Wounds In Diabetic Mice Research Articles

Abstract 5688: HMGB1 is a Novel Substrate of Dypeptidyl Peptidase IV

Introduction: High Mobility Group Box 1 (HMGB1) is a cytokine with a key role in tissue regeneration and angiogenesis. HMGB1 levels are reduced in diabetic skin and topical application of HMGB1 to skin wounds of diabetic mice enhanced vessel density and accelerated wound healing, suggesting that diabetes may affect endogenous HMGB1 functions. CD26 is a protease which selectively removes the N-terminal X-Pro or X-Ala containing motifs from a number of chemokines modulating their activity. High levels of CD26 have been detected in plasma of type II diabetic patients and its inhibition has proved effective in the treatment of diabetes. Since HMGB1 contains potential CD26 cleavage sites we determined whether HMGB1 may be a substrates for CD26 and, eventually, whether CD26-mediated cleavage may alter its biologic activity. Methods and results: Recombinant HMGB1 (5 uM) was incubated overnight with CD26 (2.2 uM). RP-HPLC analysis on Aquapore C18 column yielded a peptide with a molecular weigh lower than 2000 kDa. The CD26-mediated deletion of the N-terminal region of HMGB1 was confirmed by western blot analysis: time-dependent disappearance of the molecular weigh band corresponding to HMGB1 was detected with an anti-HMGB1 antibody which recognizes its first 16 N-terminal amino-acids of HMGB1. It was previously found that HMGB1 promoted endothelial cell migration through the activation of c-Jun N-terminal kinase (JNK) and extracellular regulated kinase (ERK) signaling pathways. To evaluate whether CD26 altered HMGB1 functions, endothelial cells were induced to migrate in presence of HMGB1 and CD26. HMGB1-mediated chemotactic activity was reduced by CD26 in a dose-dependent manner. Similarly, CD26 abolished HMGB1-induced JNK and ERK activation. Since diabetes is associated with the increased CD26 activity, both CD26 activity and HMGB1 levels were measured in serum of diabetic patients. Although HMGB1 serum levels did not significantly differ between diabetic and normoglycemic patients, increased N-terminal deleted HMGB1 was detected in serum from diabetic patients. Conclusions: CD26 cleaves HMGB1 and, via this mechanism, it inhibits its functions. These findings suggest that CD26 may account for the inhibition of HMGB1 effects found in diabetes.

Overexpression of SDF-1alpha promotes bone marrow progenitor cell recruitment to a dermal wound in both diabetic and nondiabetic mice

Overexpression of SDF-1alpha promotes bone marrow progenitor cell recruitment to a dermal wound in both diabetic and nondiabetic mice

Poly-N-Acetyl Glucosamine Nanofibers

In several fields of surgery, the treatment of complicated tissue defects is an unsolved clinical problem. In particular, the use of tissue scaffolds has been limited by poor revascularization and integration. In this study, we developed a polymer, poly-N-acetyl-glucosamine (sNAG), with bioactive properties that may be useful to overcome these limitations. To develop a scaffold-like membrane with bioactive properties and test the biologic effects in vitro and in vivo in diabetic wound healing. In vitro, cells-nanofibers interactions were tested by cell metabolism and migration assays. In vivo, full thickness wounds in diabetic mice (n = 15 per group) were treated either with sNAG scaffolds, with a cellulosic control material, or were left untreated. Wound healing kinetics, including wound reepithelialization and wound contraction as well as microscopic metrics such as tissue growth, cell proliferation (Ki67), angiogenesis (PECAM-1), cell migration (MAP-Kinase), and keratinocyte migration (p 63) were monitored over a period of 28 days. Messenger RNA levels related to migration (uPAR), angiogenesis (VEGF), inflammatory response (IL-1beta), and extracellular matrix remodeling (MMP3 and 9) were measured in wound tissues. sNAG fibers stimulated cell metabolism and the in vitro migratory activity of endothelial cells and fibroblasts. sNAG membranes profoundly accelerated wound closure mainly by reepithelialization and increased keratinocyte migration (7.5-fold), granulation tissue formation (2.8-fold), cell proliferation (4-fold), and vascularization (2.7-fold) compared with control wounds. Expression of markers of angiogenesis (VEGF), cell migration (uPAR) and ECM remodeling (MMP3, MMP9) were up-regulated in sNAG treated wounds compared with controls. The key mechanism of the bioactive membranes is the cell-nanofiber stimulatory interaction. Engineering of bioactive materials may represent the clinical solution for a number of complex tissue defects.

