Summary

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

Published: March 24, 2017
doi:

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

この研究は、米国立衛生研究所の実験動物の管理と使用に関するガイドの推奨事項に厳密に従って行きました。プロトコルは、施設内動物管理使用委員会(:AUAの#1517プロ​​トコル)によって承認されました。マウスを含むすべての研究はPHSポリシーに準拠して行きました。 デコイペプチドの1デザイン IRF5の3D構造を検索し、その上にデザインをベースにします。 17マーを設?…

Representative Results

図1で実証結果は、ペプチドを設計する方法を示しています。 図1、左上には、多くのキナーゼによってリン酸化されるIRF5中の領域(2黄色の矢印の間、アミノ酸(AA)425から436)を示しています。 図1、右上は、IRF5のリン酸化されたドメインが結合黄色の楕円形を示しています。 3DSHの二量体構造は、ヘリックス2(aa303-312)の左?…

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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Cite This Article
Weihrauch, D., Krolikowski, J. G., Jones, D. W., Zaman, T., Bamkole, O., Struve, J., Pagel, P. S., Lohr, N. L., Pritchard, Jr., K. A. Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice. J. Vis. Exp. (121), e55036, doi:10.3791/55036 (2017).

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