Insulin resistance impairs cutaneous wound healing in mice

Obesity is associated with significant changes in skin combined with metabolic alterations such as insulin resistance. Our aim was to investigate the effects of insulin resistance induced by a high‐fat diet on cutaneous wound healing. Male C57BL/6 mice were fed standard chow (SC group) or high‐fat chow (HFC group) for 30 weeks. On day 0 (28th week), an excisional wound was performed. After 14 days (30th week), the mice were euthanized. Starting from the 8th week, the HFC group had a higher body weight. The HFC group became intolerant to glucose, resistant to insulin, and presented increased plasma cholesterol and triglyceride levels. The wound area was greater in the HFC group. The inflammatory infiltrate and the amount of “fibroblast‐like” cells increased in superficial regions of the lesions in the HFC group. The collagen fibers were more organized and denser in the SC group. Hydroxyproline levels were lower in the HFC group. The nitric oxide synthase‐2‐positive cells were higher in the HFC group. Lipid peroxidation and protein carbonyl levels were higher in the HFC group. The expression levels of alpha‐smooth muscle actin and transforming growth factor‐β were higher in the HFC group. These findings support the hypothesis that insulin resistance leads to delayed cutaneous wound healing.     

Renal dysfunction aggravated impaired cutaneous wound healing in diabetic mice

Renal dysfunction has been associated with poor outcomes of wound healing in the diabetic population. The purpose of this study was to create an excisional wound healing model in diabetic mice with renal dysfunction to investigate the combined effects of diabetes and nephropathy on cutaneous ulcers. Renal impairment was introduced in diabetic db/db mice through unilateral nephrectomy and electrocoagulation of the contralateral kidney. Renal function was subsequently monitored with assays of blood urea nitrogen and spot urinary protein/creatinine ratio. After 8 weeks, splinted, full‐thickness excisional wounds were created on the dorsal skin and harvested on postoperative days 7 and 14 for further evaluation of wound healing. Renal injury promoted the increase of blood urea nitrogen 3 weeks after initial operation, which was maintained at double the control level throughout the study, concomitantly leading to a significant increase of spot urinary protein excretion. Diabetic mice with renal injury displayed notably impaired wound healing processes, concurrent with reductions in cellular proliferation and angiogenesis, as well as increases in M1 polarized macrophages, infiltrated neutrophils, oxidative stress, and cellular apoptosis. Furthermore, quantitative polymerase chain reaction (qPCR) results displayed corresponding changes of related genes (TNF‐α, IL‐1β, SOD2) in the wounds of renal injured db/db mice. Renal manipulation in this study accelerated the progress of renal impairment, which was demonstrated to aggravate impaired cutaneous wound healing in diabetic mice.     

Ozonated sesame oil enhances cutaneous wound healing in SKH1 mice

Ozone is well recognized as a bactericidal agent and its beneficial effect on wound healing could be a consequence of this property. Because ozone itself does not penetrate the cells but immediately reacts with polyunsaturated fatty acids, its effects should be the results of oxidative reaction. For this reason, ozonated oils could be a way to deliver ozone messengers to the skin. This paper evaluated the therapeutic effects of three different grades of ozonated sesame oil in acute cutaneous wounds made in the skin of SKH1 mice. Specifically, wound closure rate, histological parameters, and the level of key proteins such as vascular endothelial growth factors and cyclin D1 have been analyzed in relation to the peroxide level present in the ozonated oil. Treatment with moderately ozonated sesame oil—expressed as peroxide value about 1,500)—has a faster wound closure rate in the first 7 days than treatment with oil containing either lower or higher peroxide value, and even with controls. Moreover, under the same treatment, an earlier and higher response of cells involved in wound repair, a higher angiogenesis, as well as an enhanced vascular endothelial growth factors and cyclin D1 expression were observed. The present study shows the validity of ozonated sesame oil in cutaneous wound healing and emphasizes the importance of the ozonation grade.     

