Vasodilatasjon av isolerte kar og isolering av den ekstracellulære matrise av 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 av cellevekst og celledødsimmunresponser er sentral for rollen av transkripsjonsfaktor familien av interferon-regulerende faktorer. IRF5 markeres som å være avgjørende for regulering av immunresponser mellom type 1, en inflammatorisk fremme reaksjon og type 2, en immunrespons rettet mot vevsreparasjon. IRF5 er nøkkelen i kreft 1, og autoimmunitet 2, 3, 4, 5.
Den tett hud mus (Tsk / +) er en modell for vev fibrose og sklerodermi på grunn av en duplisering mutasjon i fibrillin-1 genet. Denne mutasjonen resulterer i en tett hud og en økning i bindevev. Disse musene utvikler myokardial inflammasjon, fibrose og endelig hjertesvikt 5, 6, 7,> 8, 9. Sklerodermi er en autoimmun fibrotisk lidelse som berører ca 150 000 pasienter i USA seks. Kjennetegnene på denne sykdommen er fibrose av indre organer, inkludert hjertet 7, 8, 9, 10, 11.
Naturen av studien krevde utformingen av et inhiberende peptid. Programvaren tilnærmingen ble valgt fremfor en tradisjonell måte ved hjelp av en fag-display. Programvaren tilnærmingen er enklere og mindre tidkrevende. Den RCSB Databanken brukes til å identifisere aktuelle bindingssteder 12. For å studere samspillet mellom den nydesignede peptid med rekombinant protein og å fokusere på de bindende parametrene, ble en teknikk som kalles biolag interferometri brukt. Biolag interferometri er en biosensor basert technique som bestemmer bindende affinitet, tilknytning og dissosiasjon ved hjelp av en biosensor og en bindende prøve. Den biosensor kan være fluorescerende, luminescently, radiometrisk og kolorimetrisk merket. Målingen er basert på masse tillegg eller uttømming likner forening og dissosiasjon 13, 14. Målet med denne studien var å forstå hvilken rolle IRF5 i myocardial betennelse og fibrose. Målet var å få innsikt i rollen som IRF5 i utviklingen av vev fibrose og sklerodermi.
Protocol
Representative Results
Discussion
Målet var å designe en IRF5 inhibitor å belyse rollen IRF5 på betennelse, fibrose, og vaskulær funksjon i hjertene til TSK / + mus. Funnene er at IRF5D ikke induserer spredning eller apoptose. Videre inflammasjon ble redusert og vaskulær funksjon forbedret. Disse dataene tyder på at IRF5 spiller en viktig mekanistisk rolle i utviklingen av inflammasjon og fibrose i sentrum av TSK / + mus, og at det har potensiale til å tjene som et terapeutisk mål.
Første skritt var å designe en i…
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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