877. Non-Viral Gene Therapy Using Arginine Modified Polyamidoamine Dendrimer (PAMAM-RG4) and Minicircle VEGF DNA into the Skin Wound of Diabetic Mice Successfully Improved the Healing and Microarchitecture

877. Non-Viral Gene Therapy Using Arginine Modified Polyamidoamine Dendrimer (PAMAM-RG4) and Minicircle VEGF DNA into the Skin Wound of Diabetic Mice Successfully Improved the Healing and Microarchitecture

Akt1 Controls Insulin-Driven VEGF Biosynthesis from Keratinocytes: Implications for Normal and Diabetes-Impaired Skin Repair in Mice

Here we investigated the potential role of protein kinase B (Akt) in normal or diabetes-impaired wound healing in mice. Interestingly, Akt1 was predominant in skin, wound tissue, and human keratinocytes cell line. Acute skin repair was characterized by an increase of Akt1 phosphorylation in wound margin keratinocytes. By contrast, phosphorylated Akt1 was nearly completely absent and paralleled by a poor phosphorylation of the eucaryotic initiation factor 4E-binding protein 1 (4E-BP1) and reduced levels of vascular endothelial growth factor (VEGF) protein in chronic wounds of diabetic ob/ob mice. Inhibition of the phosphatidyl-inositol-3 kinase/Akt pathway by wortmannin and specific abrogation of Akt1 protein using small-interfering RNA revealed a regulatory function of Akt1 in insulin-mediated VEGF biosynthesis in keratinocytes. Insulin-induced VEGF protein biosynthesis in keratinocytes was mediated by Akt1 from a constitutive VEGF-encoding mRNA pool at the posttranscriptional level through a downstream phosphorylation 4E-BP1. Moreover, transfection experiments introducing a constitutively active mutant of Akt1 into keratinocytes revealed the mammalian target of rapamycin kinase as a downstream mediator of Akt1-linked 4E-BP1 phosphorylation and translational control. Our data suggest that the endocrine hormone insulin contributes to VEGF release in skin wounds through an Akt1-mediated posttranscriptional mechanism in keratinocytes.

Commentaries on Viewpoint: Effect of altitude on leptin levels, does it go up or down? Control of leptin with altitude exposure

to the editor: The Viewpoint by Sierra-Johnson et al. ([6][1]) raises a number of interesting points about the control of leptin release in response to altitude exposure. The authors correctly point out that “altitude” can comprise many stimuli to the endocrine system including hypoxia, cold

Application of the engineered CXCL12 plasmid to diabetic mouse wounds accelerates healing to near normal via stem cell trafficking

Application of the engineered CXCL12 plasmid to diabetic mouse wounds accelerates healing to near normal via stem cell trafficking

In vitro and in vivo wound healing-promoting activities of β-lapachone

Impaired wound healing is a serious problem for diabetic patients. Wound healing is a complex process that requires the cooperation of many cell types, including keratinocytes, fibroblasts, endothelial cells, and macrophages. beta-Lapachone, a natural compound extracted from the bark of the lapacho tree (Tabebuia avellanedae), is well known for its antitumor, antiinflammatory, and antineoplastic effects at different concentrations and conditions, but its effects on wound healing have not been studied. The purpose of the present study was to investigate the effects of beta-lapachone on wound healing and its underlying mechanism. In the present study, we demonstrated that a low dose of beta-lapachone enhanced the proliferation in several cells, facilitated the migration of mouse 3T3 fibroblasts and human endothelial EAhy926 cells through different MAPK signaling pathways, and accelerated scrape-wound healing in vitro. Application of ointment with or without beta-lapachone to a punched wound in normal and diabetic (db/db) mice showed that the healing process was faster in beta-lapachone-treated animals than in those treated with vehicle only. In addition, beta-lapachone induced macrophages to release VEGF and EGF, which are beneficial for growth of many cells. Our results showed that beta-lapachone can increase cell proliferation, including keratinocytes, fibroblasts, and endothelial cells, and migration of fibroblasts and endothelial cells and thus accelerate wound healing. Therefore, we suggest that beta-lapachone may have potential for therapeutic use for wound healing.

