Real-time OXPHOS capacity analysis in wounded skin from diabetic mice: A pilot study.

INTRODUCTION: Diabetes mellitus (DM) impairs wound healing. The aim was to determine whether DM influences mitochondrial respiration in wounded skin (WS) and non-wounded skin (NWS), in a pre-clinical wound healing model of streptozotocin (STZ)-induced diabetes. METHODS: Six weeks after diabetes induction, two wounds were created in the back of C57BL/J6 mice. Using high-resolution respirometry (HRR), oxygen flux was measured, in WS and NWS, using two substrate-uncoupler-inhibitor titration protocols, at baseline (day 0), day 3 and 10 post-wounding, in STZ-DM and non-diabetic (NDM) mice. Flux control ratios for the oxidative phosphorylation (OXPHOS) capacity were calculated. RESULTS: A significant increase in mitochondrial respiration was observed in STZ-DM skin compared to control skin at baseline. The OXPHOS capacity was decreased in WS under diabetes at day 3 post-wounding (inflammation phase). However, at day 10 post-wounding (remodeling phase), the OXPHOS capacity was higher in WS from STZ-DM compared to NDM mice, and compared to NWS from STZ-DM mice. A significant relative contribution of pyruvate, malate and glutamate (PMG) oxidation to the OXPHOS capacity was observed in WS compared to NWS from STZ-DM mice, at day 10, while the relative contribution of fatty acid oxidation to the OXPHOS capacity was higher in NWS. The OXPHOS capacity is altered in WS from STZ-DM compared to NDM mice across the healing process, and so is the substrate contribution in WS and NWS from STZ-DM mice, at each time point. CONCLUSION: HRR may be a sensitive tool to evaluate the underlying mechanisms of tissue repair during wound healing.

Innovative Functional Biomaterials as Therapeutic Wound Dressings for Chronic Diabetic Foot Ulcers.

The imbalance of local and systemic factors in individuals with diabetes mellitus (DM) delays, or even interrupts, the highly complex and dynamic process of wound healing, leading to diabetic foot ulceration (DFU) in 15 to 25% of cases. DFU is the leading cause of non-traumatic amputations worldwide, posing a huge threat to the well-being of individuals with DM and the healthcare system. Moreover, despite all the latest efforts, the efficient management of DFUs still remains a clinical challenge, with limited success rates in treating severe infections. Biomaterial-based wound dressings have emerged as a therapeutic strategy with rising potential to handle the tricky macro and micro wound environments of individuals with DM. Indeed, biomaterials have long been related to unique versatility, biocompatibility, biodegradability, hydrophilicity, and wound healing properties, features that make them ideal candidates for therapeutic applications. Furthermore, biomaterials may be used as a local depot of biomolecules with anti-inflammatory, pro-angiogenic, and antimicrobial properties, further promoting adequate wound healing. Accordingly, this review aims to unravel the multiple functional properties of biomaterials as promising wound dressings for chronic wound healing, and to examine how these are currently being evaluated in research and clinical settings as cutting-edge wound dressings for DFU management.

Green Antimicrobials as Therapeutic Agents for Diabetic Foot Ulcers.

Diabetic foot ulcers (DFU) are one of the most serious and devastating complications of diabetes and account for a significant decrease in quality of life and costly healthcare expenses worldwide. This condition affects around 15% of diabetic patients and is one of the leading causes of lower limb amputations. DFUs generally present poor clinical outcomes, mainly due to the impaired healing process and the elevated risk of microbial infections which leads to tissue damage. Nowadays, antimicrobial resistance poses a rising threat to global health, thus hampering DFU treatment and care. Faced with this reality, it is pivotal to find greener and less environmentally impactful alternatives for fighting these resistant microbes. Antimicrobial peptides are small molecules that play a crucial role in the innate immune system of the host and can be found in nature. Some of these molecules have shown broad-spectrum antimicrobial properties and wound-healing activity, making them good potential therapeutic compounds to treat DFUs. This review aims to describe antimicrobial peptides derived from green, eco-friendly processes that can be used as potential therapeutic compounds to treat DFUs, thereby granting a better quality of life to patients and their families while protecting our fundamental bio-resources.

Dysregulation of endoplasmic reticulum stress response in skin wounds in a streptozotocin-induced diabetes mouse model.

