Evaluation of Platelet-Derived Growth Factor in a Rat Model of Ischemic Skin Wound Healing

Introduction Chronic wounds affect an estimated two million people in the United States and are a major socioeconomic burden to the patient and the healthcare system, resulting in an estimated yearly expenditure of upwards of $3 billion.[1] The basic foundation for good wound care involves eliminating exacerbating factors, such as localized pressure in pressure ulcers and diabetic foot wounds and edema in chronic venous stasis ulcers. Standard wound care procedures include wound debridement, the use of dressings to maintain a moist environment, and topical antimicrobial agents when needed to treat wound infections. Although these concepts and regimens improve healing in many wounds, some wounds fail to heal, and this leads to the need to understand the biochemical deficits in the wound microenvironment that prevent wounds from healing. Acute and chronic wounds have different cytokine, growth factor, proteinase, and mitogenic activity profiles. Fluids obtained from different types of chronic human wounds have elevated levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1b (IL-1b) compared to healing wounds, and elevated matrix metalloproteinases (MMPs) and serine proteases, such as neutrophil elastase.[2–5] Combined with reduced levels of tissue inhibitors of metalloproteinases (TIMPs),[6] the high proteolytic microenvironment in chronic wounds is thought to reduce the ability of chronic wounds to heal by degrading essential growth factors, receptors, and extracellular matrix proteins.[7] For example, studies of specific growth factors in chronic wounds reported reductions in the endogenous levels of TGF-beta and PDGF and a down regulation of PDGF receptor and TGF-beta.[8,9] Ischemic bipedicle rat skin flap model mimics molecular abnormalities of human chronic wounds. Rapid development of therapies that target specific abnormalities in human chronic wounds is dependent on the use of animal models that mimic the biochemical and structural abnormalities that characterize human chronic wounds. An ischemic bipedicle dorsal rat skin flap model has been reported to have delayed healing and elevated levels of proinflammatory cytokines (TNF-alpha and IL-1beta) and MMPs (pro-MMP-9, active MMP-9, and active MMP-2), similar to those observed in human chronic wounds.[10,11] Thus, this animal model should be useful in evaluating the effects of treatments on altering the microenvironments of wounds and promoting healing. A logical agent to evaluate is PDGF because of its significant role in wound healing. PDGF activates macrophages and fibroblasts and stimulates the production of extracellular matrix proteins and granulation tissue.[12] Also, human recombinant PDGF-BB was shown to increase healing of diabetic foot ulcers and pressure ulcers and is approved by the FDA for the treatment of diabetic foot ulcers.[13–15] Methods Bipedicle ischemic skin flap model. Multiple topical applications of human recombinant PDGF-BB and a single application of an adenovirus vector expressing PDGF-BB (Ad-PDGF) were evaluated in the bipedicle ischemic rat skin flap model as described previously.[10,16] Briefly, a 11cm x 2.5cm bipedicle dorsal skin flap centered on the midline between the scapula and iliac crest was raised on the dorsum of 36 adult (250gm) male Sprague Dawley rats. Six full-thickness punch wounds 6mm in diameter were arranged in pairs at 2.6cm, 5.2cm, and 7.8cm from the end of the template and 3mm from each edge, and the skin flap was repositioned and secured by surgical staples. Topical treatment of wounds with rhPDGF protein. On the day of surgery (day 0) and for the next 13 days, 50µL of either vehicle gel (1% carboxymethylcellulose gel) or 0.01-percent rhPDGF* was applied topically to the wounds of each rat in the two study groups. On days 0, 1, 5, 7, 9, and 13, rats were anesthetized by isoflurane inhalation, each wound border was traced on an acetate sheet, which was digitally scanned, and wound areas were calculated using NIH Image software. Wound areas were expressed as the mean ± standard error in mm2. Three rats from each of the two treatment groups were euthanized on days 1, 5, 7, 9, and 13, and the wound sites were collected using an 8mm punch. Four of the six wound samples from each rat were independently homogenized using a frosted glass-on-glass Duall homogenizer in phosphate-buffered saline (PBS) containing 0.1-percent Triton-X-100 at a ratio of 1mL buffer per gram of tissue, centrifuged, and the supernatant solution stored at -80 degrees C until