Differential cutaneous wound healing in thermally injured MRL/MPJ mice

Adult wound repair occurs with an initial inflammatory response, reepithelialization, and the formation of a permanent scar. MRL/MpJ mice following ear‐hole punch biopsies display accelerated healing and tissue regeneration. In this study, we characterized the healing responses in both MRL/MpJ and BALB/c mice following a 15% total body surface area full‐thickness cutaneous burn injury. Macroscopic and histological observations show that delayed wound closure in MRL/MpJ mice is accompanied by an increase in edema, reduced neutrophil infiltration, and more prominent eschar. In vivo bromodeoxyuridine labeling showed no defect in keratinocyte proliferation and migration (reepithelialization). In comparison with BALB/c mice, MRL/MpJ wounds had greater collagen deposition, less granulation tissue formation, and contained fewer α‐smooth muscle actin‐positive myofibroblasts. An observed reduction in dermal neutrophil infiltration and myofibroblast development correlated with enhanced angiogenesis. Overall, BALB/c wounds contracted sooner and to a larger degree, resulting in a significant decrease in scar formation. Interestingly, MRL/MpJ mice showed overt abnormalities in hair follicle proliferation, morphogenesis, and subsequent hair regrowth postburn injury. No substantial evidence of tissue regeneration was observed in either BALB/c or MRL/MpJ wounds. Our results convincingly demonstrate that MRL/MpJ skin burn wounds heal with scar formation with delays in two critical wound healing events: wound closure, and myofibroblast development.     

Impaired wound healing secondary to bevacizumab

Bevacizumab is a monoclonal antibody that exerts its antitumor activity by inhibiting vascular endothelial growth factor. Consequently, it suppresses endothelial cell proliferation, vascular permeability, and angiogenesis. This inhibitory effect contributes to tumour size reduction but causes wound‐healing delay, specifically during the proliferative phase, in patients receiving bevacizumab. Although surgical wound‐healing complications (WHC) associated with bevacizumab have been extensively reported, there is limited literature on peripheral WHC. More importantly, the histopathology of bevacizumab‐associated WHC has not been described. We present the histopathology findings of a non‐healing ulcer in a patient receiving bevacizumab, providing insight into the possible aetiology of this drug's adverse reaction. Furthermore, our patient's positive response to hyperbaric oxygen suggests its possible use for treatment of bevacizumab‐associated non‐healing wounds.     

Absence of inhibition of cutaneous wound healing in mice by oral doxycycline

Temporally controlled expression of genes in transgenic mice has advanced our understanding of many physiological processes. One of the more common modes of acutely altering gene levels involves the doxycycline-regulated "tet-on/tet-off" systems. There has been concern that the administration of doxycycline in itself might compromise many aspects of wound repair. Here we report that oral ad libitum administration of doxycycline (2 mg/ml in drinking water) to mice does not alter dermal or epidermal wound healing. The healing of both full- and partial-thickness skin wounds proceeded similarly regardless of doxycycline administration; in fact, collagen organization and tensile strength development appeared to accelerate in the presence of doxycycline. These data suggest that wound healing studies incorporating transgene expression can utilize tet-on/tet-off regulation of gene expression without interference from doxycycline.     

Impaired cutaneous wound healing in transforming growth factor‐β inducible early gene1 knockout mice

Transforming growth factor‐β inducible early gene (TIEG) is induced by transforming growth factor‐β (TGF‐β) and acts as the primary response gene in the TGF‐β/Smad pathway. TGF‐β is a multifunctional growth factor that affects dermal wound healing; however, the mechanism of how TGF‐β affects wound healing is still not well understood because of the complexity of its function and signaling pathways. We hypothesize that TIEG may play a role in dermal wound healing, with involvement in wound closure, contraction, and reepithelialization. In this study, we have shown that TIEG1 knockout (TIEG1^–/–) mice have a delay in wound closure related to an impairment in wound contraction, granulation tissue formation, collagen synthesis, and reepithelialization. We also found that Smad7 was increased in the wounds and appeared to play a role in this wound healing model in TIEG1^–/– mice.     

基因治疗与皮肤创面愈合

皮肤创面愈合过程是一个多因素参与的复杂过程,慢性、难愈创面严重影响了人们的生活质量.重组生长因子已经在临床用于改善创面愈合,但由于半衰期短、生物利用度低等原因,使得其临床疗效欠佳.近年来,随着基因工程技术的不断发展,基因治疗已经成为改善创面愈合的一种新的治疗方法.本文就目前基因治疗方法的优缺点、治疗性基因选择和正在进行的临床试验等方面进行了综述.     

