Name: vasodilation of isolated vessels and the isolation of the extracellular matrix of tight-skin mice
Uploaded: 2017-03-24T13:00:00
Description: Watch this Scientific Journal Video about Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice at JoVE.com

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.

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (Protocol: AUA#1517). All research involving mice was conducted in conformity with PHS policy.
1. Design of Decoy Peptide
<ol>
	<li>Find the IRF5&#39;s 3D structure and base the design on it. Design a 17 mer, termed IRF5D (ELDWDADDIRLQIDNPD), where aspartate (D) is substit...

<ol>
	<li>Bi, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+interferon+regulatory+factor+5+(IRF5)+expression+in+human+ductal+carcinoma+correlates+with+disease+stage+and+contributes+to+metastasis.">Loss of interferon regulatory factor 5 (IRF5) expression in human ductal carcinoma correlates with disease stage and contributes to metastasis.</a> Breast Cancer Res. 13 (6), 111 (2011).</li><li>Dideberg, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+insertion-deletion+polymorphism+in+the+interferon+regulatory+Factor+5+(IRF5)+gene+confers+risk+of+inflammatory+bowel+diseases.">An insertion-deletion polymorphism in the interferon regulatory Factor 5 (IRF5) gene confers risk of inflammatory bowel diseases.</a> Hum Mol Genet. 16 (24), 3008-3016 (2007).</li><li>Graham, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+haplotype+of+interferon+regulatory+factor+5+(IRF5)+regulates+splicing+and+expression+and+is+associated+with+increased+risk+of+systemic+lupus+erythematosus.">A common haplotype of interferon regulatory factor 5 (IRF5) regulates splicing and expression and is associated with increased risk of systemic lupus erythematosus.</a> Nat.Genet. 38 (5), 550-555 (2006).</li><li>Krausgruber, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRF5+promotes+inflammatory+macrophage+polarization+and+TH1-TH17+responses.">IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses.</a> Nat. Immunol. 12 (3), 231-238 (2011).</li><li>Eames, H. L., Corbin, A. L., Udalova, I. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochip+analysis+identifies+multiple+susceptibility+Loci+for+systemic+sclerosis.">Immunochip analysis identifies multiple susceptibility Loci for systemic sclerosis.</a> Am J Hum Genet. 94 (1), 47-61 (2014).</li><li>Dimitroulas, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-and+Macrovascular+Treatment+Targets+in+Scleroderma+Heart+Disease.">Micro-and Macrovascular Treatment Targets in Scleroderma Heart Disease.</a> Curr Pharm Des. , (2013).</li><li>Botstein, G. R., LeRoy, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+heart+disease+in+systemic+sclerosis+(scleroderma):+advances+in+clinical+and+pathologic+features,+pathogenesis,+and+new+therapeutic+approaches.">Primary heart disease in systemic sclerosis (scleroderma): advances in clinical and pathologic features, pathogenesis, and new therapeutic approaches.</a> Am Heart J. 102 (5), 913-919 (1981).</li><li>Oram, S., Stokes, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heart+in+scleroderma.">The heart in scleroderma.</a> Br Heart J. 23 (3), 243-259 (1961).</li><li>Xu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4F+decreases+IRF5+expression+and+activation+in+hearts+of+tight-skin+mice.">4F decreases IRF5 expression and activation in hearts of tight-skin mice.</a> PLoS One. 7 (12), 52046 (2012).</li><li>Steen, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heart+in+systemic+sclerosis.">The heart in systemic sclerosis.</a> Curr.Rheumatol.Rep. 6 (2), 137-140 (2004).</li><li>Deshpande, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RCSB+Protein+Data+Bank:+a+redesigned+query+system+and+relational+database+based+on+the+mmCIF+schema.">The RCSB Protein Data Bank: a redesigned query system and relational database based on the mmCIF schema.</a> Nucleic Acids Res. 33, D233-D237 (2005).</li><li>Concepcion, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+detection+of+biomolecular+interactions+using+biolayer+interferometry+for+kinetic+characterization.">Label-free detection of biomolecular interactions using biolayer interferometry for kinetic characterization.</a> Comb Chem High Throughput Screen. 12 (8), 791-800 (2009).