Vasodilatation des vaisseaux isolés et l'isolement de la matrice extracellulaire de souris Tight-peau
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
La régulation de la croissance cellulaire et les réponses immunitaires de mort cellulaire est essentielle pour le rôle de la famille des facteurs de transcription de facteurs de régulation de l'interféron. IRF5 est soulignée comme étant cruciale pour la régulation des réponses immunitaires entre le type 1, une réponse inflammatoire et la promotion de type 2, une réparation tissulaire réponse immunitaire ciblage. IRF5 est la clé 1 dans le cancer, l' auto – immunité et 2, 3, 4, 5.
La souris étanche peau (Tss / +) est un modèle pour la fibrose tissulaire et la sclérodermie due à une mutation de duplication dans le gène de la fibrilline-1. Cette mutation se traduit par une peau étanche et une augmentation du tissu conjonctif. Ces souris développent une inflammation du myocarde, la fibrose et finalement une insuffisance cardiaque 5, 6, 7,> 8, 9. La sclérodermie est une maladie auto – immune affectant fibrotique environ 150.000 patients aux Etats-Unis 6. Les caractéristiques de cette maladie sont une fibrose d'organes internes , y compris le cœur 7, 8, 9, 10, 11.
La nature de l'étude a demandé la conception d'un peptide inhibiteur. L'approche du logiciel a été choisi sur une approche traditionnelle en utilisant un phage display. L'approche du logiciel est plus facile et moins consommatrice de temps. La banque de données RCSB est utilisé pour identifier les sites de liaison appropriés 12. Pour étudier l'interaction du peptide nouvellement conçu avec la protéine recombinante et de se concentrer sur les paramètres de liaison, une technique appelée interférométrie biocouche a été utilisée. Biocouche interférométrie est un techniq biocapteur baséue qui détermine l'affinité de liaison, d'association et de dissociation en utilisant un biocapteur et un échantillon de liaison. Le biocapteur peut être fluorescence, luminescence, radiométrique et colorimétriquement marqué. La mesure est basée sur l' addition ou l' épuisement de masse ressemblant à l' association et la dissociation 13, 14. Le but de cette étude était de comprendre le rôle de IRF5 dans l'inflammation du myocarde et de la fibrose. L'objectif était de mieux comprendre le rôle de IRF5 dans le développement de la fibrose tissulaire et la sclérodermie.
Protocol
Representative Results
Discussion
L'objectif était de concevoir un inhibiteur de IRF5 pour élucider le rôle de IRF5 sur l'inflammation, la fibrose et la fonction vasculaire dans le cœur des Tsk / + souris. Les conclusions sont que IRF5D n'a pas induit la prolifération ou l'apoptose. En outre, l'inflammation a été réduite et la fonction vasculaire améliorée. Ces données suggèrent que IRF5 joue un rôle mécanistique important dans le développement de l'inflammation et de la fibrose dans le cœur de souris + / Tsk, et …
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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