Original Article

Journal of Investigative Dermatology (1999) 113, 375–383; doi:10.1046/j.1523-1747.1999.00705.x

Adenoviral-Mediated Overexpression of Platelet-Derived Growth Factor-B Corrects Ischemic Impaired Wound Healing

Kenneth W Liechty*, Mark Nesbit†, Meenhard Herlyn†, Antoneta Radu*, N Scott Adzick* and Timothy M Crombleholme*,†

  1. *The Children's Institute for Surgical Science at The Children's Hospital of Philadelphia, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A.
  2. †The Wistar Institute, Philadelphia, Pennsylvania, U.S.A.

Correspondence: Dr Timothy M. Crombleholme, The Children's Institute for Surgical Science, Abramson Pediatric Research Center 1102C, The Children's Hospital of Philadelphia, 34th Street and Civic Center Blvd., Philadelphia, PA 19104, U.S.A. E-mail: Crombleholme@chop.edu

Received 8 September 1998; Revised 29 March 1999; Accepted 28 May 1999.

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Abstract

Chronic wounds represent a major clinical problem with significant morbidity and healthcare expenditures, but no effective therapies. Topical platelet-derived growth factor-BB trials have required large and repeated doses to achieve only a modest effect. We examined the ability of an adenovirus containing the platelet-derived growth factor-B transgene to improve the rate of wound healing through induction of platelet-derived growth factor-B overexpression in cells participating in the wound healing response. We treated excisional wounds in the ischemic rabbit ear, which have a 60% delay in healing, with vehicle, 106, or 108 plaque-forming units of an adenovirus containing the platelet-derived growth factor-B per wound (n = 19). At 7 d this resulted in a decrease in the epithelial gap from 3.4 plusminus 1 mm (mean plusminus SD) in vehicle-treated wounds to 1.9 plusminus 1.8 mm (mean plusminus SD, p < 0.05) when treated with 106 plaque-forming units of an adenovirus containing the platelet-derived growth factor-B, and 0.7 plusminus 1.1 mm (mean plusminus SD, p < 0.001) when treated with 108 plaque-forming units of an adenovirus containing the platelet-derived growth factor-B. Ischemic excisional wounds treated with 108 plaque-forming units of an adenovirus containing the platelet-derived growth factor-B even healed more rapidly than non-ischemic excisional wounds treated with vehicle (p < 0.05). In contrast, 5 mug of platelet-derived growth factor-BB protein (n = 2) resulted in only modest granulation tissue at the margin, but no significant differences in epithelial gap (3 plusminus 0.6 mm, mean plusminus SD). Plaque-forming units (106 or 108) of an adenovirus containing the beta-galactosidase transgene (n = 4) impaired wound re-epithelialization with an epithelial gap of 5.11 plusminus 0.69 mm, mean plusminus SD, p < 0.004, and 3.8 plusminus 0.57 mm, mean plusminus SD, p < 0.07, respectively. Adenoviral-mediated gene transfer of platelet-derived growth factor-B overcame the ischemic defect in wound healing and offers promise in the treatment of chronic nonhealing wounds. The vulnerary effects of platelet-derived growth factor-B overexpression were sufficient to overcome the adverse effects of the adenovirus or transgene on wound healing.

Keywords:

adenovirus, gene therapy, skin

Abbreviations:

Ad-PDGF-B, adenovirus containing the PDGF-B transgene; Ad-LacZ, adenovirus containing the beta-galactosidase transgene; PFU, particle forming units

Chronic nonhealing wounds, including ischemic ulcers, decubitus ulcers, and diabetic foot ulcers can result in prolonged hospitalization and lost time from work for as many as 5 million patients in the USA and an estimated healthcare expense of as much as 11 billion dollars a year worldwide (Mastov 1994). If complications from other causes of impaired wound healing, including the use of corticosteroids, chemotherapy, radiation therapy, or severe malnutrition, are considered, the scope of this problem takes on even greater proportions. To date, effective therapies for both chronic nonhealing wounds and impaired wound healing have been lacking.

Whereas the etiology of wound healing impairment in chronic wounds appears to be multifactorial, growth factor deficiencies have been shown to be important. Chronic dermal ulcers have been shown to be deficient in platelet derived growth factor (PDGF) and in patients with chronic ulcers that eventually heal, increased serum levels of PDGF have been detected (Chleboun et al.1995;Pierce et al.1995).

PDGF plays an important part in each stage of wound healing beginning with its release from the alpha granules of platelets during formation of the wound hemostatic plug (Kaplan et al.1979). Although the predominant PDGF released is the AB heterodimer, the most potent form of PDGF by in vitro testing is the BB homodimer (Seppa et al.1982). PDGF has been shown to stimulate fibroblast proliferation and chemotaxis, as well as the synthesis of extracellular matrix (Kaplan et al.1979;Seppa et al.1982;Heldin et al.1989). PDGF is also secreted by fibroblasts, activated macrophages, and endothelial cells (Shimokado et al.1985;Harlan et al.1986;Paulsson et al.1987). In addition to autocrine and paracrine stimulation of its own production (Paulsson et al.1987), PDGF stimulates production of other growth factors and cytokines important in wound healing, such as transforming growth factor-beta (Pierce et al.1989), vascular endothelial growth factor (Sato et al.1993), keratinocyte growth factor (Li & Tseng 1997), and monocyte chemotactic protein-1 (Poon et al.1996). The central role of PDGF in all phases of wound healing has resulted in both experimental and clinical interest in exogenous PDGF protein application to chronic nonhealing wounds.

