Gene Expression and Release of Growth Factors During Delayed Wound Healing: A Review of Studies in Diabetic Animals and Possible Combined Laser Phototherapy and Growth Factor Treatment to Enhance Healing

Abstract

Objective: The purposes of this study were: to review studies of growth factors in cutaneous wounds of animals with diabetes to identify those factors with altered gene expression and content compared with nondiabetic animals; and to explore which deficiencies of growth factors in diabetic wounds may or may not be improved by laser irradiation. Background data: Wound healing is compromised in diabetes. Decreased production and/or increased destruction of growth factors may be responsible. Laser irradiation can increase the gene expression and release of certain growth factors by cells. Methods: Research articles investigating growth factor expression in wounds of nondiabetic and diabetic mice and rats published through September 2011 were retrieved from library sources, PubMed databases, reference lists of articles, and searches of relevant journals. Results: Vascular endothelial growth factor (VEGF), placental growth factor (PlGF), keratinocyte growth factor (KGF), fibroblast growth factor 1 (FGF-1), FGF-2, insulin-like growth factor 1 (IGF-1), IGF-2, transforming growth factor beta (TGF-β), and nerve growth factor (NGF) had decreased gene expression and content in early phases of healing for diabetic wounds. Gene expression of KGF, IGF-1, and IGF-2 was delayed, whereas that of FGF-1 and FGF-2 occurred earlier, in diabetic compared with nondiabetic wounds. Conclusions: Growth factor administration combined with laser irradiation may provide an effective therapy to maximize healing of diabetic wounds.

Introduction

C utaneous wound healing is compromised in diabetes with re-epithelization and the formation of granulation tissue and extracellular matrix being impaired.¹ Growth factors and cytokines play important roles in directing the processes of wound healing and include members of the fibroblast growth factor (FGF) family, for example keratinocyte growth factor (KGF or FGF-7), acidic FGF (aFGF or FGF-1), basic FGF (bFGF or FGF-2), and other growth factors such as transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), and platelet-derived growth factor (PDGF). It is known that the levels of certain growth factors are reduced in wounds of diabetic animals because of decreased production and/or increased destruction, with an alteration in the level of one growth factor likely to affect the production of other growth factors and cytokines. The administration of exogenous growth factors stimulated wound healing in diabetic rats and mice, which is consistent with an impairment in the gene expression and release of certain growth factors in the wounds. Laser irradiation at red or infrared wavelengths has been shown to increase wound healing in diabetic animals.² However, a preliminary search of the literature has revealed only one study in which the expression of growth factors in cutaneous wounds of diabetic animals treated by laser irradiation was reported. Irradiation 632 nm 4 J/cm²/day for 4 days of full-thickness skin wounds in an animal model of type 2 diabetes (Sand Rat) stimulated wound healing and increased bFGF expression at 36 h post-wounding.³ Interestingly, laser irradiation at 632.8 nm of incisional wounds in rat gingiva inhibited the gene expression of interleukin-1β (IL-1β) and interferon-γ (IFN-γ), increased the gene expression of PDGF and TGF-β, and had no effect on the gene expression of bFGF and tumor necrosis factor-α (TNF-α).⁴ There may also be differential effects on gene expression of growth factors and cytokines in cutaneous wounds in response to laser irradiation. Successful clinical application of lasers systems has been demonstrated, for example, to facilitate superficial wound healing⁵ and to treat diabetic foot ulcers in human patients.^6
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Irradiation of cells with red or infrared laser light can stimulate gene expression and release of growth factors from cells in culture.⁹ Experiments in which supernatants of macrophage-like cells irradiated with a 820 nm laser diode were transferred to fibroblasts also growing in culture demonstrated for the first time laser-induced secretion of growth factors resulting in increased fibroblast proliferation.¹⁰ The laser photostimulation of wound healing in splinted wounds of diabetic mice described previously^11,12 could be the result of restoring the levels of certain growth factors in these wounds. It would be helpful to know which deficiencies of growth factors in diabetic wounds might be improved by laser irradiation, and also to identify those that are not likely to be increased by laser therapy. Application of the latter to wounds in combination with laser irradiation may provide a way of maximizing the healing of diabetic wounds.

The expression of growth factors and cytokines in the wounds of nondiabetic and diabetic animals reflects in part the cellular elements present in the wounds at different stages of healing. One approach to designing a protocol of combination therapy for diabetic wounds is to first identify those growth factors that are reduced in the wounds of diabetic animals. This could be the result of a delayed recruitment or inhibition of various cell types in the wounds. The gene expression and release of certain growth factors and cytokines by specific cell types involved in wound healing in response to laser irradiation have been described previously.⁹ By this means, it may be possible to identify those growth factors or cytokines whose expression is lowered in wounds of diabetic animals and which might not be increased by laser irradiation. Such growth factors could be applied to diabetic wounds in combination with laser irradiation to further enhance healing.

It seemed pertinent to review studies examining the gene expression and release of growth factors during cutaneous wound healing of nondiabetic and diabetic animals to identify deficiencies in diabetic animals. Important processes that occur in wound healing are: (1) formation of a platelet- and fibrin-containing plug at the wound site; (2) invasion of the wound by neutrophils and monocytes/macrophages; (3) proliferation and migration of keratinocytes from the wound edge to begin re-epithelization of the wound; (4) proliferation of fibroblasts at the dermal edge; (5) formation of granulation tissue comprising fibroblasts, macrophages, lymphocytes, plasma cells, and an extracellular matrix containing collagen fibers and macromolecular components such as glycoproteins, and the formation of new blood vessels; and (6) maturation of granulation tissue and collagen fibers, and vascularization of wound tissue. The processes involving inflammation, re-epithelization, granulation tissue and extracellular matrix formation, angiogenesis, and tissue remodeling are controlled by growth factors and cytokines produced by the cellular elements present in the wounds and also contained in the blood. Cytokines regulate inflammatory cells during the early stages of wound healing and also influence the gene expression and release of growth factors that are important in the proliferation and differentiation of cells and the synthesis of an extracellular matrix. The presence of receptors or binding proteins for specific growth factors on cells is also important in determining cellular responses.

This article critically reviews those articles published up to September 2011. It focuses on experimental studies that have investigated the gene expression and release of growth factors in the cutaneous wounds of nondiabetic and diabetic animals. These studies have been performed mostly in mice and rats, and only studies with these two animals have been included. The aim of this article was to identify deficiencies of growth factors in the wounds of diabetic animals, and restoration of the levels of some which might be achieved by laser irradiation. Those growth factors whose levels might not be restored by laser irradiation at the appropriate stage of healing could be applied to the wound as part of a combined therapy with laser light.

Methods

Original research articles investigating growth factor expression in skin wounds of nondiabetic and diabetic mice and rats and published up to September 2011 were retrieved and used for this review. Relevant articles were sought and obtained from library sources and the online database PubMed using EndNote XV (Thomson Reuters, Carlsbad). Search terms were growth factor, expression, temporal expression, upregulation, downregulation, wound healing, skin, and diabetes. Additional secondary sources of information included reference lists from retrieved articles, and pertinent articles identified by hand searches of relevant journals not found from the databases.

We included studies that met the following criteria:

1. Studies involving skin wounds

2. Studies performed in nondiabetic (normal) and/or diabetic mice or rats

3. Studies examining gene expression and/or release of growth factors or cytokines from wounds

4. At least one growth factor or cytokine identified as the dependent variable

Studies excluded from this study were:

1. Studies performed on animals other than mouse or rat

2. Studies involving wounds in tissues other than skin (e.g., cornea, gingiva)

3. Reviews and meta-analyses

4. Studies for which only an abstract was available

5. Studies reported in languages for which no English language translation was available

Results

In vivo studies

Results from the literature search are summarized in Fig. 1. There were 23 searched studies on growth factors in nondiabetic and diabetic wounds (n=15, mouse; n=8, rat).^{13

–35} A variety of acute wound models was used in these studies (burn ulcer, incisional, excisional, subcutaneous PVA sponge, and stainless steel mesh chamber). Further details regarding these studies are given in Tables 1 –5. One of the studies also examined the effect of serum growth factors on VEGF mRNA expression by human keratinocytes in vitro ^,31 and this has been included in Table 5.

FIG. 1.

Flowchart of literature search: studies of gene expression of growth factors in skin wounds of nondiabetic and diabetic mice and rats.

Table 1.

Expression of Growth Factors in Skin Ulcer Wounds of Nondiabetic and Diabetic Animals

Authors, reference	Species, status	Wound model and study design	Outcomes measured and time after wounding	Comments on study design & findings
Xie et al.¹³	Rat, male Sprague- Dawley 160–180 g, nondiabetic (control, n=12) and STZ-diabetic (n=26); diabetic ulcer rats divided into two subgroups, diabetic ulcer (n=14) and FGF-1 (aFGF) (n=12) groups.	Skin ulcer made by placing a hot round iron diameter 2.0 cm water-boiled beforehand on each side of the back for 30 sec, respectively, to make deep II scald. The whole skin layer of scald was cut off 3 days later to make the ulcer model (day 0). Animals in the FGF-1 group treated with 6μg FGF-1 in saline to cover wound surface on days 3 and 7 after establishment of ulcer model. Wounds were not covered.	Immunohistochemical staining of TGF-β in formaldehyde-fixed ulcer skin tissues on days 7 and 14 after establishment of ulcer model.	Expression amounts of TGF-β cell proliferation proteins in diabetic group on day 7 after establishment of ulcer model were significantly reduced compared with control group. On day 14 after establishment of ulcer model, expression amounts of TGF-β proliferation proteins were increased in diabetic group, whereas their amounts were significantly reduced in control group.

STZ, streptozotocin; FGF, fibroblast growth factor; TGF-β, transforming growth factor beta.

Table 2.

