Angiogenesis In Diabetic Wounds Research Articles

Bone marrow mesenchymal stem cell-derived exosomal miR-221-3p promotes angiogenesis and wound healing in diabetes via the downregulation of forkhead box P1.

Impaired wound healing in patients with diabetes can develop into nonhealing ulcerations. Because bone marrow mesenchymal stem cells (BMSCs) exosomes can promote wound healing, this study aims to investigate the mechanism of BMSCs-isolated exosomal miR-221-3p in angiogenesis and diabetic wound healing. To mimic diabetes invitro, human umbilical vein endothelial cells (HUVECs) were subjected to high glucose (HG). Exosomes were derived from BMSCs and identified by transmission electron microscopy (TEM), western blot analysis and dynamic light scattering (DLS). The ability to differentiate BMSCs was assessed via Oil red O staining, alkaline phosphatase (ALP) staining and alizarin red staining. The ability to internalise PKH26-labelled exosomes was assessed using confocal microscopy. Migration, cell viability and angiogenesis were tested by scratch, MTT and tube formation assays separately. The miRNA and protein levels were analysed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) or western blotting. The relationship among miR-221-3p, FOXP1 and SPRY1 was determined using the dual-luciferase reporter, ChIP and RIP assays. Exosomal miR-221-3p was successfully isolated from BMSCs and delivered into HUVECs. HG was found to suppress the angiogenesis, cell viability and migration of HUVECs and exosomal miR-221-3p separated from BMSCs inhibited the above phenomenon. FOXP1 could transcriptionally upregulate SPRY1, and the silencing of FOXP1 reversed the HG-stimulated angiogenesis inhibition, cell viability and migration in HUVECs via the downregulation of SPRY1. Meanwhile, miR-221-3p directly targeted FOXP1 and the overexpression of FOXP1 reversed the positive effect of exosomal miR-221-3p on HUVEC angiogenesis. Exosomal miR-221-3p isolated from BMSCs promoted angiogenesis in diabetic wounds through the mediation of the FOXP1/SPRY1 axis. Furthermore, the findings of this study can provide new insights into probing strategies against diabetes.

A bioenergetically-active ploy (glycerol sebacate)-based multiblock hydrogel improved diabetic wound healing through revitalizing mitochondrial metabolism.

Diabetic wounds impose significant burdens on patients' quality of life and healthcare resources due to impaired healing potential. Factors like hyperglycemia, oxidative stress, impaired angiogenesis and excessive inflammation contribute to the delayed healing trajectory. Mounting evidence indicates a close association between impaired mitochondrial function and diabetic complications, including chronic wounds. Mitochondria are critical for providing energy essential to wound healing processes. However, mitochondrial dysfunction exacerbates other pathological factors, creating detrimental cycles that hinder healing. This study conducted correlation analysis using clinical specimens, revealing a positive correlation between mitochondrial dysfunction and oxidative stress, inflammatory response and impaired angiogenesis in diabetic wounds. Restoring mitochondrial function becomes imperative for developing targeted therapies. Herein, we synthesized a biodegradable poly (glycerol sebacate)-based multiblock hydrogel, named poly (glycerol sebacate)-co-poly (ethylene glycol)-co-poly (propylene glycol) (PEPGS), which can be degraded in vivo to release glycerol, a crucial component in cellular metabolism, including mitochondrial respiration. We demonstrate the potential of PEPGS-based hydrogels to improve outcomes in diabetic wound healing by revitalizing mitochondrial metabolism. Furthermore, we investigate the underlying mechanism through proteomics analysis, unravelling the regulation of ATP and nicotinamide adenine dinucleotide metabolic processes, biosynthetic process and generation during mitochondrial metabolism. These findings highlight the therapeutic potential of PEPGS-based hydrogels as advanced wound dressings for diabetic wound healing.

