単離された血管の血管拡張およびタイト皮膚マウスの細胞外マトリックスの単離
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
細胞増殖と細胞死の免疫応答の調節は、インターフェロン調節因子の転写因子ファミリーの役割の中心です。 IRF5は、タイプ1の間の免疫応答の調節、炎症促進応答および2型、免疫応答のターゲット組織修復のために重要であると強調表示されます。 IRF5癌1、及び自己免疫2、3、4、5のキーです。
タイト皮膚マウス（TSK / +）は、組織線維症とフィブリリン1遺伝子における重複変異に起因する強皮症のモデルです。タイト皮膚および結合組織の増加でこの変異は結果。これらのマウスは、心筋の炎症、線維化および最終的に心不全5、6、7を開発します> 8,9。強皮症は、米国6で約15万人の患者に影響を与える自己免疫線維性疾患です。この疾患の特徴は、心臓7、8、9、10、11を含む内臓の線維症です。
研究の性質は、阻害ペプチドの設計を要求しました。ソフトウェアアプローチは、ファージディスプレイを使用する従来のアプローチを介して選択しました。ソフトウェアのアプローチが容易になり、より少ない時間のかかるものです。 RCSBのデータバンクは、適切な結合部位12を同定するために使用されます。組換えタンパク質で新たに設計したペプチドの相互作用を研究し、結合パラメーターに焦点を当てて、干渉生物層と呼ばれる技術を使用しました。生物層の干渉は、バイオセンサベースのtechniqですバイオセンサーと結合サンプルを使用して結合親和性、会合及び解離を決定するUE。バイオセンサーは、蛍光、ルミネセンス、放射量および比色標識することができます。測定は、質量付加または枯渇が会合及び解離13、14に似ているに基づいています。本研究の目的は、心筋の炎症や線維症におけるIRF5の役割を理解することでした。目標は、組織線維症および強皮症の開発にIRF5の役割への洞察を得ることでした。
Protocol
Representative Results
Discussion
目標は、TSK / +マウスの心臓の炎症、線維症、および血管機能にIRF5の役割を解明するためにIRF5阻害剤を設計することでした。知見はIRF5Dの増殖またはアポトーシスを誘導しなかったことです。また、炎症を低減し、血管機能を改善しました。これらのデータは、IRF5はTSK / +マウスの中心部に炎症や線維症の発症に重要な機構的な役割を果たしていることを示唆し、それが治療標的として機能す…
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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