Expression of Growth Factors in Skin Incisional Wounds of Nondiabetic and Diabetic Animals

Authors, reference	Species, status	Wound model and study design	Outcomes measured and time after wounding	Comments on study design & findings
Zubaldi et al.¹⁴	Rat, male Sprague- Dawley 200–250 g, 40 animals with wounds (n=8/group) and 8 animals as controls; all animals were normal (nondiabetic)	Incisional wound made in skin, fascia, small intestine and colon (day 0).	Cytokine content of wounds measured at 0, 1, 3, 5, 7, and 14 days.	Levels of IL-1β were significantly increased in skin wounds on days 5 and 7 compared with controls (day 0). At all of the time points examined there were no significant differences in levels of TNF-α or TGF-β in skin wounds compared with controls. Levels of IL-10 were significantly decreased in skin wounds at all of the time points compared with controls (day 0).
Galeano et al.¹⁵	Mouse, female C57BL/KsJ diabetic and nondiabetic, 14 weeks of age at beginning of experiment. Animals were divided into 4 groups (n=21/group): first and third groups consisting respectively of nondiabetic and diabetic animals were given polydeoxyribonucleotide i.p. for 12 days, whereas the second and fourth groups of nondiabetic and diabetic animals were given vehicle for 12 days.	Incisional wounds produced by making two full-thickness longitudinal incisions 4 cm on dorsum and wound edges closed with surgical sutures (day 0). Seven animals in each group were killed after 3, 6, and 12 days, and wounds divided into three segments for histology and biochemical and molecular analysis.	VEGF mRNA measured in wound samples by real-time PCR. VEGF protein measured in wound samples by Western blot analysis.	A marked induction of VEGF mRNA was found at days 3 and 6 in wounds of nondiabetic animals given vehicle compared with basal values, and thereafter declined at day 12. VEGF mRNA was markedly lower in wounds of diabetic animals given vehicle at days 3 and 6 compared with nondiabetic animals, and low levels were still detectable at day 12. VEGF content in wounds of nondiabetic animals given vehicle was raised on day 6 after wounding. VEGF content was markedly lower in wounds of diabetic animals given vehicle at day 3, day 6, and day 12 compared with nondiabetic animals. Comment on study design: Making multiple wounds on individual animals may lead to one wound influencing the healing of another.
Bitto et al.¹⁶	Mouse, female C57BL/KsJ diabetic and nondiabetic, 14 weeks of age at beginning of experiment.	Incisional wounds produced by making two full-thickness longitudinal incisions 4 cm on dorsum and wound edges closed with surgical sutures (day 0). Ten animals for each group were killed after 7 and 14 days, and wounds harvested and divided into three segments for histology and immunohistochemical and molecular analysis. Animals were randomized to received a single dose of either rAAV-Aug-1 (recombinant adeno-associated virus-angiopoietin-1 gene) or rAAV-LacZ (recombinant adeno-associated virus-beta-galactosidase reporter gene).	VEGF mRNA measured in wound samples by real-time PCR. VEGF protein measured in wound samples by ELISA.	VEGF mRNA in wounds of diabetic animals administered rAAV-LacZ was markedly lower than in nondiabetic animals at both day 7 and day 14. rAAV-LacZ-treated nondiabetic animals had a marked induction of VEGF mRNA at day 7, which returned to normal values at day 14 after wounding. VEGF protein in wounds of diabetic animals administered rAAV-LacZ was markedly lower than in nondiabetic animals at both days 7 and 14. rAAV-LacZ-treated nondiabetic animals had a marked increase in VEGF protein at day 7, which declined to normal values at day 14 after wounding. Comment on study design: Making multiple wounds on individual animals may lead to one wound influencing the healing of another.
Bitto et al.¹⁷	Mouse, female 10 weeks of age at beginning of experiment, nondiabetic and diabetic.	Incisional wounds produced by making two full-thickness longitudinal incisions 4 cm on dorsum and wound edges closed with surgical sutures (day 0). Ten animals for each group were killed after 3, 6, and 12 days, and wounds harvested. It would appear that one wound was treated with saline whereas the other received simvastatin. Wounds were not covered.	VEGF mRNA measured in wound samples by real-time PCR. VEGF protein measured in wound samples by Western blot analysis.	VEGF mRNA was highly expressed in skin wounds of nondiabetic animals on days 3 and 6, and thereafter declined at day 12. The expression of the mature VEGF protein began to rise at day 3, and reached its maximum increase at day 6. By day 12, the VEGF protein expression had returned to very low levels. VEGF mRNA expression in skin wounds of diabetic animals on days 3 and 6 was significantly lower than for nondiabetic animals. The expression of mature VEGF protein in skin wounds of diabetic animals was significantly less than for nondiabetic animals, and did not show the maximum increase on day 6 that had been found for nondiabetic animals. Comment on study design: The strain of mouse used was not reported. Making multiple wounds on individual animals may lead to one wound influencing the healing of another.
Nogami et al.¹⁸	Rat, male Sprague- Dawley 6–9 weeks of age, nondiabetic.	Incisional wounds produced by making linear incisions 5–7 cm to the depth of the subcutaneous tissue on dorsum (day 0). Wounds were not sutured or covered. After 1, 3, and 7 days, wounds and untreated areas were excised. At least 8 animals for each group.	VEGF mRNA measured in wounds by real-time PCR. VEGF protein measured in wounds by ELISA.	VEGF mRNA expression in skin wounds of nondiabetic animals was significantly increased 24 h after wounding, compared with 3 and 7 days after wounding. VEGF protein content in skin wounds of nondiabetic animals was significantly higher at 1 and 3 days after wounding compared with unwounded skin of nondiabetic animals.
Galeano et al.¹⁹	Mouse, female C57BL/KsJ nondiabetic and diabetic, 14 weeks of age at start of experiment. Animals divided into four groups (n=21/group): first and third groups consisting respectively of nondiabetic and diabetic animals were given erythropoietin s.c., whereas the second and fourth groups of nondiabetic and diabetic animals were given vehicle.	Incisional wounds produced by making two full-thickness longitudinal incisions 4 cm on dorsum and wound edges closed with skin clips (day 0). Seven animals in each group were killed after 3, 6, and 12 days, and wounds divided into three segments for histology and biochemical and molecular analysis.	Expression of VEGF in wounds by PCR. VEGF protein in wounds measured by ELISA.	In wounds of vehicle-treated nondiabetic animals, a marked induction of VEGF mRNA occurred at days 3 and 6 after wounding, and declined on day 12. In wounds of vehicle-treated diabetic animals, induction of VEGF mRNA was markedly lower than in nondiabetic animals at days 3 and 6 after wounding, but although weak, was still detectable at day 12. The content of VEGF protein in unwounded skin of nondiabetic and diabetic animals was low or undetectable. The content of VEGF protein in vehicle-treated wounds of nondiabetic animals was increased on days 3 and 6, declining thereafter to baseline by day 12. In wounds of vehicle-treated diabetic animals, the content of VEGF protein was markedly reduced on days 3 and 6 compared with nondiabetic animals, and was also slightly present on day 12. Comment on study design: Making multiple wounds on individual animals may lead to one wound influencing the healing of another.
Altavilla et al.²⁰	Mouse, female C57BL/KsJ nondiabetic and diabetic, 1 week of age at start of experiment. Animals divided into four groups (n=21/group): first and second groups consisting respectively of diabetic and nondiabetic animals were given raxofelast (an inhibitor of lipid peroxidation), whereas the third and fourth groups of diabetic and nondiabetic animals were given vehicle.	Incisional wounds produced by making two full-thickness longitudinal incisions 4 cm on dorsum and wound edges closed with skin clips (day 0). Seven animals in each group were killed after 3, 6, and 12 days and wounds divided into three segments for histology and biochemical and molecular analysis.	Expression of VEGF in wounds by PCR. VEGF protein in wounds measured by ELISA.	In wounds of vehicle-treated nondiabetic animals, a strong induction of VEGF mRNA occurred between days 3 and 6 after wounding, but was not detectable on day 12. In wounds of vehicle-treated diabetic animals, induction of VEGF mRNA was markedly reduced at days 3 and 6 after wounding, but although weak was still detectable at day 12. The content of VEGF protein in unwounded skin of nondiabetic and diabetic animals was low or undetectable. The content of VEGF protein in vehicle-treated wounds of nondiabetic animals was increased on days 3 and 6, declining thereafter to baseline by day 12. In wounds of vehicle-treated diabetic animals, the content of VEGF protein was markedly decreased on days 3 and 6 compared with nondiabetic animals, and was also slightly present on day 12. Comment on study design: Making multiple wounds on individual animals may lead to one wound influencing the healing of another.
Gartner et al.²¹	Rat, male Fischer 175–200 g, all animals were normal (nondiabetic)	Incisional wound produced by making a 4 cm full-thickness linear incision on dorsum of animals and closed with surgical clips (day 0). Wounds were harvested after 0.5, 1, 3, 5, 10, and 21 days.	Expression of IGF-1, IGF-2 in incisional wounds by real-time PCR	IGF-1 mRNA expression was low in unwounded skin and at 0.5 day after wounding. The level in incisional wound started to increase at 1 day and peaked at 10 days after wounding. The expression remained at a high level through to 21 days after wounding. IGF-2 mRNA expression was low in unwounded skin and at 0.5 day after wounding. The level in incisional wound increased during the early stages of wound healing and reached a peak at 10 days after wounding. The expression then decreased, although the levels at 15 and 21 days were still significantly higher than for unwounded skin and at 0.5 day after wounding. It is likely that fibroblasts were both secreting and responding to IGF-1 and IGF-2. Also proliferating blood vessels may have accounted for a portion of the increased IGF-1 mRNA expression at the later time points, as IGF-1 has been detected in the proliferating endothelial cells at sites of angiogenesis.

IL, interleukin; TNF-α, tumor necrosis factor-α; TGF-β, transforming growth factor beta; VEGF, vascular endothelial growth factor; PCR, polymerase chain reaction; IGF, insulin-like growth factor.

Table 3.

