Vascular Endothelial Growth Factor-C Accelerates Diabetic Wound Healing

MMP-13 Plays a Role in Keratinocyte Migration, Angiogenesis, and Contraction in Mouse Skin Wound Healing

Impaired wound healing is a serious late-diabetic complication resulting in significant morbidity. MicroRNAs (miRNAs), post-transcriptional gene-regulators, are differentially expressed in several conditions, such as diabetes. To assess the mechanistic action of miRNAs in diabetic wound healing streptozotocin (STZ)-induced diabetic and control mice (n=6/group, 6 weeks of diabetes) were subjected to 6-mm full-thickness excisional wounds. Biopsies were obtained at days 3 and 10 post-wounding. Wound healing was delayed in diabetic mice (p<0.05). The skin miRNA profile was determined for up to 561 unique miRNAs using Taqman MicroRNA array cards. We identified 288 different robustly expressed miRNA in skin. While half of them were decreased more than 1.5 fold, 41 were increased more than 1.5 fold, during wound healing. MiR-155-5p was increased in diabetic skin (15.8 fold (95% CI: 3.5-28.1), however, it was 9.7 fold (95% CI: 4.2-15.2) decreased at day 10 post wounding, as compared to nondiabetic mouse skin. Scratch assays in HaCaT cells showed increased scratch closure after 24 hours, when miR-155 was inhibited (Ctrl: 31%, miR-155 inhibitor: 8.1% of scratch remaining, P<0.001). In vivo, topical treatment of diabetic mouse wounds with miR-155 inhibitor dose-dependently accelerated wound closure with maximal effect at 2.5pmol (P<0.03). Interestingly, reporter-gene analysis identified fibroblast growth factor (FGF) 7 mRNA as a target of miR-155-5p. Moreover, miR-155 inhibitor treated mouse wounds showed significant increase in FGF7 protein expression by immunofluorescence. We present evidence that miR-155-5p expression is altered in diabetic mouse skin and its suppression significantly improves wound healing under diabetic conditions, indicating its potential as a novel chronic diabetic foot ulcer treatment. Disclosure L.T. Dalgaard: Employee; Spouse/Partner; Novo Nordisk A/S. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. E.C. Leal: None. R. Svendsen: None. J. Moura: None. A.E. Sørensen: Research Support; Self; Danish Diabetes Academy, Merck Serono. P.T. Jørgensen: None.

Intragastric administration of Pien Tze Huang enhanced wound healing in diabetes by inhibiting inflammation and improving energy generation

Intelligent drug delivery-wound healing integrated hydrogel dressing for Type 2 Diabetes Mellitus wounds with wound microenvironment modulation.

The treatment of diabetic wounds is a considerable clinical challenge. In this study, mouse dermal fibroblasts retrovirally transduced with the human platelet-derived growth factor B (PDGF-B) gene were used to treat diabetic mouse wounds. The PDGF-B gene was obtained from human umbilical vein endothelial cells, cloned into retroviral vectors, and introduced into diabetic mouse C57B1/ks-db/db dermal fibroblasts. In vitro results demonstrated production of PDGF-B protein by these transduced cells at steady-state levels of 1000 ng PDGF-B/10(6) cells/24 hours, and expression of PDGF-B mRNA. These cells were seeded onto polyglycolic acid scaffold matrices and used to treat diabetic mouse 20-mm x 20-mm full-thickness excisional dorsal skin wounds. Measurement of the residual epithelial gap at 21 days showed significantly accelerated healing (P < 0.05) of wounds treated with PDGF-transduced cells (epithelial gap 10.46 +/- 1.20 mm) compared with untreated wounds (14.66 +/- 0.591 mm), wounds treated with polyglycolic acid alone (14.80 +/- 0.575 mm), or wounds treated with negative control LNCX-transduced cells (13.76 +/- 0.831 mm). Immunohistochemical staining showed intense staining for PDGF in wounds treated with PDGF-B-transduced cells. This study demonstrates the promising potential for gene therapy in diabetic wound healing.

