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

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

1. Find the IRF5'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'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.
2. 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).

2. Biolayer Interferometry (BLI)

NOTE: The purification for recombinant IRF5 was outsourced.¹⁶

1. 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.
   1. Add 1 mL of 2 mg/mL IRF5 to 13 µL 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.
2. 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.
3. 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 described^10,17,18.

3. Apoptosis and Proliferation Assays

1. Proliferation assay via bioreduction of the tetrazolium
   1. Administer IRF5D diluted in PBS (1 mg/kg/d, inject a volume 20 µL 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.
   2. 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.
   3. 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.
   4. 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 µg/mL and add 30 µL of the C57Bl/6J cardiac to each well. Let it settle overnight at 37 °C in the incubator.
   5. 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 µg/mL into the media (volumes were between 0 and 50 µL/mL).
   6. 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.
   7. As the MTS tetrazolium compound is stored frozen at -20 °C, thaw the compound slowly over approximately 90 minutes at room temperature. Pipette 20 µL into each well of a 96 well plate, and add 100 µL of culture media to it. Incubate plate at 37 °C for 1 to 4 h in a humidified, 5% CO₂ chamber.
   8. Read absorbance at a wavelength of 490 nm.
2. Apoptosis assay via caspase activity
   NOTE: The experimental set-up is the same as above 3.1.1 to 3.1.5.
   1. Stimulate the cells with increasing concentrations of IRF5D between 0 – 100 µg/mL into the media.
   2. Add green detection to the Caspase 3/7 substrate to the ECs and incubate it for 30 min at 37 °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.

4. Assessment of the IRF5 and ICAM-1 Expression in the Heart

1. Administer IRF5D diluted in PBS (1 mg/kg/d, inject a volume 20 µL 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.
2. 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.
3. 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.
   1. Perform a western blot on lysates of hearts. Dilute the samples to 25 µg total protein and a volume of 40 µL 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.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. 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.
   7. Apply chemiluminescence to the blot after mixing components according to manufacturer'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.

5. Assessment of Inflammatory Cell Numbers after IRF5 Inhibition

1. 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.
2. 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.
3. Mount the frozen hearts on tissue holders appropriate to the cryostat in use. Cut 10 µm thick frozen sections on a cryostat for immunohistochemistry.
4. 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.
5. Dilute anti-rabbit CD64, a monocyte/macrophage marker 1:200 in PBS. Apply to the fixed sections and incubate for 30 min at 37 °C.
6. Wash slides in PBS for 5 min three times at room temperature.
7. Dilute anti-rat NIMP, a primary neutrophil marker 1:200 in PBS. Apply to the fixed sections and incubate for 30 min at 37 °C.
8. Wash the slides for 5 min three times with PBS at room temperature.
9. Dilute the secondary antibodies anti-rabbit Alexa 488 conjugated and anti-rat Alexa 488 conjugated 1:200 in PBS.
10. Apply anti-rabbit Alexa 488 conjugated and anti-rat Alexa 488 conjugated secondary antibodies to the sections for 30 min at 37 °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 µL.
11. Use 4',6-diamidino-2-phenylindole (DAPI) as a nuclear stain in a 1:1,000 dilution in PBS. Add 1 µL 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.
12. 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.

6. Cell Dependent Vasodilation after IRF5 Inhibition

1. 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.
   1. 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.
2. 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.
   1. 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.
   2. Dissect the facialis artery by removing the soft tissue around it.
   3. Caution: Do not injure the vessel or tug on facialis artery while keeping the exposed tissue bathed in MOPS buffer (2.0 mM CaCl₂·2H₂O, 0.02 mM EDTA, 5.0 mM glucose, 4.7 mM KCl, 1.17 mM MgSO₄·7H₂O, 3.0 mM MOPS, 145 mM NaCl, 1.2 mM NaH₂PO₄·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.
3. Isolate the facialis artery.
   1. 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.
   2. 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.
   3. 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.
   4. Cannulate the vessels by tying them onto glass pipettes with a terminal diameter of 125 – 175 µm. Preload the glass pipettes and buffer reservoirs with MOPS buffer.
      Caution: Prevent air bubbles from forming in the pipettes.
   5. Tie the vessels onto the pipettes using ophthalmic sutures.
4. 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
   1. 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.
   2. Once pressurized at 60 mmHg, allow the vessels to equilibrate for 30 min.
5. 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.

7. Isolation of Cardiac Extracellular Matrix

1. 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.
2. 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.
3. 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.
4. Snap-freeze the extracellular matrix in liquid nitrogen and powderize the frozen cardiac extracellular matrix with a pre-cooled mortar and pestle.
5. Make a suspension of the pulverized matrix in PBS containing 1% antibiotic. The volume added in most cases is 300 µL. 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.
6. 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.

8. Assessment of Nuclear Translocation by Immunohistochemistry 

1. To coat dishes and slides use a final concentration of 20 µg/mL of the cardiac extracellular matrix in PBS. Pipet 500 µL of the cardiac extracellular matrix solution into a 100 mm dish for coating and 150 µL of the final extracellular matrix solution onto a slide.
2. Inhibit IRF5 by adding IRF5D in a 50 µg/mL concentration for 24 h to rat neonatal cardiac myocytes in culture. Leave control myocyte cultures untreated.
3. 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
4. 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 °C and follow with three 5-min wash steps. The secondary antibody was Alexa 488-labeled goat anti-mouse IgG antibody.
5. Dilute the secondary antibody 1:1,000 dilution in PBS. Incubate the secondary antibody for 30 min at 37 °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.
6. 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.
7. 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.

9. Statistics

1. Perform analysis of variance (ANOVA) for repeated measures within and between groups. Follow ANOVA with Bonferroni's modification of Students t-test. Express data as the standard error of the mean.