Expression of Growth Factors in Skin Excisional Wounds of Nondiabetic and Diabetic Animals

Authors, reference	Species, status	Wound model and study design	Outcomes measured and time after wounding	Comments on study design & findings
Liu et al.²²	Mouse, female BKS.Cg-m^+/+ Lepr^db/J nondiabetic and diabetic.	Excisional wounds produced by making two or four 5 mm-diameter full-thickness wounds on dorsum (day 0). Wounds were not covered. On days 0, 5, 7, and 10, wound eschar was carefully removed.	HIF-1α mRNA, PlGF mRNA, PDGF mRNA, and VEGF mRNA measured by real-time PCR in wounds of diabetic animals 8 weeks old on days 3 and 5 after wounding.	Expression of mRNAs encoding for HIF-1α, PDGF-B, and VEGF were significantly increased on day 5 compared with day 3 in wounds of 8-week-old diabetic animals. Expression of PlGF mRNA on day 5 was not significantly different to that on day 3. Comment on study design: Making multiple wounds on individual animals may lead to one wound influencing the healing of another.
Cianfarani et al.²³	Mouse, male C57BL/6 5 weeks of age, nondiabetic, and STZ-diabetic.	Excisional wound produced by making a 6mm-diameter full-thickness wound on dorsum (day 0). Wounds were not covered. One day after wounding, wounds of diabetic animals were treated with saline or vector encoding for human PlGF or β-galactosidase delivered under the clot covering wound. Wounds of nondiabetic animals were treated with saline.	PlGF and VEGF measured in protein extracts of wounds from nondiabetic and diabetic animals on days 0, 1, 3, 5, 7, and 9 after wounding by ELISA. PlGF mRNA and VEGF mRNA measured in wound samples by real-time reverse transcriptase PCR on days 0, 1, 3, 5, 7, and 9 after wounding.	In wounds of saline-treated nondiabetic animals PlGF, mRNA induction began on day 1, and reached a maximum on day 3 after wounding, concomitant with the initiation of granulation tissue formation. Levels of PlGF mRNA remained higher than those in unwounded skin at least up to day 9 after wounding. In wounds of saline-treated-diabetic animals, PlGF mRNA induction was significantly reduced compared with nondiabetic animals on days 1, 3, and 5 after wounding, with there being a 5-fold difference on day 3. By in situ hybridization analysis a strong PlGF signal was detected at day 3 on migrating keratinocytes at wound margin in nondiabetic animals, whereas in diabetic wounds, a much weaker signal on keratinocytes was found. In wounds of saline-treated nondiabetic animals, PlGF protein induction started to increase on day 1, reached highest levels on day 3, and remained upregulated until day 9 after wounding. In wounds of saline-treated diabetic animals, PlGF protein induction was significantly reduced compared with nondiabetic animals from days 1 to 7 after wounding, with a 3.5-fold difference on day 3. VEGF mRNA expression was induced in wounds of saline-treated nondiabetic animals, with a maximum on day 3 after wounding. In wounds of saline-treated diabetic animals, VEGF mRNA expression was reduced compared with saline-treated nondiabetic animals, reaching a more than 3-fold difference on days 3 and 5 after wounding. A decreased amount of VEGFR-1 mRNA was found in wounds of saline-treated diabetic animals compared with saline-treated nondiabetic animals at all time points examined (up to 9 days after wounding). Induction of PDGF-B mRNA, FGF-2 mRNA, and VEGF mRNA was strongly decreased in wounds of diabetic animals compared with nondiabetic animals, whereas induction of TGF-β1 mRNA was less affected.
Komi-Kuramochi et al.²⁴	Mouse, male hairless (^hr/hr) 8 weeks of age, all animals were normal (nondiabetic). Hairless mice were chosen to eliminate effect of hair growth cycle on FGF expression, as earlier studies had shown that many of the FGFs play important roles in hair dynamics (Kawano et al. J Invest Dermatol 2005, 124: 877–885).	Excisional wounds produced by making four full-thickness circular wounds 6mm diameter on dorsum of each animal (day 0). Wounds were not covered. On days 2, 4, 7, 15, and 21 after wounding four mice were killed, and full-thickness skin samples (four 8×8 mm squares per mouse containing the healing wounds) were excised and processed for mRNA isolation.	FGF mRNAs measured in the skin samples by real-time PCR. Also TGF-β mRNA and HGF mRNA measured during wound healing.	FGF-7 (KGF-1) mRNA and FGF-10 (KGF-2) mRNA were strongly expressed in skin. Levels of mRNAs encoding FGF-7 and FGF-10 were significantly upregulated at various times during wound healing. As compared with the levels on day 0, FGF-7 mRNA and FGF-10 mRNA were increased on days 2 and 15 after wounding. TGF-β mRNA and HGF mRNA were expressed at high levels in skin and were then upregulated during wound healing. Like that of FGF-7 and FGF-10, the expression of TGF-β mRNA peaked on days 2 and 15, whereas expression of HGF mRNA was upregulated on day 2 and then remained elevated until day 15. Comment on study design: Making multiple wounds on individual animals may lead to one wound influencing the healing of another.
Chen et al.²⁵	Mouse, female nonobese diabetic (NOD) 16–20 weeks of age. Age and weight matched nondiabetic animals used as controls.	Excisional wounds produced by making four full-thickness circular wounds 8mm diameter on dorsum of each animal (day 0). The wounds were separated by 1cm skin. Wounds were transfected with tissue factor (TF) or vehicle and covered with an adhesive Tegaderm dressing for 12 h.	VEGF mRNA measured by real-time PCR.	Induction of VEGF mRNA occurred in vehicle-treated wounds of nondiabetic animals rising ∼2-fold above the baseline within 12 h, but not reaching statistical significance. Levels of VEGF transcripts were maintained for 24 h, thereafter slowly decreasing at day 7 after wounding when wound was almost completely healed. The wounds of diabetic mice showed a small but not significant induction of VEGF mRNA not before 12 h after wounding, and increased at day 3 after wounding. Therefore, VEGF mRNA induction in wounds of nondiabetic animals contrasted with lack of induction in diabetic animals within the first hours of wounding. Comment on study design: Making multiple wounds on individual animals may lead to one wound influencing the healing of another.
Rajasekaran et al.²⁶	Rat, male Wistar, mean wt 160 g. Alloxan (i.p.) used to induce type 1 diabetes, control animals received saline.	Excisional wound produced by making excision wound 1.5×1.5 cm on dorsum of animals on 15th day after induction of diabetes (day 0). Wounds were not covered. Wounds were collected at 0, 1, 4, 7, 10, and 15 days after wounding	NGF levels in wounds were measured by ELISA.	A very low or undetectable level of NGF was found in skin tissues obtained during wound creation (0 h). Wounding resulted in increased NGF levels. A maximum level of NGF was found in wounds of control animals on day 1 and slowly declined to day 15 after wounding. The level of NGF in wounds of diabetic animals was maximal on day 1, and levels were lower than for wounds of control animals on days 1, 4, and 7 after wounding. A higher level of NGF in wounds of diabetic animals than for control animals was found on day 15 after wounding.
Graiani et al.²⁷	Mouse, male CD1 nondiabetic and STZ-diabetic animals were used in experiment at 4 weeks from induction of diabetes (12 weeks of age).	Excisional wounds produced by making two full-thickness circular wounds 4mm diameter in interscapular region of each animal (day 0). One wound was treated daily with PBS, whereas the other wound was treated with NGF, continuing for 3 days. Wounds were not covered. Wounds and surrounding skin were removed and cut into two halves. One half was for molecular biology studies performed on specimens collected on day 3 after wounding. The other half was for histology and/or immunohistochemical analysis on specimens collected on days 3 and 14 after wounding.	VEGF-A expression in wounds measured by real-time PCR. NGF was measured by ELISA.	NGF content in PBS-treated wounds of diabetic animals was significantly lower than that in PBS-treated wounds of nondiabetic animals on day 3 after wounding. Expression of VEGF-A mRNA in PBS-treated wounds of diabetic animals was significantly higher than that in PBS-treated wounds of nondiabetic animals on day 3 after wounding. Comment on study design: Making multiple wounds on individual animals may lead to one wound influencing the healing of another.
Stallmeyer et al.²⁸	Mouse, female nondiabetic C57BL/6 and diabetic C57BL/6J-ob/ob, 8 weeks of age	Excisional wounds produced by making six full-thickness skin wounds 4–6 mm in diameter, 3–4 mm apart on dorsum (day 0). The wounds were allowed to dry to form a scab. Wounds in diabetic-ob/ob mice were covered with 20 μl PBS applied directly to surface of the scabs. Wound samples of 7–8 mm in diameter, which included the scab and complete epithelial margins, were collected at 1, 3, 5, 7, and 13 days after wounding.	Expression of VEGF in wounds by RNase protection assays. VEGF₁₆₄ protein measured in wounds by ELISA.	Induction of VEGF mRNA in wounds for both nondiabetic and PBS-treated diabetic ob/ob animals was highest on day 1 after wounding, with the level for diabetic animals being slightly lower than for nondiabetic animals. Expression of VEGF protein in wounds of nondiabetic animals was maximal on days 3, 5, and 7 after wounding, with days 5–7 representing the peak of angiogenesis in the granulation tissue. The level of expression of VEGF protein in wounds of PBS-treated diabetic ob/ob animals was lower on days 1 and 3 after wounding compared with wounds of nondiabetic animals, but reached a maximum on day 7 after wounding when it was higher than for nondiabetic animals. Comment on study design: Making multiple wounds on individual animals may lead to one wound influencing the healing of another.
Moulin et al.²⁹	Rat, male 250–300 g, nondiabetic and STZ-diabetic	Excisional wounds produced by making two full-thickness skin wounds 1×1.5 cm on dorsum (day 0). Wounds were filled with resorbable collagen plasters soaked with physiological buffer. Wounds were covered with an occlusive dressing. Wound samples of 1×1.5 cm were collected after 3, 7, 15, or 30 days.	FGF-1 (aFGF) and FGF-2 (bFGF) in sample extracts measured by EIA. Active TGF-β in sample extracts measured by bioassay.	FGF-1 content was significantly lower in unwounded diabetic skin compared with nondiabetic skin. FGF-1 content in wounds of diabetic animals was significantly lower than in wounds of nondiabetic animals on day 7 after wounding, but was higher than in wounds of nondiabetic animals on day 30 after wounding. FGF-2 content was significantly lower in unwounded diabetic skin compared with nondiabetic skin. FGF-2 content in wounds of diabetic animals was significantly lower than in wounds of nondiabetic animals on days 7 and 15 after wounding. Active TGF-β content was significantly lower in unwounded diabetic skin compared to nondiabetic skin. Active TGF-β content in wounds of diabetic animals was significantly lower than in wounds of nondiabetic animals on days 7 and 30 after wounding. Comment on study design: Strain of animals was not indicated. The exact number of samples at each time point was not indicated.
Brown et al.³⁰	Mouse, male C57BL/KsJ diabetic and nondiabetic.	Excisional wounds produced by making full-thickness skin wounds 1.5×1.5 cm on dorsum (day 0). Wounds were covered with transparent, semipermeable dressing. Four animals per time point were killed at 0.5, 1, 3, 5, 10, 14, 20, and 28 days after wounding. Wounds were harvested including a margin of ∼0.5 cm of unwounded skin. One-half of each wound was fixed in paraformaldehyde for immunohistochemistry and in situ hybridization, and the other half was flash-frozen for quantification of mRNA by real time PCR and of protein.	Localization of IGF-1 and IGF-2 in wounds. Gene expression of IGF-1 and IGF-2 in wounds.	No IGF-1 mRNA was found in unwounded nondiabetic and diabetic skin. At 12 h, IGF-1 mRNA was induced in wounds of nondiabetic animals and at 3 days after wounding there was a 50-fold increase in IGF-1 mRNA compared with the level at 12 h. The level then slowly decreased to steady state levels at 28 days after wounding. In wounds of diabetic animals, IGF-1 mRNA levels rose minimally at 5 days but did not peak until 14 days after wounding, with the levels being 2-fold lower than those of the peak at 3 days after wounding in the nondiabetic animals. IGF-1 protein levels in nondiabetic wounds were approximately double those of diabetic wounds. A sharp increase in IGF-1 protein concentration occurred at 5 days in the nondiabetic wounds, which declined slowly thereafter to 28 days after wounding. IGF-1 protein levels in the diabetic wounds maintained a blunted expression until 21 days after wounding, when they rose to nondiabetic levels. IGF-2 mRNA was not detected in skin before wounding. IGF-2 mRNA levels peaked in nondiabetic wounds at 3 days after wounding. In diabetic wounds there was a delay in IGF-2 mRNA expression with peak levels occurring at 10 days after wounding. Peak levels of IGF-2 mRNA in diabetic wounds were 4-fold greater than in nondiabetic wounds. IGF-2 protein levels could not be measured satisfactorily because of substances in the extract interfering with IGF-2 measurement.
Frank et al.³¹	Mouse, BALB/c 8–12 weeks of age at start of experiments, diabetic and non-diabetic.	Excisional wounds produced by making six full-thickness circular wounds 6mm diameter, 3–4 mm apart on dorsum of each animal (n=20/experiment) (day 0). Wounds were not covered. Wounds from four animals were harvested at 1, 3, 5, 7, and 13 days after wounding. An area of 7 mm diameter that included the complete epithelial margin was excised at each time point. A similar amount of skin from dorsum of three unwounded diabetic animals was used as a control.	Expression of VEGF mRNA in wounds by RNase protection analysis.	A strong induction of VEGF mRNA occurred in wounds of nondiabetic animals within 24 h after wounding and highest levels were found between day 3 and 7 after wounding. This is the period of granulation tissue formation and the data suggest that induction of VEGF mRNA might play a role in wound angiogenesis. After an initial increase in VEGF mRNA levels in wounds of diabetic animals within 24 h after wounding, expression levels declined markedly and were barely detectable at day 5 after wounding. Comment on study design: Sex of animals was not indicated. Making multiple wounds on individual animals may lead to one wound influencing the healing of another.
Werner et al.³²	Mouse, C57BL/KsJ 8–12 weeks of age, diabetic and nondiabetic.	Excisional wounds produced by making six full-thickness circular wounds 6mm diameter, 3–4 mm apart on dorsum of each animal (n=20/experiment) (day 0). Wounds were not covered. Wounds from four animals were harvested at 0.5, 1, 3, 5, and 7 days after wounding. An area of 7mm diameter that included the complete epithelial margin was excised at each time point. A similar amount of skin from the dorsum of three unwounded diabetic animals was used as a control.	Expression of KGF mRNA, FGF-1 mRNA, and FGF-2 mRNA in wounds by RNase protection analysis.	Similar levels of KGF mRNA were found in unwounded skin of diabetic animals and nondiabetic animals. As in nondiabetic animals, there was an induction of KGF mRNA during wound healing in diabetic animals, but it was only 5- to 10-fold compared with 160-fold in nondiabetic animals. There was a delay in KGF induction in wounds of diabetic animals, with the maximal levels of KGF expression only obtained between 3 and 5 days after wounding, whereas expression of KGF mRNA in wounds of nondiabetic animals reached a peak at 24 h after wounding. These findings show that KGF mRNA expression is low in diabetic animals during the time when re-epithelization normally occurs, and therefore might underlie the delayed keratinocyte proliferation in wounds of diabetic animals. Induction of both FGF-1 mRNA and FGF-2 mRNA occurred earlier in wounds of diabetic animals compared with nondiabetic animals, and expression of both FGF-1 mRNA and FGF-2 mRNA returned to basal levels within 3 days after wounding. In contrast, enhanced expression of FGF-1 mRNA and FGF-2 mRNA in wounds of nondiabetic animals occurred during the first 7 days after wounding. FGF-2 mRNA expression in wounds of diabetic animals was induced 3.5-fold and peaked within 24 h after wounding. In wounds of nondiabetic animals, FGF-2 mRNA expression was induced 4-fold with the peak being reached at day 5 after wounding. FGF-1 mRNA expression in wounds of diabetic animals was induced less than 2-fold, being high during the first 24 h after wounding. Expression of FGF-1 mRNA declined rapidly after the first day, and very low levels were found at later stages of wound healing. A 10-fold induction of FGF-1 mRNA expression occurred in wounds of nondiabetic mice and remained elevated within the first 3 days after wounding. Therefore, FGFs seem to be expressed in a limited fashion in wounds of diabetic animals during the period when re-epithelization and granulation tissue formation normally occur. Comment on study design: Sex of animals was not indicated. Making multiple wounds on individual animals may lead to one wound influencing the healing of another.