133 Synthetic, Cyclodextrin-Based Polymer Matrix for Local Regional Drug Delivery

Various matrices containing both synthetic or natural polymers are currently used for local regional drug delivery to a number of disease locations such as chronic cutaneous wounds, joints, bone, muscle, and nerves. Insert Therapeutics has developed a novel synthetic matrix which consists of a copolymer of β-cyclodextrin and polyethylene-glycol (PEG). Addition of di- or multifunctionalized adamantane-PEG molecules results in noncovalent cross-linking through inclusion complex formation. The resulting hydrogel showed rheological characteristics amenable to topical application or local-regional injection into a variety of tissues (Bellocq et al. (2004) Bioconjug Chem 15(6): 1201–1211). This matrix was shown to be biocompatible, allowing for cellular growth and migration in vitro. It also allowed for efficient delivery of an adenovirus gene therapy vector to dermal fibroblast cells. In vivo, the matrix demonstrated efficient delivery of biotherapeutics such as recombinant adenovirus and non-viral gene therapy vectors after intradermal injection. The matrix was well tolerated and was as efficient as collagen in promoting wound healing when an adenoviral gene therapy vector expressing PDGF-bb was delivered to cutaneous wounds of diabetic mice. The matrix can additionally be modified to incorporate therapeutic small molecules, peptides, or proteins, either through inclusion complex formation or chemical linkage. Using biodegradable linker chemistry, the release rate of covalently attached molecules can be controlled. The cyclodextrin-PEG matrix is therefore an attractive alternative to existing matrices that is biocompatible, biodegradable, tunable with regard to its physicochemical properties, and can be designed to deliver multiple therapeutic agents to a variety of tissues in a controlled fashion.

High-Mobility Group Box 1 Protein in Human and Murine Skin: Involvement in Wound Healing

High-mobility group box 1 (HMGB1) protein is a multifunctional cytokine involved in inflammatory responses and tissue repair. In this study, it was examined whether HMGB1 plays a role in skin wound repair both in normoglycemic and diabetic mice. HMGB1 was detected in the nucleus of skin cells, and accumulated in the cytoplasm of epidermal cells in the wounded skin. Diabetic human and mouse skin showed more reduced HMGB1 levels than their normoglycemic counterparts. Topical application of HMGB1 to the wounds of diabetic mice enhanced arteriole density, granulation tissue deposition, and accelerated wound healing. In contrast, HMGB1 had no effect in normoglycemic mouse skin wounds, where endogenous HMGB1 levels may be adequate for optimal wound closure. Accordingly, inhibition of endogenous HMGB1 impaired wound healing in normal mice but had no effect in diabetic mice. Finally, HMGB1 had a chemotactic effect on skin fibroblasts and keratinoyctes in vitro. In conclusion, lower HMGB1 levels in diabetic skin may play an important role in impaired wound healing and this defect may be overcome by the topical application of HMGB1.

Research of PDGF-BB Gel on the Wound Healing of Diabetic Rats and Its Pharmacodynamics

One of the leading causes of impaired wound healing is diabetes mellitus. In diabetic patients, a minor skin wound often leads to serious complications. Many experiments had demonstrated that the expression of platelet-derived growth factor (PDGF) and its receptor was decreased in wounds of healing-impaired diabetic mice, indicating that a certain expression level of PDGF is essential for normal repair. The diabetic rats was induced by a single i.p. injection of streptozotocin and a 1.8 cm diameter full-thickness wound was made on each side of the rat mid-back. Then the rats were randomly divided into five groups, with eight animals in each group as follows: blank control, vehicle control, 3.5 microg PDGF-BB/cm(2) treatment group, 7 microg PDGF-BB/cm(2) treatment group and 14 microg PDGF-BB/cm(2) treatment group for either 7 or 14 consecutive days after wounding. Re-epithelialization area was measured by computerized planimetry, percentage wound closure and percentage wound contraction was calculated, granulation tissue and collagen formation was assessed by Masson trichrome, cell proliferation (proliferating cell nuclear antigen staining) and angiogenesis (Factor VIII related antigen staining) was assessed by immunohistological methods. PDGF-BB treatment improved healing quality, enhanced angiogenesis, cell proliferation and epithelialization, and formed thicker and more highly organized collagen fiber deposition in full-thickness excisional wound of diabetic rats. The effects of topically applied PDGF-BB were dose-dependent. PDGF-BB is an important future clinical tool, particularly for stimulating soft tissue repair in patients with an impaired capacity for wound healing.