</li><li>Matthew, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+biosensor+technologies+in+drug+discovery.">Current biosensor technologies in drug discovery.</a> Drug Discovery. , 69 (2006).</li><li>Doppelt-Azeroual, O., Moriaud, F., Adcock, S. A., Delfaud, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+MED-SuMo+applications.">A review of MED-SuMo applications.</a> Infect Disord Drug Targets. 9 (3), 344-357 (2009).</li><li>Kim, S., Jang, J., Yu, J., Chang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+mucosal+immunization+of+recombinant+adenovirus-based+vaccine+expressing+F1+protein+fragment+induces+protective+mucosal+immunity+against+respiratory+syncytial+virus+infection.">Single mucosal immunization of recombinant adenovirus-based vaccine expressing F1 protein fragment induces protective mucosal immunity against respiratory syncytial virus infection.</a> Vaccine. 28 (22), 3801-3808 (2010).</li><li>Frenzel, D., Willbold, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+Titration+Series+with+Biolayer+Interferometry.">Kinetic Titration Series with Biolayer Interferometry.</a> PloS one. 9 (9), 106882 (2014).</li><li>Ou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-4F,+an+apolipoprotein+A-1+mimetic,+dramatically+improves+vasodilation+in+hypercholesterolemia+and+sickle+cell+disease.">L-4F, an apolipoprotein A-1 mimetic, dramatically improves vasodilation in hypercholesterolemia and sickle cell disease.</a> Circulation. 107 (18), 2337-2341 (2003).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal.Biochem. 72 (1-2), 248-254 (1976).</li><li>Bauer, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> J.Biol.Chem. 278 (17), 14841-14849 (2003).</li><li>Weihrauch, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+IRF5+Decoy+Peptide+Reduces+Myocardial+Inflammation+and+Fibrosis+and+Improves+Endothelial+Cell+Function+in+Tight-Skin+Mice.">An IRF5 Decoy Peptide Reduces Myocardial Inflammation and Fibrosis and Improves Endothelial Cell Function in Tight-Skin Mice.</a> PLoS One. 11 (4), 0151999 (2016).</li><li>Hoogenboom, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+phage+display+technology+and+its+applications.">Antibody phage display technology and its applications.</a> Immunotechnology. 4 (1), 1-20 (1998).</li><li>Roehm, N. W., Rodgers, G. H., Hatfield, S. M., Glasebrook, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+colorimetric+assay+for+cell+proliferation+and+viability+utilizing+the+tetrazolium+salt+XTT.">An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT.</a> J Immunol Methods. 142 (2), 257-265 (1991).</li><li>Van Tonder, A., Joubert, A. M., Cromarty, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> BMC Res Notes. 8 (1), 1 (2015).</li><li>Ott, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat Med. 14 (2), 213-221 (2008).</li><li>Ou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-4F,+an+apolipoprotein+A-1+mimetic,+dramatically+improves+vasodilation+in+hypercholesterolemia+and+sickle+cell+disease.">L-4F, an apolipoprotein A-1 mimetic, dramatically improves vasodilation in hypercholesterolemia and sickle cell disease.</a> Circulation. 107 (18), 2337-2341 (2003).</li><li>Weihrauch, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+D-4F+on+vasodilation,+oxidative+stress,+angiostatin,+myocardial+inflammation,+and+angiogenic+potential+in+tight-skin+mice.">Effects of D-4F on vasodilation, oxidative stress, angiostatin, myocardial inflammation, and angiogenic potential in tight-skin mice.</a> Am J Physiol Heart Circ Physiol. 293 (3), 1432-1441 (2007).</li><li>Roy, S., P, P., Mavragani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRF-5+-+A+New+Link+to+Autoimmune+Diseases.">IRF-5 - A New Link to Autoimmune Diseases.</a> Autoimmune Disorders - Pathogenetic Aspects. , 35 (2011).</li></ol>

The goal was to design an IRF5 inhibitor to elucidate the role of IRF5 on inflammation, fibrosis, and vascular function in the hearts of Tsk/+ mice. The findings are that IRF5D did not induce proliferation or apoptosis. Moreover, inflammation was reduced and vascular function improved. These data suggest that IRF5 plays an important mechanistic role in the development of inflammation and fibrosis in the heart of Tsk/+ mice and that it has the potential to serve as a therapeutic target.
The fir...

