo fue aprobado por el Cuidado de Animales institucional y el empleo (Protocolo: AUA # 1517). Todas las investigaciones con ratones se llevó a cabo de conformidad con la política PHS. 1. Diseño de Decoy Péptido <ol><li> Encuentra estructura 3D de la IRF5 y basar el diseño en él. Diseño de un 17 mer, denominado IRF5D (E...

<ol>
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El objetivo era diseñar un inhibidor IRF5 para dilucidar el papel de IRF5 sobre la inflamación, fibrosis y la función vascular en los corazones de TSK / + ratones. Los resultados son que IRF5D no indujo la proliferación o la apoptosis. Por otra parte, la inflamación se redujo y la función vascular mejorada. Estos datos sugieren que IRF5 juega un papel mecanicista importante en el desarrollo de la inflamación y fibrosis en el corazón de TSK / + ratones y que tiene el potencial de servir como una diana terapéutic...

<html> Regulación del crecimiento celular y la respuesta inmune de muerte celular es fundamental para el papel de la familia de factores de transcripción de factores reguladores de interferón. IRF5 se pone de relieve como crucial para la regulación de la respuesta inmune entre el tipo 1, la promoción de una respuesta inflamatoria y tipo 2, una reparación de tejidos respuesta inmune de orientación. IRF5 es clave en el cáncer de 1, y la autoinmunidad 2, 3, 4, 5. El ratón apretado-piel (Tsk / +) es un modelo para la fibrosis del tejido y la esclerodermia debido a una mutación de duplicación en el gen de la fibrilina-1. Esta mutación da como resultado un apretado-piel y un aumento en el tejido conectivo. Estos ratones desarrollan inflamación miocárdica, fibrosis e insuficiencia finalmente corazón 5, 6, 7,&gt; 8, 9. La esclerodermia es un trastorno fibrótico autoinmune que afecta a aproximadamente 150.000 pacientes en los Estados Unidos 6. Las características de esta enfermedad son la fibrosis de los órganos internos, incluyendo el corazón 7, 8, 9, 10, 11. La naturaleza del estudio exigía el diseño de un péptido inhibidor. El enfoque de software fue elegido sobre un enfoque tradicional usando una visualización de fagos. El enfoque de software es más fácil y menos consume mucho tiempo. El banco de datos RCSB se utiliza para identificar sitios de unión apropiados 12. Para estudiar la interacción del péptido de nuevo diseño con la proteína recombinante y centrarse en los parámetros de unión, se utilizó una técnica llamada interferometría capa biológica. Biocapa interferometría es un techniq basado biosensorue lo que determina la afinidad de unión, de asociación y disociación utilizando un biosensor y una muestra de unión. El biosensor puede ser fluorescente, luminiscentemente, etiquetado radiométricamente y colorimétricamente. La medición se basa en la adición de masa o el agotamiento se asemeja asociación y disociación 13, 14. El objetivo de este estudio fue comprender el papel de IRF5 en la inflamación y la fibrosis miocárdica. El objetivo era profundizar en el papel de IRF5 en el desarrollo de la fibrosis del tejido y la esclerodermia.