HIF, hypoxia-inducible factor; PlGF, placental growth factor; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; PCR, polymerase chain reaction; STZ, streptozotocin; FGF, fibroblast growth factor; TGF, transforming growth factor; HGF, hepatocyte growth factor; NGF, nerve growth factor; PBS, phosphate-buffered saline; EIA, enzyme immunoassay; IGF, insulin-like growth factor; KGF, keratinocyte growth factor.

Table 4.

Expression of Growth Factors in Subcutaneous PVA Sponge or Stainless Steel Mesh Chamber of Nondiabetic and Diabetic Animals

Authors, reference	Species, status	Wound model and study design	Outcomes measured and time after wounding	Comments on study design & findings
Bitar³³	Rat, male Wistar 175–220 g, nondiabetic and STZ-diabetic. Diabetic animals were randomly divided into two groups: one group received no antidiabetic treatment, whereas the other was subjected to a daily s.c. injection of 5–8 U of insulin. A hypercortisolemic state was induced in nondiabetic control rats by s.c. implantation of slow-releasing pellets containing a supraphysiologic dose of glucocorticoids. One group of hypercortisolemic animals was subjected to treatment with the gluocorticoid receptor blocker mifepristone RU486, whereas the other received placebo pellets by same s.c. route. RU486 was also given to a group of diabetic animals that had been injected with STZ 5 days earlier.	Subcutaneous PVA sponge or stainless steel mesh chamber wound model. At 20 days after insertion of a placebo or slow-releasing pellets, a single 5 cm skin incision was made on dorsum down to the level of the panniculus carnosus (day 0). Subcutaneous pockets were created at the poles and margins of wounds into which 4 sterile PVA sponges or 4 stainless steel mesh chambers were inserted. Four sponges were used for each animal. Wounds were closed with stainless steel sutures. At 7 days after wounding animals were killed. PVA sponges were dissected free of connective tissue and used for biochemical testing. Wound fluid derived from stainless steel mesh chambers was used for biochemical testing.	Genetic expression of IGF-1, IGF-1 R, and IGF-BP₃ by real-time PCR using PVA sponges. IGF-1 protein measured in wound fluid.	Levels of mRNA expression of IGF-1, IGF-1 R, and IGF-BP₃ in PVA sponges of diabetic rats were decreased by 50%, 33%, and 32%, respectively, when compared with their corresponding nondiabetic values at 7 days after wounding. The level of IGF-1 and IGF-BP₃ in the wound fluid of diabetic rats was significantly reduced compared with that of nondiabetic animals at 7 days after wounding.
Bitar and, Labbad ³⁴	Rat, male Sprague Dawley 150–175 g at beginning of experiment, nondiabetic and STZ-diabetic animals used 21–30 days after injection of citrate buffer or STZ, respectively.	Subcutaneous PVA sponge or stainless steel mesh chamber wound model. A single 5cm skin incision was made on dorsum down to the level of the panniculus carnosus (day 0). Subcutaneous pockets were created at the poles and margins of wounds into which sterile PVA sponges or stainless steel mesh chambers were inserted. Four to six sponges or four stainless steel mesh chambers were used for each animal. Wounds were closed with stainless steel sutures. At 7 days after wounding, animals were killed. PVA sponges were dissected free of connective tissue and used for biochemical testing. Wound fluid derived from stainless steel mesh chambers was used for biochemical testing.	IGF-1 protein and TGF-β protein measured in wound fluid. TGF-β protein measured by a proliferation-based bioassay. IGF-BP measured in wound fluid.	IGF-1 protein levels in wound fluid and serum of diabetic animals were reduced by 41% and 48%, respectively, compared with nondiabetic animals at 7 days after wounding. TGF-β protein level in wound fluid of diabetic animals was decreased by 55% compared with nondiabetic animals at 7 days after wounding. There was a marked reduction in the level of a 456 kDa IGF-BP in the wound fluid and serum of diabetic animals compared with nondiabetic animals at 7 days after wounding.
Gartner et al.²¹	Rat, male Fischer 175–200 g nondiabetic.	Subcutaneous PVA sponge wound model. Ten sterile 1×1 cm PVA sponges were inserted subcutaneously in dorsum of animals (day 0). Seven animals were used for each time point. Cellular infiltrates were collected from the sponges on 0.5, 1, 3, 5, 10, and 21 days after wounding.	IGF-1 message and IGF-2 message in cellular infiltrates.	The relative copy number for IGF-I message in cellular infiltrate samples varied over the time course of wound healing. There was low copy number at 12 h after wounding, which started to increase on day 1 and continued to increase through day 21 after wounding. The copy number for the expected 383-bp PCR product for IGF-II in the cellular infiltrate samples showed a trend of increasing message expression over time course of wound healing. There also appeared to be a temporal variation in the proportion of a larger (650 bp) transcript. This transcript was identified at all time points in the cellular infiltrate samples.

STZ, streptozotocin; IGF, insulin-like growth factor; TGF, transforming growth factor.

Table 5.