A novel designed nanofibrous mat based on hydroxypropyl methyl cellulose incorporating mango peel extract for potential use in wound care system

Skin tissue is damaged by factors such as burns, physical injuries and diseases namely diabetes. Infection and non-healing of burn wounds and lack of angiogenesis in diabetic wounds lead to extensive injuries and death. Therefore, the design of wound dressings with antibacterial and restorative capabilities is very important. In this study, nanofibers (NFs) including polyurethane (PU) and hydroxypropyl methyl cellulose (HPMC) were prepared with different ratios and Mango peel extract (MPE) loaded into NFs by electrospinning method. The morphology, chemical structure, porosity, degradation, water vapor permeability, mechanical properties, wettability, antioxidant activity and some cell studies and evaluation of their antibacterial properties were investigated. The optimal mat (PU90/HPMC10) had a defect-free morphology with homogeneous NFs. Furthermore, it showed improved biodegradability, water vapor permeability and porosity compared to other Mats. All NFs were non-toxic with hydrophilic behavior in the cellular environment and had acceptable hemocompatibility. The PU90/HPMC10/20 % optimal scaffold had significantly higher cell viability and proliferation than other samples and also had a higher antibacterial ability against pathogenic bacteria S. aureus (17 mm) and E. coli (11 mm). All these findings confirm that the produced NF mats, especially those loaded with MPE, have a high potential to be used as an effective wound dressing.

Mesenchymal stem cell-derived extracellular vesicles accelerate diabetic wound healing by inhibiting NET-induced ferroptosis of endothelial cells.

Impaired angiogenesis is a major factor contributing to delayed wound healing in diabetes. Dysfunctional mitochondria promote the formation of neutrophil extracellular traps (NETs), obstructing angiogenesis during wound healing. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown promise in promoting tissue repair and regeneration in diabetes; however, the precise pathways involved in this process remain unclear. In this study, NET-induced ferroptosis of endothelial cells (ECs) and angiogenesis were assessed in diabetic wound samples from both patients and animal models. In vitro and in vivo experiments were performed to examine the regulatory mechanisms of NETs in ECs using specific inhibitors and gene-knockout mice. MSC-EVs encapsulating dysfunctional mitochondria were used to trigger mitochondrial fusion and restore mitochondrial function in neutrophils to suppress NET formation. Angiogenesis in wound tissue was evaluated using color laser Doppler imaging and vascular density analysis. Wound healing was evaluated via macroscopic analysis and histological evaluation of the epithelial gap. NET-induced ferroptosis of ECs was validated as a crucial factor contributing to the impairment of angiogenesis in diabetic wounds. Mechanistically, NETs regulated ferroptosis by suppressing the PI3K/AKT pathway. Furthermore, MSC-EVs transferred functional mitochondria to neutrophils in wound tissue, triggered mitochondrial fusion, and restored mitochondrial function, thereby reducing NET formation. These results suggest that inhibiting NET formation and EC ferroptosis or activating the PI3K/AKT pathway can remarkably improve wound healing. In conclusion, this study reveals a novel NET-mediated pathway involved in wound healing in diabetes and suggests an effective therapeutic strategy for accelerating wound healing.

Asiaticoside Prevents Oxidative Stress and Apoptosis in Endothelial Cells by Activating ROS-dependent p53/Bcl-2/Caspase-3 Signaling Pathway.

Asiaticoside (AC) is a triterpenoid saponin found in Centella asiatica (L.) urban extract that has a wide range of pharmacological properties. Our previous study demonstrated that AC could promote angiogenesis in diabetic wounds, but the specific mechanisms remain unknown. This study aimed to examine the effectiveness and mechanism of AC on human umbilical vein endothelial cells (HUVECs) exposed to tert-butyl hydroperoxide (t-BHP) toxicity. Senescence was confirmed using senescence-associated betagalactosidase (SA-β-gal) activity and expression of the cell cycle phase markers p16 and p21. The levels of SOD, NO, MDA, GSH-Px, and ROS were tested. Furthermore, several cell death-related genes and proteins (p53, Bax, Bcl-2 and Caspase-3) were assessed with RT-qPCR and Western blotting. AC significantly reduced SA-β-gal activity, with both the suppression of cellcycle inhibitors p16 and p21. We also found that the induced oxidative stress and apoptosis caused by t-BHP treatment resulted in the decrease of antioxidant enzymes activities, the surge of ROS and MDA, the up-regulation of p53, Bax and caspase-3, and the decrease of SOD, NO, GSH-Px and Bcl-2. These biochemical changes were all reversed by treatment with varying doses of AC. AC alleviates t-BHP-induced oxidative injury and apoptosis in HUVECs through the ROS-dependent p53/Bcl-2/Caspase-3 signaling pathway. It may be a potential antioxidant applied in metabolic disorders and pharmaceutical products.

