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 substituted for serine (S) to mimic phosphorylation in IRF5 at 421 - 438 (ELSWSADSIRLQISNPD). Analysis of 3D sulfhydryl groups and knowledge of IRF5&#39;s mechanism of activation, i.e., serine phosphorylation, suggested that one strategy for targeting IRF5 was to generate a decoy peptide. See Figure 1. To identify the best region in the 3D structure, use a molecular modeling software for designing small molecules and peptides15. 
	NOTE: The critical step in this process is the availability of the 3D structure of the target protein and its possible binding sites. The 3D structure of IRF5 accurately represents how IRF5 dimerizes after phosphorylation. The substitution of aspartate for serine or threonine is outsourced. The peptide is synthesized separately without being linked to IRF5.</li>
	<li>Replicate the phospho-domain simply by substituting D for S. Based on the original sequence (ELSWSADSIRLQISNPD, make a new peptide IRF5S with the amino acid sequence of Ac-ELDWDADDIRLQIDNPD-NH2 (IRF5D).</li>
</ol>
2. Biolayer Interferometry (BLI)
NOTE: The purification for recombinant IRF5 was outsourced.16
<ol>
	<li>Biotin label IRF5 in a molar ratio of 1:1 of biotin to ligand with a biotinylation reagent incorporating a long-chain linker. The long chain linker will ensure a more active and homogenous ligand surface.
	<ol>
		<li>Add 1 mL of 2 mg/mL IRF5 to 13 &#181;L of 20 mM biotin reagent or increase the volumes if needed. Incubate on ice for 2 h. After labeling, use a desalting spin column to remove the reaction mixture from the labeled protein.</li>
	</ol>
	</li>
	<li>Incubate the biotin-labeled ligand with the biosensors for 15 min. Avoid over-labeling and adjust the 1:1 molar ratio. After labeling, use a desalting spin column to remove the reaction mixture from the labeled protein.</li>
	<li>Incubate the biotin labeled IRF5 biosensors for 10 min in different concentrations of the IRF5 decoy peptide. The association and dissociation rates were determined as described10,17,18.</li>
</ol>
3. Apoptosis and Proliferation Assays
<ol>
	<li>Proliferation assay via bioreduction of the tetrazolium
	<ol>
		<li>Administer IRF5D diluted in PBS (1 mg/kg/d, inject a volume 20 &#181;L in a 20 g mouse) subcutaneously with a 27-gauge needle by a standard scruffing technique or by PBS alone to Tsk/+ and C57Bl/6J mice for 21 days.</li>
		<li>Anesthetize the mice with isoflurane (5%) and perform a cervical dislocation. Excise the hearts by cutting open the chest along the sternum. Lift up the heart with forceps and remove the heart.</li>
		<li>Lyse the hearts with a protein lysis buffer containing 50 mM Tris-HCl pH 7.4, 0.5% NP-40, 250 mM NaCl, 5 mM EDTA, 50 mM NaF10 and determine protein concentration by Bradford assay19.</li>
		<li>Coat 96-well plates with C57Bl/6J cardiac matrix isolated as described in section 7. Dilute the PBS suspended C57Bl/6J cardiac matrix to a concentration of 20 &#181;g/mL and add 30 &#181;L of the C57Bl/6J cardiac to each well. Let it settle overnight at 37 &#176;C in the incubator.</li>
		<li>Culture human umbilical vein cells using 500 mL of endothelial basal media with 0.4% bovine brain extract, 0.1% ascorbic acid, 0.1% hydrocortisone, 0.1% human epidermal growth factor, 2% fetal bovine serum and 0.1% gentamicin/amphotericin B added to it. Culture the cells on 96 well plates at 5% CO2 and 95% room air with a density of 25,000 cells per well. Stimulate with increasing concentrations of IRF5D between 0 - 100 &#181;g/mL into the media (volumes were between 0 and 50 &#181;L/mL).</li>
		<li>Determine the cell proliferation after three days and assess differences in absorbance on a microplate reader. The reaction is a bioreduction of MTS tetrazolium compound. NADPH or NADH is produced by dehydrogenase enzymes in metabolically active cells.</li>
		<li>As the MTS tetrazolium compound is stored frozen at -20 &#176;C, thaw the compound slowly over approximately 90 minutes at room temperature. Pipette 20 &#181;L into each well of a 96 well plate, and add 100 &#181;L of culture media to it. Incubate plate at 37 &#176;C for 1 to 4 h in a humidified, 5% CO2 chamber.</li>
		<li>Read absorbance at a wavelength of 490 nm.</li>
	</ol>
	</li>
	<li>Apoptosis assay via caspase activity 
	NOTE: The experimental set-up is the same as above 3.1.1 to 3.1.5.
	<ol>
		<li>Stimulate the cells with increasing concentrations of IRF5D between 0 - 100 &#181;g/mL into the media.</li>
