隔离容器的血管扩张和细胞外基质的紧皮肤小鼠隔离
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
细胞生长和细胞死亡的免疫应答的调节是至关重要的转录因子家族的干扰素调节因子的作用。 IRF5被高亮显示为用于免疫反应类型1之间的调控，炎症促进反应和2型，免疫应答靶向组织修复的关键。 IRF5是在癌症1，和自身免疫2，3，4，5键。
紧皮肤小鼠（TSK / +）为组织纤维化和硬皮病一个模型由于在原纤蛋白-1基因的复制突变。此突变导致一个紧皮肤和增加结缔组织。这些小鼠发展心肌炎症，纤维化和最后心脏衰竭5，6，7，> 8,9。硬皮病是影响美国6约有15万名患者的自身免疫性疾病纤维化。这种疾病的特点是内部器官包括心脏7，8，9，10，11的纤维化。
该研究的天性，需要抑制肽的设计。被选择了该软件的方法利用噬菌体展示传统的方法。的软件的方法是更容易和耗时更少。该RCSB数据库是用来确定适当的结合位点12。研究新设计的肽与重组蛋白的相互作用和集中的结合参数，使用一种技术，称为生物层干涉测量法。生物层干涉测量是基于生物传感器techniqUE决定使用生物传感器和具有约束力的样本结合亲和力，关联和解除关联。该传感器可以是荧光，luminescently，辐射测量和比色标记。测量是基于质量增加或耗竭类似关联和解除关联13,14。本研究的目的是要了解IRF5的心肌炎症和纤维化中的作用。目标是洞察IRF5的在组织纤维化和硬皮病的发展中的作用。
Protocol
Representative Results
Discussion
的目标是设计一个IRF5抑制剂阐明IRF5的炎症，纤维化和血管功能在啧/ +小鼠的心脏的作用。调查结果是IRF5D不诱导增殖或凋亡。此外，炎症减少和血管功能的改善。这些数据表明，IRF5在炎症和纤维化的啧/ +小鼠的心脏的发展的重要机械作用，它具有作为治疗靶的潜力。
第一步是设计的抑制剂。当肽被设计，有几点必须考虑到：序列长度，二级结构的残基容易发生氧化，氨基酸…
Divulgations
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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