Dysfunction in key cellular organelles has been linked to diabetic complications. This study intended to investigate the alterations in the unfolded protein response (UPR), autophagy, and mitochondrial function, which are part of the endoplasmic reticulum (ER) stress response, in wound healing (WH) under diabetes conditions. WH mouse models were used to evaluate the UPR, autophagy, mitochondrial fusion, fission, and biogenesis as well as mitophagy in the skin of control and diabetic mice at baseline and 10 days after wounding. The autophagic flux in response to high-glucose conditions was also evaluated in keratinocyte and fibroblast cell cultures. WH was impaired in the diabetic mouse model, and we found that the UPR and autophagy pathways were activated in skin wounds of control mice and in the non-wounded skin of diabetic mice. Moreover, high-glucose conditions induced autophagy in the keratinocyte and fibroblast cell cultures. However, mitophagy did not change in the skin of diabetic mice or the wounded skin. In addition, mitochondrial fusion was activated in control but not in the skin wounds of diabetic mice, while mitochondrial biogenesis is downregulated in the skin of diabetic mice. In conclusion, the activation of the UPR, autophagy, and mitochondrial remodeling are crucial for a proper WH. These results suggest that the increase in ER stress and autophagy in the skin of diabetic mice at baseline significantly escalated to pathological levels after wounding, contributing to impaired WH in diabetes.

Heme Oxygenase-1 as Therapeutic Target for Diabetic Foot Ulcers.

A diabetic foot ulcer (DFU) is one of the major complications of diabetes. Wound healing under diabetic conditions is often impaired. This is in part due to the excessive oxidative stress, prolonged inflammation, immune cell dysfunction, delayed re-epithelialization, and decreased angiogenesis present at the wound site. As a result of these multifactorial impaired healing pathways, it has been difficult to develop effective therapeutic strategies for DFU. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in heme degradation generating carbon monoxide (CO), biliverdin (BV) which is converted into bilirubin (BR), and iron. HO-1 is a potent antioxidant. It can act as an anti-inflammatory, proliferative, angiogenic and cytoprotective enzyme. Due to its biological functions, HO-1 plays a very important role in wound healing, in part mediated through the biologically active end products generated by its enzymatic activity, particularly CO, BV, and BR. Therapeutic strategies involving the activation of HO-1, or the topical application of its biologically active end products are important in diabetic wound healing. Therefore, HO-1 is an attractive therapeutic target for DFU treatment. This review will provide an overview and discussion of the importance of HO-1 as a therapeutic target for diabetic wound healing.

Putative Biomarkers in Tears for Diabetic Retinopathy Diagnosis.

Purpose: Tear fluid biomarkers may offer a non-invasive strategy for detecting diabetic patients with increased risk of developing diabetic retinopathy (DR) or increased disease progression, thus helping both improving diagnostic accuracy and understanding the pathophysiology of the disease. Here, we assessed the tear fluid of nondiabetic individuals, diabetic patients with no DR, and diabetic patients with nonproliferative DR (NPDR) or with proliferative DR (PDR) to find putative biomarkers for the diagnosis and staging of DR. Methods: Tear fluid samples were collected using Schirmer test strips from a cohort with 12 controls and 54 Type 2 Diabetes (T2D) patients, and then analyzed using mass spectrometry (MS)-based shotgun proteomics and bead-based multiplex assay. Tear fluid-derived small extracellular vesicles (EVs) were analyzed by transmission electron microscopy, Western Blotting, and nano tracking. Results: Proteomics analysis revealed that among the 682 reliably quantified proteins in tear fluid, 42 and 26 were differentially expressed in NPDR and PDR, respectively, comparing to the control group. Data are available via ProteomeXchange with identifier PXD033101. By multicomparison analyses, we also found significant changes in 32 proteins. Gene ontology (GO) annotations showed that most of these proteins are associated with oxidative stress and small EVs. Indeed, we also found that tear fluid is particularly enriched in small EVs. T2D patients with NPDR have higher IL-2/-5/-18, TNF, MMP-2/-3/-9 concentrations than the controls. In the PDR group, IL-5/-18 and MMP-3/-9 concentrations were significantly higher, whereas IL-13 was lower, compared to the controls. Conclusions: Overall, the results show alterations in tear fluid proteins profile in diabetic patients with retinopathy. Promising candidate biomarkers identified need to be validated in a large sample cohort.

Extracellular vesicles enriched with an endothelial cell pro-survival microRNA affects skin tissue regeneration.

