Vasodilatazione dei vasi isolati e l'isolamento della matrice extracellulare di topi Tight-pelle
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
Regolazione della crescita cellulare e la morte cellulare risposte immunitarie è fondamentale per il ruolo del fattore di trascrizione famiglia di fattori regolatori interferone. IRF5 è evidenziato come cruciale per la regolazione delle risposte immunitarie tra il tipo 1, una risposta infiammatoria promuovendo e di tipo 2, una riparazione dei tessuti risposta immunitaria targeting. IRF5 è fondamentale nel cancro 1, e l'autoimmunità 2, 3, 4, 5.
Il mouse tight-pelle (Tsk / +) è un modello per la fibrosi dei tessuti e la sclerodermia a causa di una mutazione duplicazione nel fibrillin-1 gene. Questa mutazione si traduce in una stretta-pelle e un aumento del tessuto connettivo. Questi topi sviluppano infiammazione del miocardio, fibrosi e infine insufficienza cardiaca 5, 6, 7,> 8, 9. La sclerodermia è una malattia fibrotica autoimmune che colpisce circa 150.000 pazienti negli Stati Uniti 6. Le caratteristiche di questa malattia sono la fibrosi degli organi interni compreso il cuore 7, 8, 9, 10, 11.
La natura dello studio richiedeva la progettazione di un peptide inibitorio. L'approccio software è stato scelto per un approccio tradizionale utilizzando un phage display. L'approccio è il più semplice e meno che richiede tempo. La banca dati RCSB viene utilizzato per identificare opportuni siti di legame 12. Per studiare l'interazione del peptide di nuova concezione con la proteina ricombinante e di concentrarsi sui parametri di legame, è stata utilizzata una tecnica chiamata interferometria biolayer. Biolayer interferometria è un biosensore basato technique che determina affinità di legame, di associazione e dissociazione utilizzando un biosensore e un campione vincolante. Il biosensore può essere fluorescente, luminescently, radiometricamente e colorimetricamente etichettato. La misura si basa sulla aggiunta di massa o l'esaurimento simile associazione e dissociazione 13, 14. Lo scopo di questo studio è stato quello di comprendere il ruolo di IRF5 in infiammazione del miocardio e fibrosi. L'obiettivo era quello di ottenere una conoscenza approfondita del ruolo della IRF5 nello sviluppo della fibrosi dei tessuti e la sclerodermia.
Protocol
Representative Results
Discussion
L'obiettivo era quello di progettare un inibitore IRF5 per chiarire il ruolo di IRF5 sulla infiammazione, la fibrosi e la funzione vascolare nei cuori di Tsk / + topi. I risultati sono che IRF5D non ha indotto la proliferazione o l'apoptosi. Inoltre, l'infiammazione si è ridotta e la funzione vascolare migliorata. Questi dati suggeriscono che IRF5 svolge un importante ruolo meccanicistica nello sviluppo di infiammazione e fibrosi nel cuore di Tsk / + topi e che ha il potenziale per servire come un obiettivo…
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 |
References
- Bi, X., et al. Loss of interferon regulatory factor 5 (IRF5) expression in human ductal carcinoma correlates with disease stage and contributes to metastasis. Breast Cancer Res. 13 (6), 111 (2011).
- Dideberg, V., et al. An insertion-deletion polymorphism in the interferon regulatory Factor 5 (IRF5) gene confers risk of inflammatory bowel diseases. Hum Mol Genet. 16 (24), 3008-3016 (2007).
- Graham, R. R., et al. A common haplotype of interferon regulatory factor 5 (IRF5) regulates splicing and expression and is associated with increased risk of systemic lupus erythematosus. Nat.Genet. 38 (5), 550-555 (2006).
- Krausgruber, T., et al. IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses. Nat. Immunol. 12 (3), 231-238 (2011).
- Eames, H. L., Corbin, A. L., Udalova, I. A. Interferon regulatory factor 5 in human autoimmunity and murine models of autoimmune disease. Transl Res. 167 (1), 167-182 (2016).