Despite data demonstrating a favorable effect of topical PDGF-BB on wound healing, large and repeated doses were required for even a modest clinical effect (Mustoe et al.1991,1994;Pierce et al.1991;Wieman et al.1998). Although disappointing these clinical studies demonstrate that PDGF-BB can favorably affect wound healing, but a more efficient means of delivery of PDGF-BB directly to cells participating in the wound healing response is needed. Gene transfer is an appealing strategy that may allow direct delivery of growth factor to the wound through the induction of growth factor gene expression in cells participating in the wound healing response.

We hypothesized that a replication deficient adenovirus could achieve efficient delivery of the PDGF-B transgene directly to cells participating in the wound healing response, and that the induction of PDGF-B overexpression could overcome an ischemic wound healing defect. The ischemic rabbit ear model of impaired excisional wound healing (Ahn & Mustoe 1990) was used to test the effect of adenoviral-mediated overexpression of the PDGF-B transgene on the rate of re-epithelialization, wound closure, and the production of granulation tissue and extracellular matrix.

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MATERIALS AND METHODS

Animal model

[cf1]An ischemic ear was created in one ear of 10–12 wk old female New Zealand white rabbits (Ace Animals, Boyletown, PA) as previously described (Ahn & Mustoe 1990). One day following creation of the ischemic ear a sterile surgical punch biopsy instrument (Miltex Instrument, Lake Success, NY) was used to create two 6 mm wounds on the ventral surface of both the ischemic ear and the contralateral non-ischemic ear.

Adenoviral construction

The PstI/EcoRI DNA fragment containing the open-reading frame derived from the full length human PDGF-B cDNA (provided by B. Westermark, University of Uppsala, Sweden), was cloned into the PstI/EcoRI restriction site of pSL301 (Invitrogen, Carlsbad, CA). The resulting plasmid was cut with NotI (Promega, Madison, WI) and the fragment subcloned into the adenoviral vector pADCMV-Link.1 (Davis & Wilson 1994). The resulting plasmid was cut with EcoRI (Promega) to determine the orientation of the PDGF-B gene.

The pADCMV plasmid containing the sense oriented PDGF-B gene was cut with NheI (Promega) and calcium-precipitated together with ClaI-digested adenoviral DNA (dL7001 Ad5) lacking the E1 and E3 genes (Ranheim et al.1993). DNA precipitates were incubated with human embryonic 293 cells containing the E1 genes of Ad5 for 5 h. Following which the 293 cells were subjected to 10% glycerol shock for 2 min in serum-free Dulbecco's modified Eagle's media and then grown overnight in Dulbecco's modified Eagle's media with 10% fetal bovine serum. Eagle media (Gibco/BRL, Gaithersburg, MD) with 2% fetal bovine serum, 0.8% agar (Gibco), and 0.5 mM MgCl2 was then placed over the cells. Fresh overlay was added every 4 d until the appearance of individual plaques. Plaques were expanded in 293 cells grown in Dulbecco's modified Eagle's media containing 10% fetal bovine serum until cytopathic effects were observed. Cells were then harvested and adenoviral DNA isolated as previously described (Hirt 1967). Adenoviral DNA was verified to contain the human PDGF-B gene by southern blot analysis (Sambrook et al.1989), using the PstI/EcoRI fragment of the PDGF-B gene as a probe. Rabbit fibroblasts infected with the recombinant adenovirus containing the PDGF-B transgene (Ad-PDGF-B) were verified to produce human PDGF-BB protein by western blot analysis as previously described (Sambrook et al.1989). In addition, biologic activity of the transgene product was verified by the ability to stimulate proliferation of rabbit fibroblasts.

Adenovirus containing the beta-galactosidase transgene (Ad-LacZ) was obtained from the Institute for Human Gene Therapy (University of Pennsylvania, Philadelphia, PA), and expanded to obtain a large preparation of Ad-LacZ. Ad-LacZ and Ad-PDGF-B were then purified by cesium chloride centrifugation and then desalted using a DG-10 column (Bio-Rad Laboratories, Hercules, CA). The particle number for Ad-PDGF-B and Ad-LacZ were determined by absorption at A260 using a DU-640 spectrophotometer (Beckman Instruments, Fullerton, CA) and the final plaque-forming units (PFU) determined by titration on 293 cells under an agar overlay (Graham et al.1973). The final particle/PFU ration was 25 : 1 for the Ad-LacZ vector and 1000:1 for the Ad-PDGF-B vector.