Endothelial cell (EC) activity is essential for tissue regeneration in several (patho)physiological contexts. However, our capacity to deliver in vivo biomolecules capable of controlling EC fate is relatively limited. Here, we screened a library of microRNA (miR) mimics and identified 25 miRs capable of enhancing the survival of ECs exposed to ischemia-mimicking conditions. In vitro, we showed that miR-425-5p, one of the hits, was able to enhance EC survival and migration. In vivo, using a mouse Matrigel plug assay, we showed that ECs transfected with miR-425-5p displayed enhanced survival compared with scramble-transfected ECs. Mechanistically, we showed that miR-425-5p modulated the PTEN/PI3K/AKT pathway and inhibition of miR-425-5p target genes (DACH1, PTEN, RGS5, and VASH1) phenocopied the pro-survival. For the in vivo delivery of miR-425-5p, we modulated small extracellular vesicles (sEVs) with miR-425-5p and showed, in vitro, that miR-425-5p-modulated sEVs were (1) capable of enhancing the survival of ECs exposed to ischemia-mimic conditions, and (2) efficiently internalized by skin cells. Finally, using a streptozotocin-induced diabetic wound healing mouse model, we showed that, compared with miR-scrambled-modulated sEVs, topical administration of miR-425-5p-modulated sEVs significantly enhanced wound healing, a process mediated by enhanced vascularization and skin re-epithelialization.

Bioactive Antimicrobial Peptides as Therapeutic Agents for Infected Diabetic Foot Ulcers.

Diabetic foot ulcer (DFU) is a devastating complication, affecting around 15% of diabetic patients and representing a leading cause of non-traumatic amputations. Notably, the risk of mixed bacterial-fungal infection is elevated and highly associated with wound necrosis and poor clinical outcomes. However, it is often underestimated in the literature. Therefore, polymicrobial infection control must be considered for effective management of DFU. It is noteworthy that antimicrobial resistance is constantly rising overtime, therefore increasing the need for new alternatives to antibiotics and antifungals. Antimicrobial peptides (AMPs) are endogenous peptides that are naturally abundant in several organisms, such as bacteria, amphibians and mammals, particularly in the skin. These molecules have shown broad-spectrum antimicrobial activity and some of them even have wound-healing activity, establishing themselves as ideal candidates for treating multi-kingdom infected wounds. Furthermore, the role of AMPs with antifungal activity in wound management is poorly described and deserves further investigation in association with antibacterial agents, such as antibiotics and AMPs with antibacterial activity, or alternatively the application of broad-spectrum antimicrobial agents that target both aerobic and anaerobic bacteria, as well as fungi. Accordingly, the aim of this review is to unravel the molecular mechanisms by which AMPs achieve their dual antimicrobial and wound-healing properties, and to discuss how these are currently being applied as promising therapies against polymicrobial-infected chronic wounds such as DFUs.

Diabetes and Cannabinoid CB1 receptor deficiency promote similar early onset aging-like changes in the skin.

BACKGROUND: The cannabinoid receptor type-1 (CB1R) is a major regulator of metabolism, growth and inflammation. Yet, its potential role in the skin is not well understood. Our aim was to evaluate the role of CB1R in aging-like diabetic skin changes by using a CB1R knockout mouse model. METHODS: We evaluated several signals of skin aging in wild-type control (WT), WT streptozotocin-induced type 1 diabetic mice (WT DM), CB1R knockout (CB1RKO) and CB1RKO DM mice. We quantified markers of inflammation, angiogenesis, antioxidant enzymes and collagen content. Moreover, we evaluate reactive oxygen species (ROS) levels and macrophage phenotype, M1 and M2. RESULTS: CB1R expression is decreased in the skin of WT DM mice and collagen levels are decreased in the skin of WT DM, CB1RKO and CB1RKO DM mice. Additionally, the absence of CB1R correlated with higher expression of pro-inflammatory markers, also evident in WT DM or CB1RKO DM mice. Moreover, the M1/M2 macrophage ratio and ROS levels were significantly elevated but in the diabetic WT and the CB1RKO mice, consistent with a significant decrease in the antioxidant capacity of the skin. CONCLUSIONS: Our results indicate that CB1R deficiency in the skin may lead to accelerated skin aging due to the increased production of ROS, a decrease in the antioxidant defenses and a higher pro-inflammatory environment. A significant decrease in the CB1R expression may be a significant contributing factor to the early aging-like changes in diabetes.

