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. Author manuscript; available in PMC: 2012 Mar 1.
Published in final edited form as: J Vasc Surg. 2011 Jan 6;53(3):774–784. doi: 10.1016/j.jvs.2010.10.056

Inhibition of SDF-1α further impairs diabetic wound healing

Dustin M Bermudez 1,2, Junwang Xu 3, Benjamin J Herdrich 1, Antoneta Radu 2, Marc E Mitchell 3, Kenneth W Liechty 3,*
PMCID: PMC3058337  NIHMSID: NIHMS248806  PMID: 21211927

Abstract

Objective

Impaired diabetic wound healing is associated with abnormal SDF-1α production, decreased angiogenesis, and chronic inflammation. Lentiviral-mediated overexpression of SDF-1α can correct the impairments in angiogenesis and healing in diabetic wounds. We hypothesized that SDF-1α is a critical component of the normal wound healing response and that inhibition of SDF-1α would further delay the wound-healing process.

Design of study

Db/Db diabetic mice and Db/+ non-diabetic mice were wounded with an 8mm punch biopsy and the wounds treated with a lentiviral vector containing either the GFP or SDF-1α inhibitor transgene. The inhibitor transgene is a mutant form of SDF-1α that binds, but does not activate, the CXCR4 receptor. Computerized planimetry was used to measure wound size daily. Wounds were analyzed at 3 and 7 days by histology and for production of inflammatory markers using real-time PCR. The effect of the SDF-1α inhibitor on cellular migration was also assessed.

Results

Inhibition of SDF-1α resulted in a significant decrease in the rate of diabetic wound healing, (3.8 cm2/day versus 6.5 cm2/day in GFP-treated wounds p=0.04), and also impaired the early phase of non-diabetic wound healing. SDF-1α inhibition also resulted in fewer small-caliber vessels, less granulation tissue formation, and increased proinflammatory gene expression (IL-6 and MIP-2) in the diabetic wounds.

Conclusions

The relative level of SDF-1α in the wound plays a key role in the wound healing response. Alterations in the wound level of SDF-1α, as seen in diabetes or by SDF-1α inhibition, impair healing by decreasing cellular migration and angiogenesis, leading to increased production of inflammatory cytokines and inflammation.

Introduction

Diabetes has reached pandemic proportions in the United States and across the globe. Expenditures on diabetes care in the US surpassed $174 billion in 2007 (1). An ulcer of the lower extremity precedes 84% of all diabetic lower extremity amputations and is the primary cause for hospitalization amongst diabetics (2). Despite the increasing prevalence of diabetes, the incidence of amputation and lower extremity ulcers has not significantly abated with current therapies.

Hyperbaric oxygen, recombinant platelet-derived growth factor (PDGF) and biosynthetic grafts all have been extensively studied as alternative methods to promote the wound healing process (3–10). While promising, they have not been widespread in their use. Hyperbaric oxygen has been demonstrated to increase numbers of endothelial progenitor cells (EPCs) in animal studies as well as in vitro studies but not in human subjects (11). Recombinant PDGF application to diabetic wounds demonstrates some reproducible improvement of the wound-healing process (7). Apligraf or Dermagraft are both biosynthetic grafts used to promote wound closure in diabetic patients (9,10). While there is evidence to support these therapies’ benefit when used clinically, widespread use is tempered by their prohibitive costs (12).

The molecular physiology that underlies the diabetic wound healing defect remains unclear. It is known there are fewer EPCs, greater inflammation, and fewer growth factors present in the wounds of diabetics (13–19). Specifically, deficiencies in growth factors such as PDGF, keratinocyte growth factor (KGF), transforming growth factor-beta (TGF-β), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF), have all been implicated in the delayed healing rates observed in chronic diabetic wounds (13–21). Chronic diabetic wounds have been shown to have deficiencies in the cellular response to growth factors, including decreased cellular recruitment and migration, decreased angiogenesis and granulation tissue production, impaired re-epithelialization, decreased extracellular matrix (ECM) production, and impaired wound contraction (13–21).

Stromal-derived factor-1α (SDF-1α), a CXC chemokine implicated in the wound healing process (22–25) in the Leprdb mouse model of type II diabetes can correct this wound healing defect when overexpressed (26). Greater granulation tissue, smaller epithelial gap and smaller wound size all were found on histologic analysis. To examine the role of SDF-1α in impaired and non-impaired wound healing, we injected a lentiviral vector that expresses a mutant form of SDF-1α that binds, but does not activate, CXCR4 and measured its effect on granulation tissue formation, angiogenesis, inflammation, cell migration, and wound healing.

