Adult female Yorkshire swine weighing 50–60 kg were made diabetic by intravenous administration of streptozotocin (STZ, Teva Pharmaceuticals, Petah Tikva, Israel) at a dose of 150 mg/kg as described . Blood glucose levels were obtained twice daily using a OneTouch UltraMini glucometer (LifeScan, Inc., Milpitas, CA), with diabetes confirmed by glucose levels greater than 200 mg/dL; insulin was administered twice daily on a sliding scale to maintain levels between 250 and 600 mg/dL, to model poor glycemic control.
Full thickness excisional wounding and post-injury treatments
Following 3–6 weeks of diabetic induction, animals underwent Tufts University IACUC-approved dorsal wounding surgeries: after anesthesia induction with a telazol/ketamine/xylazine cocktail, dorsal hair was removed with depilatory cream, followed by shaving. Under general anesthesia with isoflurane, the paraspinal area was prepared and draped sterilely, and full-thickness (5.0 mm deep) excisional wounds were created in the animals’ backs using sterile, stainless steel biopsy punches (8.0 mm-diameter) or a sterile No. 11 surgical blade (2.0 cm × 2.0 cm square wounds). Following hemostasis by manual compression, wounds were covered using 10 % compound tincture of benzoin adhesive and Tegaderm transparent dressing (3 M, St. Paul, MN) and animals were placed in protective jackets. Comb1 (1.0 mg/mL) and UN3 (284 μg/mL) peptides (synthesized by Anaspec, Fremont, CA) were suspended in autoclaved 4 % carboxymethylcelluose and administered to wounds daily by injection beneath the Tegaderm bandage, for a period of 3–7 days. Sterile saline served as a negative control.
Immediately following euthanasia, dorsal wounds, including the underlying subcutaneous tissue and fascia, together with an approximately 1 cm perimeter of uninjured cutaneous tissue bordering the wound, were harvested. Wounds were bisected using a sterile surgical blade. Half of each wound was fixed in buffered 4 % formaldehyde and reserved for histology. A 1 mm-thick section was removed from the remaining half of each wound, partitioned into granulation tissue, hypodermis, and adjacent, non-wounded dermis and epidermis using a sterile, stainless steel razor blade, and each piece of tissue was placed in RNAlater RNA stabilization reagent (Qiagen, Venlo, NL). The remaining tissues were placed in large cryovials, snap-frozen in liquid nitrogen, and reserved for immunohistochemistry.
Quantitative histology and immunohistochemistry of healing responses
Histology, immunohistochemistry, and microscopy of fixed, paraffin-embedded tissues
After fixation in buffered formaldehyde for 72 h, wounds were embedded in paraffin, cut into 5 μm-thick sections, and stained with hematoxylin and eosin (H&E) or Masson’s trichrome. Microscopic imaging of H&E and trichrome-stained sections was performed as described .
Immunhistochemistry and microscopy of fresh frozen tissues
Flash frozen pieces of diabetic porcine wounds were re-equilibrated in PBS-buffered 0.5 % formaldehyde and 15 % sucrose for 2 days at 4C, followed by 2 days in PBS-buffered 0.5 % formaldehyde and 30 % sucrose, and embedded in Tissue-Tek OCT compound (VWR, Radnor, PA). 18 μm-thick sections were cut and placed on Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA). Fluorescent immunohistochemistry using mouse anti-CD31 (1:200, Novus Biologicals, Littleton, CO) and rabbit anti-HSPG (1:200), and subsequent microscopy were performed as described .
Labeling tissue with FITC-conjugated Comb1
18 μm-thick sections of fresh frozen saline-treated porcine wounds were blocked with PBS containing 5 % normal goat serum (NGS) for 1 h at room temperature in a humidified chamber, then incubated with 100 nM FITC-Comb1 in 5 % NGS, protected from light. Fluorescent immunohistochemistry using rabbit anti-FITC primary antibody (1:100, Life Technologies) and subsequent microscopy were performed using methods described . Slides incubated with 5 % NGS alone served as negative controls.
