Vasodilation af isoleret Fartøjer og isoleringen af den ekstracellulære matrix af Tight-hud Mus
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
Regulering af cellevækst og celledød immunreaktioner er centralt for den rolle af transkriptionsfaktoren familien af interferon regulatoriske faktorer. IRF5 fremhæves som værende af afgørende betydning for reguleringen af immunreaktioner mellem type 1, en inflammatorisk fremme respons og type 2, et immunrespons rettet mod væv reparation. IRF5 er nøglen i kræft 1, og autoimmunitet 2, 3, 4, 5.
Den stramme hud mus (Tsk / +) er en model for væv fibrose og sklerodermi på grund af en overlapning mutation i fibrillin-1-genet. Denne mutation resulterer i en tæt hud og en stigning i bindevæv. Disse mus udvikler myocardial inflammation, fibrose og endelig hjertesvigt 5, 6, 7,> 8, 9. Sklerodermi er en autoimmun fibrotisk sygdom, der påvirker ca. 150.000 patienter i USA 6. De kendetegnende for denne sygdom er fibrose af indre organer, herunder hjertet 7, 8, 9, 10, 11.
Arten af studiet krævede udformningen af en inhibitorisk peptid. Softwaren fremgangsmåde blev valgt frem for en traditionel fremgangsmåde ved anvendelse af en fag-display. Softwaren fremgangsmåde er lettere og mindre tidskrævende. Den RCSB databank bruges til at identificere egnede bindingssteder 12. For at undersøge interaktionen af nyudviklede peptid med det rekombinante protein og for at fokusere på de bindende parametre, blev en teknik kaldet biolayer interferometri anvendes. Biolayer interferometri er en biosensor baseret Technique der afgør bindende affinitet, forenings- og afstandtagen ved hjælp af en biosensor og en bindende prøve. Biosensoren kan være fluorescens, luminescens, radiometrisk og kolorimetrisk mærket. Målingen er baseret på masse tilføjelse eller udtømning ligner forening og afstandtagen 13, 14. Formålet med denne undersøgelse var at forstå den rolle IRF5 i myocardial inflammation og fibrose. Målet var at få indsigt i den rolle, IRF5 i udviklingen af væv fibrose og sklerodermi.
Protocol
Representative Results
Discussion
Målet var at designe en IRF5 inhibitor at belyse den rolle, IRF5 på inflammation, fibrose, og vaskulær funktion i hjertet på TSK / + mus. Resultaterne er, at IRF5D ikke inducerer proliferation eller apoptose. Endvidere inflammation blev reduceret og vaskulær funktion forbedres. Disse data antyder, at IRF5 spiller en vigtig mekanistisk rolle i udviklingen af inflammation og fibrose i hjertet af TSK / + mus, og at det har potentiale til at tjene som et terapeutisk mål.
Det første …
Divulgations
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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