Abstract: A novel recombinant protein (rSVEP), originally discovered in insect saliva and known to increase blood flow, was tested with the hypothesis it would improve healing of surgically created wounds in the skin of beagle dogs. This hypothesis was tested in an experimental protocol that included: 1) use of laser Doppler perfusion imaging (LDPI) to determine dose-response and duration of blood flow increase to rSVEP injected intradermally; 2) creation of sutured and open wounds in skin of six dogs and treatment of “matched pair” wounds in each dog with rSVEP or physiologically buffered saline (PBS-Control) injected intradermally or subcutaneously; 3) objective determination of effects of treatment. Objective parameters that yielded significant data were: a) measurement of breaking strength of sutured wounds using tensiometry and b) rate of open wound healing calculated by sequential digitization of open wound area. Blood flow increased eight fold and lasted up to 72 hours in response to rSVEP. The median breaking strength of matched sutured wounds, tested on day 5, increased by 48 percent when treated with rSVEP applied by either method. Healing of open wounds was enhanced by 14 percent at Day 21 but only if the time 0 dose was given intradermally. These results support the hypothesis and indicate a novel new wound treatment.

Disclosure: Portions of this work were presented at the 16th Annual Symposium on Advanced Wound Care and 13th Medical Research Forum on Wound Repair in Las Vegas, Nevada, USA, April, 2003. This work was supported by research grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, a part of the National Institutes of Health, and the Auburn University Scott-Ritchey Research Center. Drs. Cupp and Swaim are co-inventors on an Auburn University patent application for the use of rSVEP to treat wounds.

Introduction

In the past several years, studies have revealed a number of novel and highly potent factors in the saliva of blood-feeding insects that affect vertebrate blood flow.[1] In addition to factors that target platelet activation and blood clotting, others work to increase blood flow in peripheral circulation (vasodilation) and thus enhance the ability of insects to obtain blood. Because of their specificity and potency, these molecules (mostly small molecular weight proteins) are strong candidates for therapeutic use in animal and human medicine, including treatment to increase blood supply to wound tissues. A 15 KDa protein that is a potent inducer of vasodilation was isolated from the salivary glands of the black fly, Simulium vittatum.[2] Analysis of the cDNA, cloned from an S. vittatum salivary gland cDNA library, revealed a unique molecule (GenBank accession #U94515) that was named Simulium vittatum erythema protein (SVEP).[3] To investigate its therapeutic potential for improving wound healing, a recombinant form (rSVEP) was produced in a baculovirus expression system and isolated to high purity using differential centrifugation and reversed phase high performance liquid chromatography (HPLC).[3]

Wound Healing and Blood Supply

Rapid, strong healing of open and closed wounds is the primary goal of wound management. An adequate supply of oxygen and nutrients is required for normal wound healing and is delivered to the wound via the vascular system.[4] Thus, blood supply to tissue is a major determinant in the rate and strength of wound healing. One factor that can limit blood supply to wounded tissues is local vascular system impairment as a result of either local damage due to trauma or some type of generalized process.[4]

Oxygen plays a vital role in the healing process. It influences the rate of fibroblast proliferation[5] and is necessary for the hydroxylation of lysine and proline in collagen synthesis.[6] When this is impaired due to hypoxia, diminished wound strength results.[4] In addition, lack of oxygen impairs the body’s defense mechanisms against bacterial invasion since oxygen is required by neutrophils for the creation of free oxygen radicals to kill bacteria.[7]

In addition to oxygen and nutrient provision in wounds, the blood supply delivers the cells to wounds that are vital in the healing process. In the early stage of healing, platelets, neutrophils, and macrophages are important. Platelets play a key role in hemostasis, while neutrophils and macrophages provide wound clean up. Macrophages also produce the majority of the cytokines and growth factors involved in progression from the inflammatory to the repair stage of healing.[8,9] Thus, the authors postulated that increased tissue perfusion provided by vasodilation associated with rSVEP would provide a unique means of increasing delivery of oxygen, nutrients, cells, and growth factors to healing wounds.