Replication competent adenovirus was excluded from both adenoviral constructs as previously described (Davis & Wilson 1994). In brief, the human lung carcinoma cell line A549 (American Type Culture Collection, Rockville, MD), a cell line known to be very susceptible to adenoviral infection, was incubated with either the adenoviral construct alone, replication competent adenovirus alone, or a combination of the two. Cells were observed twice a week for 2 wk for cytopathic effects. At 14 d the cells were scraped and a cell lysate formed with multiple freeze–thaw cycles. This lysate was then used to infect new A549 cells. The cells were incubated for 1 wk and observed for cytopathic effects. The cells were again lysed and the cycle repeated for 1 more week. Replication competent adenovirus was considered to be excluded if no cytopathic effects were noted during these 28 d of incubation. The replication competent adenovirus assay was sensitive to detect one replication competent adenovirus per 1011 adenoviral particles.

Administration of adenovirus

After the creation of the ischemic or non-ischemic wounds, 106 or 108 PFU of Ad-PDGF-B or Ad-LacZ in 50 mul of vehicle (20 mM HEPES/150 mM NaCl, pH 7.8, containing 10% glycerol), or vehicle, was injected intradermally around the wound using a Hamilton 50 mul syringe and 30 gauge needle (Hamilton Company, Reno, NV). Injection of 5 mug per wound of recombinant PDGF-BB (Sigma, St Louis, MO) was also performed for comparison. Following injection the wounds were dressed with Tegaderm.

Tissue processing

At 3, 7, and 16 d after wounding, the wounds were harvested and either fixed overnight in 10% neutral buffered formalin at 4°C and paraffin embedded or cryopreserved in 20% sucrose overnight at 4°C followed by embedding in OCT media (Miles, Elkhart, IN) as previously described (Culling, 1974). Full thickness skin was excised in a 8 mm radius centered on the site of the original wound, snap frozen in liquid nitrogen for later isolation of total RNA or DNA.

Histology

Serial 5 mum sections were obtained from the paraffin embedded wounds using a 30/50 microtome (Leica, Heidelberg, Germany) and stained with hematoxylin and eosin as previously described (Culling, 1974). On microscopic examination the epithelial gap, defined as the distance between encroaching epidermal elements, was measured using a stage micrometer (Leica). Granulation tissue volume was determined in square millimeters using Image-Pro Plus 3.0 software (Media Cybernetics, L.p., Silver Spring, MD). The boundaries of the granulation tissue bed were defined laterally by the transition zone from normal epidermis to hypertrophic epidermis, inferiorly by the ear cartilage, and superiorly by epithelial basement membrane or the open wound surface.

Immunohistochemistry

Rehydrated, serial paraffin sections (5 mum) were immersed in Tissue Unmasking Fluid (Signet Laboratories, Dedham, MA) to reactivate hidden or masked epitopes and then placed in a microwave (Ted Pella, Redding, CA) for 5 min on high power. Samples were blocked with nonimmune goat serum followed by incubation with a polyclonal goat anti-human PDGF-B (R&D Systems, Minneapolis, MN) at 4°C. The slides were washed with phosphate-buffered saline (PBS) and endogenous peroxidase activity blocked with methanol containing 0.3% hydrogen peroxide. Slides were rinsed with PBS and then incubated with biotinylated horse antigoat (Vector Labs, Burlingame, CA). The slides were washed with PBS and avidin–biotin complex (Vector Labs) added. The slides were rinsed in PBS, developed with chromagen 3,3'-diaminobenzidine (Sigma) and lightly stained with hematoxylin.

In situ hybridization

Paraffin sections (4 mum) were deparaffinated and rehydrated through a graded alcohol series to deionized water and allowed to air dry completely. Slides were then treated with proteinase K (10 mug per ml, Boehringer Mannheim), washed in PBS, and postfixed in 4% paraformaldehyde. Sections were covered with hybridization solution containing 50 ng biotinylated DNA probe, 50% foramide, 20% dextran sulfate, and 1 times sodium citrate/chloride buffer. Slides were heated for 10 min at 95°C and hybridized overnight in a humid chamber at 37°C. Slides were washes four times with 1 times sodium citrate/chloride buffer for 15 min at 50°C. The slides were then blocked with 0.1 M Tris-buffered saline (pH 7.4) containing 0.1% bovine serum albumin (Sigma) and 1 mM levamisole (Sigma). Streptavidin–alkaline phosphatase conjugate (1:75 dilution, Amersham, Arlington Heights, IL) was added to the slides for 30 min at 37°C. Slides were washed with 0.1 M Tris-buffered saline for 20 min at room temperature. Bound streptavidin–alkaline phosphatase conjugate was detected by incubating the slides at 37°C in alkaline phosphatase substrate solution (Gibco/BRL) for 2 h. Slides were counterstained with nuclear fast red 1% (Sigma).

RNA isolation and reverse transcription

Total cellular RNA from the wounds was isolated using Tri Reagent (Molecular Resource Center, Cincinnati, OH). as previously described (Chomczynski & Sacchi 1987). First-strand cDNA was prepared from 1 mug total cellular RNA, as previously described (Sambrook et al.1989). In brief, 1 mug of total cellular RNA in 9 mul total volume was added to each 0.65 ml microcentrifuge tube and placed on ice. A master mix was prepared and added on ice such that the final concentration of reagents for each sample was 200 U Moloney Murine Leukemia Virus Reverse Transcriptase (Gibco/BRL), 40 U RNAsin (Promega), 100 pmol of random hexamers (Boehringer Mannheim), 1 mM dithiothreitol, 500 muM deoxytriphosphates (Gibco/BRL), 50 mM KCl, 10 mM Tris–Cl (pH 8.3 at 22°C), 2.5 mM MgCl2, and 0.01% gelatin. After 1 h at 37°C the reverse transcriptase reaction was heat inactivated by incubation for 5 min at 94°C.