Methods

Lentiviral vector construction and fibroblast transduction

The SDF mutant we generated binds the CXCR4 receptor but does not activate it, based on studies by Choi et al (27), utilizing site-directed mutagenesis to evaluate the effect of specific mutations in the SDF-1α gene on CXCR4 mediated signal transduction. Replacement of the C-terminal proline amino acid with glycine generates a mutated form of SDF-1α, which binds to the CXCR4 receptor but does not activate it.

A cDNA library was prepared from mouse tissues using Trizol and Superscript (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Sequence analysis was used to confirm the murine SDF-1α cDNA as well as the SDF-1α inhibitor.

The CS-CG HIV-1 transfer plasmid, modified as previously described (28, 29) was used to generate a self-inactivating lentiviral vector. This lentiviral vector allows expression of the GFP reporter gene (Clontech Laboratories, Mountain View, CA) or the mutant SDF-1α construct with the GFP reporter gene as a single transcript under the control of a CMV promoter. VSV-G protein pseudotyped viral particles were generated by transfection into a 293T cell line and titered as previously described (30).

To test the ability of our viral construct to efficiently infect cells and produce transgene protein, we incubated passage 5 dermal fibroblasts with our lentiviral construct at a multiplicity of infection of 100 for 24 hours. Transduced fibroblasts were then plated in 12-well tissue culture plates at a seeding density of 5×105 cells per well. Tissue culture supernatants were aspirated from the plates 24 hours after transfection then frozen at −80°C. SDF-1α protein content was determined from thawed supernatants using a Quantikine enzyme-linked immunosorbent assay (ELISA) kit for murine CXCL12/SDF-1α (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. All transduced cells produced > 100 ng/mL of SDF-1α inhibitor.

Experimental animals and surgical procedure

All experimental protocols employed in this study were approved by the Institutional Animal Care and Use Committee at the Children’s Hospital of Philadelphia. Genetically diabetic, 10- to 12-week-old, female C57BKS.Cg-m/Leprdb mice and age-matched non-diabetic, heterozygous mice (The Jackson Laboratory, Bar Harbor, ME) were utilized in this study. At the time of wounding, Db/Db mice weighed greater than 45 g with blood glucose levels in excess of 400 mg/dL and Db/+ heterozygote mice weighed less than 25 g with blood glucose levels less than 250 mg/dL. Animals were given standard rodent chow and water ad libitum.

Each mouse was shaved and depilated prior to wounding. The dorsal skin was swabbed with alcohol and betadine. Each mouse underwent a single dorsal full-thickness wound (including panniuclus carnosum) with a 8mm punch biopsy (Miltex Inc., York, PA). After wounding, a Hamilton syringe was used to deliver 50μL of either the lentiviral SDF-1α inhibitor vector or a lentiviral GFP vector, as a control. 10μL were injected intradermally at 12, 3, 6 and 9 o’clock and at the wound base. Approximately 1×108 plaque-forming units (PFUs) of the lentiviral constructs were used on each mouse. Next, a sterile Tegaderm (3M) was placed over the wound and left in place for 48 hours after which it was removed.

Planimetry and wound size calculation

Photographs were obtained with a Nikon camera using a ruler for each image. Image J (http://rsbweb.nih.gov/ij/) was used to calculate the wound area of each mouse daily. The same observer measured the size of each wound. Wound area was plotted as a function of time on a daily basis.

Immunohistochemistry and histologic analysis

Wounds were harvested at days 3 and 7 and immediately fixed in 10% neutral buffered formalin (Sigma, St. Louis, MO). The tissue was then processed using a histoprocessor (Leica TP1050, GMI, Inc., Ramsey, MN). Paraffin sections were cut 4μm thick and mounted then incubated on slides (Fisher Scientific, Pittsburgh, PA) overnight. Slides were deparaffinized then washed in ethanol. Endogenous peroxidase was quenched with 0.3% hydrogen peroxide for 30 minutes at room temperature. Slides were pre-treated with proteinase K (Dako, Carpinteria, CA) for 10 minutes and rinsed with distilled water. Primary murine α-smooth muscle actin, CD31 and CD45 antibodies (BD Biosciences, San Jose, CA) were applied at 1:200, 1:50 and 1:50 dilutions respectively. They were developed with a Vectastain Elite ABC kit with secondary antibody (Vector Laboratories, Burlingame, CA). A blinded observer analyzed granulation tissue area, α-smooth muscle actin-positive vessel density, and numbers of CD31-positive and CD45-positive cells per high-power field. A total of three high-power fields per slide were examined.

