Wounds In Diabetic Mice Research Articles

Regulation of hypoxia-induced angiogenesis: a chaperone escorts VEGF to the dance.

Regulation of hypoxia-induced angiogenesis: a chaperone escorts VEGF to the dance.

Development of angiotensin (1‐7) as an agent to accelerate dermal repair

Angiotensin II has been shown to be a potent agent in the acceleration of wound repair. Angiotensin (1-7), a fragment of angiotensin II that is not hypertensive, was found to be comparable to angiotensin II in accelerating dermal healing. This activity was evaluated in four models: rat and diabetic mouse full-thickness excisional wounds; rat random flap; and guinea pig partial thickness thermal injury. In all models, angiotensin (1-7) was comparable to angiotensin II. Angiotensin (1-7) accelerated the closure of wounds in diabetic mice and rats. In diabetic mice the resultant tissue at day 25 after injury was more comparable to normal tissue than the fibrotic scar observed in placebo-treated wounds. In the random flap model, angiotensin (1-7) was comparable to angiotensin II in maintaining flap viability (approximately 82%) and flap survival (40%). Finally, angiotensin (1-7) increased proliferation in the hair follicles at the edge of the wound and site of thermal injury, and the number of patent blood vessels on day 7 after partial thickness thermal injury. These data may be partially explained by the effect of angiotensin II and angiotensin (1-7) on keratinocyte proliferation. While platelet-derived growth factor had no effect on keratinocyte proliferation, angiotensin II and angiotensin (1-7) significantly increased keratinocyte proliferation. These data show that angiotensin(1-7) is comparable to angiotensin II in accelerating skin repair. Furthermore, the hypertensive and wound healing effects can be separated within the family of angiotensin peptides.

Expressional Regulation of Angiopoietin-1 and -2 and the Tie-1 and -2 Receptor Tyrosine Kinases during Cutaneous Wound Healing: A Comparative Study of Normal and Impaired Repair

It has become evident that a closely regulated presence of vascular endothelial growth factor (VEGF) and angiopoietin (Ang) factors determines the fate of blood vessel formation during angiogenesis. As angiogenesis is central to a normal wound-healing process, we investigated the regulation of Ang-1 and -2 and the related tyrosine kinase with immunoglobulin and epidermal growth factor homology (Tie)-1 and -2 receptors during normal repair in Balb/c mice and diabetes-impaired wound healing conditions in genetically diabetic (db/db) mice. For both normal and impaired healing conditions, we observed a constitutive expression of Ang-1, which was paralleled by an increase of Ang-2 upon injury. Whereas the observed Ang-2 expression declines from Day 7 after injury in control mice, diabetic-impaired healing was characterized by still increasing amounts of Ang-2 at these time points. Furthermore, Tie-1 was strongly induced during repair with a prolonged expression in diabetic mice, whereas Tie-2 expression was constitutive during normal repair but completely absent in diabetes-impaired healing. The overexpression of Ang-2 in the presence of markedly reduced VEGF in wounds of diabetic mice was associated with a dramatic decrease in endothelial cell numbers compared with normal healing as assessed by analysis of the endothelium-specific markers CD31 and von Willebrand factor, whereas the lymphatic endothelium remained stable as determined by expression of VEGF receptor-3 (VEGFR-3/Flt-4).

Expression and localization of p53 and bcl‐2 in healing wounds in diabetic and nondiabetic mice

In a healing wound, inflammatory cells undergo apoptosis immediately beneath the leading edge of migrating epithelium. A potential mediator of this apoptosis pattern is p53, a protein with antiproliferative effects. Another protein, bcl-2, is antagonistic to p53 and prevents apoptosis. The purpose of this study was to determine the expression and location of p53 and bcl-2 mRNA and protein in healing wounds of normal and genetically diabetic mice. At various time points, full-thickness skin wounds from nondiabetic and diabetic mice were evaluated for p53 and bcl-2 by immunohistochemistry and in situ hybridization. Apoptosis patterns were also determined using the TUNEL method. Messenger RNA for p53 and bcl-2 were quantitated by competitive reverse transcriptase-polymerase chain reaction. Protein and mRNA for p53 were expressed in the leading edge of migrating epithelium, with apoptosis patterns closely following those of p53 production. p53 mRNA levels decreased soon after wounding, but after a few days, levels increased to greater than baseline. bcl-2 was localized to the wound epithelium, but relative amounts tended to oppose levels of p53, i.e, when p53 increased, bcl-2 decreased and vice versa. Wounds in diabetic animals showed a delayed onset of p53 mRNA expression but had persistently greater levels for longer periods of time. bcl-2 mRNA expression was further delayed in diabetic mice and did not develop to levels as high as p53. Production of both proteins was delayed, consistent with the mRNA expression. Our data show that immediately after wounding, bcl-2 increases and p53 decreases to allow for the cellular proliferation that is required for tissue repair. Over time, bcl-2 levels decrease while p53 levels increase to shut down the inflammatory process and down-regulate the proliferative response. Diabetic animals appear to lose the indirect relationship between p53 and bcl-2. This loss may contribute to the altered apoptosis patterns observed in diabetic healing.