DNA isolation

Total DNA from the wounds was isolated using DNAzol (Molecular Resource Center). In brief, approximately 100 mg of tissue was homogenized in 1 ml of DNAzol. The DNA was precipitated with 0.5 ml of 100% ethanol. The DNA precipitate was pelleted by centrifugation and then washed twice with 95% ethanol. The DNA pellet was then dissolved in sterile water.

Polymerase chain reaction (PCR) analysis

Specific primers for human PDGF-B and rabbit beta-actin were selected based on the published human PDGF-B cDNA (Collins et al.1985) and rabbit beta-actin cDNA sequences (Sakai et al.1995). The human PDGF-B primer pair used was upstream primer 5'-TGGGCGCTCTTCCTGTCTCTC and downstream primer 5'-CTCGGCCCCATCTTCCTCTCC resulting in a 165 base pair product. The rabbit beta-actin primer pair used was upstream primer 5'-TGGGCAGAAGGACTCGTA and downstream primer 5'-CGCAGCTCGTTGTAGAAG resulting in a 144 base pair product. PCR was performed on aliquots of the cDNA or total DNA prepared above as previously described (Gilliland et al.1990). In brief, either 5 mul of the reverse transcriptase reaction or 0.5 mug total DNA was added to each 0.65 ml microcentrifuge tube and placed on ice. A master mix was prepared and added on ice such that the final concentration of reagents for each sample was 2.5 U Amplitaq Gold DNA polymerase (Perkin Elmer, Norwalk, CT), 200 muM deoxytriphosphates (dNTP, Pharmacia, Piscataway, NJ), 50 mM KCl, 10 mM Tris–Cl (pH 8.3 at 22°C), 1.5 mM MgCl2, 0.01% gelatin, and 1 muM upstream and downstream primers. The samples were kept on ice until the thermocycler block (Hybaid Limited, Manchester, U.K.) was at 94°C, when the samples were immediately placed into the block for 9 min. Samples were amplified using 30 cycles for cDNA and 45 cycles for DNA of 30 s at 94°C followed by 30 s annealing at 58°C for PDGF-B or 54°C for beta-actin in separate tubes followed by 1 minute of extension at 72°C. Upon completing the final cycle, samples were incubated for 5 min at 72°C. The PDGF-B primers were shown to be specific for human PDGF-B and not to amplify rabbit PDGF-B (Data not shown).

PCR was used to generate a biotinylated probe for in situ hybridization using the PstI/EcoRI fragment of the PDGF-B gene as a template. The PCR primers and conditions were the same as described for PDGF-B with the exception of the dNTP concentrations, which were 80 muM for dATP, dGTP, and dCTP with 52 muM for dTTP and 28 muM for biotin-16–2'-deoxyuridine-5'-triphosphate (dUTP, Boehringer Mannheim). The 165 base pair PCR product, corresponding to base pairs 136–288 of the coding sequence of the PDGF-B gene, was separated from unincorporated nucleotides using a G-50 Sephadex spin column (Boehringer Mannheim).

Statistical analysis

Differences in the epithelial gap between the ischemic and non-ischemic wounds were analyzed by the nonpaired t test. Differences in adenoviral and vehicle treated groups were assessed by analysis of variance and the nonpaired t test with the Bonferroni correction.

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RESULTS

Effect of ischemia on wound histology and re-epithelialization

In order to confirm wound healing impairment in our model, wound healing was examined histologically and quantitated by measuring the epithelial gap in ischemic and non-ischemic rabbit ears. At 7 d ischemic wounds (n = 14) had minimal re-epithelialization and absent granulation tissue in the wound bed (Figure 1a). In contrast, at 7 d non-ischemic wounds (n = 16) have re-epithelialized 60% of the original wound defect and have a modest amount of granulation tissue (Figure 1b). Ischemic wounds at 7 d had an epithelial gap of 3.4 plusminus 1 mm vs 2.3 plusminus 1.4 mm (mean plusminus SD) in the non-ischemic wounds (p < 0.006, Figure 2). By 16 d the ischemic wounds (n = 4) still have not completely re-epithelialized with an epithelial gap of 1.15 plusminus 0.35 mm, but do show a moderate amount of granulation tissue (Figure 1c). In contrast, at 16 d the non-ischemic wounds (n = 4) have all completely re-epithelialized and show maturation of granulation tissue (Figure 1d).

Figure 1.
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Histologic analysis of vehicle-treated wounds. Six millimeter excisional wounds in ischemic and non-ischemic rabbit ears were treated with an intradermal, circumferential injection of the vehicle used to resuspend the adenovirus. The wounds were then harvested at 7 and 16 d. Hematoxylin and eosin staining of the vehicle-treated ischemic wounds (A) and non-ischemic wounds (B) at 7 d is shown. The edge of the encroaching epithelium is indicated by the arrows. Also shown is the hematoxylin and eosin staining of vehicle-treated ischemic wounds (C) and non-ischemic wounds (D) at 16 d. Scale bar: 1 mm.