Dietary supplementation with sulforaphane ameliorates skin aging through activation of the Keap1-Nrf2 pathway.

Visible impairments in skin appearance, as well as a subtle decline in its functionality at the molecular level, are hallmarks of skin aging. Activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-pathway, which is important in controlling inflammation and oxidative stress that occur during aging, can be triggered by sulforaphane (SFN), an isothiocyanate found in plants from the Brassicaceae family. This study aimed to assess the effects of SFN intake on age-related skin alterations. Male C57BL6 young (2 months) and old (21 months) mice were treated for 3 months with SFN diet (442.5 mg per kg) or control diet. The antioxidant capacities of the skin were increased in old SFN-treated animals as measured by mRNA levels of Nrf2 (P<.001) and its target genes NQO1 (P<.001) and HO1 (P<.01). Protein expression for Nrf2 was also increased in old SFN fed animals (P<.01), but not the protein expression of NQO1 or HO1. Additionally, ROS and MMP9 protein levels were significantly decreased (P<.05) in old SFN fed animals. Histopathological analysis confirmed that there was no difference in epidermal thickness in old, when compared to young, SFN treated animals, while the dermal layer thickness was lower in old vs. young, treated animals (P<.05). Moreover, collagen deposition was improved with SFN treatment in young (P<.05) and structurally significantly improved in the old mice (P<.001). SFN dietary supplementation therefore ameliorates skin aging through activation of the Nrf2-pathway.

Improved diabetic wound healing by LFcinB is associated with relevant changes in the skin immune response and microbiota.

Bovine lactoferricin (LFcinB) has antimicrobial and immunomodulatory properties; however, the effects on diabetic wound healing remain poorly understood. The wound healing potential of LFcinB was investigated with in vitro, ex vivo, and in vivo models. Cell migration and proliferation were tested on keratinocytes and on porcine ears. A type 1 diabetic mouse model was also used to evaluate wound healing kinetics, bacterial diversity patterns, and the effect of LFcinB on oxidative stress, macrophage phenotype, angiogenesis, and collagen deposition. LFcinB increased keratinocyte migration in vitro (p < 0.05) and ex vivo (p < 0.001) and improved wound healing in diabetic mice (p < 0.05), though not in normoglycemic control mice. In diabetic mouse wounds, LFcinB treatment led to the eradication of Bacillus pumilus, a decrease in Staphylococcus aureus, and an increase in the Staphylococcus xylosus prevalence. LFcinB increased angiogenesis in diabetic mice (p < 0.01), but this was decreased in control mice (p < 0.05). LFcinB improved collagen deposition in both diabetic and control mice (p < 0.05). Both oxidative stress and the M1-to-M2 macrophage ratios were decreased in LFcinB-treated wounds of diabetic animals (p < 0.001 and p < 0.05, respectively) compared with saline, suggesting a downregulation of inflammation in diabetic wounds. In conclusion, LFcinB treatment demonstrated noticeable positive effects on diabetic wound healing.

Development of an optimized and scalable method for isolation of umbilical cord blood-derived small extracellular vesicles for future clinical use.

Extracellular vesicles (EV) are a promising therapeutic tool in regenerative medicine. These particles were shown to accelerate wound healing, through delivery of regenerative mediators, such as microRNAs. Herein we describe an optimized and upscalable process for the isolation of EV smaller than 200 nm (sEV), secreted by umbilical cord blood mononuclear cells (UCB-MNC) under ischemic conditions and propose quality control thresholds for the isolated vesicles, based on the thorough characterization of their protein, lipid and RNA content. Ultrafiltration and size exclusion chromatography (UF/SEC) optimized methodology proved superior to traditional ultracentrifugation (UC), regarding production time, standardization, scalability, and vesicle yield. Using UF/SEC, we were able to recover approximately 400 times more sEV per mL of media than with UC, and upscaling this process further increases EV yield by about 3-fold. UF/SEC-isolated sEV display many of the sEV/exosomes classical markers and are enriched in molecules with anti-inflammatory and regenerative capacity, such as hemopexin and miR-150. Accordingly, treatment with sEV promotes angiogenesis and extracellular matrix remodeling, in vitro. In vivo, UCB-MNC-sEV significantly accelerate skin regeneration in a mouse model of delayed wound healing. The proposed isolation protocol constitutes a significant improvement compared to UC, the gold-standard in the field. Isolated sEV maintain their regenerative properties, whereas downstream contaminants are minimized. The use of UF/SEC allows for the standardization and upscalability required for mass production of sEV to be used in a clinical setting.