Local low-frequency vibration accelerates healing of full-thickness wounds in a hyperglycemic rat model.

Local low-frequency vibration (LLFV) promotes vasodilation and blood flow, enhancing wound healing in diabetic foot ulcers with angiopathy. However, vibration-induced vasodilation does not occur, owing to chronic hyperglycemia and inflammation. We hypothesized that LLFV improves glycometabolism and inflammation, leading to vasodilation and angiogenesis in diabetic wounds. Therefore, this study investigated the effect of LLFV on wound healing in hyperglycemic rats, primarily focusing on glycometabolism, inflammation, vasodilation, and angiogenesis. Streptozotocin-induced hyperglycemic Sprague-Dawley rats were used in this study. We applied LLFV to experimentally-induced wounds at 50 Hz and 0, 600, 1,000 or 1,500 mVpp for 40 min/day from post-wounding days (PWD) 1-14. The relative wound areas in the 600 and 1,000 mVpp groups on PWD 5-7 were significantly smaller than those at 0 mVpp. The expression of Glo-1 (1,500 mVpp) and Slc2A4 (1,000 and 1,500 mVpp) was upregulated on PWD 4 and 14, respectively. However, there was no difference in methylglyoxal expression levels in any group until PWD 14. At 1,000 mVpp, the expression of Tnfa on PWD 4, and that of Ptx3 and Ccl2 on PWD 14 was downregulated. Furthermore, the M1/M2 macrophage ratio was considerably decreased on both days. The expression of Nos3, Vegfa and vascular endothelial growth factor A was upregulated on PWD 4. In addition, vasodilation and angiogenesis were more obvious on PWD 14 with 1,000 mVpp. The results suggest that LLFV promotes wound healing, improves glycometabolism and inflammation, and enhances vasodilation and angiogenesis in hyperglycemic wounds.

Effects of microRNAs on angiogenesis in diabetic wounds.

Diabetes mellitus is a morbid condition affecting a growing number of the world population, and approximately one third of diabetic patients are afflicted with diabetic foot ulcers (DFU), which are chronic non-healing wounds that frequently progress to require amputation. The treatments currently used for DFU focus on reducing pressure on the wound, staving off infection, and maintaining a moist environment, but the impaired wound healing that occurs in diabetes is a constant obstacle that must be faced. Aberrant angiogenesis is a major contributor to poor wound healing in diabetes and surgical intervention is often necessary to establish peripheral blood flow necessary for healing wounds. Over recent years, microRNAs (miRNAs) have been implicated in the dysregulation of angiogenesis in multiple pathologies including diabetes. This review explores the pathways of angiogenesis that become dysregulated in diabetes, focusing on miRNAs that have been identified and the mechanisms by which they affect angiogenesis.

Metformin coordinates with mesenchymal cells to promote VEGF-mediated angiogenesis in diabetic wound healing through Akt/mTOR activation

IntroductionCell therapy with mesenchymal stem cells (MSCs) and biomaterials holds great potential for the treatment of diabetic ulceration; however, the underlying mechanism as well as its compatibility with the first-line anti-diabetic drug, metformin (MTF), has not been well elucidated. MethodsMSCs derived from the umbilical cord were labeled with fluorescent proteins, followed by transplantation in a fibrin scaffold (MSCs/FG) onto the STZ-induced diabetic wound in a C57BL6/J mouse model. MTF was administered by oral gavage at a dose of 250 mg/kg/day. The wound healing rate, epithelization, angiogenesis, and underlying mechanism were evaluated in MSCs/FG- and MTF-treated diabetic wounds. Moreover, the dose-dependent effects of MTF and involvement of the Akt/mTOR pathway were analyzed in keratinocyte and fibroblast cultures. ResultsMSCs/FG significantly promoted angiogenesis in diabetic wound healing without signs of differentiation or integration. The recruitment of fibroblasts and keratinocytes by MSCs/FG promotes migration and vascular endothelial growth factor (VEGF) expression in an Akt/mTOR-dependent manner. MTF, which is generally considered a mTOR inhibitor, displayed dose-dependent effects on MSC-unregulated Akt/mTOR and VEGF expression. Oral administration of MTF at an anti-diabetic dosage synergistically acted with MSCs/FG to promote Akt/mTOR activation, VEGF expression, and subsequent angiogenesis in diabetic wounds; however, it reduced the survival of MSCs. ConclusionsOur study identifies that MTF coordinates with mesenchymal cells to promote Akt/mTOR activation and VEGF-mediated angiogenesis during diabetic wound healing. These findings offer new insights into MSCs engraftment in FG scaffolds for diabetic wound healing and provide support for the promotion of MSCs therapy in patients prescribed with MTF.

