Vasodilatation isolierter Gefäße und die Isolierung der extrazellulären Matrix von Fest-Haut Mäuse
Summary
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.
Abstract
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 ± 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 µg/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 µg/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.
Introduction
Regulation von Zellwachstum und Zelltod-Immunantworten ist, um die Rolle des Transkriptionsfaktor-Familie von Interferon regulatorischer Faktoren zentral. IRF5 wird als entscheidend für die Regulation von Immunantworten zwischen Typ 1, eine entzündliche Reaktion fördern und Typ 2, eine Immunantwort Targeting Gewebereparatur hervorgehoben. IRF5 ist der Schlüssel in der Krebs – 1 und Autoimmunität 2, 3, 4, 5.
Die straffe Haut Maus (Tsk / +) ist ein Modell für Gewebefibrose und Sklerodermie aufgrund einer Verdopplungsmutation im Fibrillin-1-Gen. Diese Mutation führt zu einer tight-Haut und eine Erhöhung in Bindegewebe. Diese Mäuse entwickeln myokardiale Entzündung, Fibrose und schließlich Herzinsuffizienz 5, 6, 7,> 8, 9. Sklerodermie ist eine Autoimmun fibrotischen Erkrankung beeinflussen etwa 150.000 Patienten in den Vereinigten Staaten von Amerika 6. Die Kennzeichen dieser Krankheit sind Fibrose der inneren Organe einschließlich des Herzens 7, 8, 9, 10, 11.
Die Art der Studie forderte die Gestaltung eines hemmenden Peptids. Der Software-Ansatz wurde eine traditionelle Ansatz unter Verwendung einer Phagen-Display ausgewählt über. Der Software-Ansatz ist einfacher und weniger zeitraubend. Die RCSB Datenbank wird verwendet , 12 geeignete Bindungsstellen zu identifizieren. Um die Interaktion des neu gestalteten Peptid mit dem rekombinanten Protein zu studieren und zu den Bindungsparameter zu fokussieren, eine Technik, genannt Bioschicht Interferometrie verwendet wurde. Bioschicht Interferometrie ist ein Biosensor basiert technique, die bestimmt, Bindungsaffinität, Assoziation und Dissoziation einen Biosensor und eine Bindungsprobe unter Verwendung. Der Biosensor kann fluoreszierend, lumineszierend sein, radiometrisch und kolorimetrisch gekennzeichnet. Die Messung basiert auf Masse zusätzlich oder Abreicherung ähnelt Assoziation und Dissoziation 13, 14. Das Ziel dieser Studie war es, die Rolle der IRF5 in myokardiale Entzündung und Fibrose zu verstehen. Das Ziel war, einen Einblick in die Rolle der IRF5 in der Entwicklung von Gewebefibrose und Sklerodermie zu gewinnen.
Protocol
Representative Results
Discussion
Das Ziel war es, ein IRF5 Inhibitor zu entwerfen, die Rolle der IRF5 auf Entzündung, Fibrose und Gefäßfunktion in den Herzen der Tsk / + Mäuse aufzuklären. Die Ergebnisse sind, dass IRF5D haben Proliferation oder Apoptose nicht induziert. Darüber hinaus wurde die Entzündung reduziert und Gefäßfunktion verbessert. Diese Daten legen nahe, dass IRF5 spielt eine wichtige mechanistische Rolle bei der Entwicklung der Entzündung und Fibrose im Herzen von Tsk / + Mäusen und daß sie das Potential als therapeutisches …
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Triton X 100 | Sigma Aldrich | X100- 100ml | |
| Alexa 488-labeled goat anti-mouse IgG antibody | Thermo Fisher | A11001 | |
| Bardford reagent | Thermo Fisher | 23200 | Pierce |
| Biosensors | Forte-Bio | MR18-0009 | |
| CD64 (H-250) | Santa Cruz Biotechnologies | sc-15364 | |
| CellEvent Caspase-3/7 Substrate | Thermo Fisher/Life Technologies | C10427 | |
| CellTiter AQueous One Solution Cell Proliferation Assay kit | Promega | G3580 | Promega |
| DAPI (4′,6-diamidino-2-phenylindole) | Thermo Fisher | D-1306 | 1:1000 dilution in PBS |
| donkey anti rat Alexa 488 | Thermo Fisher | A-21208 | 1:1000 dilution in PBS |
| ECL plus | GE healthcare/Amersham | RPN2133 | After a lot of trial and error we came back to this one |
| Eclipse TE 200-U microscope with EZ C1 laser scanning software | Nikon | ||
| goat anti rabbit Alexa 488 | Thermo Fisher | A-11008 | 1:1000 dilution in PBS |
| HRP anti-goat | Santa Cruz Biotechnologies | sc-516086 | !:10000 dilution in TBS |
| HRP donkey anti-mouse | Santa Cruz Biotechnologies | sc-2315 | 1:10000 dilution in TBS |
| ICAM-1 antibody | Santa Cruz Biotechnologies | sc-1511 | 1:200 dilution in PBS |
| IRF5 antibody (H56) | Santa Cruz Biotechnologies | sc-98651 | |
| Micro plate reader Elx800 | Biotek | ||
| NIMP neutrophil marker | Santa Cruz Biotechnologies | sc-133821 | 1:200 dilution in PBS |
| Octet RED | Forte Bio | protein-protein binding | |
| Peptide design Medit SA software | RCSB.org | ||
| Recombinant IRF5 protein synthesis | TopGene Technologies | protein expression, synthesis service | |
| sodium dodecyl phosphate | Sigma Aldrich | 436143 | detergent |
| Ketamine | Pharmacy | Schedule III controlled substance, presciption required | |
| Xylazine | MedVet | ||
| 3.5X-45X Trinocular Dissecting Zoom Stereo Microscope with Gooseneck LED Lights | Am Scope | SKU: SM-1TSX-L6W | |
| Zeba Desalting Columns | Thermofisher | 2161515 | |
| Endothelial Basal Media EBM Bullet kit | Lonza | CC-3124 | kit contains growth supplemets |
| VIA-100K | Boeckeler Instruments | ||
| 4-15% TGX gel | Bio-Rad | 5671081 | |
| MedSuMo software | Medit, Palaiseau, France | ||
| Laemmli Buffer | BioRad |
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