Category: Basic Sciences/Biologics, Diabetes Introduction/Purpose: Approximately 50% of the 170 million people worldwide with diabetes experience complications associated with delayed cutaneous wound healing. The current widely used treatment for DFU is topical platelet-derived growth factor (PDGF); however, it has significant side-effects. Preclinical studies have shown that a small molecular weight opioid receptor antagonist, blocks the interactions between the growth inhibitory peptide Opioid Growth Factor (OGF) and its receptor OGFr and accelerates cell proliferation and enhances angiogenesis and wound closure. The hypothesis of this research is that the topical formulation of opioid receptor antagonist is an effective therapy for healing of cutaneous wounds in diabetic rats, with healing times comparable to the standard care and will promote enhanced cell replication and accelerated angiogenesis. Methods: Fourteen diabetic rats per group with four excisional cutaneous wounds were positioned dorsally on type 1 diabetic rats and randomly treated topically with an opioid receptor antagonist (0.03% Naltrexone), topical platelet derived growth factor (Regranex®), or placebo moisturizing cream alone. Wound closure, DNA synthesis (bromodeoxyuridine incorporation-BrdU labeling), and production of the cytokines PDGF and vascular endothelial growth factor (VEGF) were monitored. Results: Naltrexone and Regranex® treatment resulted in more than 65% closure on days 8 and 10 respectively. On day 12, only one Naltrexone- and two Regranex-treated wounds remained (~1%), whereas the control group had 50% visible wounds. DNA synthesis in Regranex® treated wounds was 21% in comparison to vehicle-treated wounds at 28%; Naltrexone-treated diabetic wounds had 42% BrdU labeling comparable to the normal vehicle treated wounds. Naltrexone treatment significantly increased cell replication in comparison to Regranex treatment and to vehicle-only treated wounds (p&lt;0.001). Tissue immunostained at 1, 2, and 4 days after surgery with anti-PDGF antibodies indicated that both Regranex® and Naltrexone increase PDGF+ labeling at 1 day, with Naltrexone treatment (but not Regranex®) continuing to enhance PDGF production at 4 days Conclusion: Topical opioid receptor antagonist has been shown to enhance wound healing by increasing cell replication, accelerating angiogenesis, and matrix formation and targets an underlying pathophysiology of diabetes and enhances cell replication at all stages of wound remodeling. Topical opioid receptor antagonist is safe and effectively targets the disease-modifying pathways in this comparison study of topical platelet derived growth factor for wound closure in Type 1 diabetic rats.These data suggest that blockade of the OGF-OGFr axis, utilizing 0.03% Naltrexone is a safe and effective alternative for treatment for diabetic wounds by targeting several phases of wound repair.

Clinic-oriented injectable smart material for the treatment of diabetic wounds: Coordinating the release of GM-CSF and VEGF

Acceleration of wound healing activity with syringic acid in streptozotocin induced diabetic rats

Diabetes, one of the most common diseases in the world, causes a decrease in the formation of macrophages, endothelial cells, keratinocytes, platelets, and, fibroblasts, which play a role in the cellular response to tissue damage in wound healing, and a decrease in the cytokines they secrete, inflammatory mediators, or growth factors, making wound healing difficult. As the healing period of the wounds increases, the psychology of the patients is adversely affected by the inability to return to their daily routines, the loss the of workforce increases, and the country's economy is adversely affected. In the last decades, because of the increase in many diseases such as diabetes and diabetes-related wounds, the use of calorie-free foods and herbal treatment methods has been preferred. Plants are increasingly being used as a source of pharmaceutical medications since they are simple to obtain, have no negative side effects, are affordable, and are simple to utilize. In order to meet these needs, sweeteners containing stevia extracts or components, obtained from the Stevia rebaudiana plant, that are calorie-free and 300 fold sweeter than cane sugar, have been on the market for some time. Despite the fact that Stevia rebaudiana, often known as sugar grass, is not a commonly utilized plant around the globe, it is possible to find literature that claims it is used as a preventive and curative for a variety of diseases. Although Stevia rebaudiana has various biological effects such as antioxidant, antidiabetic, antitumor, and antimicrobial, knowledge of diabetes and its effects on diabetic wound healing are limited. The purpose of this review is to evaluate how effectively Stevia rebaudiana treats diabetes and diabetic wounds.