		<li>Add green detection to the Caspase 3/7 substrate to the ECs and incubate it for 30 min at 37 &#176;C. Assess the fluorescence on the microplate reader simultaneously with an absorption/emission wavelength of 502/530 nm. Run the experiments in triplicate. 
		NOTE: The green fluorescent agent is a four amino acid peptide bound to a nucleic acid binding dye. This reagent becomes fluorescent when cleaved after caspase 3 and 7 activation. This fluorescence is measured. The advantage is a reduction in wash steps.</li>
	</ol>
	</li>
</ol>
4. Assessment of the IRF5 and ICAM-1 Expression in the Heart
<ol>
	<li>Administer IRF5D diluted in PBS (1 mg/kg/d, inject a volume 20 &#181;L in a 20 g mouse) subcutaneously with a 27-gauge needle by a standard scruffing technique or by PBS alone to Tsk/+ and C57Bl/6J mice once per day for 21 days.</li>
	<li>Anesthetize the mice with isoflurane (5%) and perform a cervical dislocation. Excise the hearts by cutting open the chest along the sternum. Lift up the heart with forceps and remove the heart.</li>
	<li>Lyse the hearts with a protein lysis buffer containing 50 mM Tris-HCl pH 7.4, 0.5% NP-40, 250 mM NaCl, 5 mM EDTA, 50 mM NaF10 and determine protein concentration by Bradford assay19.
	<ol>
		<li>Perform a western blot on lysates of hearts. Dilute the samples to 25 &#181;g total protein and a volume of 40 &#181;L with Laemmli sample buffer containing 5% mercaptoethanol. Run the samples on a 4 - 15% TGX gel at 50 mV for 5 min. Increase voltage to 100 mV for 1 h until the dye front runs out.</li>
		<li>Place the gel in 1x transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol, adjust pH to 8.3 if necessary) and incubate for 10 - 15 min.</li>
		<li>Assemble the transfer sandwich (sponge, filter, gel, cellulose membrane, filter, sponge). Make sure no bubbles are trapped in between. The blot should be on the cathode and the gel on the anode. Transfer for 1 h in the cold room at 100 mA.</li>
		<li>The next day, rinse the blot and then block the background in 5% nonfat dry milk for 30 min at room temperature. Rinse the blot with Tris buffered saline containing 5% Tween three times for 5 min each.</li>
		<li>Incubate overnight with the primary antibodies using primary polyclonal antibodies to IRF5 or ICAM-1. Dilute the antibodies in Tris buffered saline in a 1:1,000 dilution.</li>
		<li>Dilute the secondary antibodies in Tris buffered saline and incubate the blot for 1 h at room temperature. For secondary antibodies, use horseradish peroxidase conjugated donkey anti-mouse IgG (1:10,000) and horseradish peroxidase donkey anti-goat IgG (1:10,000), respectively.</li>
		<li>Apply chemiluminescence to the blot after mixing components according to manufacturer&#39;s protocol and incubate for 3 min to luminesce the bands of interest. Expose the blot to an X-ray film. Use ImageJ to measure density. Normalize the density values to the control bands10.</li>
	</ol>
	</li>
</ol>
5. Assessment of Inflammatory Cell Numbers after IRF5 Inhibition
<ol>
	<li>Administer IRF5D diluted in PBS (1 mg/kg/d) subcutaneously with a 27-gauge needle by a standard scruffing technique or by PBS alone to Tsk/+ and C57Bl/6J mice for 21 days.</li>
	<li>Anesthetize the mice with isoflurane (5%) and perform a cervical dislocation. Excise the hearts by cutting open the chest along the sternum. Lift up the heart with forceps and remove the heart. Snap freeze and store the excised hearts until analysis.</li>
	<li>Mount the frozen hearts on tissue holders appropriate to the cryostat in use. Cut 10 &#181;m thick frozen sections on a cryostat for immunohistochemistry.</li>
	<li>Fix the frozen sections with 1% paraformaldehyde in phosphate buffered saline for 20 min at room temperature. Permeabilize the sections with 0.5% Triton-X 100 in PBS for 5 min. 
	Caution: Paraformaldehyde is a regulated carcinogen and needs to be handled with care.</li>
	<li>Dilute anti-rabbit CD64, a monocyte/macrophage marker 1:200 in PBS. Apply to the fixed sections and incubate for 30 min at 37 &#176;C.</li>
	<li>Wash slides in PBS for 5 min three times at room temperature.</li>
	<li>Dilute anti-rat NIMP, a primary neutrophil marker 1:200 in PBS. Apply to the fixed sections and incubate for 30 min at 37 &#176;C.</li>
	<li>Wash the slides for 5 min three times with PBS at room temperature.</li>
	<li>Dilute the secondary antibodies anti-rabbit Alexa 488 conjugated and anti-rat Alexa 488 conjugated 1:200 in PBS.</li>