Expression of HSP70 in Healing Wounds of Diabetic and Nondiabetic Mice

Background. Heat shock proteins (HSPs) stabilize intracellular processes of cells under stress. Little is known about the role of HSPs in wound healing, or whether their expression is altered by systemic disease. The focus of this study was to examine the local heat shock response to wounding in diabetic mice.Methods. Congenitally diabetic and phenotypically normal mice underwent standardized full-thickness cutaneous wounding. Mice were sacrificed at sequential time points and the wound beds excised. Tissues underwent immunohistochemical (IHC) and RT-PCR analyses for inducible HSP70.Results. HSP70 protein expression in the wound bed by IHC peaked at 24 h in the nondiabetic mice. Expression of HSP70 was delayed in the diabetic mice until Day 3, which correlates with the clinical delay in healing seen in this model. The protein was especially prominent in the epithelium and in inflammatory cells migrating into the granulation tissue matrix. RT-PCR demonstrated upregulation of HSP70 mRNA within 12 h after wounding, lasting until Day 3, and decreasing thereafter in both the nondiabetic and the diabetic animals.Conclusion. Cutaneous wounding produces a HSP response in inflammatory cells, and expression of inducible HSP70 is delayed in diabetic mice. This delay may be related to the impaired inflammatory response of diabetics, and may contribute to impaired wound healing. The wound may be a continuing source of the heat shock response in inflammatory cells after injury.

Role of nerve growth factor in cutaneous wound healing: accelerating effects in normal and healing-impaired diabetic mice.

Four full-thickness skin wounds made in normal mice led to the significant increase in levels of nerve growth factor (NGF) in sera and in wounded skin tissues. Since sialoadenectomy before the wounds inhibited the rise in serum levels of NGF, the NGF may be released from the salivary gland into the blood stream after the wounds. In contrast, the fact that messenger RNA and protein of NGF were detected in newly formed epithelial cells at the edge of the wound and fibroblasts consistent with the granulation tissue produced in the wound space, suggests that NGF was also produced at the wounded skin site. Topical application of NGF into the wounds accelerated the rate of wound healing in normal mice and in healing-impaired diabetic KK/Ta mice. This clinical effect of NGF was evaluated by histological examination; the increases in the degree of reepithelialization, the thickness of the granulation tissue, and the density of extracellular matrix were observed. NGF also increased the breaking strength of healing linear wounds in normal and diabetic mice. These findings suggested that NGF immediately and constitutively released in response to cutaneous injury may contribute to wound healing through broader biological activities, and NGF improved the diabetic impaired response of wound healing.

163 Analysis of healing process of MRSA-infected cutaneous wounds in normal and diabetic mice

163 Analysis of healing process of MRSA-infected cutaneous wounds in normal and diabetic mice

Apoptosis down-regulates inflammation under the advancing epithelial wound edge: Delayed patterns in diabetes and improvement with topical growth factors

Background . Wound healing is involved in many aspects of care, ranging from anastomoses and skin incisions to foot ulcers and decubitus. Clinical healing failures are a major challenge to the physician and cause significant morbidity and mortality in select patient populations. As we are starting to understand more fully the mechanisms of impaired wound healing, the diabetic mouse (C5 7BL/KsJ-db/db) has been a good model for research. The diabetic wound exhibits significant delays in healing, previously identified as impaired cellular infiltration and granulation tissue formation. Apoptosis, or programmed cell death, is intimately involved in the regulation of inflammation and ultimately should play a role in the inflammatory phase of wound healing. Methods . To examine its role in wound healing, patterns of apoptosis in large, full-thickness cutaneous wounds were compared between groups of diabetic and nondiabetic mice. Results . Initially apoptosis was mainly limited to the wound edge and followed the advancing epithelial edge toward the center of the wound as healing progressed. Significant delays in the appearance of the apoptotic pattern were noted in the diabetic mice. Wounds in diabetic mice were then treated with topical application of growth factors. The delay in apoptotic pattern was reversed after treatment with the combination of insulin-like growth factor—II and platelet-derived growth factor, approaching levels in nondiabetic animals. Conclusions . Apoptosis appears concurrently with reepithelialization of the wound and may signal the end of the inflammatory phase of healing at that site in the wound. One can speculate that a signal for apoptosis and down-regulation of inflammation in the wound is derived from the epithelium.