Initial tests of rSVEP treatment of surgically created wounds using New Zealand White Rabbits and diabetic Zucker Rats revealed a positive correlation with decreased tissue necrosis of healing wounds although no detectable enhancement of wound repair in open or closed wounds was noted when treated by subcutaneous injection (unpublished observation). These observations, coupled with the subsequent discovery of enhanced potency of rSVEP in dog skin, recommended further evaluation of rSVEP treatment as a novel and clinically applicable means to improve healing using dogs as the more sensitive model.

Materials and Methods

All animal procedures were approved by the Auburn University Institutional Animal Care and Use Committee (IACUC), and production and use of recombinant protein was approved by the Auburn University Institutional Biosafety Committee.

Dose/response of peripheral blood flow to rSVEP. rSVEP was produced in a baculovirus culture expression system using SF9 cells. Recombinant protein was isolated from cell culture supernatant by differential centrifugation using 100 and 10KDa Centriplus membranes, followed by final separation on a C-18 reversed-phase HPLC column. HPLC-pure protein was lyopholized and reconstituted in Tris buffer, pH 8.3, 0.15MNaCl. Purity was confirmed by SDS/PAGE silver-stained gels and concentration determined using a micro-Lowry protein assay. Dosing solutions were prepared in PBS, pH 7.4, then sterile-filtered using 0.22µ syringe filters and stored at 4 degrees C.

A dose/response of blood flow to rSVEP in beagle dog skin was established using four purpose-bred beagle dogs. Four paired sites on each side of the dogs’ trunks were marked with a fine-tipped lab marker to indicate the sites where rSVEP would be administered. Laser Doppler perfusion imaging (LDPI) was used to noninvasively serially document changes in skin perfusion that were induced by rSVEP. Baseline blood flow was measured at each site using LDPI, then the full range of dosages (0, 5, 10, and 20µg in 50µl PBS) was injected on each side, with the order of lowest to highest dose reversed in the cranial to caudal direction in order to account for any endogenous cranial to caudal perfusion differences. Blood perfusion was measured again at 0.5, 24, 48, and 72 hours post-injection to determine the degree and duration of vasodilation response.

Sums of the numerical values (number of pixels generated by LDPI detection of peripheral blood perfusion) that correlated to blood flow volume were calculated using a spread sheet. Change in blood flow in response to treatment was calculated as a percentage of time zero using the formula: (Sum of pixels post-treatment ÷ Sum of pixels at time 0) x 100 = % of time zero flow

Under general anesthesia, aseptic surgery was performed on six purpose-bred beagles. Three pairs of small wounds were created on the trunk with matched wounds on either side of the dorsal midline. Two pairs of wounds were open square wounds measuring 2cm on each side (Figure 1).The third pair of wounds was 3cm-long sutured incisions (Figure 1). Immediately following wound creation, a 200µl volume of PBS control solution or PBS containing 5µg rSVEP (25µg/mL) was applied to wounds by injecting equal aliquots (50µl each) to the four sides of the open wounds or to the two sides (100µl each) of the sutured wounds. Wounds on one side served as PBS controls with the matching wound on the other side being treated with rSVEP. Three dogs received injections subcutaneously, and three had intradermal injections in the initial treatment with dogs under general anesthesia. In subsequent treatments, a change was made to subcutaneous injections for all six dogs. For each dog, one pair of open wounds was used for histopathologic and electron microscopic evaluation.The second pair of open wounds was used for LDPI analysis of wound perfusion and planimetric evaluation. The third pair of sutured wounds was used for LDPI analysis and breaking strength analysis (Figure 1).

Figure 1

This diagram shows the placement of wounds, injection frequency, and evaluation parameters for surgically created wounds in beagle dogs. Three pairs of small wounds were created on the trunk with matched wounds on either side of the dorsal midline. Two pairs of wounds were open square wounds measuring 2cm on each side. The third pair of wounds were 3cm-long sutured incisions. Immediately following wound creation, a 200µl volume of PBS control solution or PBS containing 5µg rSVEP (25µg/mL) was applied to wounds by injecting equal aliquots (50µl each) to the four sides of the open wounds or to the two sides (100µl each) of the sutured wounds. Wounds on one side served as PBS controls with the matching wound on the other side being treated with rSVEP.

