Generation of Wounds

Wound experiments were performed in 6- to 8-week-old male Sprague-Dawley rats and BALB/c mice. Rats were anesthetized with an intraperitoneal injection of 50/50% ketamine-xylaxine, and an intraperitoneal injection of 2.5% avertin (Sigma-Aldrich) was used for mice. Skin was shaved, cleaned, and disinfected with povidone-iodine (Betadine; Sigma-Aldrich) and 70% alcohol. All animal experiments were approved by the Institutional Animal Care and Use Committee of Burnham Institute for Medical Research.

Two types of injuries were used with patellar tendons. For phage screening in rats, patellar tendons were exposed through small skin incisions placed on the lateral side of the joint, so that the skin wound and tendon wound were not in direct contact with each other. Six longitudinal, full-length incisions were made into the tendon. Full-thickness incision wounds, 1.5 cm in length, were made in skin on the back of the animal. The skin wounds were left uncovered without a dressing. For quantification of phage homing and peptide injections, two size 11 surgical scalpels were placed side-by-side and the central third of the patellar tendon was removed analogous to the graft used in anterior cruciate ligament reconstruction. Achilles tendons were wounded by making four longitudinal, full-length incisions into the tendon. Skin wounds were 8-mm circular, full-thickness excision wounds, made to the skin with a biopsy punch. None of the procedures prevented the animals from bearing weight and moving immediately after the operation and without a noticeable limp.

Phage Libraries, Library Screening, and Individual Phage Clones

The libraries were prepared by using NNK-oligonucleotides encoding a random library of cyclic peptides of the general structure CX₇C (C, cysteine; X, any amino acid). The oligonucleotide mixture was cloned into the T7Select 415-1 vector according to the manufacturer’s instructions (Novagen, Madison, WI). This vector displays peptides in all 415 copies of the phage capsid protein as a C-terminal fusion. Libraries with this structure have yielded numerous high-affinity cell-binding peptides.6,11,16,17

The screening process involved three in vivo selection rounds, performed essentially as described.17 Eight-week-old Sprague-Dawley rats were injected with the library [1 to 5 × 10¹¹ phage in 1.5 ml of phosphate-buffered saline (PBS)] through the tail vein or intracardially and were perfused 12 minutes later through the heart with 1% bovine serum albumin in Dulbecco’s modified Eagle’s medium to remove unbound intravascular phage. The short circulation time focused the screening on the blood vessels because the intact phage cannot readily access extravascular tissue. It also minimized any neutralization or processing of the phage in the tissues. The first in vivo round included 19 animals with both patellar tendon and skin wounds, which were separately pooled. The second round used separate sets of three animals for tendon and skin wound screening, and the third round was performed with one wound of each kind.

The following primers, expressing the indicated peptides, were used to prepare phage: CAR, 5′-AATTCCTGCGCGCGTTCGAAGAATAAGGATTGCTA-3′ and 5′-AGCTTAGCAATCCTTATTCTTCGAACGCGCGCAGG-3′; CRK, 5′-AATTCCTGCCGGAAGGATAAGTGCTA-3′ and 5′-AGCTTAGCACTTATCCTTCCGGCAGG-3′; CAQSNNKDC, 5′-AATTCCTGCGCGCAGTCGAACAATAAGGATTGCTA-3′ and 5′-AGCTTAGCAATCCTTATTGTTCGACTGCGCGCAGG-3′; CAR2, 5′-AATTCCTGCGCTAGGTCT- ACTGCTAAGACTTGCTA-3′ and 5′-AGCTTAGCAAGTCTTAGCAGTAGACCTAGCGCAGG-3′; and CRASKC, 5′-AATTCCTGCCGGGCATCTAAGTGCTA-3′ and 5′-AGCTTAGCACTTAGATGCCCGGCAGG-3′. To test single phage clones in vivo, 1 × 10¹⁰ phage in 1.5 ml of PBS were injected through the tail vein. After the circulation period and perfusion, tissue samples were harvested and weighed. Phage titers per mg of wet tissue were then determined, and the results were expressed as the ratio between the titer of the test clone and nonrecombinant control phage in the same tissue.

