Procyanidin B2 improves endothelial progenitor cell function and promotes wound healing in diabetic mice via activating Nrf2

One of the major reasons for the delayed wound healing in diabetes is the dysfunction of endothelial progenitor cells (EPCs) induced by hyperglycaemia. Improvement of EPC function may be a potential strategy for accelerating wound healing in diabetes. Procyanidin B2 (PCB2) is one of the major components of procyanidins, which exhibits a variety of potent pharmacological activities. However, the effects of PCB2 on EPC function and diabetic wound repair remain elusive. We evaluated the protective effects of PCB2 in EPCs with high glucose (HG) treatment and in a diabetic wound healing model. EPCs derived from human umbilical cord blood were treated with HG. The results showed that PCB2 significantly preserved the angiogenic function, survival and migration abilities of EPCs with HG treatment, and attenuated HG-induced oxidative stress of EPCs by scavenging excessive reactive oxygen species (ROS). A mechanistic study found the protective role of PCB2 is dependent on activating nuclear factor erythroid 2-related factor 2 (Nrf2). PCB2 increased the expression of Nrf2 and its downstream antioxidant genes to attenuate the oxidative stress induced by HG in EPCs, which were abolished by knockdown of Nrf2 expression. An in vivo study showed that intraperitoneal administration of PCB2 promoted wound healing and angiogenesis in diabetic mice, which was accompanied by a significant reduction in ROS level and an increase in circulating EPC number. Taken together, our results indicate that PCB2 treatment accelerates wound healing and increases angiogenesis in diabetic mice, which may be mediated by improving the mobilization and function of EPCs.     

Monotropein promotes angiogenesis and inhibits oxidative stress‐induced autophagy in endothelial progenitor cells to accelerate wound healing

Attenuating oxidative stress‐induced damage and promoting endothelial progenitor cell (EPC) differentiation are critical for ischaemic injuries. We suggested monotropein (Mtp), a bioactive constituent used in traditional Chinese medicine, can inhibit oxidative stress‐induced mitochondrial dysfunction and stimulate bone marrow‐derived EPC (BM‐EPC) differentiation. Results showed Mtp significantly elevated migration and tube formation of BM‐EPCs and prevented tert‐butyl hydroperoxide (TBHP)‐induced programmed cell death through apoptosis and autophagy by reducing intracellular reactive oxygen species release and restoring mitochondrial membrane potential, which may be mediated viamTOR/p70S6K/4EBP1 and AMPK phosphorylation. Moreover, Mtp accelerated wound healing in rats, as indicated by reduced healing times, decreased macrophage infiltration and increased blood vessel formation. In summary, Mtp promoted mobilization and differentiation of BM‐EPCs and protected against apoptosis and autophagy by suppressing the AMPK/mTOR pathway, improving wound healing in vivo. This study revealed that Mtp is a potential therapeutic for endothelial injury‐related wounds.     

Role of Endothelial Progenitor Cells and Inflammatory Cytokines in Healing of Diabetic Foot Ulcers

Background

To evaluate changes in endothelial progenitor cells (EPCs) and cytokines in patients with diabetic foot ulceration (DFU) in association with wound healing.

Methods

We studied healthy subjects, diabetic patients not at risk of DFU, at risk of DFU and with active DFU. We prospectively followed the DFU patients over a 12-week period. We also investigated similar changes in diabetic rabbit and mouse models of wound healing.

Results

All EPC phenotypes except the kinase insert domain receptor (KDR)⁺CD133⁺ were reduced in the at risk and the DFU groups compared to the controls. There were no major EPC differences between the control and not at risk group, and between the at risk and DFU groups. Serum stromal-cell derived factor-1 (SDF-1) and stem cell factor (SCF) were increased in DFU patients. DFU patients who healed their ulcers had lower CD34⁺KDR⁺ count at visits 3 and 4, serum c-reactive protein (CRP) and granulocyte-macrophage colony-stimulating factor (GM-CSF) at visit 1, interleukin-1 (IL-1) at visits 1 and 4. EPCs tended to be higher in both diabetic animal models when compared to their non-diabetic counterparts both before and ten days after wounding.

Conclusions

Uncomplicated diabetes does not affect EPCs. EPCs are reduced in patients at risk or with DFU while complete wound healing is associated with CD34⁺KDR⁺ reduction, suggesting possible increased homing. Low baseline CRP, IL-1α and GM-CSF serum levels were associated with complete wound healing and may potentially serve as prognostic markers of DFU healing. No animal model alone is representative of the human condition, indicating the need for multiple experimental models.     