Real-time quantitative PCR

Wounds were harvested at days 3 and 7 post-wounding and frozen in liquid nitrogen. Samples were homogenized in TRIzol (Invitrogen, Carlsbad, CA) and total RNA was extracted and purified following the manufacturer’s instructions. RNA was converted into cDNA using the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA). Real-time PCR was performed with the ABI 7900 real-time PCR thermal cycler (Applied Biosystems, Foster City, CA) to amplify samples in triplicate. All samples were amplified using TaqMan prepared primer assays. The average of the triplicate gene product for each gene was compared to 18S ribosomal RNA expression to generate a relative fold expression. Results were reported as means ± SEM. A total of 19 mice (with four groups of five mice each) were sacrificed to perform PCR analysis with a total of 57 PCR reactions carried out.

Cellular migration

A standard 24-well colorimetric assay was utilized to assess cellular migration. Boyden chambers (Chemicon, Santa Cruz, CA) equipped with 8μm pore diameter polycarbonate filters were used. Cells were suspended in migration medium (1μg/mL heparin/0.1% bovine serum albumin in Dulbecco’s Modified Eagle Medium) and 2×105 cells were loaded into the upper chamber. The lower chambers were plated 24 hours prior to the migration assay with one of five conditions: 1) fibroblasts alone, 2) fibroblasts transfected with lenti-SDF-1α, 3) fibroblasts transfected with lenti-SDF-1α and fibroblasts transfected with the lenti-SDF-1α inhibitor at a ratio of 1:1, 4) fibroblasts transfected with lenti-SDF-1α and fibroblasts transfected with the lenti-SDF-1α inhibitor at a ratio of 1:2, and 5) no cells. The cells plated prior to migration were done so with 2×105 cells. This lower chamber was washed with PBS and filled with migration medium. After 16 hours, cells in the migration medium were removed and quantified under a light microscope at 20× following staining with 0.5% crystal violet. All migration experiments were done in triplicate.

Statistical methods

Data are presented as mean ± SEM to two significant figures. Statistical analysis was performed with one-way ANOVA using SPSS 16.0 (SPSS, Inc., Chicago, IL). Values were transformed (ln) when required to achieve normality of sampling. When significant differences (p<0.05) were found, a Least Significant Difference post-hoc test, Mann-Whitney U, was used to identify differences between individual means.

Results

Wound healing

Treatment of diabetic wounds with the lenti-SDF-1α inhibitor resulted in a significant and further impairment in wound healing. Diabetic wounds treated with lentiviral-SDF inhibitor were significantly larger at day 7 (0.25 cm2 ± 0.006) compared to diabetic wounds treated with lentiviral GFP (0.16 cm2 ± 0.05, p = 0.009), Figure 1A. Treatment of diabetic wounds with the lenti-SDF-1α inhibitor also resulted in a dramatic delay in the mean number of days to wound closure when compared to the lenti-GFP group (25 ± 2.5 days to closure, versus 15.1 ± 1.3 days to closure, p = 2.1×10−5). In addition, treatment of diabetic wounds with the lenti-SDF-1α inhibitor resulted in a dramatic decrease in the rate of wound closure (3.8 mm2/day ± 0.94 mm2 in the lenti-SDF-1α inhibitor group n = 5, versus 6.5 mm2/day ± 2.2 mm2 in the lenti-GFP group, n = 5, p = 0.04), Figure 1B.

Figure 1.

Figure 1

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Rates of wound healing. A: Diabetic wounds at day 7 in both GFP-treated and SDFi-treated groups. B: mean wound area for diabetic mice treated with either the SDFi or control GFP vector at each day post-wounding. Each data point represents an n=5. C: Heterozygote mice in both experimental groups at day 7. D: mean wound area for heterozygous mice in both groups on a daily basis until closure. Asterisks above the graph indicate post-wounding days at which there was a significant difference in wound size between the two groups.

