Vasodilatatie van geïsoleerde Schepen en de isolatie van de extracellulaire matrix van Tight-skin Muizen
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
Regulatie van celgroei en celdood immuunresponsen staat centraal in de rol van de transcriptiefactor familie van interferon regulerende factoren. IRF5 staat aangegeven als cruciaal voor de regulatie van immuunresponsen tussen type 1, een inflammatoire respons stimuleren en type 2, een immuunreactie gericht weefselherstel. IRF5 is belangrijk bij kanker 1, en autoimmuniteit 2, 3, 4, 5.
De strakke huid muis (Tsk / +) is een model voor weefsel fibrose en sclerodermie als gevolg van een verdubbeling mutatie in het fibrillin-1-gen. Deze mutatie resulteert in een strakke huid en een toename van bindweefsel. Deze muizen ontwikkelen myocardiale ontsteking, fibrose en uiteindelijk hartfalen 5, 6, 7,> 8, 9. Scleroderma is een auto-fibrotische aandoening die ongeveer 150.000 patiënten in de Verenigde Staten 6. De kenmerken van deze ziekte fibrose van organen zoals het hart 7, 8, 9, 10, 11.
De aard van de studie eiste het ontwerp van een remmend peptide. De software aanpak werd verkozen boven een traditionele benadering waarbij een faagdisplay. De software benadering is eenvoudiger en minder tijdrovend. De RCSB databank wordt gebruikt om geschikte bindingsplaatsen 12 identificeren. De interactie van de nieuw ontworpen peptide met het recombinante eiwit te bestuderen en te focussen op de binding parameters, werd een techniek genaamd biolayer interferometrie gebruikt. Biolayer interferometrie is een biosensor gebaseerd techniqUE die bepaalt bindingsaffiniteit, vereniging en dissociatie met behulp van een biosensor en een bindend monster. De biosensor kan fluorescent, luminescent, radiometrisch en colorimetrisch gelabeld. De meting is gebaseerd op massa toevoeging of depletie lijkt associatie en dissociatie 13, 14. Het doel van deze studie was de rol van IRF5 myocardiale ontsteking en fibrose begrijpen. Het doel was om inzicht te krijgen in de rol van IRF5 in de ontwikkeling van weefsel fibrose en sclerodermie.
Protocol
Representative Results
Discussion
Het doel was om een IRF5 remmer te ontwerpen om de rol van IRF5 op ontsteking, fibrose, en vasculaire functie in de harten van Tsk / + muizen helderen. De bevindingen zijn dat IRF5D geen proliferatie of apoptose induceerde. Bovendien werd inflammatie verminderd en vasculaire functie verbeterd. Deze gegevens suggereren dat IRF5 speelt een belangrijke mechanistische rol in de ontwikkeling van ontsteking en fibrose in het hart van Tsk / + muizen en dat het de potentie om te dienen als een therapeutisch doelwit.
<…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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