Regulation of cell growth and cell death immune responses is central to the role of the transcription factor family of interferon regulatory factors. IRF5 is highlighted as being crucial for the regulation of immune responses between type 1, an inflammatory promoting response and type 2, an immune response targeting tissue repair. IRF5 is key in cancer1, and autoimmunity2,3,4,5.
The tight-skin mouse (Tsk/+) is a model for tissue fibrosis and scleroderma due to a duplication mutation in the fibrillin-1 gene. This mutation results in a tight-skin and an increase in connective tissue. These mice develop myocardial inflammation, fibrosis and finally heart failure5,6,7,8,9. Scleroderma is an autoimmune fibrotic disorder affecting approximately 150,000 patients in the United States6. The hallmarks of this disease are fibrosis of internal organs including the heart7,8,9,10,11.
The nature of the study demanded the design of an inhibitory peptide. The software approach was chosen over a traditional approach using a phage display. The software approach is easier and less time consuming. The RCSB data bank is used to identify appropriate binding sites12. To study the interaction of the newly designed peptide with the recombinant protein and to focus on the binding parameters, a technique called biolayer interferometry was used. Biolayer interferometry is a biosensor based technique that determines binding affinity, association and disassociation using a biosensor and a binding sample. The biosensor can be fluorescently, luminescently, radiometrically and colorimetrically labeled. The measurement is based on mass addition or depletion resembling association and disassociation13,14. The aim of this study was to understand the role of IRF5 in myocardial inflammation and fibrosis. The goal was to gain insight into the role of IRF5 in the development of tissue fibrosis and scleroderma.

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			<td>&#160;Triton X 100</td>
			<td>Sigma Aldrich</td>
			<td>X100- 100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 488-labeled goat anti-mouse IgG antibody&#160;</td>
			<td>Thermo Fisher</td>
			<td>A11001</td>
			<td></td>
		</tr>
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			<td>Bardford reagent</td>
			<td>Thermo Fisher</td>
			<td>23200</td>
			<td>Pierce&#160;</td>
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			<td>Biosensors</td>
			<td>Forte-Bio</td>
			<td>MR18-0009</td>
			<td></td>
		</tr>
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			<td>CD64 (H-250)</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-15364</td>
			<td></td>
		</tr>
		<tr>
			<td>CellEvent Caspase-3/7 Substrate</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>C10427</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter AQueous One Solution Cell Proliferation Assay kit</td>
			<td>Promega</td>
			<td>G3580</td>
			<td>Promega</td>
		</tr>
		<tr>
			<td>DAPI (4&#8242;,6-diamidino-2-phenylindole)</td>
			<td>Thermo Fisher</td>
			<td>D-1306</td>
			<td>1:1000 dilution in PBS</td>
		</tr>
		<tr>
			<td>donkey anti rat Alexa 488</td>
			<td>Thermo Fisher</td>
			<td>A-21208</td>
			<td>1:1000 dilution in PBS</td>
		</tr>
		<tr>
			<td>ECL plus</td>
			<td>GE healthcare/Amersham</td>
			<td>RPN2133</td>
			<td>After a lot of trial and error we came back to this one</td>
		</tr>
		<tr>
			<td>Eclipse TE 200-U microscope with EZ C1 laser scanning software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti rabbit Alexa 488</td>
			<td>Thermo Fisher</td>
			<td>A-11008</td>
			<td>1:1000 dilution in PBS</td>
		</tr>
		<tr>
			<td>HRP&#160; anti-goat</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-516086</td>
			<td>!:10000 dilution in TBS</td>
		</tr>
		<tr>
			<td>HRP donkey anti-mouse</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-2315</td>
			<td>1:10000 dilution in TBS</td>
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		<tr>
			<td>ICAM-1 antibody</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-1511</td>
			<td>1:200 dilution in PBS</td>
		</tr>
		<tr>
			<td>IRF5 antibody (H56)</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-98651</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro plate reader Elx800</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIMP neutrophil marker</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-133821</td>
			<td>1:200 dilution in PBS</td>
		</tr>
		<tr>
			<td>Octet RED</td>
			<td>Forte Bio</td>
			<td></td>
			<td>protein-protein binding</td>
		</tr>
		<tr>
			<td>Peptide design&#160; Medit SA software</td>
			<td>RCSB.org</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant IRF5 protein synthesis</td>
			<td>TopGene Technologies</td>
			<td></td>
			<td>protein expression, synthesis service</td>
		</tr>
		<tr>
			<td>sodium dodecyl phosphate</td>
			<td>Sigma Aldrich</td>
			<td>436143</td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Pharmacy</td>
			<td></td>
			<td>Schedule III controlled substance, presciption required&#160;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>MedVet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3.5X-45X Trinocular Dissecting Zoom Stereo Microscope with Gooseneck LED Lights</td>
			<td>Am Scope</td>
			<td>SKU: SM-1TSX-L6W</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeba Desalting Columns</td>
			<td>Thermofisher</td>
			<td>2161515</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Basal Media EBM Bullet kit</td>
			<td>Lonza</td>
			<td>CC-3124</td>
			<td>kit contains growth supplemets</td>
		</tr>
		<tr>
			<td>VIA-100K</td>
			<td>&#160;Boeckeler Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-15% TGX gel</td>
			<td>Bio-Rad</td>
			<td>5671081</td>
			<td></td>
		</tr>
		<tr>
			<td>MedSuMo software</td>
			<td>Medit, Palaiseau, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli Buffer</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
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vasodilation of isolated vessels and the isolation of the extracellular matrix of tight-skin mice