Novel ROS-scavenging hydrogel with enhanced anti-inflammation and angiogenic properties for promoting diabetic wound healing

Accelerating angiogenesis of diabetic wounds is crucial to promoting wound healing. Currently, vascular endothelial growth factor (VEGF), an angiogenesis-related bioactive molecule, is widely used in clinic to enhance wound angiogenesis, but it faces problems of inactivation and low utilization due to harsh microenvironment. Here, we developed a novel reactive oxygen species (ROS)-scavenging hydrogel aimed to polarize macrophages toward an anti-inflammatory phenotype, inducing efficient angiogenesis in diabetic wounds. This composite hydrogel with good biosafety and mechanical properties showed sustainable release of bioactive VEGF. Importantly, it could significantly reduce ROS level and rapidly improve wound microenvironment, which ensured the activity of VEGF in vitro and in vivo and successful healing eventually. At the same time, the composite hydrogel exhibited excellent antibacterial properties. In vivo results confirmed good anti-inflammatory, stimulated vascularization and accelerated wound healing attributed to the novel ROS-scavenging hydrogel, which might serve as a promising wound dressing in diabetic wound healing.

LRG1 Accelerates Wound Healing in Diabetic Rats by Promoting Angiogenesis via the Wnt/β-Catenin Signaling Pathway.

Patients with diabetic wounds may end with lower extremity amputation or death. Leucine-rich α-2-glycoprotein 1 (LRG1) is an effective regulator of angiogenesis and essential for timely wound healing. However, its role in regulating angiogenesis in diabetic wounds remains unclear. This study aimed to investigate the pro-angiogenic function of exogenous LRG1 in diabetic wound healing and explore possible mechanisms. LRG1 expression patterns following injury in normal and diabetic wounds were determined by western blotting. Local injection of LRG1 was used to verify the effects on angiogenesis and wound healing in diabetic rats. Immunohistochemical staining for CD31 was used to analyze the vessel density. Human umbilical vein endothelial cells (HUVECs) cultured in hyperglycemia were used to explore how LRG1 promotes angiogenesis in diabetic wound healing. We found that the expression peak of LRG1 around the wounds was delayed in diabetic rats compared with that in normal rats. Exogenous administration of LRG1 significantly accelerated the wound closure rate and promoted angiogenesis in diabetic rats. In addition, exogenous LRG1 effectively restored the proliferation, migration, and tube formation ability of HUVECs under hyperglycemia. Mechanistically, LRG1 promoted angiogenesis and diabetic wound healing mainly by activating the Wnt/β-catenin pathway, which is inhibited in diabetic wounds. This research suggests that LRG1 promotes angiogenesis and wound closure in diabetic rats by improving angiogenesis via activation of the Wnt/β-catenin pathway. Hence, LRG1 may be a possible therapeutic strategy for diabetic foot treatment.