Mechanistic Actions of microRNAs in Diabetic Wound Healing.

Wound healing is a complex biological process that is impaired under diabetes conditions. Chronic non-healing wounds in diabetes are some of the most expensive healthcare expenditures worldwide. Early diagnosis and efficacious treatment strategies are needed. microRNAs (miRNAs), a class of 18-25 nucleotide long RNAs, are important regulatory molecules involved in gene expression regulation and in the repression of translation, controlling protein expression in health and disease. Recently, miRNAs have emerged as critical players in impaired wound healing and could be targets for potential therapies for non-healing wounds. Here, we review and discuss the mechanistic background of miRNA actions in chronic wounds that can shed the light on their utilization as specific wound healing biomarkers.

Protein tyrosine phosphatase 1B inhibition as a potential therapeutic target for chronic wounds in diabetes.

Non-healing diabetic foot ulcers (DFUs) are a serious complication in diabetic patients. Their incidence has increased in recent years. Although there are several treatments for DFUs, they are often not effective enough to avoid amputation. Protein tyrosine phosphatase 1B (PTP1B) is expressed in most tissues and is a negative regulator of important metabolic pathways. PTP1B is overexpressed in tissues under diabetic conditions. Recently, PTP1B inhibition has been found to enhance wound healing. PTP1B inhibition decreases inflammation and bacterial infection at the wound site and promotes angiogenesis and tissue regeneration, thereby facilitating diabetic wound healing. In summary, the pharmacological modulation of PTP1B activity may help treat DFUs, suggesting that PTP1B inhibition is an outstanding therapeutic target.

Lack of lymphocytes impairs macrophage polarization and angiogenesis in diabetic wound healing.

AIMS: This study aimed to investigate the effect of lymphocytes in wound healing and the underlying mechanisms, in diabetic and non-diabetic mice, using Balb/c recombination activating gene (Rag)-2 and interleukin 2 receptor gamma (IL-2Rγ) double knockout (KO) (RAG2-/- IL-2Rγ-/-) mice. MAIN METHODS: Wound healing in vivo was performed in control and STZ-induced diabetic mice, in both KO and WT mice. Inflammation and ROS production were evaluated by immunofluorescence microscopy analysis, antioxidant enzymes and angiogenesis were evaluated by quantitative PCR and immunofluorescence microscopy analysis, and wound closure kinetics evolution was evaluated by measurement of acetate tracing of the wound area. KEY FINDINGS: Wound closure was significantly delayed in KO mice, where the M1/M2 macrophage ratio and basal ROS levels were significantly increased, while antioxidant defenses and angiogenesis were significantly decreased. Moreover, the expected increase in matrix metallopeptidase (MMP)-9 protein levels in diabetic conditions was not observed in KO mice, suggesting that the mechanisms leading to the increase in MMP-9 observed in diabetic wounds may in part be lymphocyte-dependent. SIGNIFICANCE: Our results indicate that lack of lymphocytes compromises wound healing independent of diabetes. The lack of these cells, even in non-diabetic mice, mimics the phenotype observed in wounds under diabetic conditions. Moreover, the combination of diabetes and the lack of lymphocytes, further impair the wound healing conditions, indicating that when the innate regulatory function is lost in these KO mice, excessive M1 polarization, poor angiogenesis and impaired wound healing are worsen.

The Kinetics of Small Extracellular Vesicle Delivery Impacts Skin Tissue Regeneration.

Small extracellular vesicles (SEVs) offer a promising strategy for tissue regeneration, yet their short lifetime at the injured tissue limits their efficacy. Here, we show that kinetics of SEV delivery impacts tissue regeneration at tissue, cellular, and molecular levels. We show that multiple carefully timed applications of SEVs had superior regeneration than a single dose of the same total concentration of SEVs. Importantly, diabetic and non-diabetic wounds treated with a single time point dose of an injectable light-triggerable hydrogel containing SEVs demonstrated a robust increase in closure kinetics relative to wounds treated with a single or multiple doses of SEVs or platelet-derived growth factor BB, an FDA-approved wound regenerative therapy. The pro-healing activity of released SEVs was mediated at the tissue/cell level by an increase in skin neovascularization and re-epithelization and at the molecular level by an alteration in the expression of 7 miRNAs at different times during wound healing. This includes an alteration of has-miR-150-5p, identified here to be important for skin regeneration.

microRNA-155 inhibition restores Fibroblast Growth Factor 7 expression in diabetic skin and decreases wound inflammation.