Potential Use of Blood Bank Platelet Concentrates to Accelerate Wound Healing of Diabetic Ulcers

Many clinical trials have shown the effectiveness of platelet releasates on diabetic wound healing, but large volumes of blood must be aspirated from patients and a platelet separator is required. This study was undertaken to investigate the potential of blood bank platelet concentrate (BBPC) for accelerating diabetic wound healing. Platelet-derived growth factor-BB (PDGF-BB) contents in BBPC were determined by enzyme-linked immunosorbent assay in vitro, and the in vivo study involved comparing extents of wound healing in BBPC-treated and control groups using diabetic mouse wound models. In the in vitro study, 5.2 +/- 1.2 pg of PDGF-BB was found to be released by 1 million platelets in fresh BBPC, and adding thrombin to BBPC significantly increased the levels of PDGF-BB released. Our in vivo study in diabetic mice revealed that BBPC treatment greatly accelerated wound healing. Our results suggest that BBPC has potential to accelerate the healing of diabetic ulcers.

Lentiviral Gene Transfer of SDF-1α to Wounds Improves Diabetic Wound Healing

Chronic wounds continue to be a major clinical problem and novel therapeutic approaches are needed. We have previously demonstrated that treatment of diabetic mouse wounds with local application of stromal progenitor cells results in improved healing and increased production of stromal-derived growth factor-1alpha (SDF-1alpha). We hypothesized that lentiviral-mediated increased production of SDF-1alpha in the wound environment could also improve diabetic wound healing. Full-thickness excisional wounds were created in Db-/Db- mice and immediately treated with 10(6), 10(8), or 10(9) plaque-forming units of a lentiviral construct containing GFP-SDF-1alpha or GFP alone. At 7 and 14 days post wounding, wounds were harvested for histological and molecular analysis. At 7 days, Db-/Db- wounds treated with lenti GFP-SDF-1alpha exhibited a decrease in wound surface area for all doses tested. Morphologically, SDF-treated wounds were more cellular with increased granulation tissue volume compared to controls (P < 0.05). GFP expression was maintained in treated tissue at 7 days post wounding, but little expression was observed at 14 days. While we did not observe a difference in the gross wound surface area at 14 days, histological analysis revealed that SDF-treated wounds were fully epithelialized (n = 6) compared to only one of six controls. Lentiviral-mediated overproduction of SDF-1alpha is sufficient to correct the pathophysiologic abnormalities in diabetic wound healing resulting in complete epithelialization at 2 weeks. SDF-1alpha-mediated improvement in diabetic wound healing has significant implications for the development of novel therapeutic strategies to facilitate wound closure which target progenitor cell mobilization and recruitment.

Sustained expression of Hif‐1α in the diabetic environment promotes angiogenesis and cutaneous wound repair

Impaired wound healing in diabetic patients is associated with deficiencies in the production of factors involved in cell proliferation and migration, such as vascular endothelial growth factor. However, it remains unclear how the transcriptional regulation of the genes encoding these factors is affected by the diabetic environment. Hypoxia-inducible factor-1alpha (Hif-1alpha), the regulatory subunit of the Hif-1 transcription factor, plays an important role in activating many of these genes. Therefore, we tested whether Hif-1alpha function is impaired in the diabetic wound environment and whether restoring Hif-1 function improves wound healing. Here, we show that Hif-1alpha protein levels are dramatically reduced in wounds of leptin receptor-deficient diabetic mice compared with nondiabetic littermates. Reduction in Hif-1alpha levels results in decreased DNA-binding activity and in decreased expression of several Hif-1 target genes, including vascular endothelial growth factor, heme oxygenase-1, and inducible nitric oxide synthase. Furthermore, we demonstrate that sustained expression of Hif-1alpha in leptin receptor-deficient diabetic wounds restores expression of these factors, enhances angiogenesis, and significantly accelerates wound healing. Taken together, these results suggest that Hif-1alpha function plays a significant role in wound healing and reduced levels of Hif-1alpha may contribute to impaired healing.

Topical application of laminin-332 to diabetic mouse wounds

Keratinocyte migration is essential for wound healing and diabetic wound keratinocytes migrate poorly. Keratinocyte migration and anchorage appears to be mediated by laminin-332 (LM-332). Impaired diabetic wound healing may be due to defective LM-332 mediated keratinocyte migration. To evaluate LM-332 expression in diabetic (db/db) and control (db/-) mice and to test LM-332 wound healing effects when applied to mouse wounds. LM-332 expression in mouse wounds was evaluated using immunohistochemistry. LM-332 wound healing effects were evaluated by directly applying soluble LM-332, a LM-332 biomaterial, or a control to mouse wounds. Percent wound closure and histology score, based on healing extent, were measured. Precursor LM-332 expression was markedly reduced in db/db when compared to db/- mice. In vitro, soluble LM-332 and LM-332 biomaterial demonstrated significant keratinocyte adhesion. In vivo, soluble LM-332 treated wounds had the highest histology score, but significant differences were not found between wound treatments (p>0.05). No differences in percentage wound closure between treatment and control wounds were found (p>0.05). The db/db wounds express less precursor LM-332 when compared to db/-. However, LM-332 application did not improve db/db wound healing. LM-332 purified from keratinocytes was primarily physiologically cleaved LM-332 and may not regulate keratinocyte migration. Application of precursor LM-332 rather than cleaved LM-332 may be necessary to improve wound healing, but this isoform is not currently available in quantities sufficient for testing.

