İzole Kapların vazodilatasyon ve Sıkı cilt Fare Ekstraselüler Matrix İzolasyonu
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
hücre büyümesi ve hücre ölümü, bağışıklık karşılıklarının düzenlenmesinde, interferon ayarlayıcı faktör transkripsiyon faktörü ailesinin rolü için esastır. IRF5 tip 1 arasında, bağışıklık karşılıklarının düzenlenmesinde, bir enflamatuar teşvik yanıt ve tip 2, bir bağışıklık tepkisi hedefleme doku onarımı için çok önemli olarak vurgulanır. IRF5 kanseri 1 ve otoimmünite, 2, 3, 4, 5 anahtardır.
Sıkı cilt fare (Tsk / +) nedeniyle fibrillin-1 genindeki bir mutasyon çoğaltma doku fibrozis ve skleroderma için bir modeldir. sıkı cilt Bu mutasyon sonuçları ve bağ dokusu bir artış. Bu fareler, miyokardiyal inflamasyon, fibrozis ve son olarak, kalp yetmezliği 5, 6, 7 geliştirilmesi> 8, 9. Skleroderma Amerika Birleşik Devletleri'nde 6 yaklaşık 150.000 hasta etkileyen bir otoimmün fibrotik bozukluktur. Bu hastalığın diğerlerinden ayıran kalp 7, 8, 9, 10, 11 dahil olmak üzere, iç organların fibroz bulunmaktadır.
Çalışmanın doğası inhibitör peptid tasarımını talep etti. yazılım yaklaşımı bir faj ekranı kullanarak geleneksel bir yaklaşım üzerinde seçildi. yazılım yaklaşımı daha kolay ve daha az zaman alıcıdır. RCSB veri bankası, uygun bağlanma bölgelerini 12 tanımlamak için kullanılır. rekombinant protein ile yeni tasarlanmış peptid etkileşimini incelemek ve bağlayıcı parametreleri üzerinde odaklanmak, bir teknik denilen biolayer enterforemetre kullanıldı. Biolayer interferometri, bir biyosensor göre TECHNIQ olanue bir biyosensör ve bağlayıcı bir örneği kullanarak bağlanma afinitesi, dernek ve ayrılmayı belirler. biyosensör floresan, luminescently, radyometrik ve kolorimetrik etiketli olabilir. Ölçüm kütle eklenmesi veya tükenmesi birleşme ve ayrışma 13, 14 benzer dayanır. Bu çalışmanın amacı, miyokard inflamasyon ve fibrozis IRF5 rolünü anlamak oldu. hedef doku fibrozis ve skleroderma gelişiminde IRF5 rolünü anlamak için oldu.
Protocol
Representative Results
Discussion
Gol Tsk / + farelerin kalplerinde inflamasyon, fibrozis, ve vasküler fonksiyon üzerine IRF5 rolünü aydınlatmak için bir IRF5 inhibitörü tasarlamak oldu. Bulgular IRF5D proliferasyonu veya apoptosisi teşvik etmediğini bulunmaktadır. Ayrıca, iltihabın azalır ve damar fonksiyon geliştirildi. Bu veriler IRF5 Tsk / + farelerin kalp inflamasyon ve fibrozis gelişiminde önemli bir mekanik rol oynadığını düşündürmektedir ve bir tedavi hedefi olarak hizmet potansiyeline sahip olduğunu.
<p class="jove…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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