Peptides

Peptides were synthesized with an automated peptide synthesizer by using standard solid-phase fluorenylmethoxycarbonyl chemistry. During synthesis, the peptides were labeled with fluorescein using an amino-hexanoic acid spacer as described.18 Each individual fluorescein-conjugated peptide was injected intravenously into the tail vein of rats or mice with wounds. The peptides were allowed to circulate for different periods of time, followed by heart perfusion. Tissues were embedded into OCT (Tissue-Tek; Sakura Finetek U.S.A., Inc., Torrance, CA) and processed for microscopy.

Immunohistochemistry

Frozen tissue sections were fixed in acetone for 10 minutes and incubated with 0.5% blocking reagent for 1 hour (NEN Life Sciences, Boston, MA). Tissue sections were incubated with the primary antibody overnight at 4°C. The following monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were used: rabbit anti-T7-phage affinity-purified pAb (1:100),17 rat anti-mouse CD31 mAb (1:200; BD Pharmingen, San Diego, CA) and rabbit anti-fluorescein isothiocyanate (FITC) pAb (1:200; Invitrogen, Carlsbad, CA). The primary antibodies were detected with labeled secondary antibodies, and each staining experiment included sections stained with species-matched immunoglobulins as negative controls. The sections were washed several times with PBS, mounted in Vectashield mounting medium with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA) and visualized under an inverted fluorescent or light microscope.

Cell Culture

Chinese hamster ovary cells (CHO-K) were obtained from the American Type Culture Collection (Rockville, MD). The pgsA-745 mutant cell line,19 which is derived from CHO-K, was kindly provided by Dr. J. Esko (University of California at San Diego, La Jolla, CA). Cells were maintained in α-minimal essential medium and Earle’s salt supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin sulfate, 100 U of penicillin G/ml, and 292 μg/ml l-glutamine (Invitrogen).

Cell Binding Assays

Cells were detached with 0.5 mmol/L ethylenediaminetetraacetic acid solution (Irvine Scientific, Santa Ana, CA), washed with PBS, and resuspended in 1% bovine serum albumin plus α-minimal essential medium. For the phage binding experiments, ~1 × 10¹⁰ phages were added to 15 ml of culture media containing ~1 × 10⁶ cells in a test tube. The samples were rotated for 2 hours at +4°C. The cells were then washed six times and transferred to a new tube. After a final wash, the cells were counted and cell-bound phage titers were determined. Heparinase treatment of the CHO-K cells was performed using 1.5 IU/ml heparinase I and 1.25 IU/ml heparinase III in serum-free culture media for 2 hours.

Peptide binding to cells was studied essentially as described above for phage. Peptides were tested at 5 μmol/L concentration, with or without 5 × 10⁹ phage. After incubation on ice for 30 minutes, the cells were washed and resuspended with PBS containing 2 μg/ml of propidium iodide (Invitrogen) and analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA).

To study peptide internalization, CHO-K cells or human umbilical vein endothelial cells seeded on plastic coverslips were incubated with 10 μmol/L fluorescein-conjugated peptides for 30 minutes to 72 hours, washed three times with PBS, and fixed with 4% paraformaldehyde for 20 minutes at room temperature. After several washes with PBS, the nuclei were visualized by staining with DAPI, and the slides were mounted with ProLong Gold anti-fade reagent (Invitrogen). The images were acquired using Olympus IX81 inverted and Olympus Fluoview FV1000 confocal microscopes (Olympus, Melville, NY). Z-stack images were taken by confocal microscope every 1 μm through the cells.