Topical 1% propranolol cream promotes cutaneous wound healing in spontaneously diabetic mice

Diabetic foot ulcers (DFUs) are a constant threat to diabetic patients and can lead to amputations and even death. Intralesional administration of propranolol in diabetic wounds has not been reported previously. This study aimed to investigate the efficacy of propranolol cream in diabetic wounds. Fifty‐six spontaneously diabetic mice were divided into the propranolol group and the control group. After preparing full‐thickness wounds on the back of the mice, 1% propranolol cream was topically applied to wounds in the experimental group and 0% propranolol cream in controls. The wound sizes were measured and calculated against the original area. The wounds were analyzed up to 21 days after injury. At all evaluation time‐points, the wound size (%) in the propranolol group was significantly smaller than in the controls. Epidermal growth factor (EGF) protein expression increased in the experimental vs. control group. Vascular endothelial growth factor (VEGF) expression was significantly lower in the experimental vs. control group whereas NG2 proteoglycan was increased throughout the study. However, matrix metallopeptidase (MMP)‐9 expression was at first significantly higher in the experimental vs. control group then the MMP‐9 protein level in the control group increased and surpassed that in the experimental group. In conclusion, intralesional administration of 1% propranolol cream promotes reepithelialization and regulates abnormal angiogenesis in diabetic wounds. Propranolol cream may become a new drug for the treatment of DFUs.     

Involvement of DPP IV/CD26 in cutaneous wound healing process in mice

Dipeptidyl peptidase IV (DPP IV/CD26) is a widely distributed multifunctional protein that plays a significant role in different physiological as well as pathological processes having a broad spectrum of bioactive substrates and immunomodulative properties. It has potential influence on different processes crucial for wound healing, including cell adhesion, migration, apoptosis, and extracellular matrix degradation. However, despite its known enzymatic and immunomodulative functions, limited data characterize the role of DPP IV/CD26 in cutaneous wound healing mechanisms. The aim of this study was to investigate the process of wound healing in conditions of CD26 deficiency in order to obtain better insights on the role of DPP IV/CD26 in cutaneous regeneration. Experimental wounds were made on the dorsal part of CD26 deficient (CD26^?/?) and wild‐type mice (C57BL/6). The process of cutaneous wound healing was monitored on defined time schedule postwounding by macroscopic, microscopic, and biochemical analyses. Obtained results revealed a better rate of wound closure, revascularization and cell proliferation in CD26^?/? mice, with enhanced local expression of hypoxia‐inducible factor 1α and vascular endothelial growth factor. CD26 deficiency induced prompt macrophage recruitment at the site of skin damage but did not influence mobilization of T‐cells in comparison with wild‐type mice. CD26^?/? mice have significantly higher values of IP‐10 in serum and control skins compared with wild‐type mice but values in wounds did not differ significantly on days 2, 4, and 7 of wound healing. DPP IV/CD26 activity was found to be decreased 4 days postwounding in serum and 2, 4, and 7 days postwounding in wounds of wild‐type animals compared with control skins. These findings contribute to better understanding of wound healing mechanisms and could give a support in finding new therapeutic approaches for wound healing and tissue regeneration.     

Acceleration of cutaneous wound healing by brassinosteroids

Brassinosteroids are plant growth hormones involved in cell growth, division, and differentiation. Their effects in animals are largely unknown, although recent studies showed that the anabolic properties of brassinosteroids are possibly mediated through the phosphoinositide 3‐kinase/protein kinase B signaling pathway. Here, we examined biological activity of homobrassinolide (HB) and its synthetic analogues in in vitro proliferation and migration assays in murine fibroblast and primary keratinocyte cell culture. HB stimulated fibroblast proliferation and migration and weakly induced keratinocyte proliferation in vitro. The effects of topical HB administration on progression of wound closure were further tested in the mouse model of cutaneous wound healing. C57BL/6J mice were given a full‐thickness dermal wound, and the rate of wound closure was assessed daily for 10 days, with adenosine receptor agonist CGS‐21680 as a positive control. Topical application of brassinosteroid significantly reduced wound size and accelerated wound healing in treated animals. mRNA levels of transforming growth factor beta and intercellular adhesion molecule 1 were significantly lower, while tumor necrosis factor alpha was nearly suppressed in the wounds from treated mice. Our data suggest that topical application of brassinosteroids accelerates wound healing by positively modulating inflammatory and reepithelialization phases of the wound repair process, in part by enhancing Akt signaling in the skin at the edges of the wound and enhancing migration of fibroblasts in the wounded area. Targeting this signaling pathway with brassinosteroids may represent a promising approach to the therapy of delayed wound healing.     