During the course of the study, all wounds were covered with a sterile semiocclusive bandage pad. A secondary absorbent bandage wrap extending from the axillary to the inguinal area covered the primary bandage, with 2-inch–wide adhesive tape being used for the tertiary bandage layer. Bandage wrap and tape were used in a cris-cross pattern in the cranial pectoral region to prevent the bandage from sliding caudally. Bandages were changed daily for 10 days then every other day until Day 21.The dogs received systemic antibiotic of Cefadroxil (22mg/kg q 12 hrs, per os).

Breaking strength of the sutured wounds was assessed by applying tensile force to the excisedclosed wounds. Computer-aided planimetry was used to serially document reduction in open wound size using calculated values of percent contraction, epithelialization, and total healing (combined contraction and epithelialization).
The skin segment for breaking strength analysis was rectangular, 5cm long, and 2cm wide with the incision bisecting the segment across its short dimension. This segment was obtained aseptically, and the skin defect that remained was surgically sutured. Planimetric evaluation and breaking strength analysis yielded significant information for assessing the effect of treatment.

Statistical analysis. Breaking strength of rSVEP-treated and PBS-control sutured wounds of each dog was tested using the Wilcoxon Signed Ranks Test. Percent of total wound healing, derived from sequential measurements of wound area over a 21-day period for rSVEP-treated and PBS-control open wounds, was compared using the paired t test.

Results

Dose/response effects of rSVEP on blood perfusion are shown in Figure 2. Blood flow was increased in response to rSVEP at the first time of measurement post-treatment (0.5 hr) and continued to increase until maximum levels at 24 to 48 hours. It then declined but remained elevated at 72 hours. Blood flow did not change in response to PBS and remained at baseline levels throughout the 72-hour period (Figure 2).

Figure 2

This graph shows the dose/response increase in volume and duration of blood perfusion in skin of beagle dogs in response to intradermal injection with rSVEP. Following baseline blood flow measurement using laser Doppler perfusion imaging (LDPI), a range of dosages of rSVEP (0, 5, 10, 20µg in 50µl PBS) was injected on each side of the dog, with the order of lowest to highest dose reversed in the cranial to caudal direction in order to account for any endogenous cranial to caudal perfusion differences. Blood perfusion was measured again at 0.5, 24, 48, and 72 hours post-injection to determine the degree and duration of vasodilation response.

The breaking strength of sutured wounds was tested at Day 5 post-surgery after the wounds had been treated twice with PBS or rSVEP on Days 0 and 3. Individual percent increases in force required to break an rSVEP-treated wound over the PBS-treated one, removed from the same dog, are shown in Figure 3. Although the amount of force required was quite variable among dogs, all rSVEP-treated wounds were stronger than their PBS-treated matching wound (Wilcoxon Signed Ranks Test, n=5, p=0.043). Data for the sixth dog was omitted as an outlier, although the breaking strength of the rSVEP-treated wound was markedly stronger than that of the PBS-treated control wound, because the authors could not exclude the possibility that the apparent difference was affected by the size of the closed wound segments removed for measurement. The skin samples from this dog may have accidentally been harvested with some unincised skin in the area of the wound.

Figure 3

This graph shows the effect of rSVEP treatment on the breaking strength of closed wounds. Following two treatments with 5µg rSVEP or PBS control solution, median breaking strength of excised wounds at Day 5 was increased 48% by rSVEP treatment. (Wilcoxon Signed Ranks Test, n=5, p=0.043).

Planimetric differential effects of treatment on the healing of open wounds was greatest at the first time point of measurement, Day 7, but continued with a similar differential pattern at each measurement time throughout the observation period of 21 days (Figure 4). The increase in percent of total wound healing in response to intradermal administration of rSVEP can be seen graphically in Figure 4. Treatment with rSVEP given initially by intradermal injection, followed by subsequent subcutaneous injections (ID/SC), resulted in a significant overall improvement in wound healing (mean=14%, paired t test, rSVEP [ID/SC] vs. PBS [ID/SC], p=0.011). Interestingly, initial intradermal injection of solution followed by subsequent subcutaneous injections improved wound healing compared to wounds treated with all subcutaneous injections (SC/SC) for both PBS-treated (mean=18%, paired t test, p= 0.045) and rSVEP-treated wounds (mean=36%, paired t test, p=0.022), even though the method of application differed only for the first treatment at Day 0. Figure 5 shows the bilateral wounds of a dog treated ID/SC with rSVEP (left side) and PBS (right side). There is less open wound (exposed granulation tissue in the rSVEP treated wound.