Treatment for chronic diabetic foot ulcers is limited by the inability to simultaneously address the excessive inflammation and impaired re-epithelization and remodeling. Impaired re-epithelization leads to significantly delayed wound closure and excessive inflammation causes tissue destruction, both enhancing wound pathogen colonization. Among many differentially expressed microRNAs, miR-155 is significantly upregulated and fibroblast growth factor 7 (FGF7) mRNA (target of miR-155) and protein are suppressed in diabetic skin, when compared to controls, leading us to hypothesize that topical miR-155 inhibition would improve diabetic wound healing by restoring FGF7 expression. In vitro inhibition of miR-155 increased human keratinocyte scratch closure and topical inhibition of miR-155 in vivo in wounds increased murine FGF7 protein expression and significantly enhanced diabetic wound healing. Moreover, we show that miR-155 inhibition leads to a reduction in wound inflammation, in accordance with known pro-inflammatory actions of miR-155. Our results demonstrate, for the first time, that topical miR-155 inhibition increases diabetic wound fibroblast growth factor 7 expression in diabetic wounds, which, in turn, increases re-epithelization and, consequently, accelerates wound closure. Topical miR-155 inhibition targets both excessive inflammation and impaired re-epithelization and remodeling, being a potentially new and effective treatment for chronic diabetic foot ulcers.

Calcium Dobesilate Is Protective against Inflammation and Oxidative/Nitrosative Stress in the Retina of a Type 1 Diabetic Rat Model.

Calcium dobesilate (CaD) has been prescribed to some patients in the early stages of diabetic retinopathy to delay its progression. We previously reported that the treatment of diabetic animals (4 weeks of diabetes) with CaD, during the last 10 days of diabetes, prevents blood-retinal barrier breakdown. Here, we aimed to investigate whether later treatment of diabetic rats with CaD would reverse inflammatory processes in the retina. Diabetes was induced with streptozotocin, and 6 weeks after diabetes onset, CaD (100 mg/kg/day) was administered for 2 weeks. The treatment with CaD significantly increased glial fibrillary acidic protein (GFAP) levels in the retina of nondiabetic animals (138.6 ± 12.8% of control) and enhanced the diabetes-induced increase in GFAP levels (174.8 ± 5.6% of control). In addition, CaD prevented the increase in mRNA and protein expression of tumor necrosis factor and interleukin-1ß, as well as the formation of oxidized carbonyl residues and the increase in nitrotyrosine immunoreactivity, particularly in the ganglion cell layer of diabetic animals. We demonstrate that the treatment of diabetic animals with CaD can reverse the established proinflammatory processes in the retina. These beneficial effects appear to be attributed, at least partially, to the antioxidant properties of CaD.

Mast Cells Regulate Wound Healing in Diabetes.

Diabetic foot ulceration is a severe complication of diabetes that lacks effective treatment. Mast cells (MCs) contribute to wound healing, but their role in diabetes skin complications is poorly understood. Here we show that the number of degranulated MCs is increased in unwounded forearm and foot skin of patients with diabetes and in unwounded dorsal skin of diabetic mice (P < 0.05). Conversely, postwounding MC degranulation increases in nondiabetic mice, but not in diabetic mice. Pretreatment with the MC degranulation inhibitor disodium cromoglycate rescues diabetes-associated wound-healing impairment in mice and shifts macrophages to the regenerative M2 phenotype (P < 0.05). Nevertheless, nondiabetic and diabetic mice deficient in MCs have delayed wound healing compared with their wild-type (WT) controls, implying that some MC mediator is needed for proper healing. MCs are a major source of vascular endothelial growth factor (VEGF) in mouse skin, but the level of VEGF is reduced in diabetic mouse skin, and its release from human MCs is reduced in hyperglycemic conditions. Topical treatment with the MC trigger substance P does not affect wound healing in MC-deficient mice, but improves it in WT mice. In conclusion, the presence of nondegranulated MCs in unwounded skin is required for proper wound healing, and therapies inhibiting MC degranulation could improve wound healing in diabetes.