A highly stretchable, adhesive, and antibacterial hydrogel with chitosan and tobramycin as dynamic cross-linkers for treating the infected diabetic wound

Skin lesions and ulcerations are severe complications of diabetes that often result in leg amputations. In this study we investigated the function of the cytoskeletal protein flightless I (FLII) in diabetic wound healing. We hypothesised that overexpression of FLII would have a negative effect on diabetic wound closure and modulation of this protein using specific FLII-neutralising antibodies (FnAb) would enhance cellular proliferation, migration and angiogenesis within the diabetic wound. Using a streptozotocin-induced model of diabetes we investigated the effect of altered FLII levels through Flii genetic knockdown, overexpression or treatment with FnAb on wound healing. Diabetic wounds were assessed using histology, immunohistochemistry and biochemical analysis. In vitro and in vivo assays of angiogenesis were used to assess the angiogenic response. FLII levels were elevated in the wounds of both diabetic mice and humans. Reduction in the level of FLII improved healing of murine diabetic wounds and promoted a robust pro-angiogenic response with significantly elevated von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF)-positive endothelial cell infiltration. Diabetic mouse wounds treated intradermally with FnAb showed improved healing and a significantly increased rate of re-epithelialisation. FnAb improved the angiogenic response through enhanced formation of capillary tubes and functional neovasculature. Reducing the level of FLII led to increased numbers of mature blood vessels, increased recruitment of smooth muscle actin-α-positive cells and improved tight junction formation. Reducing the level of FLII in a wound may be a potential therapeutic approach for the treatment of diabetic foot ulcers.

Context Diabetic wounds (DW) are a complication of diabetes and slow wound healing is the main manifestation. Methylene blue (MB) has been shown to exhibit therapeutic effects on diabetes-related diseases. Objective To investigate the mechanisms of action of MB-nanoemulsion (NE) in the treatment of DW. Materials and methods The concentration of MB-NE used in the in vivo and in vitro experiments was 0.1 mg/mL. Streptozocin-induced diabetic mice were used as models. The mice were separated into nondiabetic, diabetic, MB-NE treated, and NE-treated groups. Intervention of high glucose-induced human umbilical vein endothelial cells using MB-NE. The mechanism by which MB-NE promotes DW healing is investigated by combining histological analysis, immunofluorescence analysis, TUNEL and ROS assays and western blotting. Results In diabetic mice, the MB-NE accelerated DW healing (p < 0.05), promoted the expression of endothelial cell markers (α-SMA, CD31 and VEGF) (p < 0.05), and reduced TUNEL levels. In vitro, MB accelerated the migration rate of cells (p < 0.05); promoted the expression of CD31, VEGF, anti-apoptotic protein Bcl2 (p < 0.05) and decreased the expression of the pro-apoptotic proteins cleaved caspase-3 and Bax (p < 0.05). MB upregulated the expression of Nrf2, catalase, HO-1 and SOD2 (p < 0.05). In addition, MB reduced the immunofluorescence intensity of TUNEL and ROS in cells and reduced apoptosis. The therapeutic effect of MB was attenuated after treatment with an Nrf2 inhibitor (ML385). Discussion and conclusion This study provides a foundation for the application of MB-NE in the treatment of DW.