Figure 4

This graph shows the rate of healing of surgically created open wounds. Newly created wounds were injected on each side with equal aliquots (50µl to the four sides of the open wounds) of a 200µl volume of PBS control solution or PBS containing 5µg rSVEP (concentration of 25µg/mL). Three dogs received injections subcutaneously (SC) and three had injections intradermally (ID) in the initial treatment when dogs were under general anesthesia. In subsequent treatments, carried out without anesthesia, a change was made to subcutaneous injections for all six dogs. Key: SC/SC: injections subcutaneous, Day 0, subcutaneous, Days 3, 6, 9, 12; ID/SC: injections intradermal, Day 0, subcutaneous, Days 3, 6, 9, 12; Results: (1) ID/SC: rSVEP 14% more healing than PBS, Day 21 (paired t test, p=0.011); (2) ID/SC vs SC/SC: PBS, ID/SC 18% more healing than SC/SC (paired t test, p=0.045); rSVEP, ID/SC 36% more healing than SC/SC (paired t test, p=0.022).

Figure 5

Figure 5. This photograph shows paired open wounds of beagle dogs treated with rSVEP or PBS control solution ID/SC at Day 19. There is more total wound healing (less open granulation tissue) on the rSVEP wound (open arrow) than on the PBS wound (closed arrow).

Discussion

Saliva of blood-feeding insects contains a variety of molecules with high potency and specificity in modulating vertebrate hemostasis. One such molecule that causes vasodilation (SVEP) is found in black fly saliva.

The potency of SVEP to increase blood flow in skin suggested that it might have practical utility as a therapeutic agent for human and animal wounds with compromised blood flow, including surgically created, accidentally acquired, or chronic metabolically related wounds. Thus, this initial study was designed to test the hypothesis by treating surgically created wounds with rSVEP or PBS control solutions and using objective measures to determine rate and strength of repair. This test was believed to be stringent in that the surgical wounds were created and treated under conditions that would optimize normal healing even without intervention. With rSVEP treatment, sutured wounds were stronger when tested at Day 5, after only two treatments, and the percent of total wound healing was greater for open wounds treated every three days through Day 12. These results demonstrate the positive effects of rSVEP to enhance the normal healing of skin wounds. Further studies are warranted to determine if treatment with rSVEP would improve healing of chronic and other wounds that have a compromised blood supply.

The somewhat surprising outcome of this study was the differential effect of administering treatment intradermally at the initial treatment, Day 0. This observation suggests that difference in potency due to site of administration may account for the absence of detectable effect of subcutaneously administered rSVEP on rate and strength of wound repair in skin of NZW rabbits and diabetic Zucker Rats tested in preliminary work. It further suggests that intradermal administration to dogs at each treatment in the present study may have yielded an even greater positive effect. Because proteins are not readily absorbed through the epidermis into the dermis, injection was the only practical means of assuring delivery of rSVEP to the dermis near the wound edge. Intradermal injection, however, is associated with a degree of discomfort. Thus, the initial dose of rSVEP or PBS that was given to three dogs intradermally was administered while the dogs were under anesthesia for wound creation. Because subcutaneous injections caused no or little discomfort, subsequent injections on these dogs and all injection on other dogs were subcutaneous. It appears that application in a manner that would facilitate transdermal delivery may provide the best means for therapeutic delivery of rSVEP to the optimum site of action for wound healing—the dermal tissue. The transdermal delivery of rSVEP near a wound edge may hold great potential for stimulating the healing of chronic wounds associated with extensive cellular necrosis and compromised blood supply such as decubital ulcers or diabetic foot wounds, as well as helping assure perfusion of severely traumatized wound tissues. Additionally, the vasoactive properties of rSVEP may promote the delivery of systemic antibiotics to these and other wounds with a compromised blood supply.