Mouse models to study wound closure and topical treatment of infected wounds in healing‐impaired and normal healing hosts

Full-thickness wounds were made on the backs of wound healing-impaired diabetic mice and their normally healing litter mates. The wounds were then inoculated with 10(4) colony-forming units of Pseudomonas aeruginosa. In both cases, the inoculum increased rapidly to between 10(9) and 10(10) colony-forming units/wound area. The infection caused a significant decrease in wound closure in the normally healing mice. In the wound healing-impaired diabetic mice, infection increased the size of the wound area over 100% by day 21. The wound became filled with inflammatory cells and serous fluid, and the mice lost significant amounts of weight, an additional sign of severe, ongoing infection. Early antimicrobial treatment of infected wounds in diabetic mice (1 hour after wounding and microbial inoculation) reversed the increase in wound size area, improved wound closure, and reduced to a significant degree the weight loss observed in untreated control mice. Delay in treatment for as little as 8 hours significantly reduces the efficacy of antimicrobial treatment. These models can be used to study the effects of infection as well as to determine the efficacy of topical antimicrobial and/or wound healing-enhancing substances on these wounds in both normally healing and healing-impaired hosts.

Transfection with aFGF cDNA Improves Wound Healing

Somatic gene therapy is a potentially useful strategy for the delivery of growth factors or cytokines to enhance wound healing. Experimental excisional and incisional wounds in impaired-healing diabetic mice (db/db) were treated with aFGF and with a plasmid coding for aFGF. A eukaryotic expression plasmid composed of the Hst signal peptide sequence in-frame with the human aFGF sequence was used. Transfection of tissues was accomplished either by direct plasmid uptake or by uptake facilitated with cationic liposomes. The results show that the closure of excisional wounds was significantly accelerated (p < 0.05) by topical application of human recombinant aFGF or by transfection with the aFGF plasmid but not by vehicle or control plasmid not containing the aFGF sequence. In incisional wounds, aFGF or transfection with the plasmid significantly increased the wound-breaking strength compared to their corresponding controls (p < 0.05). Quantitative histology of the plasmid-treated incisional wound sections revealed improved wound quality. The transcription of mRNA from human aFGF cDNA in the incisional wound tissue extracts was confirmed by RT-PCR, and the expressed aFGF was detected by immune dot blot and immunohistochemistry assays. The transfection was a transient process with a peak at 9 d in db/+ (littermates of the diabetic mice) incisional wounds, at 36 d in db/db incisional wounds, and at 27 d in db/db excisional wounds. Cells transfected with human aFGF occupied up to 6.4% of the transectional area in the wound sites. Thus, aFGF gene delivery resulted in both gene expression and a functional improvement in healing.

Acidic fibroblast growth factor: evaluation of topical formulations in a diabetic mouse wound healing model.

The efficacy of topical formulations of acidic fibroblast growth factor (aFGF) in healing of full-thickness wounds has been studied in a diabetic db+/db+ mouse model. The effect of several formulation variables, dose, and application frequency was examined. It was found that wound healing in diabetic animals treated with aFGF or placebo was slower than in their nondiabetic littermates. The availability of aFGF from the viscous vehicle employed in this study (1% hydroxyethyl cellulose) was demonstrated in vitro using diffusion cells. The viscous formulation of aFGF was equally effective in wound healing as a nonviscous formulation in phosphate-buffered saline. A formulation containing heparin (necessary for full biological and conformational stability of aFGF) at a mass ratio of 3:1 to aFGF was more efficacious than formulations with lower heparin: aFGF ratios. Wounds treated with three doses of 3.0 micrograms/cm2 aFGF healed faster than those treated with a single dose of 3.0 micrograms/cm2 aFGF. Three applications of 3.0 or 0.6 microgram/cm2 a FGF were equally effective in accelerating wound healing.