Heparin Binding

To measure phage binding to heparin, heparin-coated acrylic beads 10% (v/v) were suspended in 20 mmol/L Na₂HPO₄ buffer, pH 7.2, containing 0.2 mol/L NaCl.20 Approximately 5.0 × 10⁹ phage particles were incubated with the beads for 1 hour at room temperature. The beads were washed, transferred to new tube, and bound phage was eluted with 1.2 mol/L NaCl (pH 7.2) and titrated.20

Sequence Specificity of Wound Homing by CAR and CRK

In the same tendon wound screen that produced the CAR sequence, we identified a peptide with a somewhat related sequence, CARSTKATC (CAR2). We generated a CAR mutant phage by changing two basic amino acids to neutral ones (CARSKNKDC mutated to CAQSNNKDC). Both CAR2 and the mutant phage showed impaired wound-homing properties: the CAR2 phage had ~20% of the homing activity of CAR and the mutant phage was essentially inactive in homing (Supplemental Figure 3, A–C, see http://ajp.amjpathol.org). We also generated a mutant CRK phage by changing two amino acids (from CRKDKC to CRASKC). The mutant phage had also almost completely lost the homing ability (Supplemental Figure 3, A–C, see http://ajp.amjpathol.org). The loss of activity as a result of the sequence changes emphasizes the role of the basic amino acids in the homing activity and attests to the specificity of the homing.

Synthetic CAR and CRK Peptides Accumulate in Wounds

We synthesized the CAR and CRK peptides as fluorescein (FITC) conjugates and tested their tissue distribution after intravenous injection to mice and rats with tendon and skin wounds. Both peptides produced strong fluorescent signal in the wounds that partially co-localized with CD31-positive cells at 4 and 8 hours after the injection (Figures 2 and 3). CAR2, even though slightly active at the phage-homing level, produced no accumulated fluorescence, and an unrelated five amino acid control peptide (KAREC) also gave no detectable signal in the wounds. Because of an autofluorescent background in wounds, the fluorescein-labeled peptides were also detected with an anti-FITC antibody, and the signal was thus amplified and converted to a nonfluorescent dye. The antibody staining confirmed the localization of the fluorescein label in the granulation tissue, particularly at later time points when the fluorescent signal was weak. The fluorescent signal produced by CAR and CRK in nontumor tissues (liver, kidney, lung, and spleen) did not differ from that of the fluorescein-labeled CAR2 or KAREC peptides used as controls (Supplemental Figure 4A, see http://ajp.amjpathol.org). In agreement with the phage results, no CAR or CRK peptide accumulated in normal skin (Supplemental Figure 4B, see http://ajp.amjpathol.org) or tendons (not shown).

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Figure 2

CAR peptide homes to blood vessels and granulation tissue in tendon and skin wounds. Fluorescein-conjugated peptides CAR (A, C, and E) or CAR2 (B, D, and F) were intravenously injected into rats (A–D) or mice (E and F) with 5-day-old wounds in Achilles tendons (A–D) or skin (E and F). Wound tissue was collected 4 hours later, which allowed the peptide to penetrate into the tissue, and examined for the presence of the peptides. In A–D, rabbit anti-FITC followed by FITC-conjugated (A and B) or biotin-conjugated anti-rabbit IgG and streptavidin peroxidase (C and D; brown), were used to detect the signal from the fluorescein-labeled peptide. Blood vessels were stained with CD-31 antibody (magenta in C and D) and the nuclei were stained with DAPI (blue). Original magnifications, ×200.

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Figure 3

CRK peptide homes to blood vessels and granulation tissue in wounds. Fluorescein-conjugated peptides CRK (A, C, and E) or KAREC (control peptide: B, D, and F), were intravenously injected as in Figure 2 into rats (A and B) or mice (C–F) with 5-day-old wounds in the skin (A–D) or patellar tendons (E and F). The peptides, blood vessels, and nuclei were detected as in Figure 2. Original magnifications, ×200.

Healing Stage-Dependent Changes in Phage Homing

To determine the extent our wound-homing peptides would home to wounds at times other than the 5-day time point, we injected individual phage clones at days 7, 10, and 14, after wounding. The total number of homing phage rescued from the wounds decreased by two orders of magnitude as wound healing progressed (not shown), presumably reflecting the pruning that occurs in wound vasculature during the maturation of granulation tissue into scar tissue.3 However, comparison with control phage revealed selective homing even at the late (day 14) stages of wound healing (Figure 4). The CRK phage, which homed less strongly than CAR on day 5, was generally the more efficient homing peptide at the later stages of wound healing. Phage staining showed that both phage clones co-localized with blood vessels at all stages in wound healing (Supplemental Figures 1 and 2, see http://ajp.amjpathol.org), and in agreement with the phage homing data, little CAR phage was detected in 14-day wounds, whereas the CRK phage still gave relatively strong staining at this stage (Supplemental Figure 5, see http://ajp.amjpathol.org). The co-localization of the phage staining indicates that the molecular change reflected in the relative homing of CAR and CRK resides in the vasculature and that this change is related to the maturation of the vasculature in wounds.