Tyrosine phosphorylation of EGF-R and PDGF-R proteins during acute cutaneous wound healing process in mice

The effect of topical application of epidermal growth factor (EGF) and platelet-derived growth factors (PDGFs) on the levels of EGF-R and PDGF-R proteins and their tyrosine phosphorylation were analyzed during an acute cutaneous wound healing process in mice. The growth factor-treated wounds had optimum levels of receptor proteins as early as day 1 compared with the control, which had only a basal level. Analysis of the tyrosine phosphorylation of the receptor proteins in control and growth factor-treated wounds indicated that they were phosphorylated until day 5 after wounding. Only the mature forms of α-PDGF-R and β-PDGF-R proteins were phosphorylated and not their precursors. Our results show that rapid attainment of maximum levels of growth factor receptor proteins and their tyrosine phosphorylation as early as day 1 and the maintenance of the same until day 3 appear to aid faster and better wound healing. Topical application of PDGF-AA alone did not facilitate the wound healing process and it also antagonized the EGF-medicated wound healing when applied premixed with EGF or within 30 minutes after EGF application. Under these conditions, the receptor proteins were not phosphorylated. Thus, an increased and sustained level of EGF-R and PDGF-R proteins and their tyrosine phosphorylation appear to accelerate the wound healing process.     

Acupuncture accelerates wound healing in burn-injured mice

The effects of acupuncture on healing of deep second degree burns were compared to the conventional hydrocolloid dressing, Duoderm in mice. The expression level of inflammatory protein-1α (MIP-1α) was significantly reduced in the injured skin and the number of eosinophils in blood decreased significantly following the acupuncture treatment compared to the Duoderm dressing at 7 days after the burn. In addition, the acupuncture treatment was more effective in decreasing the wound size and inducing epidermal regeneration. Histological findings also revealed that there was less leukocyte infiltration and a greater reduction in the immunohistochemical reaction to MIP-2 in the wounds treated with acupuncture versus Duoderm. Furthermore, the numbers of BrdU- and basic fibroblast growth factor (bFGF)-positive cells were significantly increased by the acupuncture treatment, compared to the Duoderm treatment at 7 days. Moreover, in the acupuncture treated mice, the expression of fibronectin was increased and α-SMA was decreased at 7 days. Thus, this present study demonstrates that acupuncture accelerates the skin regeneration process following deep second degree burns.     

A role for decorin in cutaneous wound healing and angiogenesis

Decorin is known to influence tissue tensile strength and cellular phenotype. Therefore, decorin is likely to have an impact on tissue repair, including cutaneous wound healing. In this study, cutaneous healing of both excisional and incisional full‐thickness dermal wounds was studied in decorin‐deficient (Dcn^?/?) animals. A statistically significant delay in excisional wound healing in the Dcn^?/? mice occurred at 4 and 10 days postwounding and, in incisional wounds at 4, 10, and 18 days when compared with wild‐type (Dcn^?/?) controls. Fibrovascular invasion into polyvinylalcohol sponges was significantly increased by day 18 in Dcn^?/? mice relative to Dcn^+/+ mice. The 18‐day sponge implants in the Dcn^?/? mice showed a marked accumulation of biglycan when compared with the corresponding implants in Dcn^+/+ mice. Thus, regulated production of decorin may serve as an excellent therapeutic approach for modifying impaired wound healing and harmful foreign body reactions.     

Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats

Background

Diabetes is one of the most prevalent human metabolic diseases. Wound healing in diabetes is frequently impaired and treatment remains challenging. Sphingolipid metabolites play important roles in the regulation of glucose metabolism. SPK1 is the key enzyme in the sphingolipid metabolic pathway. S1P/SPK plays a pivotal role in the signalling pathways of diverse cellular processes including proliferation, differentiation, migration, apoptosis in diverse cell types.

Methods

To investigate the role of sphingosine kinase 1 (SPK1) in skin injury, plasmids containing the SPK1 gene (pcDNA3-FLAG-SPK1) were applied to cutaneous wounds on a streptozotocin-induced diabetic rat model over a 21-day period. The wound area and rate of wound healing were determined. The histopathological features of the healed wounds were also observed, and SPK1 expression in the skin was detected by immunohistochemistry.

Results

There was a significant decrease in wound area in diabetic rats treated with 125 and 60 μg/wound pcDNA3-FLAG-SPK1 (P < 0.001–0.01). The mean sizes of the wounds were 0.67 ± 0.15 cm², 0.83 ± 0.18 cm², and 1.09 ± 0.23 cm² in both treated and diabetic control group at the 7th day post-treatment respectively. In addition, wound healing in diabetic rats of test group was accelerated. At the 7th day, the mean rates of healing were 73.2 ± 5.7% and 66 ± 7.3% in test group of 125 and 60 μg/wound respectively, and 55.4 ± 9.9% in diabetic control group (P < 0.001–0.01). Histology revealed that tissue sections from the treated diabetic rats contained more granulation tissue and capillaries than that of the control rats. There was high SPK1 expression in the skin of the treated diabetic rats.

Conclusions

SPK1 gene therapy may represent a novel approach to cutaneous wound healing.