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Figure 2.
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Measurements of epithelial gap in vehicle-treated wounds. Wounds were harvested 7 d after wounding from 6 mm ischemic and non-ischemic rabbit ear wounds treated with an intradermal, circumferential injection of vehicle. Tissue sections from the wounds were hematoxylin and eosin stained and the epithelial gap measured as the distance between the epithelial margins measured using a stage micrometer. Vehicle treated ischemic rabbit ears (n = 14) are demonstrated by the open bar and non-ischemic wounds (n = 16) by the solid bar.

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Effect of Ad-PDGF-B on wound histology

In order to assess the effect of Ad-PDGF-B on wound healing, all wounds were examined histologically and wound healing quantitated by measuring the epithelial gap and granulation tissue volume. Treatment of ischemic wounds with a single intradermal injection of 106 PFU of Ad-PDGF-B per wound (n = 10) resulted in correction of the wound healing impairment at 7 d, with formation of a large amount of granulation tissue and partial re-epithelialization (Figure 3b), indicated by a decrease in the epithelial gap to 1.9 plusminus 1.8 mm (p < 0.05 versus vehicle control, Figure 4a). Treatment of ischemic wounds with 108 PFU of Ad-PDGF-B per wound (n = 9) resulted in an even more dramatic improvement in wound healing at 7 d, with a large amount of granulation tissue and complete (n = 6) or near complete (n = 3) re-epithelialization (Figure 3c), as indicated by a decrease in the epithelial gap to 0.7 plusminus 1.1 mm (p < 0.001 versus vehicle control, Figure 4a). By 16 d after wounding, the ischemic wounds treated with 108 PFU Ad-PDGF-B demonstrated resolution of the exuberant granulation tissue response (Figure 3d) with an appearance similar to the non-ischemic vehicle-treated wounds (Figure 3e). Treatment of non-ischemic wounds with 106 PFU of Ad-PDGF-B per wound (n = 9) resulted in a slight improvement in wound re-epithelialization at 7 d as indicated by a decrease in the epithelial gap to 1.3 plusminus 1.5 mm vs 2.3 plusminus 1.4 mm in the vehicle-treated non-ischemic wounds (Figure 4b). Treatment of non-ischemic wounds with 108 PFU of Ad-PDGF-B per wound (n = 9) resulted an epithelial gap of 2.8 plusminus 1.4 mm. Treatment of non-ischemic wounds with 106 or 108 PFU of Ad-PDGF-B, however, did not significantly improve wound re-epithelialization. By 16 d all ischemic and non-ischemic wounds treated with Ad-PDGF-B had completely re-epithelialized. A single intradermal administration of 5 mug recombinant human PDGF-BB protein into ischemic wounds (n = 2) only resulted in a slight increase in granulation tissue at the wound margin at 7 d, but no differences in granulation tissue in the wound base (Figure 3f) or in re-epithelialization (Figure 4a), with an epithelial gap of 3 plusminus 0.6 mm. No beta-galactosidase transgene expression was detected histochemically in ischemic wounds treated with 106 PFU of Ad-LacZ (n = 4, Figure 3g). Treatment of ischemic wounds with the higher dose of 108 PFU Ad-LacZ (n = 4) resulted in beta-galactosidase transgene expression at 7 d, evidenced by the blue cells at the wound margin. Treatment with either 106 or 108 PFU of Ad-LacZ did not improve wound granulation tissue formation and actually impaired wound re-epithelialization with increase in epithelial gap to 5.11 plusminus 0.69 mm (Figure 4c, p < 0.004 versus vehicle) and 3.8 plusminus 0.57 mm, respectively (p < 0.07 versus vehicle). At 7 d ischemic wounds treated with 108 PFU of Ad-PDGF-B had a significantly higher granulation tissue volume of 7.0 plusminus 1.8 mm2 (p < 0.000001, Figure 4d) compared with the ischemic wounds treated with vehicle (2.1 plusminus 0.8 mm2).

Figure 3.
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Histologic analysis of wounds treated with Ad-PDGF-B, Ad-LacZ, or PDGF-BB protein. The 6 mm excisional wounds in the ischemic rabbit ear were treated with an intradermal, circumferential injection of either vehicle, 106 or 108 PFU of either Ad-PDGF-B or Ad-LacZ, or 5 mug of recombinant PDGF-BB protein. The wounds were harvested 7 or 16 d after wounding. Hematoxylin and eosin staining of vehicle treated ischemic excisional wounds demonstrates the epithelial defect (A), indicated by the arrows, and the absence of granulation tissue in the wound base. Hematoxylin and eosin staining of ischemic wounds treated with 106 PFU of Ad-PDGF-B per wound (n = 10) is shown in (B). The improvement in wound healing, complete re-epithelialization, and exuberant granulation tissue response of ischemic wounds treated with 108 PFU of Ad-PDGF-B per ischemic wound (n = 9) is shown in (C). Part (D) demonstrates the complete re-epithelialization and maturation of granulation tissue seen 16 d after treatment of ischemic wounds treated with 108 PFU Ad-PDGF-B. An hematoxylin and eosin stain of a non-ischemic vehicle-treated wound at 16 d is shown for comparison (E). Part (F) demonstrates the minimal granulation tissue at the wound margin, but lack of improvement in granulation tissue in the wound base or in re-epithelialization seen in ischemic wounds treated with 5 mug recombinant human-PDGF-BB. beta-galactosidase staining of ischemic wounds treated with 106 PFU of Ad-LacZ at 7 d demonstrates no transgene expression seen in these wounds (G). beta-galactosidase staining of ischemic wounds treated with 108 PFU of Ad-LacZ demonstrates beta-galactosidase transgene expression, as evidenced by blue cells in the wound margin (H). The edge of epithelium is indicated by the arrows. Scale bar: (A–E) 1 mm; (F–H) 200 mum.