	<li>Apply anti-rabbit Alexa 488 conjugated and anti-rat Alexa 488 conjugated secondary antibodies to the sections for 30 min at 37 &#176;C accordingly. The volume of antibody varies according to the size of the section. The amount should cover the section generously. In most cases the volume is between 100 and 200 &#181;L.</li>
	<li>Use 4&#39;,6-diamidino-2-phenylindole (DAPI) as a nuclear stain in a 1:1,000 dilution in PBS. Add 1 &#181;L into 1 mL PBS and use the DAPI in the last wash step. Add aqueous mounting media to a cover slip and put it onto the slide.</li>
	<li>Analyze the fluorescently labeled sections by confocal microscopy using a 40X oil objective. Use excitation wavelengths 488 and emission wavelengths of greater than 530 nm for CD64 and NIMP. DAPI was excited with 360 nm and emitted at 460 nm in blue. Distinguish monocytes/macrophages and neutrophils (n = 10 images per antibody, from 3 different mice in each group) by their different labeling and count them. Express data as number of counted cells.</li>
</ol>
6. Cell Dependent Vasodilation after IRF5 Inhibition
<ol>
	<li>Anesthetize C57Bl/6J control mice and Tsk/+ mice with and without peptide treatment with a ketamine (80 - 100 mg/kg)/xylazine (10 - 12.5 mg/kg) mixture. Inject the ketamine/xylazine mixture intraperitoneally. Restrain the mouse manually.
	<ol>
		<li>Use a 25 gauge or smaller needle. Insert the needle into the lower right quadrant of the abdomen. Withdraw the plunger before injection to ensure that the needle does not enter a blood vessel or the bowel.</li>
	</ol>
	</li>
	<li>Once a sufficient level of anesthesia has been reached, secure the mouse in the supine position, wipe the fur with alcohol soaked gauze pad, and using a pair of scissors open the thoracic cavity and remove the heart. 
	NOTE: Exsanguination will euthanize the mouse and make dissection easier by relieving pressure in the vasculature thus minimizing bleeding when cutting blood vessels.
	<ol>
		<li>Open the skin using a pair of scissors, cutting up from the chest lateral to the jawbone with care as to not disturb the underlying tissues toward the front of the jaw.</li>
		<li>Dissect the facialis artery by removing the soft tissue around it.</li>
		<li>Caution: Do not injure the vessel or tug on facialis artery while keeping the exposed tissue bathed in MOPS buffer (2.0 mM CaCl2&#183;2H2O, 0.02 mM EDTA, 5.0 mM glucose, 4.7 mM KCl, 1.17 mM MgSO4&#183;7H2O, 3.0 mM MOPS, 145 mM NaCl, 1.2 mM NaH2PO4&#183;H2O, 2.0 mM pyruvic acid) to prevent desiccation. Perform under a binocular stereo zoom dissecting microscope (magnification of 3.5X to 45X) and a fiber optic light source.</li>
	</ol>
	</li>
	<li>Isolate the facialis artery.
	<ol>
		<li>Grasp the facialis proximal to the first bifurcation and distal to where the artery will be cut. Cut the artery from the parent artery, the external carotid artery. Remove the facialis arteries from control mice, Tsk/+ mice with and without IRF5D treatment.</li>
		<li>While still holding the artery in the forceps, cut the two branches coming off the facialis, allowing the vessel to be gently lifted while any uncut surrounding tissue can be identified and severed.</li>
		<li>Continue this way until the bifurcation is reached cutting both daughter arteries to free the vessel. There is no need to clamp the vessels prior to cutting due to relieved pressure in the vasculature from removing the heart. Put the vessel in MOPS buffer while the other facialis artery is dissected and isolated.</li>
		<li>Cannulate the vessels by tying them onto glass pipettes with a terminal diameter of 125 - 175 &#181;m. Preload the glass pipettes and buffer reservoirs with MOPS buffer. 
		Caution: Prevent air bubbles from forming in the pipettes.</li>
		<li>Tie the vessels onto the pipettes using ophthalmic sutures.</li>
	</ol>
	</li>
	<li>Mount the vessel chambers on an inverted triocular microscope with a video camera attachment. Run the video through a video caliper measurement system for on screen measurements of the vessels.20
	<ol>
		<li>Pressurize them in a two-step process. First, with the MOPS-filled reservoirs at a height above the vessel equal to 20 mmHg pressure, manually open the stopcock valves in the reservoir lines to the vessel. Inspect the vessel for signs of leaking around the ties and down the length of the vessel. Second, raise the reservoirs to 60 mmHg and reinspect the vessel.</li>