Engineering Bacteria‐Activated Multifunctionalized Hydrogel for Promoting Diabetic Wound Healing

AbstractEngineering therapeutic angiogenesis in impaired tissues is critical for chronic wound healing. Materials can be engineered to deliver specific biological cues that enhance angiogenesis. However, currently available materials have limitations for use in angiogenesis engineering since the complex inflammation environment of wounds requires spatiotemporal control. Immune cells are the central component of wound microenvironment and orchestrate immune responses to wound healing. This study presents a novel approach of using a delivery system comprising living Lactococcus, incorporated in a heparin‐poloxamer thermoresponsive hydrogel, designed to bioengineer the wound microenvironment and enhance the angiogenesis in a highly dynamic‐temporal manner. The living system can produce and protect vascular endothelial growth factor (VEGF) to increase proliferation, migration, and tube formation of endothelial cells, as well as secrete lactic acid to shift macrophages toward an anti‐inflammatory phenotype, resulting in successful angiogenesis in diabetic wounds. Further, the delivery system confines the bacterial population to wounds, thereby minimizing the risk of systemic toxicities. Therefore, this living hydrogel system can be harnessed for safe and efficient delivery of therapeutics that drive the wound microenvironment toward rapid healing and may serve as a promising scaffold in regenerative medicine.

Extracellular Vesicles from Human Umbilical Cord Mesenchymal Stem Cells Facilitate Diabetic Wound Healing Through MiR-17-5p-mediated Enhancement of Angiogenesis.

Endothelial dysfunction caused by persistent hyperglycemia in diabetes is responsible for impaired angiogenesis in diabetic wounds. Extracellular vehicles (EVs) are considered potential therapeutic tools to promote diabetic wound healing. The aim of this study was to investigate the effects of EVs secreted by human umbilical cord mesenchymal stem cells (hucMSC-EVs) on angiogenesis under high glucose (HG) conditions in vivo and in vitro and to explore the underlying mechanisms. In vivo, local application of hucMSC-EVs enhanced wound healing and angiogenesis. In vitro, hucMSC-EVs promoted proliferation, migration, and tube formation by inhibiting phosphatase and tensin homolog (PTEN) expression and activating the AKT/HIF-1α/VEGF pathways. MiR-17-5p was found to be highly enriched in hucMSC-EVs. In vitro, MiR-17-5p agomirs downregulated the expression of PTEN and activated the AKT/HIF-1α/VEGF pathway to promote proliferation, migration, and tube formation in HG-treated HUVECs. In vivo, miR-17-5p agomirs mimicked the effects of hucMSC-EVs on wound healing and angiogenesis, whereas miR-17-5p inhibitors reversed their effects. Our findings suggest that hucMSC-EVs have regenerative and protective effects on HG-induced endothelial cells via transfer of miR-17-5p targeting PTEN/ AKT/HIF-1α/VEGF pathway, thereby accelerating diabetic wound healing. Thus, hucMSC-EVs may be promising therapeutic candidates for improving diabetic wound angiogenesis.

Attenuation of Flightless I Increases Human Pericyte Proliferation, Migration and Angiogenic Functions and Improves Healing in Murine Diabetic Wounds.

Pericytes are peri-vascular mural cells which have an important role in the homeostatic regulation of inflammatory and angiogenic processes. Flightless I (Flii) is a cytoskeletal protein involved in regulating cellular functions, but its involvement in pericyte activities during wound healing is unknown. Exacerbated inflammation and reduced angiogenesis are hallmarks of impaired diabetic healing responses, and strategies aimed at regulating these processes are vital for improving healing outcomes. To determine the effect of altering Flii expression on pericyte function, in vitro and in vivo studies were performed to assess the effect on healing, inflammation and angiogenesis in diabetic wounds. Here, we demonstrated that human diabetic wounds display upregulated expression of the Flii protein in conjunction with a depletion in the number of platelet derived growth factor receptor β (PDGFRβ) +/ neural glial antigen 2 (NG2) + pericytes present in the dermis. Human pericytes were found to be positive for Flii and attenuating its expression in vitro through siRNA knockdown led to enhanced proliferation, migration and angiogenic functions. Genetic knockdown of Flii in a streptozotocin-induced murine model of diabetes led to increased numbers of pericytes within the wound. This was associated with dampened inflammation, an increased rate of angiogenic repair and improved wound healing. Our findings show that Flii expression directly impacts pericyte functions, including proliferation, motility and angiogenic responses. This suggests that Flii regulation of pericyte function may be in part responsible for the changes in pericyte-related processes observed in diabetic wounds.