- Mayes, M. D., et al. Immunochip analysis identifies multiple susceptibility Loci for systemic sclerosis. Am J Hum Genet. 94 (1), 47-61 (2014).
- Dimitroulas, T., et al. Micro-and Macrovascular Treatment Targets in Scleroderma Heart Disease. Curr Pharm Des. , (2013).
- Botstein, G. R., LeRoy, E. C. Primary heart disease in systemic sclerosis (scleroderma): advances in clinical and pathologic features, pathogenesis, and new therapeutic approaches. Am Heart J. 102 (5), 913-919 (1981).
- Oram, S., Stokes, W. The heart in scleroderma. Br Heart J. 23 (3), 243-259 (1961).
- Xu, H., et al. 4F decreases IRF5 expression and activation in hearts of tight-skin mice. PLoS One. 7 (12), 52046 (2012).
- Steen, V. The heart in systemic sclerosis. Curr.Rheumatol.Rep. 6 (2), 137-140 (2004).
- Deshpande, N., et al. The RCSB Protein Data Bank: a redesigned query system and relational database based on the mmCIF schema. Nucleic Acids Res. 33, D233-D237 (2005).
- Concepcion, J., et al. Label-free detection of biomolecular interactions using biolayer interferometry for kinetic characterization. Comb Chem High Throughput Screen. 12 (8), 791-800 (2009).
- Matthew, A. Current biosensor technologies in drug discovery. Drug Discovery. , 69 (2006).
- Doppelt-Azeroual, O., Moriaud, F., Adcock, S. A., Delfaud, F. A review of MED-SuMo applications. Infect Disord Drug Targets. 9 (3), 344-357 (2009).
- Kim, S., Jang, J., Yu, J., Chang, J. Single mucosal immunization of recombinant adenovirus-based vaccine expressing F1 protein fragment induces protective mucosal immunity against respiratory syncytial virus infection. Vaccine. 28 (22), 3801-3808 (2010).
- Frenzel, D., Willbold, D. Kinetic Titration Series with Biolayer Interferometry. PloS one. 9 (9), 106882 (2014).
- Ou, J., et al. L-4F, an apolipoprotein A-1 mimetic, dramatically improves vasodilation in hypercholesterolemia and sickle cell disease. Circulation. 107 (18), 2337-2341 (2003).
- Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal.Biochem. 72 (1-2), 248-254 (1976).
- Bauer, P. M., et al. Compensatory phosphorylation and protein-protein interactions revealed by loss of function and gain of function mutants of multiple serine phosphorylation sites in endothelial nitric-oxide synthase. J.Biol.Chem. 278 (17), 14841-14849 (2003).
- Weihrauch, D., et al. An IRF5 Decoy Peptide Reduces Myocardial Inflammation and Fibrosis and Improves Endothelial Cell Function in Tight-Skin Mice. PLoS One. 11 (4), 0151999 (2016).
- Hoogenboom, H. R., et al. Antibody phage display technology and its applications. Immunotechnology. 4 (1), 1-20 (1998).
- Roehm, N. W., Rodgers, G. H., Hatfield, S. M., Glasebrook, A. L. An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. J Immunol Methods. 142 (2), 257-265 (1991).
- Van Tonder, A., Joubert, A. M., Cromarty, A. D. Limitations of the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays. BMC Res Notes. 8 (1), 1 (2015).
- Ott, H. C., et al. Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat Med. 14 (2), 213-221 (2008).
- Ou, J., et al. L-4F, an apolipoprotein A-1 mimetic, dramatically improves vasodilation in hypercholesterolemia and sickle cell disease. Circulation. 107 (18), 2337-2341 (2003).
- Weihrauch, D., et al. Effects of D-4F on vasodilation, oxidative stress, angiostatin, myocardial inflammation, and angiogenic potential in tight-skin mice. Am J Physiol Heart Circ Physiol. 293 (3), 1432-1441 (2007).
- Roy, S., P, P., Mavragani, C. IRF-5 – A New Link to Autoimmune Diseases. Autoimmune Disorders – Pathogenetic Aspects. , 35 (2011).