Treatment of non-diabetic wounds with the lenti-SDF-1α inhibitor demonstrated a significant impairment in the initial phase of wound healing, with increased wound area in days 1–5 versus the lenti-GFP treated group, Figure 1C, p≤0.03. However, the mean wound area after 5 days and the overall rate of wound closure for the non-diabetic wounds treated with SDF-1α inhibitor were not significantly different from GFP-treated wounds. Figure 1D displays the mean wound area over time.

Granulation Tissue

The effect of the lenti-SDF-1α inhibitor on granulation tissue was assessed at 7 days using trichrome staining and measurement of the granulation tissue volume. Representative sections of granulation tissue from both lenti-GFP and lenti-SDF inhibitor treated diabetic mice are displayed in Figures 2A and 2B, respectively. Treatment with the lenti-SDF-1α inhibitor resulted in a significant decrease in the granulation tissue volume (1.8 per mm2 ± 0.4, p = 0.031, Figure 2E) compared to the lenti-GFP treated diabetic wounds (1.1 per mm2 ± 0.46). Diabetic wounds also demonstrated significantly less granulation tissue than non-diabetic wounds (Figure 2C and D).

Figure 2.

Figure 2

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Masson’s trichrome stain of representative sections of wound tissue 7 days after initial wounding. A: diabetic wound treated with lenti-GFP. B: diabetic wound treated with lenti-SDF-1α inhibitor. C: heterozygote wound treated with GFP. D: heterozygote wound treated with lenti-SDF-1α inhibitor E: mean volume of granulation tissue at day 7. Black bar represents 200μm.

Representative sections of granulation tissue from both lenti-GFP and lenti-SDF-1α inhibitor treated non-diabetic mice are displayed in Figures 2C and 2D, respectively. The mean granulation tissue volume was not significantly different (2.4 per mm2 ± 0.32 versus 2.5 per mm2 ± 0.94, p = 0.31, figure 2E).

Angiogenesis

The effect of the lenti-SDF-1α inhibitor on angiogenesis was assessed using immunohistochemistry for CD31, an endothelial marker. Representative photos of immunoperoxidase staining for CD31 at 7 days in diabetic wounds treated with lenti-GFP or lenti-SDF-1α inhibitor are demonstrated in Figures 3A and 3B, respectively. Treatment with the SDF-1α inhibitor resulted in a significant decrease in the number of CD31-positive cells/HPF (5.1 ± 1.83 cells/HPF, n = 5, Figure 3E, p=0.005) versus lenti-GFP treated wounds (7.1 ± 1.75 cells/HPF).

Figure 3.

Figure 3

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CD31 positive cells at day 7. A: diabetic mouse treated with lenti-GFP. B: diabetic mouse treated with lenti-SDF-1α inhibitor. C: heterozygote mouse treated with GFP. D: heterozygote mouse treated with lenti-SDF-1α inhibitor. E: mean number of CD31 cells counted for both groups. Black bars represent 25 μm.

Representative photos of CD31 staining in non-diabetic wounds treated with lenti-GFP and lenti-SDF-1α inhibitor are displayed in Figures 3C and 3D, respectively. Treatment of non-diabetic wounds with the lenti-SDF-1α inhibitor resulted in a significant decrease in the number of CD31-positive cells/HPF (10.1 cells ± 4.45 cells/HPF, n=10, p=0.04, Figure 3E) compared to non-diabetic wounds treated with lenti-GFP (13.3 ± 3.75 cells/HPF). In addition, the number of CD31-positive cells/HPF was also significantly higher in non-diabetic wounds compared to diabetic wounds (p<0.05).

Vasculogenesis

The effect of the lenti-SDF-1α inhibitor on vasculogenesis was assessed using immunohistochemistry for α-smooth muscle actin (ASMA). Representative photos of immunoperoxidase staining for ASMA at 7 days in diabetic wounds treated with lenti-GFP or lenti-SDF-1α inhibitor are demonstrated in Figures 3A and 3B, respectively. Vessel density per high power field was then quantified. Treatment of diabetic wounds with the lenti-SDF-1α inhibitor resulted in a significant decrease in vessel density (15.1 ± 4.1 vessels/HPF, n = 5, p=0.018, Figure 4E) compared to lenti-GFP treated wounds (19.1 ± 4.5 vessels/HPF, n = 5).