Sphingosine 1-phosphate accelerates wound healing in diabetic mice

Blood platelets store sphingosine 1-phosphate (S1P) abundantly and release this bioactive lipid extracellularly. S1P acts as an intercellular mediator through interaction with the endothelial differentiation gene (EDG)/S1P family of G protein-coupled receptors. Of the EDG family S1P receptors, EDG-5 (S1P2) is inhibited in migration induced by S1P. Diabetes impairs numerous aspects of tissue repair. Failure of wound angiogenesis is known to delay diabetic wound healing. We examined whether S1P subcutaneous injection could improve the healing of full-thickness skin wounds in healthy and diabetic mice. We further determine if the combined S1P and EDG-5 (S1P2) antagonist injection in diabetic mice could affect wound healing. Finally, we examined the histopathological findings of the wound following S1P injection in diabetic mice. Eight- to 10-week-old BALA/c mice, diabetic db/db mice and Wister rats were used for the studies. A full-thickness wound was made on the dorsal skin of the healthy and diabetic mice. Either 10 microM or 100 microM of S1P or vehicle control (BSA/PBS) was injected into the wound bed every day. We calculated the wound area after each injection. EDG-5 (S1P2) antagonist (JTE-013) or vehicle (DMSO) was then injected in addition to the S1P around the dorsal wound of diabetic mice and the wound diameter was measured. Wound tissue samples were excised following injection for histopathological examination. Wound area in normal BALA/c mice did not significantly decrease upon S1P injection compared to S1P-untreated controls. S1P injection alone showed significant promotion of wound healing in diabetic mice compared to no S1P treatment. The combination of S1P and EDG-5 (S1P2) receptor antagonist administration induced maximal wound healing in diabetic mice. Histopathological examination revealed that S1P induces neo-vascularization potential in rats and diabetic mice wound. S1P injection in diabetic mice significantly accelerated cutaneous wound healing in the neo-vascularization process. The results demonstrate that S1P affects and sustains all key cellular processes responsible for wound repair and point to a unique potential for this molecule in the therapy of diabetic wounds, particularly as an angiogenic agent in treatment of diabetic wounds.

Autologous Bone Marrow–Derived Cultured Mesenchymal Stem Cells Delivered in a Fibrin Spray Accelerate Healing in Murine and Human Cutaneous Wounds

The nonhematopoietic component of bone marrow includes multipotent mesenchymal stem cells (MSC) capable of differentiating into fat, bone, muscle, cartilage, and endothelium. In this report, we describe the cell culture and characterization, delivery system, and successful use of topically applied autologous MSC to accelerate the healing of human and experimental murine wounds. A single bone marrow aspirate of 35-50 mL was obtained from patients with acute wounds (n = 5) from skin cancer surgery and from patients with chronic, long-standing, nonhealing lower extremity wounds (n = 8). Cells were grown in vitro under conditions favoring the propagation of MSC, and flow cytometry and immunostaining showed a profile (CD29+, CD44+, CD105+, CD166+, CD34-, CD45-) highly consistent with published reports of human MSC. Functional induction studies confirmed that the MSC could differentiate into bone, cartilage, and adipose tissue. The cultured autologous MSC were applied up to four times to the wounds using a fibrin polymer spray system with a double-barreled syringe. Both fibrinogen (containing the MSC) and thrombin were diluted to optimally deliver a polymerized gel that immediately adhered to the wound, without run-off, and yet allowing the MSC to remain viable and migrate from the gel. Sequential adjacent sections from biopsy specimens of the wound bed after MSC application showed elongated spindle cells, similar to their in vitro counterparts, which immunostained for MSC markers. Generation of new elastic fibers was evident by both special stains and antibodies to human elastin. The application of cultured cells was safe, without treatment-related adverse events. A strong direct correlation was found between the number of cells applied (greater than 1 x 10(6) cells per cm2 of wound area) and the subsequent decrease in chronic wound size (p = 0.0058). Topical application of autologous MSC also stimulated closure of full-thickness wounds in diabetic mice (db/db). Tracking of green fluorescent protein (GFP)+ MSC in mouse wounds showed GFP+ blood vessels, suggesting that the applied cells may persist as well as act to stimulate the wound repair process. These findings indicate that autologous bone marrow-derived MSC can be safely and effectively delivered to wounds using a fibrin spray system.