Induction of Expression of Growth Factors in Skin Wounds or Cultured Cells by Cytokines and Growth Factors

Authors, reference	Species, status or cell type	Study design	Outcomes measured and time after wounding	Comments on study design & findings
Xie et al.¹³	Rat, male Sprague-Dawley 160–180 g, nondiabetic (control group, n=12) and STZ-diabetic (n=26); diabetic ulcer rats divided into two subgroups, diabetic ulcer (n=14) and FGF-1 (aFGF) (n=12) groups. Saline was administered to nondiabetic animals and diabetic ulcer group.	Skin ulcer made by placing a hot round iron, diameter 2.0 cm water-boiled beforehand, on each side of the back for 30 sec to make deep II scald. The whole skin layer of scald was cut off 3 days later to make the ulcer model (day 0). Animals in the FGF-1 (aFGF) group treated with 6 μg FGF-1 in saline to cover wound surface on days 3 and 7 after establishment of ulcer model. Wounds were not covered.	Immunohistochemical staining of TGF-β in formaldehyde-fixed ulcer skin tissues on days 7 and 14 after establishment of ulcer model.	Expression amounts of TGF-β in FGF-1-treated diabetic ulcer group were not significantly different to saline-treated nondiabetic ulcer group on days 7 and 14 after establishment of ulcer model.
Liu et al.²²	Mouse, female BKS.Cg-m^+/+ Lepr^db/J nondiabetic and diabetic.	Excisional skin wounds produced by making 5 mm-diameter full-thickness wounds on dorsum (day 0). Wounds were not covered. On days 0, 5, 7, and 10, wound eschar was carefully removed. Animals subsequently received intradermal injections of a plasmid which encoded a constitutively active form of HIF-1α or of vehicle at two sites on both sides of the wound. Animals were electroporated at the site of injection within 2 min after plasmid injection using a square wave electroporator.	HIF-1α mRNA, PlGF mRNA, PDGF mRNA and VEGF mRNA measured by real-time reverse-transcriptase PCR in wounds of diabetic animals 6 month-old on days 3, 7, 14, and 21 days after electroporation.	HIF-1α mRNA levels were maximal on day 3 and were still elevated compared to non-transfected animals on day 21 after electroporation. VEGF mRNA, PlGF mRNA, and PDGF mRNA were significantly raised on day 7 after electroporation. These results show that plasmid injection with electroporation increased HIF-1α mRNA levels, leading to increased expression of genes encoding angiogenic growth factors/cytokines in wounds of diabetic mice. Comment on study design: Making multiple wounds on individual animals may lead to one wound influencing the healing of another.
Cianfarani et al.²³	Mouse, male C57BL/6 5 weeks of age nondiabetic and STZ-diabetic.	Excisional skin wound produced by making a 6 mm-diameter full-thickness wound on dorsum (day 0). Wounds were not covered. One day after wounding, wounds of diabetic animals were treated with saline or vector encoding for human PlGF or β-galactosidase. Wounds of nondiabetic animals were treated with saline.	PlGF and VEGF measured in protein extracts of wounds from nondiabetic and diabetic animals by ELISA. PlGF mRNA and VEGF mRNA measured in wound samples by real-time reverse-transcriptase PCR.	There was a significant increase in mRNA levels of PDGF-B, FGF-2 and VEGF in wounds of diabetic animals treated with vector encoding for PlGF compared with wounds in diabetic animals treated with vector encoding for β-galactosidase. PDGF-B and VEGF mRNA increase was highly significant at day 8 after treatment (day 9 after wounding, 5.5-fold increase for both growth factors) in diabetic wounds treated with vector encoding for PlGF compared with wounds treated with vector encoding for β-galactosidase. The increase was delayed and was stronger than that observed in wounds from nondiabetic animals. FGF-2 induction was significantly higher in diabetic wounds treated with vector encoding for PlGF compared with wounds treated with vector encoding for β-galactosidase starting from day 4 after treatment (day 5 after wounding) and reaching a 4.3-fold difference at day 6 (day 7 after wounding), with timing and levels comparable to those observed in nondiabetic animals.
				TGFβ-1 mRNA normally peaking on day 3 after wounding was similar for diabetic wounds treated with vector encoding for PlGF compared with wounds treated with vector encoding for β-galactosidase.
Graiani et al.²⁷	Mouse, male CD1 nondiabetic and STZ-diabetic, at 4 weeks from induction of diabetes (12 weeks of age) were used in experiment.	Excisional skin wounds produced by making two full-thickness circular wounds 4 mm diameter in interscapular region of each animal. One wound was treated daily with PBS, whereas the other wound was treated daily with 1μg NGF, starting immediately after wounding and continuing for 3 days. Wounds and surrounding skin were removed and cut into two halves. One half was for molecular biology studies performed on specimens collected on day 3 after wounding. The other half was for histology and/or immunohistochemical analysis on specimens collected on days 3 and 14 after wounding.	VEGF-A expression in wounds measured by real-time reverse-transcriptase PCR. NGF was measured by ELISA.	NGF-treated wounds of diabetic animals had a significant 5-fold increase in VEGF-A mRNA compared with PBS-treated wounds of diabetic animals on day 3 after wounding.The effect of NGF on VEGF-A expression in wounds of nondiabetic animals was less pronounced and there was no significant difference compared with PBS-treated wounds of nondiabetic animals. Comment on study design: Making multiple wounds on individual animals may lead to one wound influencing the healing of another.
Galiano et al.³⁵	Mouse, female BKS.Cg-m^+/+ Lepr^db/J, diabetic 12 weeks of age.	Excisional skin wounds produced by making two 6-mm circular full-thickness skin wounds on dorsum (day 0). A donut-shaped 12-mm splint made of 0.5 mm-thick silicone sheeting was placed around wounds and adhered to skin with cyanoacrylate glue and sutures. Recombinant human VEGF₁₆₅ protein or PBS vehicle was placed in wound bed at dose of 20 μg per wound. Wound and splint covered with an occlusive dressing. VEGF protein was applied to wounds on days 0, 2, 4, 6, and 8 after wounding. Wounds were covered with occlusive dressing after VEGF or PBS administration.Wounds were excised with a 1mm rim of surrounding skin on days 7 and 12 after wounding.	Expression of IGF-1, CTGF, TGF-β1, FGF-2 and PDGF by real-time reverse-transcriptase PCR.	Wounds treated with VEGF protein had 2.3-fold more FGF-2 mRNA and 1.7-fold more PDGF mRNA compared with PBS-treated wounds at day 7 after wounding. The upregulation in levels of FGF-2 mRNA and PDGF mRNA persisted even at day 12 after wounding.There was no significant increase in expression of IGF-1 mRNA, CTGF mRNA, and TGF-β1 mRNA for wounds treated with VEGF protein compared with PBS-treated wounds. Comment on study design: Making multiple wounds on individual animals may lead to one wound influencing the healing of another.
Moulin et al.²⁹	Rat, male 250–300 g, nondiabetic and STZ-diabetic.	Excisional skin wounds produced by making two full-thickness skin wounds 1×1.5 cm on dorsum (day 0). Wounds were filled with resorbable collagen plasters soaked with platelet releasate (PR) (corresponding to 10⁶ platelets) or physiological buffer. Wounds were covered with an occlusive dressing. Wound samples of 1×1.5 cm were collected after 3, 7, 15, or 30 days.The amount of PR in each wound was indicated as containing 5 ng PDGF and 7 ng total TGF-β (active and acid-activated).	FGF-1 and FGF-2 in sample extracts measured by EIA.Active TGF-β in sample extracts measured by bioassay.	FGF-1 content in PR-treated wounds of diabetic animals was significantly lower than in buffer-treated wounds of diabetic animals on days 15 and 30 after wounding, and significantly lower than in buffer-treated wounds of nondiabetic animals on days 7 and 15 after wounding.FGF-2 content in PR-treated wounds of diabetic animals was significantly higher than in buffer-treated wounds of diabetic animals on days 3, 7, 15, and 30 after wounding, and significantly higher than in buffer-treated wounds of nondiabetic animals on days 3 and 30 after wounding.Active TGF-β content in PR-treated wounds of diabetic animals was significantly higher than in buffer-treated wounds of diabetic animals on day 7 after wounding. Comment on study design: Making multiple wounds on individual animals may lead to one wound influencing the healing of another.
Frank et al.³¹	Human, keratinocyte cell line HaCaT.	Cells cultured in DMEM with 10% FBS and grown to confluence without changing the medium and rendered quiescent by 16 h culture in serum-starved DMEM. Cells were then incubated for varying periods in fresh DMEM with serum, purified growth factors, or cytokines. Aliquots of cells harvested before and at different time points after treatment with these reagents.	Expression of VEGF mRNA induction in keratinocytes by RNase protection analysis.	Low levels of VEGF mRNA were detected in quiescent keratinocytes. Upon the addition of 10% FBS, a large induction of VEGF mRNA occurred; within 90 min after serum stimulation, VEGF mRNA levels were 20-fold higher compared to basal level. The effect of serum on VEGF mRNA levels was long lasting, and elevated levels were still observed 8 h after serum stimulation. At 24 h after serum addition, VEGF mRNA levels returned to the basal level.Local hemorrhage is one of the initial events after skin wounding, and these findings show the potency of serum to stimulate VEGF mRNA expression.The effect of purified serum growth factors on VEGF mRNA expression was analyzed.EGF was a potent inducer of VEGF mRNA expression and increased VEGF mRNA levels 20-fold within 90 min, 50-fold within 5 h, and 60-fold within 8 h. EGF-induced VEGF mRNA expression was long lasting, and high levels of VEGF mRNA were still observed 24 h after addition of EGF.PDGF-BB had no effect on VEGF mRNA levels.In addition to EGF (a keratinocyte mitogen), TGF-β1 (a potent inhibitor of keratinocyte proliferation) also increased VEGF mRNA levels 16-fold within 2 h. TGF-β1 increased VEGF mRNA levels only transiently, and 5 h after addition of the growth factor, VEGF mRNA expression levels had significantly declined.A large induction of KGF expression occurred in the dermis during wound healing, and effect of KGF was analyzed.VEGF mRNA expression was rapidly induced by KGF and slightly elevated VEGF mRNA levels were detected as early as 15 min after addition of KGF. Within 30 min after KGF addition, VEGF mRNA levels were 5-fold higher than basal level and an 8- to 10-fold induction of VEGF mRNA was found within 2 h after stimulation with KGF. Similar to EGF-induced VEGF expression, high levels of VEGF mRNA were observed for at least 8 h after addition of KGF.Infiltration by PMNs followed by macrophages is another early event in wound healing.TNF-α (which inhibits keratinocyte growth in vitro) is a very potent activator of VEGF mRNA expression and a 50-fold increase of VEGF mRNA expression was observed within 2 h after addition of TNF-α to keratinocytes.IL-1β (which is mitogenic for keratinocytes) was much less efficient, and increased VEGF mRNA levels <5-fold.Similar to TGF-β1-induced VEGF mRNA expression, VEGF mRNA induction by these cytokines was only transient, and 5 h after cytokine addition VEGF mRNA expression levels had significantly declined.IL-6 (which like IL-1 is mitogenic for keratinocytes) had no effect on VEGF mRNA expression in keratinocytes.