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 &plusmn; 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 &#181;g/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 &#181;g/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

The results demonstrated in Figure 1 show how to design a peptide. Figure 1, upper left, shows the region (between the 2 yellow arrows, amino acids (aa) 425-436) in IRF5 that is phosphorylated by a number of kinases. Figure 1, upper right, shows a yellow oval where IRF5&#39;s phosphorylated domain binds. The dimeric structure of 3DSH was rotated to observe a cleft or valley to the left of the Helix 2 (aa303-312). This is where the phospho...

Watch this Scientific Journal Video about Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice at JoVE.com

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

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.

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 ...

The overall goal of these procedures for isolation of extracellular matrix and ex vivo vasodilation is to study IRF5 inhibitory peptide treatment in rodents. This method can help answer how changes in the extracellular matrix can augment or attenuate disease progression. The main advantage of the vasodilation studies is to give us an insight into in vivo processes without having to take all the signaling components into consideration.The isolation of cardiac extracellular matrix can be applied to other organs such as hind-limb or lung. We first had the idea when we started to focus on the duplication of fibrillin one in the tight skin mouse, and recognized the impact of altered extracellular matrix and cell behavior. After anesthetizing the mouse and preparing it for surgery, open the skin using a pair of scissors.Cutting up from the chest to lateral to the jawbone with care as to not disturb the underlying tissue towards the front of the jaw. Then dissect the fascialis artery by removing the soft tissue around it. While doing this, be careful to avoid damaging the artery.Bathe the exposed tissue in mops buffer so they remain healthy. Once the fascialis artery has been fully exposed, grasp the fascialis proximal to the first bifurcation distal to where the artery will be cut. Then, cut it away from the parent artery and take hold of the fascialis artery with the forceps.Cut the two branches coming off the fascialis and then gently lift the vessel while severing connections to surrounding tissue until the bifurcation is reached. At the bifurcation, cut both daughter arteries to free the vessel and transfer it into a dish of mops buffer where it will remain hydrated until needed. To measure vasodilation, first prepare to cannulate the vessels.Load glass pipettes with a terminal diameter of 125 to 175 microns full of mops buffer. Also fill solution reservoirs with mops. Now cannulate the isolated vessels.Tie them onto glass pipettes using opthalmic suture. Be sure to prevent air bubbles from forming in the pipettes. Next, mount the vessel chambers on an inverted microscope with the video camera attachment.Run the video through the video caliper measurement system for an on screen measurement of the vessels. Now pressurize the vessels using a two step process. First raise the mops filled reservoirs at a height above the vessel for 20 millimeters of mercury of pressure.Open the line with the mops to the vessel. As the vessel pressurizes look for signs of leaking around the ties and down the length of the vessel. If no leaks are found, raise the reservoirs to produce 60 millimeters of mercury of pressure.Then inspect the vessels for leaks again. Once the pressure is maintained without leaks, equilibrate the vessel for 30 minutes at this pressure. After being equilibrated, measure vasodilation in response to a compound of interest.First constrict the vessel by one half to three quarters by adding U46619. Let the solution bathe the vessels for six minutes to stabilize. Then, gradually add the test compound to the bath solution such as acetylcholine.Every two minutes, increase the concentration of the test solution in the bath by 10 beginning at 000001 molar and progressing to 001 molar. The changes in vasodilation raised questions about signaling. But our focus is on the heart.To reduce the use of animals, we chose to isolate cardiac extracellular matrix to focus on the signaling initiated from the outside. Begin by collecting the heart from an anesthetized mouse. Transfer it to a beaker loaded with 15 