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			<td>&#160;Triton X 100</td>
			<td>Sigma Aldrich</td>
			<td>X100- 100ml</td>
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		<tr>
			<td>Alexa 488-labeled goat anti-mouse IgG antibody&#160;</td>
			<td>Thermo Fisher</td>
			<td>A11001</td>
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			<td>Bardford reagent</td>
			<td>Thermo Fisher</td>
			<td>23200</td>
			<td>Pierce&#160;</td>
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			<td>Biosensors</td>
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			<td>MR18-0009</td>
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			<td>CD64 (H-250)</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-15364</td>
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			<td>CellEvent Caspase-3/7 Substrate</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>C10427</td>
			<td></td>
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			<td>CellTiter AQueous One Solution Cell Proliferation Assay kit</td>
			<td>Promega</td>
			<td>G3580</td>
			<td>Promega</td>
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			<td>DAPI (4&#8242;,6-diamidino-2-phenylindole)</td>
			<td>Thermo Fisher</td>
			<td>D-1306</td>
			<td>1:1000 dilution in PBS</td>
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			<td>donkey anti rat Alexa 488</td>
			<td>Thermo Fisher</td>
			<td>A-21208</td>
			<td>1:1000 dilution in PBS</td>
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			<td>ECL plus</td>
			<td>GE healthcare/Amersham</td>
			<td>RPN2133</td>
			<td>After a lot of trial and error we came back to this one</td>
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			<td>Eclipse TE 200-U microscope with EZ C1 laser scanning software</td>
			<td>Nikon</td>
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			<td></td>
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			<td>goat anti rabbit Alexa 488</td>
			<td>Thermo Fisher</td>
			<td>A-11008</td>
			<td>1:1000 dilution in PBS</td>
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			<td>HRP&#160; anti-goat</td>
			<td>Santa Cruz Biotechnologies</td>
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			<td>!:10000 dilution in TBS</td>
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			<td>HRP donkey anti-mouse</td>
			<td>Santa Cruz Biotechnologies</td>
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			<td>1:10000 dilution in TBS</td>
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			<td>Santa Cruz Biotechnologies</td>
			<td>sc-1511</td>
			<td>1:200 dilution in PBS</td>
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			<td>IRF5 antibody (H56)</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-98651</td>
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			<td>Micro plate reader Elx800</td>
			<td>Biotek</td>
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			<td></td>
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			<td>NIMP neutrophil marker</td>
			<td>Santa Cruz Biotechnologies</td>
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			<td>1:200 dilution in PBS</td>
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			<td>Octet RED</td>
			<td>Forte Bio</td>
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			<td>protein-protein binding</td>
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			<td>Peptide design&#160; Medit SA software</td>
			<td>RCSB.org</td>
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			<td></td>
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			<td>Recombinant IRF5 protein synthesis</td>
			<td>TopGene Technologies</td>
			<td></td>
			<td>protein expression, synthesis service</td>
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			<td>sodium dodecyl phosphate</td>
			<td>Sigma Aldrich</td>
			<td>436143</td>
			<td>detergent</td>
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			<td>Ketamine</td>
			<td>Pharmacy</td>
			<td></td>
			<td>Schedule III controlled substance, presciption required&#160;</td>
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			<td>Xylazine</td>
			<td>MedVet</td>
			<td></td>
			<td></td>
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		<tr>
			<td>3.5X-45X Trinocular Dissecting Zoom Stereo Microscope with Gooseneck LED Lights</td>
			<td>Am Scope</td>
			<td>SKU: SM-1TSX-L6W</td>
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			<td>Zeba Desalting Columns</td>
			<td>Thermofisher</td>
			<td>2161515</td>
			<td></td>
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			<td>Endothelial Basal Media EBM Bullet kit</td>
			<td>Lonza</td>
			<td>CC-3124</td>
			<td>kit contains growth supplemets</td>
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			<td>VIA-100K</td>
			<td>&#160;Boeckeler Instruments</td>
			<td></td>
			<td></td>
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			<td>4-15% TGX gel</td>
			<td>Bio-Rad</td>
			<td>5671081</td>
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			<td>MedSuMo software</td>
			<td>Medit, Palaiseau, France</td>
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			<td>Laemmli Buffer</td>
			<td>BioRad</td>
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vasodilation of isolated vessels and the isolation of the extracellular matrix of tight-skin mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides.
IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 &plusmn; 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 &#181;g/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 &#181;g/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

Los resultados demostrados en la Figura 1 muestran cómo diseñar un péptido. La Figura 1, parte superior izquierda, muestra la región (entre las 2 flechas amarillas, los aminoácidos (aa) 425 a 436) en IRF5 que es fosforilada por una serie de quinasas. La figura 1, parte superior derecha, muestra un óvalo de color amarillo donde dominio fosforilada de IRF5 une. La estructura dimérica de 3DSH se hizo girar para observar una hendidura...

Watch this Scientific Journal Video about Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice at JoVE.com

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide.

Vasodilatación de los vasos aislados y el aislamiento de la matriz extracelular de la piel-apretado ratones

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

Research

JoVE Journal

Immunology and Infection

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

La inducción de la hipofisitis autoinmune experimental en ratones SJL

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Investigación

Inmunología e Infección

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

La medición de la carga bacteriana y la respuesta inmune en ratones infectados con Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Introducing Shear Stress in the Study of Bacterial Adhesion

La introducción de la tensión de cizallamiento en el Estudio de la adhesión bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

Aislamiento y análisis de Brain-secuestrados Los leucocitos de<em&gt; Plasmodium berghei</em&gt; ANKA de ratones infectados

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Isolation and Th17 Differentiation of NaÃƒÆ’Ã‚Â¯ve CD4 T Lymphocytes

Aislamiento y diferenciación de Th17 Naïve linfocitos T CD4

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Measuring Local Anaphylaxis in Mice

Medición anafilaxia local en ratones

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Un ensayo de base poliacrilamida formato multi-bien para estudiar el efecto de la rigidez de la matriz extracelular en la infección bacteriana de las células adherentes

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Aislamiento y caracterización de las vesículas extracelulares de adultos Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Evaluación de las respuestas del huésped-patógeno y eficacia de la vacuna en ratones

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...