		<li>Once pressurized at 60 mmHg, allow the vessels to equilibrate for 30 min.</li>
	</ol>
	</li>
	<li>In order to assess vasodilation in response to acetylcholine (10-7 to 10-4 M), preconstrict the vessel to a diameter 50 - 75% of its maximum diameter using 1 - 3.0 x 10-8 to 10-7 M U46619. After allowing the vessel to stabilize for 6 min, add acetylcholine to the bath in 2 min duration steps such that the final concentrations in the bath are 10-7, 10-6, 10-5, 10-4. Measure the vasodilation in all three groups.</li>
</ol>
7. Isolation of Cardiac Extracellular Matrix
<ol>
	<li>Anesthetize C57Bl/6J control mice and Tsk/+ mice with and without peptide treatment with isoflurane (5%) and perform a cervical dislocation. Excise the hearts by cutting open the chest along the sternum. Lift up the heart with forceps and remove the heart.</li>
	<li>Put the removed hearts into a beaker and add 15 mL hypertonic 1% sodium dodecyl sulfate (SDS). Agitate hearts in the 1% SDS solution for 18 h at room temperature to break up the cells by adding a stir bar to the 1% SDS solution and putting the beaker on a stir plate. 
	Caution: Be aware that extracellular matrix will disintegrate if left too long in hypertonic solution. Conversely, if the hearts are not agitated long enough in hypertonic solution, then the cells will not properly dissolve and there will be cell debris left in the beaker.</li>
	<li>Wash for 30 min in 15 mL 0.5% Triton X-100. Then wash for 15 min with 15 mL PBS. Decellularize each heart individually.</li>
	<li>Snap-freeze the extracellular matrix in liquid nitrogen and powderize the frozen cardiac extracellular matrix with a pre-cooled mortar and pestle.</li>
	<li>Make a suspension of the pulverized matrix in PBS containing 1% antibiotic. The volume added in most cases is 300 &#181;L. Sonicate the suspension for 30 - 60 s on ice on the high setting. 
	NOTE: It is very important that the sample keeps cold. Be aware that the suspension is very viscous due to the high protein glycan content.</li>
	<li>Determine protein concentration using a Bradford assay. 
	NOTE: Ensure that there is anti-biotic and anti-fungal in the suspension. Penicillin/Streptomycin are the most commonly used and recommended.</li>
</ol>
8. Assessment of Nuclear Translocation by Immunohistochemistry&#160;
<ol>
	<li>To coat dishes and slides use a final concentration of 20 &#181;g/mL of the cardiac extracellular matrix in PBS. Pipet 500 &#181;L of the cardiac extracellular matrix solution into a 100 mm dish for coating and 150 &#181;L of the final extracellular matrix solution onto a slide.</li>
	<li>Inhibit IRF5 by adding IRF5D in a 50 &#181;g/mL concentration for 24 h to rat neonatal cardiac myocytes in culture. Leave control myocyte cultures untreated.</li>
	<li>Assess trafficking of IRF5 from the cytosol to the nucleus using immunofluorescence and analyze by confocal microscopy. Identify the green labeling for IRF5, identify the blue DAPI stain for the nucleus</li>
	<li>Apply the primary anti-IRF5 antibody. The primary antibody was used in a 1:200 dilution in PBS. Incubate the slides for 30 min at 37 &#176;C and follow with three 5-min wash steps. The secondary antibody was Alexa 488-labeled goat anti-mouse IgG antibody.</li>
	<li>Dilute the secondary antibody 1:1,000 dilution in PBS. Incubate the secondary antibody for 30 min at 37 &#176;C. Achieve nuclear stain with DAPI. Dilute DAPI 1:1,000 in PBS. Wash the slides a total of 3 times for 5 min and add DAPI to the third and last wash step.</li>
	<li>Capture trafficking by using confocal microscopy and measure fluorescence intensity in the nuclei and cytosol as previously described10. Identify the green labeling for IRF5, identify the blue DAPI stain for the nucleus. The cells that exhibit blue nuclei with green labeling in them contain activated IRF5, which trafficked from the cytosol to the nucleus. When IRF5 is not activated green labeling is found in the cytosol.</li>
	<li>Analyze the fluorescently labeled cells by confocal microscopy using a 40X oil objective. Use an excitation wavelength of 488 nm and emission wavelengths of greater than 530 nm for IRF5. Use an excitation wavelength of 360 nm and an emission wavelength of 460 nm for visualization of DAPI.</li>
</ol>
9. Statistics
<ol>
	<li>Perform analysis of variance (ANOVA) for repeated measures within and between groups. Follow ANOVA with Bonferroni&#39;s modification of Students t-test. Express data as the standard error of the mean.</li>
</ol>