milliliters of hypertonic one percent sodium dodecyl sulfate.Keep the solution stirring gently on a stir plate at room temperature for 18 hours. The next day, change the solution to 15 milliliters of 5 percent triton X100 for 30 minutes followed by 15 milliliters of PBS for 15 minutes. Then snap freeze the ECM and liquid nitrogen in a 1.5 milliliter microcentrifuge tube.Once frozen, transfer the tissue into a pre-cooled mortar and pestle and grind it into a powder. Suspend the powder in PBS with added antibiotic at a 1:100 concentration in a viscous solution. Sonicate the suspension for 30 to 60 seconds in an ice bath at a high sonication setting.ECM protein is now isolated and must stay cold. Before using the protein, measure its concentration using a Bradford Assay. The IRF5D peptide is an inhibitory peptide that differs greatly from normal IRF5.IRF5D has only 17 amino acids and binds to IRF5's phosphorylation site. This prevents phosphorylation and homodimerization, thus interfering with the formation of the IRF5 active state. Heterozygous tight skin mice were treated with IRF5D subcutaneously for three weeks.The number of neutrophil and CD64 were decreased as determined by immunohistochemistry in the average number of stained cells per field of view. IRF5D treatments improved acetylcholine induced vasodilation of fascialis arteries in heterozygous tight skin mice treated with IRF5D compared to those treated with phosphate buffer. In cultured myocytes derived from the cardiac tissue of the transgenic mice, there were more IRF5 positive cells in cultures from tight skin heterozygous mice than in control mice.Treatment of either culture with IRF5D resulted in a reduction of IRF5 positive nuclei. After watching this video, you should have a good understanding of how to decellularize a heart. Once mastered, this technique can be transposed to many organs and aid to answer a variety of questions And remember that it is important not to overdigest the matrix.You should also have a good understanding of how to dissect and hang the fascialis artery. Once mastered, this technique should take approximately 60 minutes and can be used on just about any vascular bed. It is important to remember how fragile the endothelium is, proceed with caution during the dissection.It is not uncommon to have healthy smooth muscle and damaged endothelium.

vasodilation-isolated-vessels-isolation-extracellular-matrix-tight

Research

JoVE Journal

Immunology and Infection

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) containing immunologically active molecules. Knowledge regarding the molecular mechanisms of vesicle biogenesis, the content of the vesicles, and their functions at the pathogen-host interface is very limited. Addressing these questions requires rigorous procedures for isolation, purification, and validation of EVs. Previously, vesicle production was found to be enhanced when M. tuberculosis was exposed to iron restriction, a condition encountered by Mtb in the host environment. Presented here is a complete and detailed protocol to isolate and purify EVs from iron-deficient mycobacteria. Quantitative and qualitative methods are applied to validate purified EVs.

Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...

Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to draining lymph nodes (LNs). Interruption of lymph flow is an important method when studying the function of lymphatic vessels. The afferent lymphatic vessels from the murine footpad to the popliteal lymph nodes (pLNs) are well-defined as the only routes for lymph drainage into the pLNs. Suturing these afferent lymphatic vessels can selectively prevent lymph flow to the pLNs. This method allows for interference in lymph flow with minimal damage to the lymphatic endothelial cells in the draining pLN, the afferent lymphatic vessels, as well as other lymphatic vessels around the area. This method has been used to study how lymph impacts high endothelial venules (HEV) and chemokine expression in the LN, and how lymph flows through the adipose tissue surrounding the LN in the absence of functional lymphatic vessels. With the growing recognition of the importance of lymphatic function, this method will have broader applications to further unravel the function of lymphatic vessels in regulating the LN microenvironment and immune responses.

A protocol to block lymph flow by surgical suturing of afferent lymphatic vessels is presented.

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to ...