पृथक वेसल्स के वैसोडायलेटेशन और चुस्त-त्वचा चूहे के बाह्य मैट्रिक्स का अलगाव
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
सेल के विकास और कोशिका मृत्यु प्रतिरक्षा प्रतिक्रिया के नियमन इंटरफेरॉन विनियामक कारकों का प्रतिलेखन कारक परिवार की भूमिका के लिए केंद्रीय है। IRF5 टाइप 1 के बीच प्रतिरक्षा प्रतिक्रिया के विनियमन, एक सूजन को बढ़ावा देने प्रतिक्रिया और टाइप 2, एक प्रतिरक्षा प्रतिक्रिया को निशाना बनाने ऊतकों की मरम्मत के लिए महत्वपूर्ण होने के रूप में प्रकाश डाला है। IRF5 कैंसर 1, और autoimmunity 2, 3, 4, 5 में महत्वपूर्ण है।
तंग त्वचा माउस (Tsk / +) fibrillin -1 जीन में उत्परिवर्तन के कारण दोहराव ऊतक फाइब्रोसिस और scleroderma के लिए एक मॉडल है। एक तंग त्वचा में इस उत्परिवर्तन के परिणाम और संयोजी ऊतक में वृद्धि हुई है। इन चूहों, दौरे सूजन, फाइब्रोसिस और अंत में दिल की विफलता 5, 6, 7 का विकास> 8, 9। Scleroderma एक autoimmune fibrotic यूनाइटेड स्टेट्स 6 में लगभग 150,000 मरीजों को प्रभावित विकार है। इस रोग की पहचान दिल 7, 8, 9, 10, 11 सहित आंतरिक अंगों की फाइब्रोसिस हैं।
अध्ययन की प्रकृति एक निरोधात्मक पेप्टाइड की डिजाइन की मांग की। सॉफ्टवेयर दृष्टिकोण एक फेज प्रदर्शन का उपयोग कर एक परंपरागत दृष्टिकोण पर चुना गया था। सॉफ्टवेयर दृष्टिकोण आसान और कम समय लगता है। RCSB डेटा बैंक उचित बाध्यकारी साइटों 12 की पहचान के लिए प्रयोग किया जाता है। पुनः संयोजक प्रोटीन के साथ नए डिजाइन पेप्टाइड की बातचीत का अध्ययन और बाध्यकारी मानकों पर ध्यान केंद्रित करने के लिए, एक तकनीक बुलाया biolayer इंटरफेरोमेट्री इस्तेमाल किया गया था। Biolayer इंटरफेरोमेट्री एक biosensor आधारित techniq हैUE कि बंधन आत्मीयता, संघ और disassociation एक biosensor और एक बाध्यकारी नमूना का उपयोग करते हुए निर्धारित करता है। biosensor fluorescently, luminescently, radiometrically और colorimetrically लेबल किया जा सकता। माप बड़े पैमाने पर अतिरिक्त या कमी एसोसिएशन और disassociation 13, 14 दिखने पर आधारित है। इस अध्ययन का उद्देश्य दौरे सूजन और फाइब्रोसिस में IRF5 की भूमिका को समझने के लिए किया गया था। लक्ष्य ऊतक फाइब्रोसिस और scleroderma के विकास में IRF5 की भूमिका में जानकारी हासिल करने के लिए किया गया था।
Protocol
Representative Results
Discussion
लक्ष्य Tsk / + चूहों के दिलों में सूजन, फाइब्रोसिस, और नाड़ी समारोह पर IRF5 की भूमिका को स्पष्ट करने के लिए एक IRF5 अवरोध डिजाइन करने के लिए किया गया था। निष्कर्ष है कि IRF5D प्रसार या apoptosis प्रेरित नहीं कर रहे हैं। इसक?…
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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