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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 authors declare that they have no competing financial interests.

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.

<table><tbody><tr><td>&amp;nbsp;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&amp;nbsp;</td><td>Thermo Fisher</td><td>A11001</td><td></td></tr><tr><td>Bardford reagent</td><td>Thermo Fisher</td><td>23200</td><td>Pierce&amp;nbsp;</td></tr><tr><td>Biosensors</td><td>Forte-Bio</td><td>MR18-0009</td><td></td></tr><tr><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&amp;prime;,6-diamidino-2-phenylindole)</td><td>Thermo Fisher</td><td>D-1306</td><td>1:1,000 dilution in PBS</td></tr><tr><td>donkey anti rat Alexa 488</td><td>Thermo Fisher</td><td>A-21208</td><td>1:1,000 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>1:1,000 dilution in PBS</td></tr><tr><td>HRP&amp;nbsp; anti-goat</td><td>Santa Cruz Biotechnologies</td><td>sc-516086</td><td>!:10,000 dilution in TBS</td></tr><tr><td>HRP donkey anti-mouse</td><td>Santa Cruz Biotechnologies</td><td>sc-2315</td><td>1:10,000 dilution in TBS</td></tr><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&amp;nbsp; 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&amp;nbsp;</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>&amp;nbsp;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></tbody></table>