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Figure 4.
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Measurements of epithelial gap in wounds treated with Ad-PDGF-B, Ad-LacZ, or PDGF-B protein. Wounds were harvested 7 d after wounding from 6 mm ischemic rabbit ear wounds treated with an intradermal, circumferential injection of vehicle, 106 or 108 PFU of Ad-PDGF-B or Ad-LacZ, or 5 mug of recombinant PDGF-BB protein. Tissue sections from the wounds were hematoxylin and eosin stained and the epithelial gap measured as the distance between the epithelial margins measured using a stage micrometer. (A) Ischemic wounds treated with vehicle is shown by the open bar, 106 PFU of Ad-PDGF-B per wound (n = 10) is shown by the solid bar, and 108 PFU of Ad-PDGF-B per wound (n = 9) by the striped bar. (B) Non-ischemic wounds treated with vehicle is shown by the open bar, 106 PFU of Ad-PDGF-B per wound (n = 9) is shown by the solid bar, and 108 PFU of Ad-PDGF-B per wound (n = 9) by the striped bar. (C) Treatment of ischemic wounds with vehicle (open bar), 5 mug recombinant PDGF-BB protein (solid bar), 106 PFU of Ad-LacZ (cross-hatched bar, n = 7), or 108 PFU of Ad-LacZ (vertical striped bar, n = 4) is shown. (D) Granulation tissue volume was quantitated using Image-Pro Plus 3.0 software in ischemic wounds treated with vehicle (open bar) or 108 PFU of Ad-PDGF-B (solid bar).

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Detection of human PDGF-BB protein by immunohistochemistry

In order to confirm PDGF-BB protein production following gene transfer, wounds were analyzed by immunohistochemistry using an antibody specific for human PDGF-BB. Ischemic wounds treated with vehicle demonstrated minimal immunohistochemical staining for human PDGF-BB protein at 3 d (Figure 5a). In contrast, there was extensive and specific immunostaining for human PDGF-BB protein in the ischemic wounds treated with 108 PFU Ad-PDGF-B (Figure 5b). This immunostaining localized to fibroblasts and endothelial cells in the wound margin participating in the wound healing response.

Figure 5.
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Immunohistochemical analysis for PDGF-BB in wounds treated with Ad-PDGF-B. Production of human PDGF-BB protein was examined in wounds harvested 7 d after wounding from ischemic rabbit ears treated with an intradermal, circumferential injection of either vehicle or 108 PFU of Ad-PDGF-B by immunohistochemistry. Immunostaining of an ischemic wound treated with either vehicle (A) or 108 PFU Ad-PDGF-B (B) is shown. Scale bar: 25 mum.

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Localization of Ad-PDGF-B infected cells by in situ hybridization

In order to confirm the presence of vector derived human DNA in cells participating in wound healing, in situ hybridization for human PDGF-B was performed on ischemic wounds 3 d after wounding. Ischemic wounds treated with vehicle demonstrated no in situ hybridization signal (Figure 6a). In contrast, ischemic wounds treated with 108 PFU Ad-PDGF-B demonstrated dense in situ hybridization signal (Figure 6b). Cells that had positive in situ hybridization appeared to be primarily fibroblasts at the wound margin which were participating in the wound healing response.

Figure 6.
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In situ hybridization for PDGF-B gene in wounds treated with Ad-PDGF-B. In situ hybridization was performed for Ad-PDGF-B on wounds harvested 3 d after wounding from ischemic rabbit ears treated with an intradermal, circumferential injection of either vehicle or 108 PFU of Ad-PDGF-B. In situ hybridization of ischemic wounds treated with either vehicle (A) or 108 PFU Ad-PDGF-B (B) is shown. Scale bar: 50 mum.

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Duration of transgene expression by reverse transcriptase–PCR

In order to assess the duration of transgene expression in the wounds, reverse transcriptase–PCR analysis for human-specific PDGF-B was performed and demonstrated significant amounts of human PDGF-B mRNA at 3 d in both the ischemic and non-ischemic wounds treated with 108 PFU Ad-PDGF-B (Figure 7a). By 7 d, very little human PDGF-B mRNA was detected in both ischemic and non-ischemic wounds receiving 106 or 108 PFU Ad-PDGF-B, consistent with clearance of transgene expression from the wounds. No significant difference was noted in rabbit specific beta-actin controls.