Quantitative analysis of wound healing responses
Composite images of H&E, trichrome, and immuno-labeled wounds were created as described . Percent wound closure was determined by dividing the total linear distance of keratinocyte migration from the wound edge by the total width of each wound, each measured using the NIH ImageJ freehand line tool. Angiogenic responses were quantified from merged immunofluorescence images: total wounded area, defined as the histologically apparent interruption in normal skin morphology, and granulation tissue area, defined as the area with dual positivity for CD31 and HSPG, were quantified using the NIH ImageJ freehand tool, and the percentage of granulation tissue occupying the wound was calculated; microvascular density within the granulation tissue was calculated by measuring CD31/HSPG co-localization intensity using NIH ImageJ , and normalizing against the granulation tissue area.
Peptide binding studies
Primary adult HMVEC (Lonza Bioscience, Walkersville, MD) were cultured in Endothelial Basal Medium-2 (Lonza) supplemented with 5 % fetal bovine serum (FBS) and the growth factors contained within the EGM2-MV kit (Lonza), according to the manufacturer’s instructions. HFF and HaCaT (gifts from the laboratory of Dr. Jonathan Garlick, Tufts) were grown in DMEM supplemented with 10 % FBS (Atlanta Biologicals, Atlanta, GA), 1 % antibiotic–antimycotic, 1 % l-glutamine, and 10 mM HEPES (Life Technologies). HMVEC were used at P5–8, HFF were used at P14–22, and HaCaT were used at P40–46.
Cellular responses to injury in vitro
Cells were seeded in 6-well plates (VWR) and allowed to grow to confluence. 7–10 days later, concentric scratch wounds were created in post-confluent monolayers using stainless steel rakes that uniformly denude ~50 % of the resting population in each well . Denuded cells (time 0) were collected, lysed in RIPA buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO), and mechanically disrupted by dounce homogenization on ice; 24 h after injury, the migrating cell populations were lysed and homogenized in the same manner. Equal amounts of protein from these homogenates were mixed with reducing sample buffer containing a final concentration of 2 % β-mercaptoethanol (Sigma-Aldrich), heated to 95 °C for 5 min, and separated by SDS-PAGE.
Peptide blot overlay
After SDS-PAGE, protein samples were transferred to Whatman Protran 0.1 μm pore size nitrocellulose membranes (GE Healthcare, Little Chalfont, UK) and blocked for 1 h at room temperature with a protein-free blocking solution (Life Technologies). Membranes were then overlaid with protein-free blocking solution (Life Technologies) containing FITC-conjugated Comb1 peptide (200 nM, synthesized by Biomatik, Cambridge, ON) and incubated for 1 hour at room temperature with end-over-end rotation, protected from light. Membranes were washed three times in PBS, and peptides were cross-linked to putative receptors using 2 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Life Technologies) and 2 mM N-hydroxysuccinimide (NHS, Life Technologies) for 15 min at room temperature. Cross-linking was quenched by addition of three volumes of 200 mM glycine, 20 mM Tris–HCl, pH 7.5, for 3 min at room temperature. Membranes were washed in TBS and blocked overnight at 4 °C in protein-free blocking solution. Western blotting was performed using goat anti-FITC (1:100, Life Technologies) overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated donkey anti-goat secondary antibody (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA), and developed using Clarity ECL western blotting substrate (BioRad, Hercules, CA). Digital imaging of Western blots was performed using a BioSpectrum Imaging System with VisionWorks software (UVP, Upland, CA).
Quantitative molecular analyses
Total RNA was isolated from compartments of diabetic porcine wounds (adjacent, unwounded dermis and epidermis, hypodermis, and granulation tissue) by homogenization in TRIzol (Life Technologies) and purification with RNEasy mini columns (Qiagen).
Quantitative real-time PCR
One microgram RNA was reverse transcribed using the Quantitect Reverse Transcription kit (Qiagen). Reactions containing 5 ng cDNA were prepared in duplicate using Taqman probes and Taqman Gene Expression Master Mix (Life Technologies) according to the manufacturer’s instructions, and PCR was performed using an iCycler iQ5 real-time PCR detection system (BioRad). Relative mRNA expression was determined following normalization to beta-actin or RPL32 using the 2−∆∆Ct method.
Data analysis and statistics
Excisional wounding experiments were performed a total of seven times; 7-days peptide treatments were performed five times, and 3-days peptide treatments were performed twice. In all quantitative analyses, mean values were compared for each treatment group on day 3 and 7 post-wounding, and all data are expressed as fold change relative to saline controls on each day ± SEM. All statistical analyses were performed using an unpaired Student’s t test. Peptide blot overlays were performed at least three times.