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Figure 4

Healing stage dependence of phage homing to wounds. CAR phage (filled bars) or CRK phage (open bars) was intravenously injected into rats 5, 7, 10, and 14 days after wounding. The phage was recovered 12 minutes after the injection and quantified. Results are expressed as fold titers relative to that of similarly injected nonrecombinant T7 phage after adjusting the output to the wet weight of the tissue samples. A: Patellar tendon wounds; B: Achilles tendon wounds; C: skin wounds. The CAR and CRK phage homing shows a different healing time dependence. Error bars represent mean ± SEM for 11, four, seven, and five separate experiments at each time point. *P < 0.05, **P < 0.01, ***P < 0.001 versus nonrecombinant T7 phage, two-way analysis of variance.

Cell Surface Heparan Sulfate as the Target Molecule for CAR

The CAR sequence homology with the heparin-binding site of BMP4 and the presence in CRK of a classical heparin-binding motif (XBBXBX; where X denotes any amino acid and B basic residue), suggested that a particular form of a glycosaminoglycan, probably a heparan sulfate, might serve as the binding site for one or both of the peptides. We used Chinese hamster ovary cells (CHO-K) and the pgsA-745 mutant CHO line that is defective in glycosaminoglycan biosynthesis19 to test the binding of CAR and CRK to cell surface glycosaminoglycans. CAR phage bound to the CHO-K cells 55-fold more than nonrecombinant control phage (P < 0.0001), but there was no specific binding to the glycosaminoglycan-deficient cells (Figure 5A). The CRK phage did not bind significantly to either cell line (Figure 5A). Pretreatment of the CHO-K cells with heparinase I and III decreased the binding of the CAR phage by almost 80% (P < 0.0001; Figure 5B). The CAR phage also bound to heparin-coated beads; 70-fold more CAR phage than nonrecombinant control phage was recovered from the beads (P < 0.0001; Figure 5C). CRK phage binding was only marginally higher than that of the control phage.

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Figure 5

Binding of CAR phage to cell surface heparan sulfate and heparin. A: CAR phage (filled bars) specifically binds to CHO-K cells, but not to the glycosaminoglycan-deficient pgsA-745 cells. CRK phage (open bars) does not bind significantly to either cell line. B: Heparinase treatment of the CHO-K cells (gray bar) suppresses the binding of the CAR phage to these cells. C: CAR phage, but not CRK phage, binds to heparin-coated beads. Error bars represent mean ± SEM for three or more separate experiments performed in duplicate. *P < 0.05; ***P < 0.001. A and B: Two-way analysis of variance; C: unpaired Student’s t-test. Brackets indicate the difference between CAR and CRK phages.

Fluorescence-activated cell sorting analysis also revealed strong binding of the synthetic CAR peptide to the CHO-K cells, but not to the pgsA-745 cells. An excess of unlabeled CAR peptide inhibited the binding in a dose-dependent manner. The binding could also be inhibited with the CAR phage. The CAR2 peptide bound only weakly to the CHO-K cells, and KAREC showed no binding to either the CHO-K or pgsA-745 cells (data not shown). These results indicate that CAR, but not CRK, has an active heparin-binding site, and that CAR binds to glycosaminoglycan moiety in cell surface heparan sulfate proteoglycans.

We thank Drs. Teppo Järvinen, Hannele Uusitalo-Järvinen, and Eva Engvall for comments on the manuscript; Dr. Yoav Altman for help with fluorescence-activated cell sorting analysis; and Robin Newlin, Kang Liu, Buddy and Adriana Charbono, and Kelly McKaig for providing excellent technical assistance.