Figure 7.
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Reverse transcriptase–PCR and PCR analysis for PDGF-B mRNA and transgene DNA in wounds treated with Ad-PDGF-B. (A) The amount and duration of human-specific PDGF-B mRNA production in wounds harvested 3 and 7 d after wounding from ischemic rabbit ears treated with an intradermal, circumferential injection of 108 PFU of Ad-PDGF-B using reverse transcriptase–PCR analysis is shown. (B) The presence of adenoviral PDGF-B transgene DNA from ischemic and non-ischemic wounds harvested 14 d after treatment with 106 or 108 PFU of Ad-PDGF-B is shown.

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Persistence of adenoviral DNA by PCR

In order to asses the duration of adenoviral DNA in the wounds, PCR was performed for the human specific PDGF-B transgene. At 14 d no adenoviral DNA was detected in ischemic wounds treated with 106 or 108 PFU Ad-PDGF-B, despite using 45 PCR cycles (Figure 7b). With 45 cycles, adenoviral DNA was detected in non-ischemic wounds treated with 106 or 108 PFU Ad-PDGF-B. There was no difference in beta-actin controls.

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DISCUSSION

Gene therapy using an adenoviral vector containing the PDGF-B transgene overcame the significant deficit in wound healing seen in ischemic rabbit ear excisional wounds. A single administration of Ad-PDGF-B resulted in correction of the ischemic impairment in wound re-epithelialization not only compared with vehicle, Ad-LacZ, or recombinant human PDGF-BB-treated ischemic wounds, but even compared with non-ischemic control wounds. Treatment of ischemic wounds with Ad-PDGF-B also significantly increased granulation tissue formation and also appears to have increased extracellular matrix production, and angiogenesis compared with vehicle, Ad-LacZ, or recombinant human PDGF-BB-treated wounds. This ischemic rabbit ear model poses a formidable challenge to wound healing, with a marked delay in wound closure (Ahn & Mustoe 1990) and minimal contribution of wound contraction to the healing response. The removal of the perichondrium in the base of the wounds requires the wound to heal by the formation of granulation tissue and re-epithelialization from the periphery (Chen et al.1992). Acceleration of wound closure observed in response to Ad-PDGF can only be achieved by increasing both the rate of granulation tissue production at the wound margin and the rate of keratinocyte migration over the wound bed.

Despite an extensive literature on the use of exogenous PDGF-BB protein in wound healing, results comparable with those achieved with Ad-PDGF-B have not been observed (Mustoe et al.1991,1994;Pierce et al.1991;Robson et al.1992;Steed 1995;Wieman et al.1998). In our experiments, granulation tissue and re-epithelialization of wounds treated with PDGF-BB protein were not significantly different from vehicle-treated wounds. Similarly, previous reports of topical application of PDGF-BB protein in the non-ischemic rabbit ear model resulted in only a modest increase in granulation tissue (53%). In addition, in the non-ischemic model limited improvement in wound closure was observed, with complete closure of 56% of the PDGF-BB-treated wounds versus 25% complete closure of vehicle-treated wounds (Mustoe et al.1991). In our study, even in the presence of ischemic wound healing impairment, Ad-PDGF-B resulted in a 66% incidence of complete wound closure. Ad-PDGF-B completely overcame the ischemic wound healing deficit, as these ischemic wounds healed even more rapidly than even non-ischemic wounds. Human clinical trials involving the topical application of PDGF-BB protein to diabetic and decubitus ulcers have thus far reported only modest improvements in wound closure, despite large repetitive doses, and a long duration of therapy (Pierce et al.1991;Mustoe et al.1994;Wieman et al.1997). Possible factors responsible for the limited effect of exogenous PDGF-B protein include binding of growth factor to macroglobulins, fibronectin, or proteoglycans in the wound base rendering them biologically unavailable (Folanga & Eaglstein 1993;Whitelock et al.1996), or increased degradation of growth factor as a result of the elevated matrix metalloproteinases in chronic wounds (Grinnell et al.1992).

The very modest improvement in wound healing observed with PDGF-BB protein demonstrates that PDGF-BB can improve chronic or impaired wound healing, but a better method of delivery is needed. Gene transfer has previously been attempted in wound healing. Application of plasmid DNA, or DNA particle bombardment, has been used, but has had limited success (Chen & Okayama 1988;Andree et al.1994;Krueger et al.1994;Benn et al.1996). In order to improve efficiency of retroviral infection, in vitro transfection of keratinocytes with retrovirus, followed by incorporation into an acellular cadaveric dermis, and subsequent transplantation, has been attempted (Eming et al.1995,1998). This increased transfection efficiency to between 40% and 80%, much lower than the 95% efficiency observed with adenovirus (Kozarsky & Wilson 1993), but also required cumbersome ex vivo transfection techniques. In addition, these ex vivo studies examined the effect of recombinant growth factor production on graft survival and did not examine the effects of growth factor on wound healing. In each instance either low efficiency of gene transfer, limited biologic effect of the transgene in wound healing, or cumbersome techniques have been responsible for the limited effect of previous gene therapy applications in wound healing. This experience lead us to the alternative of adenoviral-mediated gene transfer. Adenoviruses have been shown to be very efficient in gene transfer, infecting skin cells at more than 95% efficiency in vitro (Kozarsky & Wilson 1993). Previously, an adenovirus has been used to effect reporter gene transfer to the skin without adverse effects (Lu et al.1997), but this study did not examine the effect of adenoviral infection on wound healing. In this current study, an adenovirus containing the PDGF-B transgene, a growth factor with significant biologic effect in wound healing, was used successfully in the induction of PDGF-B gene overexpression, resulting in reversal of the ischemic impairment in wound healing and rapid re-epithelialization of the wound.