STZ, streptozotocin; FGF, fibroblast growth factor; TGF, transforming growth factor; HIF, hypoxia-inducible factor; PlGF, placental growth factor; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor ; PCR, ppolymerase chain reaction; PBS, phosphate-buffered saline; NGF, nerve growth factor; CTGF, connective tissue growth factor; EIA, enzyme immunoassay; DMEM, Dulbecco's Modified Eagle Medium; FBS, fetal bovine serum; EGF, epidermal growth factor; KGF, keratinocyte growth factor; PMN, polymorphonuclear leukocytes; TNF-α, tumor necrosis factor α; IL, interleukin.

Burn ulcer wound model (Table 1)

In the rat, one study used nondiabetic and diabetic animals, which served as an experimental model of type 1 diabetes. Expression amounts of TGF-β cell proliferation proteins in diabetic animals on day 7 after establishing the ulcer were significantly reduced compared with nondiabetic animals. Expression amounts of TGF-β proliferation proteins were increased for diabetic animals on day 14 compared with day 7, whereas for nondiabetic animals the amounts were decreased on day 14 compared with day 7.

Incisional wound model (Table 2)

In the mouse, all five studies used nondiabetic and diabetic animals, which served as an experimental model of type 2 diabetes. Two full-thickness longitudinal incisions were made in the dorsum and closed with surgical sutures or skin clips. These wounds would have healed by first intention. A marked induction of VEGF mRNA was found at days 3 and 6 in the wounds of nondiabetic animals compared with basal values, and thereafter declined at day 12 after wounding. VEGF mRNA was markedly lower in the wounds of diabetic animals at days 3 and 6 compared with nondiabetic animals, and low levels were still detectable at day 12. VEGF content in the wounds of nondiabetic animals was raised on day 6 after wounding. VEGF content was markedly lower in the wounds of diabetic animals at days 3, 6, and 12 compared with nondiabetic animals. Therefore, the expression of mature VEGF protein in skin wounds of diabetic animals was significantly less than for nondiabetic animals, and did not show the maximum increase on day 6 that had been found for nondiabetic animals.

In the rat, all three studies used nondiabetic animals only. In two of these studies, a full-thickness single linear incision was made in the dorsum, and in one study the wounds were not sutured or covered, whereas in the other study the wounds were closed with surgical clips. In the former study, the wounds healed by second intention, which is the process by which excisional wounds heal, and VEGF mRNA expression in the wounds was significantly increased 1 day after wounding compared with 3 and 7 days after wounding. VEGF protein content in the wounds was significantly higher at 1 and 3 days after wounding compared with the unwounded skin of nondiabetic animals. Therefore, mRNA expression and protein content of VEGF were high at day 1 for wounds that were not sutured.

In the latter study, the wounds healed by first intention. IGF-1 mRNA expression was low in unwounded skin and at 0.5 day after wounding. The level in the wound started to increase at 1 day and peaked at 10 days after wounding. The expression remained at a high level through to 21 days after wounding. IGF-2 mRNA expression was low in unwounded skin and at 0.5 day after wounding. The level in the wound increased during the early stages of wound healing and reached a peak at 10 days after wounding. The expression then decreased, although the levels at 15 and 21 days were still significantly higher than for unwounded skin and at 0.5 day after wounding. Therefore, mRNA expression for both IGF-1 and IGF-2 was maximal at 10 days for wounds that were sutured.

Excisional wound model (Table 3)

In the mouse, all except one of the nine studies used nondiabetic and diabetic animals. Four of the studies used an experimental model of type 1 diabetes, and four used an experimental model of type 2 diabetes. In the study with nondiabetic animals only, four full-thickness wounds were made on the dorsum and not covered, and healing would have occurred by second intention. Expression of mRNA for FGF-7 (KGF-1), FGF-10 (KGF-2) and TGF-β was increased on days 2 and 15 as compared with the levels on day 0.

For the eight studies using nondiabetic and diabetic animals, two studies made a single full-thickness wound on the dorsum and in one of these studies the wounds were covered. In the study in which a single circular wound was made, mRNA induction of placental growth factor (PlGF) in wounds of nondiabetic animals began on day 1 and reached a maximum on day 3 after wounding. In wounds of diabetic animals, PlGF mRNA induction was significantly reduced compared with nondiabetic animals on days 1, 3, and 5 after wounding. PlGF protein induction in wounds of nondiabetic animals began to increase on day 1, reached a maximum on day 3, and remained upregulated until day 9 after wounding. In wounds of diabetic animals, PlGF protein induction was significantly reduced compared with nondiabetic animals from days 1 to 7 after wounding. VEGF mRNA expression was induced in wounds of nondiabetic animals and peaked on day 3 after wounding. In wounds of diabetic animals, VEGF mRNA expression was reduced compared with nondiabetic animals, with a marked lowering on days 3 and 5 after wounding. Induction of PDGF-B mRNA, FGF-2 mRNA, and TGF-β1 mRNA was strongly decreased in wounds of diabetic animals compared with nondiabetic animals. In the study in which a single square wound was made and covered with a transparent dressing, IGF-1 mRNA was induced in wounds of nondiabetic animals at 12 h and at 3 days after wounding there was a 50-fold increase compared with the level at 12 h, with a gradual decrease occurring to 28 days after wounding In wounds of diabetic animals, IGF-1 mRNA levels rose minimally at 5 days and did not reach a maximum until 14 days after wounding, with the levels being twofold lower than those of the peak at 3 days in the nondiabetic animals. A sharp increase in IGF-1 protein concentration occurred at 5 days in the nondiabetic wounds, which declined slowly thereafter to 28 days after wounding. IGF-1 protein levels in the diabetic wounds remained low until 21 days after wounding when they rose to nondiabetic levels. IGF-2 mRNA in wounds of nondiabetic animals peaked at 3 days after wounding, whereas in wounds of diabetic animals peak levels occurred at 10 days after wounding and were fourfold greater than in nondiabetic wounds. In the other six studies, multiple wounds were made on the dorsum of each animal. In the study in which two circular wounds were made in the interscapular region, nerve growth factor (NGF) in wounds of diabetic animals was significantly lower than in wounds of nondiabetic animals on day 3 after wounding. Expression of VEGF-A mRNA was significantly higher in wounds of diabetic animals than in wounds of nondiabetic animals on day 3 after wounding. In the other studies four or six circular wounds were made on the dorsum. One of these studies showed a lack of induction of VEGF mRNA in wounds of diabetic animals within the first few hours of wounding, and expression was markedly lower and barely detectable at day 5 compared with wounds of nondiabetic animals, for which highest levels occurred between days 3 and 7 after wounding. Another study found a delay in KGF induction in wounds of diabetic animals, with maximal levels of KGF expression only obtained between 3 and 5 days after wounding, whereas expression of KGF mRNA in wounds of nondiabetic animals reached a peak at 24 h after wounding. In addition, induction of both FGF-1 mRNA and FGF-2 mRNA occurred earlier in wounds of diabetic animals compared with nondiabetic animals, and expression of both FGF-1 mRNA and FGF-2 mRNA returned to basal levels within 3 days after wounding. In contrast, enhanced expression of FGF-1 mRNA and FGF-2 mRNA in wounds of nondiabetic animals occurred during the first 7 days after wounding. FGF-2 mRNA expression in wounds of diabetic animals was induced 3.5-fold and peaked within 24 h after wounding. In wounds of nondiabetic animals, FGF-2 mRNA expression was induced fourfold with the peak being reached at day 5 after wounding.

In the rat, both studies used nondiabetic and diabetic animals, which served as an experimental model of type 1 diabetes. In one study, a single square wound was made on the dorsum, and the level of NGF in wounds of diabetic animals was maximal on day 1 and levels were lower than for wounds of nondiabetic animals on days 1, 4, and 7 after wounding. A higher level of NGF in wounds of diabetic animals than for nondiabetic animals occurred on day 15 after wounding. In the other study, two square wounds were made on the dorsum and covered with an occlusive dressing. FGF-1 content in wounds of diabetic animals was significantly lower than in wounds of nondiabetic animals on day 7 after wounding, but was higher than in nondiabetic wounds on day 30 after wounding. FGF-2 content in wounds of diabetic animals was significantly lower than in wounds of nondiabetic animals on days 7 and 15 after wounding. Active TGF-β content in wounds of diabetic animals was significantly lower than in wounds of nondiabetic animals on days 7 and 30 after wounding.

Subcutaneous PVA sponge and stainless steel mesh chamber wound model (Table 4)

In the rat, one study used nondiabetic animals only, whereas the two other studies used both nondiabetic and diabetic animals which served as an experimental model of type 1 diabetes. In the two latter studies the levels of IGF-1 mRNA expression in sponges and of IGF-1 and TGF-β in wound fluid of diabetic rats were significantly reduced compared to nondiabetic animals at 7 days after wounding.

Influence of added growth factors in vivo

Burn ulcer wound model (Table 5)

Application of FGF-1 to cover the wound surface of burn ulcer in diabetic rats, which served as an experimental model of type 1 diabetes, on days 3 and 7 after establishment of ulcer, caused an increase in expression amounts of TGF-β compared with saline-treated diabetic rats on day 7, and a decrease in amounts on day 14.

Excisional skin wound model (Table 5)

Intradermal injection of a plasmid encoding for hypoxia-inducible factor (HIF)-1α increased the expression of VEGF, PlGF, and PDGF in wounds of diabetic mice used as an experimental model of type 2 diabetes. Treatment of wounds in diabetic mice as an experimental model of type 1 diabetes with a vector encoding for PlGF increased the expression of VEGF, FGF-2, and PDGF. Treatment with NGF of wounds in diabetic mice as an experimental model of type 1 diabetes increased the expression of VEGF-A mRNA. Recombinant VEGF applied to wounds of diabetic mice as an experimental model of type 2 diabetes increased the expression of FGF-2 and PDGF.

Treatment with platelet releasate containing PDGF and TGF-β of wounds in diabetic rats as an experimental model of type 1 diabetes decreased FGF-1 content on days 15 and 30 after wounding, but increased FGF-2 content on days 3, 7, 15, and 30 and active TGF-β content on day 7 after wounding.