Compromised angiogenesis and vascular Integrity in impaired diabetic wound healing

Vascular deficits are a fundamental contributing factor of diabetes-associated diseases. Although previous studies have demonstrated that the pro-angiogenic phase of wound healing is blunted in diabetes, a comprehensive understanding of the mechanisms that regulate skin revascularization and capillary stabilization in diabetic wounds is lacking. Using a mouse model of diabetic wound healing, we performed microCT analysis of the 3-dimensional architecture of the capillary bed. As compared to wild type, vessel surface area, branch junction number, total vessel length, and total branch number were significantly decreased in wounds of diabetic mice as compared to WT mice. Diabetic mouse wounds also had significantly increased capillary permeability and decreased pericyte coverage of capillaries. Diabetic wounds exhibited significant perturbations in the expression of factors that affect vascular regrowth, maturation and stability. Specifically, the expression of VEGF-A, Sprouty2, PEDF, LRP6, Thrombospondin 1, CXCL10, CXCR3, PDGFR-β, HB-EGF, EGFR, TGF-β1, Semaphorin3a, Neuropilin 1, angiopoietin 2, NG2, and RGS5 were down-regulated in diabetic wounds. Together, these studies provide novel information about the complexity of the perturbation of angiogenesis in diabetic wounds. Targeting factors responsible for wound resolution and vascular pruning, as well those that affect pericyte recruitment, maturation, and stability may have the potential to improve diabetic skin wound healing.

<p>MicroRNA-126: a promising biomarker for angiogenesis of diabetic wounds treated with negative pressure wound therapy</p>

BackgroundNegative pressure wound therapy represents an effective therapy to treat nonhealing diabetic wounds by promoting angiogenesis, of which the mechanism hasn’t been investigated thoroughly. Growing evidence suggests that miRNAs hold great potential to be clinical biomarkers, and miR-126 is an essential angiogenesis regulator in diabetic wound repair. PurposeOur study aims to explore the effect of NPWT on the expression of miR-126 in the wound tissue and plasma of diabetic rat models and the association between circulating miR-126 and two quantitative indexes of angiogenesis. MethodsFull-thickness excisional wounds were created on the back of diabetic rats. Measure the wound closure and collect the wound tissue and blood for H&E, immunohistochemistry, Western blot and RT-PCR. Here we demonstrated that significantly increased capillary density and arteriolar density in the NPWT group at each specified time-point. ResultsIn the NPWT group, miR-126 expression was significantly increased on days 3, 5, 7, and 9 (P<0.05). Furthermore, statistically significant increases in VEGF mRNA and protein expression and p-ERK expression, as well as decreased SPRED1 expression, were noted upon treatment with NPWT on day 9. Our data revealed that miR-126 expression in the wound and plasma was significantly associated (P<0.05). Moreover, a positive correlation was also detected between increased levels of circulating miR-126 and arteriolar density, as well as capillary density (P<0.05). ConclusionThe study suggested that miR-126 was upregulated by NPWT and could represent a promising monitoring tool for angiogenesis in diabetic wounds treated with NPWT.

Abstract P2021: Cutaneous Wound Healing in Diabetic Mice is Improved by Topical Mineralocorticoid Receptor Blockade