biochemically analyzed. The remaining two wound biopsies from each rat were fixed in formalin and paraffin sections prepared for histology. Topical treatment of wounds with Ad-PDGF. Bipedicle skin flaps were created on the dorsum of 18 adult male Sprague Dawley rats as described above, but only three 6mm full-thickness wounds were created in the flap instead of six wounds. Six rats were randomly assigned to one of three treatment groups designated as Ad-PDGF, Ad-LacZ, and saline. Immediately after the flap was stapled in place, 100µl of saline containing a total of 4.4 x 10 to the 7th plaque-forming units (pfu) of Ad-PDGF, Ad-LacZ vector, or no vector (saline control) were injected at three sites in the edge of the wound. On days 1, 3, 6, 10, and 14 after surgery, rats were anesthetized; wound edges were traced on acetate sheets and digitally scanned; and wound areas were calculated using NIH Image software and expressed mean ± standard error of mm2. Measurement of TNF-alpha levels in wound biopsies. Levels of TNF-alpha in homogenates of wounds were measured by ELISA following the manufacturer’s protocols (Quantikine, R&D Systems, Minneapolis, Minnesota). Briefly, homogenate supernatants were added in duplicate to 96-well plates coated with the capture antibody, incubated, washed, incubated with anti-TNF-alpha antibody conjugated to horseradish peroxidase then washed, chromogen substrate (tetramethylbenzidine and hydrogen peroxide) added, and color measured at 450nm. Samples and TNF-alpha standards were assayed in duplicate, and the level of TNF-alpha was calculated from the standard curve and expressed as the average ± standard error of pg of TNF-alpha per µg of protein. Measurement of MMP Levels in wound biopsies. Levels of pro-MMP-9 and active MMP-9 were measured in supernatants of wound homogenates using gelatin zymography as described previously.[6,10] Briefly, supernatants of wound biopsy homogenates were electrophoresed on gelatin zymogram gels (Invitrogen, Carlsbad, California) along with MMP-2 and MMP-9 standards; the gels were renatured and developed for 24 hours at 37 degrees C. The gels were then stained with Coomassie Rapid Stain (Diversified Biotech, Boston, Massachusetts), digital photographs were analyzed by Kodak 1-D image analysis software, and the amount of protease activity calculated as ng MMP per mL of homogenate supernatant from standard curves of recombinant MMP standards. Levels of MMP activity were graphed as the average ± standard error of six samples. Results Healing of ischemic wounds is accelerated by topical treatment with PDGF. We have previously reported that healing of wounds in the bipedicle skin flap is significantly delayed compared to healing of non-ischemic wounds created in dorsal skin of rats.[10] As shown in Figure 1, daily topical treatment of ischemic wounds with PDGF significantly accelerated the rate of healing, especially on days 3, 5, and 7 after surgery. Furthermore, the improved healing persisted even through days 9 and 13. Thus, daily treatment of ischemic rat skin wounds with PDGF generated a similar effect of daily treatment of foot ulcers in diabetic patients reported by Steed and colleagues.[12] TNF-alpha levels are reduced in ischemic wounds treated with PDGF. We reported previously that TNF-alpha mRNA and protein expression were significantly elevated in ischemic rat skin wounds on day 13 after surgery compared to nonischemic skin wounds.10 In this study, the concentration of TNF-alpha protein increased approximately 14-fold on day 3 after surgery then progressively declined but remained elevated about 6-fold on day 13 compared to normal nonwounded skin (Figure 2). Levels of TNF-alpha in ischemic wounds treated daily with PDGF showed a very similar overall profile to the vehicle-treated wounds, but the levels were significantly reduced to about half the levels in vehicle-treated wounds on days 3 and 5. Furthermore, the level of TNF-alpha in PDGF-treated ischemic wounds had dropped to levels approaching that in normal non-wounded skin by day 7 after injury and remained low through day 13. These data indicate that daily topical PDGF treatment reduced the magnitude of the pro-inflammatory cytokine response during healing and more quickly returned the wound microenvironment to levels approaching nonwounded skin. MMP-9 levels are reduced in ischemic wounds treated with PDGF. All MMPs are synthesized as inactive proforms, and all undergo activation, usually by proteolytic cleavage that releases a small peptide fragment, which exposes the active site of the enzyme.