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 ...

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

Research

JoVE Journal

Immunology and Infection

8.0K Views.  Medical College of Wisconsin. 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. 

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

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

Developmental Biology

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Bioengineering

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

Medicine

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Biology

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby the permeability of the endothelium to blood cells, plasma macromolecules, and water can be adapted according to the physiological need. In certain diseases, cytokines and growth factors are released that target the endothelial barrier to transiently increase vascular permeability; however, their prolonged presence may cause chronic vascular hyperpermeability and thereby tissue-damaging edema. The Miles assay is an in vivo technique that allows researchers to study vascular hyperpermeability through the proxy measurement of vascular leakage. Here, we provide a detailed protocol on how to perform this procedure in the mouse, which is the most widely used model organism to study mammalian physiology and pathology. The procedure involves the intravenous injection of Evans blue dye to label the circulating albumin followed by multiple intradermal injections of permeability-inducing agents and vehicle control solutions into opposing flanks of the mouse. Consequently, Evans blue dye gradually leaks into the dermis, where it accumulates and can be extracted for quantification as leakage induced by the permeability-inducing agent relative to the vehicle. The Miles assay can be performed in wild type or genetically modified mouse models and may be combined with drug administration to study molecular mechanisms that regulate vascular permeability and identify agents/targets capable of inducing or blocking hyperpermeability.

Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow increases with decreasing diameter from arteries to arterioles that ultimately feed into capillaries. Due to their smaller size and location in the parenchyma, arterioles have been relatively understudied and with less reproducibility in findings than surface pial arteries. Regardless, arteriolar endothelial cell structure and function&#8212;integral to the physiology and etiology of chronic degenerative diseases&#8212;requires extensive investigation. In particular, emerging evidence demonstrates that compromised endothelial function precedes and exacerbates cognitive impairment and dementia.
In the parenchymal microcirculation, endothelial K+ channel function is the most robust stimulus to finely control the spread of vasodilation to promote increases in blood flow to areas of neuronal activity. This paper illustrates a refined method for freshly isolating intact and electrically coupled endothelial &#34;tubes&#34; (diameter, ~25 &#181;m) from mouse brain parenchymal arterioles. Arteriolar endothelial tubes are secured during physiological conditions (37 &#176;C, pH 7.4) to resolve experimental variables that encompass K+ channel function and their regulation, including intracellular Ca2+ dynamics, changes in membrane potential, and membrane lipid regulation. A distinct technical advantage versus arterial endothelium is the enhanced morphological resolution of cell and organelle (e.g., mitochondria) dimensions, which expands the usefulness of this technique. Healthy cerebral perfusion throughout life entails robust endothelial function in parenchymal arterioles, directly linking blood flow to the fueling of neuronal and glial activity throughout precise anatomical regions of the brain. Thus, it is expected that this method will significantly advance the general knowledge of vascular physiology and neuroscience concerning the healthy and diseased brain.

Intensive preparation of intact mouse cerebral endothelial &#34;tubes&#34; from cerebral parenchymal arterioles is illustrated for studying cerebral blood flow regulation. Further, we demonstrate the experimental strengths of this endothelial study model for fluorescence imaging and electrophysiology measurement of key cellular signaling pathways, including changes in intracellular [Ca2+] and membrane potential.

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow ...

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

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