Introduction: Chronic skin ulcers resulting from impaired wound healing are a serious complication of diabetes. Unresolved inflammation, associated with the dysregulation of both the phenotype and function of macrophages, is involved in the poor healing of diabetic wounds. Objective: To investigate the role of the mineralocorticoid receptor in the healing delay and specifically in the impaired angiogenesis and prolonged inflammation of diabetic wounds. Methods: 6 mm wounds were created on the back skin of female streptozotocin (STZ)-induced or db/db mice. Wounds were treated topically with canrenoate (Canre: 0.5mM) or PBS twice a day, for 5 (STZ mice) or 7 (db/db mice) days. Wound area was measured at indicated time post-wounding. Wounded skin was collected for histology and molecular analysis. Results: topical pharmacological inhibition of the MR by Canre can resolve prolonged inflammation to improve delayed skin wound healing in diabetic mouse models. The improvement is demonstrated by increasing of 39% of neo-epidermisation and 62% of epidermal keratinocyte proliferation in db/db wounds treated by Canre. Moreover, Canre could improve the reduced blood vessel density in the wound beds of STZ mice (STZ + PBS: 5.33% ± 0.32 vs STZ + Canre: 8.26% ± 0.64) and of db/db mice (db/db + PBS: 1.32% ± 0.29 vs db/db + Canre: 3.31% ± 0.33). The beneficial effect of Canre is associated with an increased ratio of anti-inflammatory M2 macrophages to pro-inflammatory M1 macrophages in diabetic wounds (ratio of M2 to M1 in db/db + PBS vs db/db + Canre: 0.72 ± 0.15 vs 1.72 ± 0.29, respectively). Furthermore, we show that MR blockade leads to downregulated expression of a MR target, lipocalin 2 (Lcn2), which may facilitate macrophage polarization towards the M2 phenotype and promote impaired angiogenesis in diabetic wounds. Indeed, diabetic Lcn2-deficient mice showed improved wound healing, associated with macrophage M2 polarization and angiogenesis. In addition, recombinant Lcn2 protein prevented IL4-induced macrophages switch from M1 to M2 phenotype. Conclusion: topical MR blockade accelerates skin wound healing in diabetic mice via Lcn2 reduction, M2 macrophage polarization, prevention of prolonged inflammation, and induction of angiogenesis.

Cutaneous Wound Healing in Diabetic Mice Is Improved by Topical Mineralocorticoid Receptor Blockade

Skin ulcers resulting from impaired wound healing are a serious complication of diabetes. Unresolved inflammation, associated with the dysregulation of both the phenotype and function of macrophages, is involved in the poor healing of diabetic wounds. Here, we report that topical pharmacological inhibition of the mineralocorticoid receptor (MR) by canrenoate or MR small interfering RNA can resolve inflammation to improve delayed skin wound healing in diabetic mouse models; importantly, wounds from normal mice are unaffected. The beneficial effect of canrenoate is associated with an increased ratio of anti-inflammatory M2 macrophages to proinflammatory M1 macrophages in diabetic wounds. Furthermore, we show that MR blockade leads to downregulation of the MR target, LCN2, which may facilitate macrophage polarization toward the M2 phenotype and improve impaired angiogenesis in diabetic wounds. Indeed, diabetic LCN2-deficient mice showed improved wound healing associated with macrophage M2 polarization and angiogenesis. In addition, recombinant LCN2 protein prevented IL-4-induced macrophage switch from M1 to M2 phenotype. In conclusion, topical MR blockade accelerates skin wound healing in diabetic mice via LCN2 reduction, M2 macrophage polarization, prevention of inflammation, and induction of angiogenesis.

SIRT1 activation promotes angiogenesis in diabetic wounds by protecting endothelial cells against oxidative stress

ObjectiveChronic wounds are a devastating complication of diabetes and can lead to amputations or even death. Current medical therapies are insufficient to accelerate its repair. The objective of this study was to explore the role of Sirtuin1 (SIRT1) in diabetic wounds. Methods and materialsPerilesional skin tissue samples from diabetic ulcers and normoglycemic trauma wounds were used to detect SIRT1 expression and oxidative stress levels. In a diabetic mouse model, SIRT1 was pharmacologically activated to attenuate angiogenesis and accelerate wound closure. Finally, in vitro experiments were performed to elucidate some of the mechanisms by which SIRT1 activation promotes angiogenesis in diabetic wound healing. ResultsWe found that skin tissue from diabetes patients showed lower expression of SIRT1 and severe oxidative stress. Decreased SIRT1 expression was observed in skin tissue from streptozocin (STZ)-induced diabetic mice and was associated with impaired wound healing. In addition, the wounds of STZ-induced diabetic mice treated with SRT1720 (a specific SIRT1 activator) demonstrated locally improved wound healing and angiogenesis. In the in vitro experiment, similar results were observed. Under hyperglycemia conditions, human umbilical vein endothelial cells (HUVECs) showed lower expression of SIRT1 and higher levels of reactive oxygen species (ROS) production. Furthermore, the migration, proliferation and in vitro tube formation ability of HUVECs were impaired under hyperglycemia conditions, and SRT1720 treatment rescued these impairments and decreased ROS production in HUVECs. ConclusionsThis study provides experimental evidence that SIRT1 activation could improve angiogenesis in wounds in vitro and in vivo and that sirtuin1 activation accelerates wound healing in diabetic mice by promoting angiogenesis. These positive therapeutic effects may be mediated by protecting vascular endothelial cells from oxidative stress injury. This study suggested that SIRT1 may serve as a potentially important and potent therapeutic target for treating diabetic ulcers.