Figure 4.

Figure 4

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α–smooth muscle actin staining at 7 days post wounding. A: Diabetic treated with lenti-GFP. B: diabetic treated with lenti-SDF-1α inhibitor. C: Heterozygote treated with lenti-GFP. D: heterozygote treated with lenti-SDF-1α inhibitor. E: Vessel at day 7. Black bar represents 25μm.

Representative photos of immunoperoxidase staining for ASMA at 7 days in non-diabetic wounds treated with lenti-GFP or lenti-SDF-1α are demonstrated in Figures 3C and 3D, respectively. There was no significant difference in vasculogenesis between lenti-GFP or lenti-SDF-1α treated non-diabetic wounds (27.9± 4.41 vessels/HPF, n = 5 versus 25± 8.52 vessels/HPF, n = 5, Figure 3E). However, diabetic wounds had a significant decrease in vessel numbers/HPF compared to non-diabetic wounds (p = 9.4 × 10−6).

Inflammation

The effect of SDF-1α inhibition on inflammation was assessed using immunohistochemistry for CD45, the common leukocyte antigen. Representative photos of immunoperoxidase staining for CD45 at 7 days in diabetic wounds treated with lenti-GFP or lenti-SDF-1α inhibitor are demonstrated in Figures 5A and 5B, respectively. Diabetic wounds treated with the lenti-SDF-1α inhibitor demonstrated a significant increase in CD45 positive cells/HPF (22 ± 12.9 cells/HPF, n=5, p<0.005, Figure 5E) compared to lenti-GFP treated diabetic wounds (18 ± 8.0, n = 5).

Figure 5.

Figure 5

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CD45 staining at day 7. A: Diabetic treated with lenti-GFP. B: diabetic treated with lenti-SDF-1α inhibitor. C: Heterozygote treated with lenti-GFP. D: heterozygote treated with lenti-SDF-1α inhibitor. E: Mean number of CD45+ cells per high-powered field.

Representative photos of immunoperoxidase for CD45 at 7 days in non-diabetic wounds treated with lenti-GFP or lenti-SDF-1α inhibitor are demonstrated in Figures 5C and 5D. There was no significant difference in inflammatory cell infiltration in the non-diabetic wounds treated with lenti-GFP or lenti-SDF-1α inhibitor, however, there were significantly fewer CD45 positive cells in the non-diabetic wounds compared to the diabetic wounds (p<0.002, Figure 5E).

Inflammatory cytokine gene expression

Real time-PCR was used to assess inflammatory cytokine gene expression in diabetic and non-diabetic wounds treated with either lenti-GFP or lenti-SDF-1α inhibitor. Diabetic wounds treated with the lenti-SDF-1α inhibitor demonstrated increased IL-6 gene expression at day 3 post-wounding versus lenti-GFP treated wounds (7.5 ± 2.1 fold versus 0.53± 0.14 fold, p = 0.016, Figure 6A). By 7 days there was no difference in IL-6 gene expression between groups. Diabetic wounds treated with the lenti-SDF-1α inhibitor also demonstrated increased gene expression of macrophage inflammatory protein-2 (MIP-2) at day 7 (1.98±0.49 fold versus 1.36±0.17 fold in lenti-GFP control, p = 0.026, Figure 6A).

Figure 6.

Figure 6

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Real-time quantitative PCR analysis of whole wounds at days 3 and 7 post wounding. Panel A represents IL-6 levels at days 3 and 7 in diabetic and non-diabetic mice. Panel B represents MIP-2 levels at days 3 and 7 post wounding in diabetic and non-diabetic mice. N = 5 for each experimental group.

At 3 days, non-diabetic wounds treated with lenti-GFP demonstrated decreased interleukin-6 (IL-6) expression (1 fold ± 0.36) compared to diabetic wounds treated with lenti-GFP (3.52 fold ± 1.2, p = 0.017). No difference in IL-6 gene expression was seen between groups treated with lenti-GFP at day 7. Non-diabetic wounds treated with lenti-GFP demonstrated decreased MIP-2 gene expression at day 3 (4.36 fold ± 0.98 versus 1 fold ± 0.20 at day 3, p = 0.004) and post-wounding day 7 (0.73 fold ± 0.17 versus 0.08 ± 0.009 at day 7, p = 0.002).