Diabetic patients exhibit dysfunction of the normal wound healing process, leading to local ischemia by vascular occlusive disease as well as sustained increases in the proinflammatory cytokines and overproduction of reactive oxygen species (ROS). Of the many sources of ROS, the enzyme xanthine oxidase (XO) has been linked to overproduction of ROS in diabetic environment, and studies have shown that treatment with XO inhibitors decreases XO overactivity and XO-generated ROS. This study evaluates the role of XO in the diabetic wound and the impact of specifically inhibiting its activity on wound healing. Treatment of diabetic wounds with siXDH (xanthine dehydrogenase siRNA) decreased XDH mRNA expression by 51.6%, XO activity by 35.9%, ROS levels by 78.1%, pathologic wound burden by 31.5%, and accelerated wound healing by 7 days (23.3%). Polymerase chain reaction analysis showed that increased XO activity in wild-type wound may be due to XDH to XO conversion and/or XO phosphorylation, but not to gene transcription, whereas increased XO activity in diabetic wounds may also be from gene transcription. These results suggest that XO may be responsible for large proportion of elevated oxidative stress in the diabetic wound environment and that normalizing the metabolic activity of XO using targeted delivery of siXDH may decrease overproduction of ROS and accelerate wound healing in diabetic patients.

The treatment of diabetic wounds is a formidable clinical challenge. In this study, lentiviral vectors carrying the human platelet-derived growth factor B (PDGF-B) gene were used to treated diabetic mouse wounds. Full-thickness 2.0-cm x 2.0-cm excisional wounds were created on the dorsa of genetically diabetic C57BL/KsJ-m+/+Lepr(db) mice. Lentiviral vectors containing the PDGF-B gene were injected into the wound margins and base. Mice were killed at 14-, 21-, and 35-day intervals. Measurement of the residual epithelial gap showed a trend towards increased healing in lentiviral PDGF-treated wounds compared with untreated and saline-treated wounds at all time points. At 21 days, there was significantly increased healing in lentiviral PDGF-treated wounds (0.98+/-0.17 cm) compared with saline-treated wounds (1.22+/-0.30 cm; P<0.05). Immunohistochemistry for CD31 revealed significantly increased neovascularization in lentiviral PDGF-treated wounds compared with untreated and saline-treated wounds at 14 and 21 days (P<0.01). Picrosirius red staining demonstrated thicker and more highly organized collagen fibers in treated wounds compared with untreated and saline-treated wounds. Quantitative analysis of collagen content showed a 3.5-fold and 2.3-fold increase in lentiviral PDGF-treated wounds versus untreated and saline-treated wounds, respectively (P<0.01). Lentiviral gene therapy with PDGF-B can sustain diabetic wound healing over time and may possess promising potential in the clinical setting.

Efficient management of difficult-to-heal diabetic wounds remains a clinical challenge owing to bacterial infections, as well as oxidative and hyperglycemic complex pathology. Therefore, developing intelligent strategies for diabetic wound healing is urgently needed. Herein, an ultrasound (US)-responsive microneedle (MN) patch (MN@GOX@TiO2-X@CO) capable of controlled delivery of carbon monoxide (CO) gas within the skin for effective treatment of diabetic infected wounds is developed. Benefiting from the specific form of microneedle (MN) patch, sonosensitizer (TiO2-X), •OH-responsive CO prodrug (MPA-CO), and glucose oxidase (GOX) can be loaded together and effectively delivered to infectious wounds. With the semi-fluidic hyaluronic acid (HA) coating under the physiological condition, CO could be released efficiently in situ and directly acted on infected wound tissue upon US triggering. Both in vitro and in vivo results showed that US-triggered CO release from MN@GOX@TiO2-X@CO not only effectively inhibited the S. aureus and MRSA infection but also promoted fibroblasts proliferation and migration under hyperglycemic physiology, thereby accelerating diabetic wound healing. Collectively, the approach effectively addresses the impaired skin regeneration function in diabetic wounds and offers a promising therapeutic strategy for the efficient healing of infected diabetic wounds.