Adenovirus as a means of gene transfer has been reported in the treatment of genetic diseases such as cystic fibrosis and hypercholesterolemia (Kozarsky et al.1993;Grubb et al.1994). Low transduction rates, short duration of adenoviral transgene expression, as well as the need for re-administration, however, have limited the success of adenovirus in gene therapy in these applications (Kozarsky & Wilson 1993). These limitations are due in part to immunologic response to both the adenovirus as well as the transgene. In the setting of wound healing, where correction of a genetic defect is not the goal, however, the limited duration of transgene expression may prove to be advantageous, with clearance of the adenovirus and transgene by the time of wound closure. Unlike other potential applications of adenoviral-mediated gene therapy, limited duration of high-level transgene expression may be all that is necessary to effect wound healing in chronic nonhealing wounds. In addition, once skin integrity is re-established, the need for re-administration is not needed. In cases where skin integrity has not been fully re-established, re-administration may be necessary; however, the duration of transgene may be affected by the immune response.

In contrast to the significant improvement in wound healing achieved by Ad-PDGF-B in ischemic wounds, Ad-Lacz adversely affected wound healing with a significant increase in the epithelial gap compared with vehicle control. Previous work in our laboratory studying gene therapy in a nonimpaired model of wound healing demonstrated an acute inflammatory response to adenoviral administration, but no adverse effect on wound healing (Sylvester et al.submitted). The adverse effect on wound healing seen with Ad-LacZ in this model of impaired wound healing may be the result of the inflammatory response to the adenovirus or the transgene product in the wound. Alternatively, the negative effect may be related to the properties of the ischemic rabbit ear model, where an already impaired wound is unable to tolerate further inflammation due to the adenoviral infection. A direct cytotoxic effect of the adenovirus is unlikely with the recombinant adenovirus used in these studies being free of cytolytic effects. This observation makes the vulnerary effects of Ad-PDGF-B all the more impressive. Not only was Ad-PDGF-B able to overcome the ischemic defect in wound healing, but it was also able to overcome the potentially negative effect of adenovirus on impaired wound healing. This negative effect was overcome, however, with the selection of a proper transgene with known biologic effects in wound healing (Kaplan et al.1979).

In the non-ischemic wounds there appeared to be improvement in wound re-epithelialization in wounds treated with 106 PFU Ad-PDGF-B, but no improvement in wounds treated with 108 PFU Ad-PDGF-B. The lack of a statistically significant effect may be due to the fact that these wounds are healing at near maximal rate. To detect a small improvement in wound healing in these nonimpaired wounds a larger sample size may be needed.

One potential concern of using adenovirus containing the human PDGF-B transgene is its homology to the gene product of the simian sarcoma virus (SSV p28v-sis), which also acts through the PDGF receptor. The effect of the SSV p28v-sis gene product is mediated through chronic activation of the PDGF pathway, resulting in persistent cell proliferation and ultimately cell transformation (Johnsson et al.1986). The SSV p28v-sis is a retrovirus that integrates into the host cell DNA, but is not immortalizing, with SSV p28v-sis transformed cells living no longer than nontransformed cells. In contrast, adenoviruses remain episomal and rarely integrate into the human genome (Bett et al.1993). The recombinant adenovirus used in this study is also replication deficient, noncytolytic, and does not induce apparent phenotypic changes in infected cells (Ranheim et al.1993). Even in the rare case of the development of a replication competent adenovirus, the cell would be lysed and the vector cleared from the tissue. In addition, PCR analysis of wounds demonstrated that PDGF-B transgene expression and adenoviral transgene DNA are no longer detected in ischemic wounds by 7 d. Further evidence for resolution of the induced PDGF-B overexpression is the observation that the proliferative changes in the base of the wound are limited and not progressive as would be expected with continued PDGF-B overexpression. At 16 d the ischemic wounds treated with Ad-PDGF-B have a similar appearance to non-ischemic wounds that were treated with vehicle, with regard to cellularity, granulation tissue, and wound closure.

Adenoviral-mediated gene transfer of the human PDGF-B transgene appears to be very effective in promoting PDGF-B overexpression resulting in reversal of the ischemia-induced wound healing impairment in the rabbit ear excisional wound model. The efficiency of gene transfer and biologic effects of adenoviral-mediated gene transfer of PDGF-B are superior to previously reported applications of exogenous PDGF-BB protein as well as other techniques of gene transfer in wound healing. Adenoviral-mediated gene transfer offers significant promise as a novel approach to efficient growth factor delivery in impaired wound healing.

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Acknowledgments

The authors gratefully acknowledge the assistance of Timothy Sablich and Bernard Martin in the performance of these studies.

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