Non-diabetic wounds treated with the lenti-SDF-1α inhibitor did not demonstrate any significant difference in IL-6 gene expression at 3 or 7 days. There was, however, significant increase in MIP-2 gene expression in non-diabetic wounds treated with the lenti-SDF-1α inhibitor at 7 days (1.37 ± 0.20 fold) versus non-diabetic wounds treated with lenti-GFP (0.08 ± 0.009 fold, p = 0.003). Figure 6B displays the relative-fold expression for IL-6 and MIP-2, respectively, in both lenti-SDF-1α inhibitor and lenti-GFP-treated non-diabetic wounds.

Cellular migration

Fibroblasts transduced with lenti-SDF-1α resulted in a significant increase in the migration of splenic leukocytes (253± 45 cells) compared to non-transduced fibroblasts (160± 39 cells, p = 0.004, figure 7) or no cells (144± 40 cells, p = 0.017). The addition of fibroblasts transduced with the lenti-SDF-1α inhibitor, without changing the number of total fibroblasts, resulted in a dose dependent decrease in the migration of splenic leukocytes.

Figure 7.

Figure 7

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Splenic leukocyte migration assay. There was a decrease in leukocyte migration with increasing concentrations of lenti-SDF-1α inhibitor transfected cells. On average, 256 ± 42 cells were counted in the group with lenti-SDF-1α cells. When the lower chamber had an increased ratio of lenti-SDF-1α inhibitor, fewer cells migrated. 126 ± 51 cells were counted when 1.5 × 105 lenti-SDF-1α inhibitor cells were plated, p < 0.05. An asterisk denotes a p-value < 0.05.

Discussion

We demonstrate that SDF-1α is a key component in the wound healing process, and that competitive inhibition of the activation of the CXCR4 receptor can alter the rate of wound healing – especially in the diabetic mouse. Competitive inhibition of SDF-1α significantly impairs the rate of wound healing, decreases angiogenesis and increases inflammation in the diabetic mouse. These are all features of a chronic wound phenotype. In non-diabetic mice, the inhibitor also decreases rate of wound-healing early on and is associated with increased inflammation. These findings suggest a delay in wound healing, with the increased duration of an open wound, may result in increased inflammatory response to the persistent wound. Non-diabetic wounds may be able to overcome this increased inflammatory response, but diabetic wounds, which already have a disordered inflammatory response, may be unable to compensate for the increased inflammation.

Diabetic wounds are deficient in SDF-1α (11) and correction of this deficiency improves healing (26). Since there is no SDF-1α deficiency in non-diabetics, the level of SDF-1α production required to overcome the effects of the inhibitory vector are lower. These findings suggest a role for SDF-1α in normal wound healing but may have an even more critical role in the pathogenesis of diabetic wound healing.

SDF-1α is a chemotactic factor regulating the migration of endothelial progenitor cells (EPCs) as well as leukocytes (31, 32). EPC migration is also regulated by activated endothelial nitric oxide synthase (eNOS). In addition to SDF-1α deficiency, diabetic wounds have also been shown to be deficient in eNOS (11). Decreased EPC recruitment and function have been implicated in the diabetic wound healing impairment (33, 34). It has recently been demonstrated that db/db mice have fewer circulating EPCs – and that subsequently wound healing was significantly improved when more EPCs were mobilized (35). These findings, combined with our observations of further wound healing impairment with SDF-1α inhibition, further supports a role for SDF-1α in wound healing and the diabetic impairment in healing.

SDF-1α is regulated, in part, through the transcriptional activator hypoxia inducible factor-1α (HIF-1α) (31). Following injury, wound-induced tissue hypoxia leads to upregulation of HIF-1α (36). Toksoy et al. has demonstrated that there is a clear increase in the expression of SDF-1α at the ischemic margin of a wound in a normal wound-healing environment (37). This coincides with the region where the majority of vasculogenesis occurs. In vitro, SDF-1α has been shown to promote the proliferation and migration of endothelial cells (38, 39), and in vivo, it augments the growth of granulation tissue and endothelial and progenitor cells (11, 26, 40).