Small molecule inhibition of dipeptidyl peptidase-4 enhances bone marrow progenitor cell function and angiogenesis in diabetic wounds

In diabetes, stromal cell-derived factor-1 (SDF-1) expression and progenitor cell recruitment are reduced. Dipeptidyl peptidase-4 (DPP-4) inhibits SDF-1 expression and progenitor cell recruitment. Here we examined the impact of the DPP-4 inhibitor, MK0626, on progenitor cell kinetics in the context of wound healing. Wildtype (WT) murine fibroblasts cultured under high-glucose to reproduce a diabetic microenvironment were exposed to MK0626, glipizide, or no treatment, and SDF-1 expression was measured with ELISA. Diabetic mice received MK0626, glipizide, or no treatment for 6 weeks and then were wounded. Immunohistochemistry was used to quantify neovascularization and SDF-1 expression. Gene expression was measured at the RNA and protein level using quantitative polymerase chain reaction and ELISA, respectively. Flow cytometry was used to characterize bone marrow-derived mesenchymal progenitor cell (BM-MPC) population recruitment to wounds. BM-MPC gene expression was assayed using microfluidic single cell analysis. WT murine fibroblasts exposed to MK0626 demonstrated increased SDF-1 expression. MK0626 treatment significantly accelerated wound healing and increased wound vascularity, SDF-1 expression, and dermal thickness in diabetic wounds. MK0626 treatment increased the number of BM-MPCs present in bone marrow and in diabetic wounds. MK0626 had no effect on BM-MPC population dynamics. BM-MPCs harvested from MK0626-treated mice exhibited increased chemotaxis in response to SDF-1 when compared to diabetic controls. Treatment with a DPP-4 inhibitor significantly improved wound healing, angiogenesis, and endogenous progenitor cell recruitment in the setting of diabetes.

Momordica charantia extracts protect against inhibition of endothelial angiogenesis by advanced glycation endproducts in vitro.

Diabetes mellitus characterized by hyperglycemia favors formation of advanced glycation endproducts (AGEs) capable of triggering vascular complications by interfering with imbalanced inflammation and angiogenesis to eventually impede wound-healing. Momordica charantia (MC, bitter melon) has been shown to prevent AGE formation and to promote angiogenesis in diabetic wounds in animal models. However, the mechanism underlying its effects on angiogenesis is unclear. We investigated the effects of methanolic extracts of MC pulp (MCP), flesh (MCF) and charantin (active component of MC) using an in vitro model of angiogenesis. MC extracts or low concentrations of bovine serum albumin-derived AGEs (BSA-AGEs) stimulated proliferation, migration (using wound-healing assay) and tube formation (using Matrigel™-embedded 3D culture) of bovine aortic endothelial cells (BAEC) together with increases in the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, the key angiogenic signaling cytoplasmic protein. Blocking the receptor for AGEs (RAGE) inhibited low BSA-AGE- and MC extract-induced ERK1/2 phosphorylation and tube formation, indicating the crucial role of RAGE in the pro-angiogenic effects of MC extracts. Moreover, inhibitory effects of high BSA-AGE concentration on cell proliferation and migration were reduced by the addition of MC extracts, which reversed the BSA-AGE anti-angiogenic effect on tube formation. Thus, MC extracts exert direct pro-angiogenic signaling mediated via RAGE to overcome the anti-angiogenic effects of high BSA-AGEs, highlighting the biphasic RAGE-dependent mechanisms involved. This study enhances our understanding of the mechanisms underlying the pro-angiogenic effects of MC extracts in improvement of diabetes-impaired wound-healing.