[17] As shown in Figure 3, levels of active MMP-9 are very low in normal nonwounded skin (day 0). However, levels of active MMP-9 rise rapidly and peak at 10-fold higher levels on day 5 after injury in vehicle-treated ischemic wounds then slowly and progressively decrease to about 6-fold elevation by day 13 compared to non-injured skin. Although the overall temporal pattern of active-MMP-9 expression in wounds treated with PDGF was similar to wounds treated with vehicle, daily topical treatment with rhPDGF significantly decreased levels of active MMP-9 to a maximum of about 4-fold elevation on day 3 after wounding. On day 13 after injury, levels of active MMP-9 in PDGF-treated wounds had decreased to less than 3-fold the levels in normal skin. Single Ad-PDGF treatment increases healing of ischemic rat skin wounds. As shown in Figure 4, healing of ischemic skin wounds treated with a single application of Ad-PDGF vector at the time of injury significantly increased the rate of healing beginning on day 3 compared to wounds treated with saline or with the nontherapeutic Ad-LacZ vector. The healing of wounds treated with saline or the Ad-LacZ gene were not significantly different, which indicates that the adenovirus vector used at this level of pfu does not have a detrimental effect on healing of ischemic wounds. Interestingly, Ad-PDGF treatment did not decrease wound area until day 3, whereas topical treatment with PDGF protein reduced wound area on day 1 after injury. The slight lag in effect on wound closure observed with Ad-PDGF compared to PDGF protein may be due to the inherent time required for the adenovirus vector to infect and express PDGF by the wound cells. Discussion The bipedicle ischemic rat skin flap model parallels several of the major characteristics of many human chronic wounds, namely ischemia and bacterial colonization of open wounds. In addition, our previous biochemical studies demonstrated important similarities between the molecular profile of cytokines and proteases in human chronic wounds and the ischemic rat skin wound model. However, for this animal model to be useful in understanding the molecular abnormalities that contribute to the failure of wounds to heal and to be useful as a screening tool to identify new agents and treatments that may promote healing of human chronic wounds, it should reproduce the molecular abnormalities of human chronic wounds and reproduce the effects of agents that are known to improve healing of human chronic wounds. Clinical studies using topical PDGF have demonstrated an improvement in the healing of diabetic foot ulcers and pressure ulcers.[12] Furthermore, other studies have reported reductions in the cytokine and proteinase levels of chronic wounds as healing progresses.[2,3] Thus, topical rhPDGF treatment should also accelerate healing of the ischemic rat wounds and alter the biochemical profiles of key cytokines and proteases that change as human chronic wounds begin to heal. Overall, the results of these initial studies showed a significant improvement in the healing rate of ischemic wounds treated daily with topical PDGF or with a single Ad-PDGF treatment. Furthermore, daily topical application of PDGF protein dramatically reduced the peak levels of TNF-alpha and active MMP-9 on days 3 and 5 after injury and also rapidly reduced cytokine and MMPs levels to values approaching those found in normal skin. These responses agree with the results from human clinical studies and support the concept that the ischemic rat’s skin wound model replicates some key biochemical characteristics and responses that occur as human chronic wounds begin to heal. However, more complete molecular analyses of the ischemic rat wounds and chronic human wounds are needed to assess how closely the rat model and human chronic wounds parallel each other. This can be accomplished by microarray analyses that assess changes in expression of thousands of genes as healing proceeds in the ischemic rat skin wounds and human chronic wounds. Transient gene therapy is a promising concept for treatment of chronic human wounds. As described in recent reviews, there are numerous theoretical advantages of gene therapy for chronic wounds, including the sustained production of physiological levels of growth factors in the wound microenvironment, fewer number of treatments, and reduced cost.[18,19] The initiation of clinical studies appears eminent, and results of transient gene therapy in animal models of impaired healing will continue to provide important data for optimizing patient treatment. *Regranex, Ortho-McNeil Pharmaceutical, Raritan, New Jersey