In vitro studies

Influence of added growth factors

The addition of 10% fetal bovine serum to quiescent human keratinocytes caused a large induction of VEGF mRNA expression within 90 min, and elevated levels were still observed 8 h after serum addition. Moreover, the addition of serum growth factors such as EGF and KGF caused a large increase in VEGF mRNA expression that was maintained for at least 8 h after addition of the growth factor. TGF-β1 also stimulated VEGF mRNA expression, but expression levels had significantly declined by 5 h after addition of the growth factor. The cytokine TNF-α caused a large increase in VEGF mRNA expression within 2 h, whereas IL-1β was much less efficient in increasing VEGF mRNA expression, and similar to TGF-β1, expression levels had significantly declined after 5 h (Table 5).

Discussion

Wound healing and metabolic effects of growth factors

Growth factors mediate the cellular interactions among a variety of cells that orchestrate the complex sequence of cell migration, proliferation, differentiation, and protein expression during wound healing. Growth factors are released at the wound site and considered to be necessary for wound healing. The families of growth factors expressed in varying levels by the cells involved with healing are EGF, KGF, FGF, VEGF, IGF, TGF, PDGF, ILs, and colony-stimulating factor (CSF). Many of these factors are responsible for metabolic responses that have a high influence on wound healing. For example, IGF-1 has a similar effect as insulin; both increase glucose uptake and oxidation, produce similar suppression of glucose production, and free fatty acid levels and fat oxidation rates. An absolute IGF-1 and relative insulin deficiency may contribute to adverse metabolic changes in middle age,³⁶ and may predispose to developing diabetes. IGF-1 has been used experimentally to treat both type 1 and type 2 diabetes.³⁷ EGF inhibits gonadal steroid production from both ovarian and testicular tissues, and granulosa cell EGF inhibits follicle-stimulating hormone (FSH) and cAMP stimulation of estrogen and progesterone production.^38
–40 Estrogen is a major regulator of wound repair.⁴¹ EGF and FGF inhibit FSH-induction of luteinizing hormone (LH) receptor in granulosa cells, whereas PDGF stimulates FSH-induction of LH receptor.⁴²

Impaired healing of diabetic wounds

The impaired healing of diabetic wounds is characterized by delayed re-epithelization, decreased cellular infiltration and granulation tissue formation, reduced angiogenesis, and reduced and disorganized collagen formation.^1,43,44 Increased levels of free radicals in diabetes cause lipid peroxidation and also compromise the metabolism of keratinocytes, fibroblasts, and endothelial cells.⁴⁵ In addition, deficiencies have been reported in the levels of various growth factors in the wounds of diabetic animals. These could be caused by altered gene expression of the growth factors, degradation by proteinases in the wounds, and/or reduced or delayed cellular infiltration into the wounds.

Experimental models and growth factors in diabetic wounds

A variety of in vivo animal models was used in the studies, with a greater number of studies performed in mice than in rats. Burn ulcer, incisional, and excisional cutaneous wounds were studied, and experimental models of type 1 and type 2 diabetes were used.

This review has highlighted significant alterations in the temporal pattern and level of gene expression of growth factors in cutaneous wounds of diabetic mice and rats. Growth factors having a decreased mRNA expression pattern and level in the early phases of healing for diabetic wounds compared with nondiabetic wounds were VEGF, PlGF, KGF, FGF-1, FGF-2, IGF-1, IGF-2, TGF-β, and NGF. There was a delay in the expression of mRNAs for KGF, IGF-1, and IGF-2, whereas the expression of FGF-1 and FGF-2 occurred much earlier, in excisional wounds of diabetic mice compared with nondiabetic mice. In addition to the levels of mRNA expression for these growth factors being reduced, the levels of these proteins were decreased in the diabetic wounds compared with the nondiabetic wounds. VEGF and PlGF belong to the family of vascular endothelial growth factors and are responsible for vascularization of the healing wound. NGF is a neurotrophin that is proangiogenic and has been shown to increase the expression of VEGF by endothelial cells.²⁷ KGF (FGF-7), FGF-1 (acidic FGF), and FGF-2 (basic FGF) are members of the fibroblast-stimulating family of growth factors. IGF-1 and IGF-2 belong to a family of proteins involved in mediating the growth and proliferation of cells. IGF-1 has the potential to stimulate cellular proliferation and collagen production by fibroblasts. TGF-β is a protein that controls proliferation and cellular differentiation in most cells.

Possible combined laser photoirradiation and growth factor administration to treat diabetic wounds

The historical supernatant transfer experiments of Rajaratnam and coworkers demonstrated for the first time the secretion of growth factors by laser-irradiated cells.¹⁰ Photoirradiation of diabetic wounds using laser light at visible red wavelengths stimulated wound healing,¹¹ and could potentially be combined with growth factor administration to maximize the healing of diabetic wounds. A recent review of the effects of laser photoirradiation on gene expression and release of growth factors by cells in culture indicated that growth factors such as FGF-2, IGF, NGF, and VEGF are increased by exposure to visible red laser light.⁹ Possible growth factors that might be considered as part of a combined therapy with laser irradiation could be TGF-β, FGF-1, PlGF, and KGF, as these have not been shown to be increased by laser light treatment. To evaluate which of these might be most suitable, the searched studies were analyzed for the effects of these growth factors on healing of diabetic wounds and the levels of expression of growth factor mRNAs in diabetic wounds and cells in vitro. The latter are summarized in Table 5.

Influence of individual growth factors on healing of diabetic wounds

When applied individually, each of the growth factors TGF-β, FGF-1, PlGF, and KGF enhanced diabetic wound healing. A single intradermal injection of TGF-β1 plasmid DNA immediately after making an excisional wound in diabetic mice was found to stimulate wound closure and the formation of a denser and more organized extracellular matrix.⁴⁶ Treatment of skin ulcer wounds in diabetic rats with FGF-1 on days 3 and 7 accelerated wound closure and increased granulation tissue and collagen formation, neoangiogenesis, and re-epithelization. The expression amounts of TGF-β in diabetic rats on days 7 and 14 were modified such that they were no longer different from nondiabetic rats.¹³ Application of PlGF as a vector 1 day after creating an excisional wound in diabetic mice stimulated wound closure, with increased formation and accelerated maturation of granulation tissue. The expression of VEGF, FGF-2, and PDGF was increased. In addition, the number of monocytes/macrophages in the granulation tissue was increased.²³ It has been found that the monocyte/macrophage density was significantly reduced in the granulation tissue of diabetic mice compared with nondiabetic mice.²³ A single intradermal injection of KGF-1 DNA plasmid after making an excisional wound in diabetic mice, followed by electroporation, accelerated wound closure, and improved the quality of healing.⁴⁷ In vitro studies with human keratinocytes showed that expression of VEGF was increased by KGF.³¹

TGF-β is produced by neutrophils and macrophages that migrate into a provisional matrix at the site of the wound, and dermal fibroblasts infiltrating from the wound edge are stimulated by TGF-β and responsible for the synthesis of a rich collagen matrix to replace the provisional matrix.⁴⁶ FGF-1 is secreted by multiple cell types and stimulates the proliferation of cells of mesodermal, ectodermal, and endodermal origin. PlGF is expressed by migrating keratinocytes and endothelial cells of small blood vessels during the angiogenic phase of wound healing, and contributes to regulating endothelial and lymphatic vessel development.⁴⁸ KGF is produced by mesenchymal cells including fibroblasts and endothelial and smooth muscle cells, and stimulates re-epithelization of wounds.⁴⁹

Future studies of combination treatment with laser light and growth factors

Quantitative assessment of wound healing parameters allows the efficacy of various growth factors and cytokines in affecting distinct stages of the wound healing process to be screened. Even though specific growth factors can be an important treatment at certain stages of wound healing, they are insufficient to promote the entire longitudinal process of wound repair.¹ Combination treatments may therefore be the preferred approach to developing future therapies for wound healing. Combinations of two growth factors have been tested and shown to act synergistically to promote wound healing in diabetic animals. They include the combinations of FGF-2 with TGF-β, PDGF with TGF-α, PDGF with IGF-2, and FGF-2 with IGF-2.^50
–52 Since laser irradiation has been shown to positively influence various stages of the wound healing process, research should be implemented on testing a combination therapy of visible red laser irradiation and growth factor administration. The most suitable growth factor would be determined by trial, but in the first instance, TGF-β, FGF-1, PlGF, and KGF could be tested, as these had not been shown to be increased by exposure of cells in culture to visible red laser light.⁹ Visible red laser irradiation with VEGF application might also be tested, as administration of VEGF protein increased angiogenesis and granulation tissue formation in diabetic mouse wounds.³⁵ Combination therapy may enable several critical healing stages to be promoted concomitantly, and reduce the effective dose of laser irradiation and growth factor administered, thereby minimizing adverse changes.

Conclusions

The reviewed studies showed that gene expression and content of certain growth factors are decreased in wounds of diabetic mice and rats. Administration of individual growth factors can enhance wound healing in diabetic animals. Possible combination therapy utilizing laser irradiation and application of specific growth factors at appropriate stages of wound healing may provide an effective means of accelerating the healing of diabetic wounds. Future studies should be designed to test the effect of laser irradiation alone and in combination with a single growth factor in an appropriate animal model of compromised wound healing. One such model is to use genetic diabetic mice in which a full-thickness skin excisional wound is splinted using an occlusive adhesive dressing to retard contraction, and for which healing occurs by re-epithelization and granulation tissue formation.¹¹

Footnotes

Author Disclosure Statement

No competing financial interests exist.

References

Braiman-Wiksman

, Solomonik

, Spira

, Tennenbaum

2007. Novel insights into wound healing sequence of events. Toxicol. Pathol., 35:767–779.

Peplow

P.V.

, Chung

T-Y.

, Baxter

G.D.

2010. Laser photobiomodulation of wound healing: a review of experimental studies in mouse and rat animal models. Photomed. Laser Surg., 28:291–325.

Byrnes

K.R.

, Barna

, Chenault

V.M.

et al. 2004. Photobiomodulation improves cutaneous wound healing in an animal model of type II diabetes. Photomed. Laser Surg., 22:281–290.

Safavi

S.M.

, Kazemi

, Esmaeili

, Fallah

, Modarresi

, Mir

2008. Effects of low-level He–Ne laser irradiation on the gene expression of IL-1β, TNF-α, IFN-γ, TGF-β, bFGF, and PDGF in rat's gingiva. Lasers Med. Sci., 23:331–335.