Inhibition of SDF-1α activity resulted in decreased CD31+ cells and vessels in the wound, and decreased granulation tissue formation, which correlated with impaired wound healing rates and delayed wound closure. These findings suggest that a lack of CXCR4 activation results in impaired wound healing through decreased recruitment of EPCs and subsequent angiogenesis. Overexpression of SDF-1α hastens wound healing in the diabetic mouse and augments angiogenesis (26). Furthermore, mesenchymal stem cells have also been shown to correct the deficiency of SDF-1α and promote granulation tissue formation and improved wound healing (40).

Diabetic wounds are known to be more abundant in pro-inflammatory cytokines and chronic inflammation has been implicated in the pathogenesis of the diabetic wound healing impairment (14, 19, 41). We hypothesized that with inhibition of the CXCR4 receptor, the delay in wound healing may be due to chronic inflammation – supported by our finding that there was greater CD45 infiltrate in not only diabetic mice treated with control vector compared with heterozygote mice treated with control vector, but also diabetic mice treated with the inhibitor. Impaired wound healing may in fact increase the proinflammatory cytokines circulating in the wound and ultimately result in a chronic inflammatory state. While no clear relationship exists at the moment between inhibition of the CXCR4 pathway and upregulation of inflammatory cytokines, these pro-inflammatory cytokines have a possible role in the development and persistence of chronic wounds (41).

We demonstrated increased inflammatory cells in diabetic wounds treated with the SDF-1α inhibitor at 7 days compared to GFP treated diabetic wounds. This increased inflammation correlates with increased expression of IL-6 at 3 days and MIP-2 at 7 days. The impaired wound healing in non-diabetic wounds treated with the SDF-1α inhibitor was early in the course of wound healing and was corrected by day 6. The correlation of this gene expression data and wound closure is complicated because of the lag between gene expression and the end result of the gene product.

Previous studies have demonstrated that expression of SDF-1α will promote cellular and leukocyte migration (38). The observation that we see a decline in leukocyte migration with greater expressed levels of mutant SDF-1α confirms this mechanism of impaired cellular migration. That our animal model is a leptin-resistant diabetic mouse may confound the picture. Leptin is known to promote angiogenesis – so the differences illustrated in our study at baseline may be exaggerated in a background of no leptin activity.

There is a lack of novel approaches to treat diabetic wound healing. The application of growth factors, as in the case of recombinant PDGF, has been utilized to improve human tissue healing but the results have not been dramatic. Biosynthetic grafts and hyperbaric oxygen therapy have been utilized as a novel therapy to improve the diabetic wound healing process. These too have been met with marginal success and at times prohibitive costs (42).

SDF-1α is a highly conserved chemokine that plays a critical and multifaceted role in the wound healing process in normal and diabetic environments. We hypothesized that further decreases in SDF-1α functional activity would result in further impairments in diabetic healing and may affect non-diabetic healing. In addition, the diabetic wound healing impairment has been associated with chronic inflammation. Chronic leg ulcers were found to be significantly more infiltrated with inflammatory cells than one would expect in a well-healing wound (43).

We hypothesized that delayed healing would result in increased production of proinflammatory cytokines and increased inflammation as seen in diabetic wounds. Figure 8 displays what we suspect is the mechanism by which our inhibitor has an effect on wound healing. Specifically, we feel that the impaired angiogenesis that occurs as a result of the inhibitor sets into play a wound that heals poorly and is stymied by chronic inflammation. Our results here appear to support these findings with a marked increase in inflammatory cells and inflammatory cytokines with further inhibition of SDF-1α activity. While clearly more studies are needed to define this relationship, SDF-1α may prove to be a key factor in the wound healing process that could be targeted to correct the diabetic wound healing defect.

Figure 8.

Figure 8

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A: Schematic of the mechanism by which the SDF-1α inhibitor has an effect on the intracellular transduction at the CXCR4 receptor. While the inhibitor competitively inhibits at CXCR4 through binding, it does not activate a series of important intracellular signaling cascades. B: Diabetic wounds may be easier to inhibit SDF-1α due to intrinsically low levels. This prevents angiogenesis and subsequently promotes a chronic wound.

Acknowledgments

The research presented in this article was supported in part by two grants from the National Institutes of Health: # R56DK080672-01A1 (R56) and DP2DK083085-01 (diabetes pathfinder grant). We thank Dr. Philip W. Zoltick of the Center for Fetal Research at the Children’s Hospital of Philadelphia for constructing the lentiviral vectors used in this project.

Footnotes

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