Vasodilatation av isolerade fartyg och isolering av den extracellulära matrisen av Tight-skin Möss
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
Reglering av celltillväxt och celldöd immunresponser är central för rollen av transkriptionsfaktorn familjen av interferon reglerande faktorer. IRF5 lyfts fram som är avgörande för regleringen av immunsvar mellan typ 1, en inflammatorisk främja respons och typ 2, ett immunsvar inriktning vävnadsreparation. IRF5 är nyckeln i cancer en, och autoimmunitet 2, 3, 4, 5.
Den täta hud mus (TSK / +) är en modell för vävnadsfibros och sklerodermi på grund av en dubbelmutation i fibrillin-1-genen. Denna mutation resulterar i en tät-hud och en ökning av bindväv. Dessa möss utvecklar myokardial inflammation, fibros och slutligen hjärtsvikt 5, 6, 7,> 8, 9. Sklerodermi är en autoimmun fibrotisk sjukdom som drabbar cirka 150.000 patienter i USA 6. Kännetecken av denna sjukdom är fibros i inre organ, inklusive hjärtat 7, 8, 9, 10, 11.
Beskaffenheten av studien krävde konstruktionen av en hämmande peptid. Tillvägagångssättet programvara valdes över en traditionell metod med hjälp av en fag display. Det tillvägagångssätt programvara är lättare och mindre tidskrävande. RCSB databank används för att identifiera lämpliga bindningsställen 12. För att studera interaktionen av nydesignade peptiden med det rekombinanta proteinet och att fokusera på de bindande parametrar, var en teknik som kallas biolayer interferometri används. Biolayer interferometri är en biosensor baserad technique som bestämmer bindningsaffiniteten, förenings- och avståndstagande genom att använda en biosensor och en bindande prov. Biosensorn kan vara fluorescerande, luminescently, radiometriskt och kolorimetriskt märkt. Mätningen baseras på mass tillägg eller utarmning liknar förening och avståndstagande 13, 14. Syftet med denna studie var att förstå betydelsen av IRF5 i hjärtinfarkt inflammation och fibros. Målet var att få inblick i rollen av IRF5 i utvecklingen av vävnadsfibros och sklerodermi.
Protocol
Representative Results
Discussion
Målet var att utforma en IRF5 hämmare att belysa rollen av IRF5 på inflammation, fibros och vaskulär funktion i hjärtat hos Tsk / + möss. Resultaten är att IRF5D inte inducera proliferation eller apoptos. Dessutom har inflammation minskade och vaskulär funktion förbättras. Dessa data tyder på att IRF5 spelar en viktig mekanistisk roll i utvecklingen av inflammation och fibros i hjärtat av Tsk / + möss och att det har potential att fungera som ett terapeutiskt mål.
Det första s…
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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