Hopkins

J.T.

, McLoda

T.A.

, Seegmiller

J.G.

, Baxter

G.D.

2004. Low-level laser therapy facilitates superficial wound healing in humans: a triple-blind, sham- controlled study. J. Athl. Train., 39:223–229.

Kleinman

, Simmer

, Braksma

1996. Low power laser therapy in patients with diabetic foot ulcers: early and long term outcome. Laser Ther., 8:205–208.

Houreld

, Abrahamse

2005. Low-level laser therapy for diabetic wound healing. Diabet. Foot Ankle, 8:182–193.

Kazemi–Khoo

2006. Successful treatment of diabetic foot ulcers with low-level laser therapy. Foot, 16:184–187.

Peplow

P.V.

, Chung

T-Y.

, Baxter

G.D.

2011. Laser photobiomodulation of gene expression and release of growth factors and cytokines from cells in culture: a review of human and animal studies. Photomed. Laser Surg., 29:285–304.

10.

Rajaratnam

, Bolton

, Dyson

1994. Macrophage responsiveness to laser therapy with varying pulsing frequencies. Laser Ther., 6:107–112.

11.

Chung

T-Y.

, Peplow

P.V.

, Baxter

G.D.

2010. Laser photobiomodulation of wound healing in diabetic and non-diabetic mice: effects in splinted and unsplinted wounds. Photomed. Laser Surg., 28:251–261.

12.

Chung

T-Y.

, Peplow

P.V.

, Baxter

G.D.

2010. Laser photobiostimulation of wound healing: defining a dose response for splinted wounds in diabetic mice. Lasers Surg. Med., 42:656–664.

13.

Xie

, Zhang

, Dong

et al. 2011. Improved refractory wound healing with administration of acidic fibroblast growth factor in diabetic rats. Diabetes Res. Clin. Prac., 93:396–403.

14.

Zubaldi

, Buie

W.D.

, Hart

D.A.

, Sigalet

2010. Temporal expression of cytokines in rat cutaneous, fascial, and intestinal wounds: a comparative study. Dig. Dis. Sci., 55:1581–1588.

15.

Galeano

, Bitto

, Altavilla

et al. 2008. Polydeoxyribonucleotide stimulates angiogenesis and wound healing in the genetically diabetic mouse. Wound Repair Regen., 16:208–217.

16.

Bitto

, Minutoli

, Galeano

M.R.

et al. 2008. Angiopoietin-1 gene transfer improves the impaired wound healing of the genetically diabetic mice without increasing VEGF expression. Clin. Sci., 114:707–718.

17.

Bitto

, Minutoli

, Altavilla

et al. 2008. Simvastatin enhances VEGF production and ameliorates impaired wound healing in experimental diabetes. Pharmacol. Res., 57:159–169.

18.

Nogami

, Hoshi

, Kinoshita

, Arai

, Takama

, Takahashi

2007. Vascular endothelial growth factor expression in rat skin incision wound. Med. Mol. Morphol., 40:82–87.

19.

Galeano

, Altavilla

, Cucinota

et al. 2004. Recombinant human erythropoietin stimulates angiogenesis and wound healing in the genetically diabetic mouse. Diabetes, 53:2509–2517.

20.

Altavilla

, Saitta

, Cucinotta

et al. 2001. Inhibition of lipid peroxidation restores impaired vascular endothelial growth factor expression and stimulates wound healing and angiogenesis in the genetically diabetic mouse. Diabetes, 50:667–674.

21.

Gartner

M.H.

, Benson

J.D.

, Caldwell

M.D.

1992. Insulin-like growth factors I and II expression in the healing wound. J. Surg. Res., 52:389–394.

22.

Liu

, Marti

G.P.

, Wei

et al. 2008. Age-dependent impairment of HIF-1α expression in diabetic mice: correction with electroporation-facilitated gene therapy increases wound healing, angiogenesis, and circulating angiogenic cells. J. Cell Physiol., 217:319–327.

23.

Cianfarani

, Zambruno

, Brogelli

et al. 2006. Placenta growth factor in diabetic wound healing. Altered expression and therapeutic potential. Am. J. Pathol., 169:1167–1182.

24.

Komi–Kuramochi

, Kawano

, Oda

et al. 2005. Expression of fibroblast growth factors and their receptors during full-thickness skin wound healing in young and aged mice. J. Endocrinol., 186:273–289.

25.

Chen

, Kasper

, Heck

et al. 2005. Tissue factor as a link between wounding and tissue repair. Diabetes, 54:2143–2154.

26.

Rajasekaran

N.S.

, Nithya

, Rose

, Chandra

T.S.

2004. The effect of finger millet feeding on the early responses during the process of wound healing in diabetic rats. Biochim. Biophys. Acta, 1689:190–201.

27.

Graiani

, Emanueli

, Desortes

et al. 2004. Nerve growth factor promotes reparative angiogenesis and inhibits endothelial apoptosis in cutaneous wounds of Type 1 diabetic mice. Diabetologia, 47:1047–1054.

28.

Stallmeyer

, Pfeilschifter

, Frank

2001. Systemically and topically supplemented leptin fails to reconstitute a normal angiogenic response during skin repair in diabetic ob/ob mice. Diabetologia, 44:471–479.

29.

Moulin

, Lawny

, Barritault

, Caruelle

J.P.

1998. Platelet releasate treatment improves skin healing in diabetic rats through endogenous growth factor secretion. Cell. Mol. Biol., 44:961–971.

30.

Brown

D.L.

, Kane

C.D.

, Chernausek

S.D.

, Greenhalgh

D.G.

1997. Differential expression and localization of insulin-like growth factors I and II in cutaneous wounds of diabetic and nondiabetic mice. Am. J. Pathol., 151:715–724.

31.

Frank

, Hubner

, Breier

, Longaker

M.T.

, Greenhalgh

D.G.

, Werner

1995. Regulation of vascular endothelial growth factor expression in cultured keratinocytes. J. Biol. Chem., 270:12,607–12,613.

32.

Werner

, Breeden

, Hubner

, Greenhalgh

D.G.

, Longaker

M.T.

1994. Induction of keratinocyte growth factor expression is reduced and delayed during wound healing in the genetically diabetic mouse. J. Invest. Dermatol., 103:469–473.

33.

Bitar

MS.

2000. Insulin and glucocorticoid-dependent suppression of the IGF-I system in diabetic wounds. Surgery, 127:687–695.

34.

Bitar

M.S.

, Labbad

Z.N.

1996. Transforming growth factor-β and insulin-like growth factor-I in relation to diabetes-induced impairment of wound healing. J. Surg. Res., 61:113–119.

35.

Galiano

R.D.

, Tepper

O.M.

, Pelo

C.R.

et al. 2004. Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells. Am. J. Pathol., 164:1935–1947.

36.

Boulware

S.D.

, Tamborlane

W.V.

, Rennert

N.J.

, Geshundheit

, Sherwin

R.S.

1994. Comparison of the metabolic effects of recombinant human insulin-like growth fasctor-1 and insulin. J. Clin. Invest., 93:1131–1139.

37.

LeRoth

, Yakar

2007. Mechanisms of disease: metabolic effects of growth hormone and insulin-like growth factor 1. Nat. Rev. Endocrinol., 3:302–310.

38.

Hsueh

A.J.

, Welsh

T.H.

, Jones

P.B.

1981. Inhibition of ovarian and testicular steroidogenesis by epidermal growth factor. Endocrinol., 108:2002–2004.

39.

Jones

P.B.

, Welsh

T.H.

, Hsueh

A.J.

1982. Regulation of ovarian progestin production by epidermal growth factor in cultured rat granulosa cells. J. Biol. Chem., 257:11,268–11,273.

40.

Knecht

, Catt

K.J.

1983. Modulation of cAMP-mediated differentiation of ovarian granulosa cells by epidermal growth factor and platelet derived growth factor. J. Biol. Chem., 258:2789–2794.

41.

Ashcroft

G.S.

, Mills

S.J.

, Lei

et al. 2003. Estrogen modulates cutaneous wound healing by downregulating macrophage inhibitory factor. J. Clin. Invest., 111:1309–1318.

42.

Mondschein

J.S.

, Schomberg

D.W.

1981. Growth factors modulate gonadotropin receptor induction in granulosa cell cultures. Science, 211:1179–1180.

43.

Yue

D.K.

, Swanson

, McLennan

et al. 1986. Abnormalities of granulation tissue and collagen formation in experimental diabetes, uraemia and malnutrition. Diabet. Med., 3:221–225.

44.

Fahey III

T.J.

, Sadaty

, Jones II

W.G.

, Barber

, Smoller

, Shires

G.T.

1991. Diabetes impairs the late inflammatory response to wound healing. J. Surg. Res., 50:308–313.

45.

White

M.J.

, Heckler

F.R.

1990. Oxygen free radicals and wound healing. Clin. Plast. Surg., 17:473–484.

46.

Chesnoy

, Lee

P-Y.

, Huang

2003. Intradermal injection of transforming growth factor-β1 gene enhances wound healing in genetically diabetic mice. Pharm. Res., 20:345–350.

47.

Marti

, Ferguson

, Wan

et al. 2004. Electroporative transfection with KGF-1 DNA improves wound healing in a diabetic mouse model. Gene Ther., 11:1780–1785.

48.

Failla

C.M.

, Odorisio

, Cianfarani

et al. 2000. Placenta growth factor is induced in human keratinocytes during wound healing. J. Invest. Dermatol., 115:388–395.

49.

Bando

, Hiroshima

, Kataoka

et al. 2010. Modulation of calprotectin in human keratinocytes by keratinocyte growth factor and interleukin-1α Immunol. Cell Biol., 88:328–333.

50.

Broadley

K.N.

, Aquino

A.M.

, Ditesheim

J.A.

et al. 1988. Growth factors bFGF and TGFβ accelerate the rate of wound repair in normal and in diabetic rats. Int. J. Tissue React., 10:345–353.

51.

Greenhalgh

D.G.

, Hummel

R.P.

, Albertson

, Breeden

M.P.

1993. Synergistic actions of platelet-derived growth factor and the insulin-like growth factors in vivo. Wound Repair Regen., 1:69–81.

52.

Brown

R.L.

, Breeeden

B.S.

, Greenhalgh

D.G.

1994. PDGF and TGF-α act synergistically to improve wound healing in the genetically diabetic mouse. J. Surg. Res., 56:562–570.