Enhanced epithelial gap closure and increased angiogenesis in wounds of diabetic mice treated with adult murine bone marrow stromal progenitor cells

The direct application of bone marrow (BM) can accelerate the healing of chronic wounds. We hypothesized that this effect is due to the presence of stromal progenitor cells (SPCs) found within whole BM preparations. To test this hypothesis, we isolated adult murine SPCs from whole BM and examined their ability to enhance impaired wound healing compared with ficoll separated BM cells in the diabetic (db/db) mouse model. SPCs significantly enhanced reepithelialization, granulation tissue formation, and neovascularization compared with control wounds treated with BM or PBS alone. Higher frequencies of donor SPC cells compared with donor BM cells were observed in treated wounds at 7 days. Transdifferentiation into GFP-positive mature endothelial cells was not observed. These observations suggest that SPCs improve wound healing through indirect mechanisms which lead to enhanced vascularization rather than through direct participation and incorporation into tissue. We conclude that topical application of BM-derived SPCs may represent an effective strategy for the treatment of chronic diabetic wounds.

Chimeric VEGF-E NZ7 /PlGF Specifically Binding to VEGFR-2 Accelerates Skin Wound Healing via Enhancement of Neovascularization

VEGF-E(NZ7)/PlGF molecules composed of Orf virus-derived VEGF-E(NZ7) and human PlGF1 were previously proven to be potent angiogenic factors stimulating angiogenesis without significant enhancement of vascular leakage and inflammation in vivo. For its future clinical application, there is a pressing need to better understand the beneficial effects of VEGF-E(NZ7)/PlGF during wound healing in adulthood. In this study, several angiogenic factors were administrated to skin punched wounds of both wild-type and diabetic mice. The treatment with VEGF-E(NZ7)/PlGF accelerated wound closure accompanied with enhanced angiogenesis, the process was occurring slightly faster than that in VEGF-A164 group. Moreover, the macrophage infiltration and lymphangiogenesis level in healed wounds were strikingly lower in VEGF-E(NZ7)/PlGF group than VEGF-A164 group, suggesting that the increased inflammation was the key issue preventing speedy wound healing of VEGF-A164-treated skin. Considering clinical safety, we further examined the antigenicity of chimeric VEGF-E(NZ7)/PlGF. Compared with the original VEGF-E(NZ7), the immunogenicity of VEGF-E(NZ7)/PlGF molecules was markedly decreased in mice and squirrel monkeys with the increase of PlGF1 humanized ratio. These results indicate that VEGF-E(NZ7)/PlGF molecules are superior to VEGF-A for the acceleration of either normal or delayed skin wound healing and might be regarded as potential drugs in therapeutic angiogenesis.

Differential Regulation of Angiogenic Genes in Diabetic Wound Healing

Wound healing is a complicated biological process that involves interactions of multiple cell types, various growth factors, their mediators, and the extracellular matrix proteins. In this study, we have studied the differential regulation of angiogenic genes during wound healing in transgenic (Lepr -/-) diabetic mice and non-diabetic mice. Under aseptic conditions, 8 mm full thickness cutaneous wounds were created on either side of the mid-dorsal. Wound tissues were studied at 4, 7, and 11 days post-wounding and healing was assessed by histology. The pathway-specific gene array data demonstrated differential regulation of growth factors, transcription factors, and other related genes, such as fibroblast growth factors and their receptors. The extracellular matrix protein osteopontin (OPN), an important component of cellular immunity and inflammation, showed higher expression in non-diabetic wounds after 4 days post-wounding, whereas its expression was at basal level in diabetic wounds. OPN expression remained upregulated in non-diabetic wounds at day 7 post-wounding and was downregulated to basal level at day 11 post-wounding. However, expression of OPN was upregulated in diabetic wounds at day 7 post-wounding and remained constitutively higher at day 11 post-wounding. OPN expression was concomitant with the extent of healing as assessed by histology at the corresponding sampling point. This finding suggests that OPN might be playing a crucial role in the early events of the wound healing and its delayed expression may be in part responsible for the delayed healing of wounds in diabetic mice.