Functional extracellular matrix hydrogel modified with MSC‐derived small extracellular vesicles for chronic wound healing

ObjectivesDiabetic wound healing remains a global challenge in the clinic and in research. However, the current medical dressings are difficult to meet the demands. The primary goal of this study was to fabricate a functional hydrogel wound dressing that can provide an appropriate microenvironment and supplementation with growth factors to promote skin regeneration and functional restoration in diabetic wounds.Materials and MethodsSmall extracellular vesicles (sEVs) were bound to the porcine small intestinal submucosa‐based hydrogel material through peptides (SC‐Ps‐sEVs) to increase the content and achieve a sustained release. NIH3T3 cell was used to evaluate the biocompatibility and the promoting proliferation, migration and adhesion abilities of the SC‐Ps‐sEVs. EA.hy926 cell was used to evaluate the stimulating angiogenesis of SC‐Ps‐sEVs. The diabetic wound model was used to investigate the function/role of SC‐Ps‐sEVs hydrogel in promoting wound healing.ResultsA functional hydrogel wound dressing with good mechanical properties, excellent biocompatibility and superior stimulating angiogenesis capacity was designed and facilely fabricated, which could effectively enable full‐thickness skin wounds healing in diabetic rat model.ConclusionsThis work led to the development of SIS, which shows an unprecedented combination of mechanical, biological and wound healing properties. This functional hydrogel wound dressing may find broad utility in the field of regenerative medicine and may be similarly useful in the treatment of wounds in epithelial tissues, such as the intestine, lung and liver.

Schematic illustration showing synthesis of the SC‐Ps scaffold dressing and nanoscale sEVs loaded SC‐Ps scaffold dressing and the potential application of the dressings in diabetic wound healing and skin reconstruction.     

糖尿病伤口愈合的分子机制

伤口愈合不良是糖尿病的一个并发症,严重时可能导致截肢,已成为糖尿病患者住院率最高的疾病,但是其确切的分子机制尚不清楚。目前研究发现糖尿病发生时有多种分子和细胞功能受损,如生长因子、一氧化氮、活性氧分子、基质金属蛋白酶、m icroRNA、内皮祖细胞等,最终导致糖尿病伤口愈合不良。本文将就其近年研究的相关分子机制予以详述。     

Circulating Endothelial Progenitor Cell and Platelet Microparticle Impact on Platelet Activation in Hypertension Associated with Hypercholesterolemia

Aim

The purpose of this project was to evaluate the influence of circulating endothelial progenitor cells (EPCs) and platelet microparticles (PMPs) on blood platelet function in experimental hypertension associated with hypercholesterolemia.

Methods

Golden Syrian hamsters were divided in six groups: (i) control, C; (ii) hypertensive-hypercholesterolemic, HH; (iii) ‘prevention’, HHin-EPCs, HH animals fed a HH diet and treated with EPCs; (iv) ‘regression’, HHfin-EPCs, HH treated with EPCs after HH feeding; (v) HH treated with PMPs, HH-PMPs, and (vi) HH treated with EPCs and PMPs, HH-EPCs-PMPs.

Results

Compared to HH group, the platelets from HHin-EPCs and HHfin-EPCs groups showed a reduction of: (i) activation, reflected by decreased integrin 3β, FAK, PI3K, src protein expression; (ii) secreted molecules as: SDF-1, MCP-1, RANTES, VEGF, PF4, PDGF and (iii) expression of pro-inflammatory molecules as: SDF-1, MCP-1, RANTES, IL-6, IL-1β; TFPI secretion was increased. Compared to HH group, platelets of HH-PMPs group showed increased activation, molecules release and proteins expression. Compared to HH-PMPs group the combination EPCs with PMPs treatment induced a decrease of all investigated platelet molecules, however not comparable with that recorded when EPC individual treatment was applied.

Conclusion

EPCs have the ability to reduce platelet activation and to modulate their pro-inflammatory and anti-thrombogenic properties in hypertension associated with hypercholesterolemia. Although, PMPs have several beneficial effects in combination with EPCs, these did not improve the EPC effects. These findings reveal a new biological role of circulating EPCs in platelet function regulation, and may contribute to understand their cross talk, and the mechanisms of atherosclerosis.     

High Calcium Bioglass Enhances Differentiation and Survival of Endothelial Progenitor Cells,Inducing Early Vascularization in Critical Size Bone Defects

Early vascularization is a prerequisite for successful bone healing and endothelial progenitor cells (EPC), seeded on appropriate biomaterials, can improve vascularization. The type of biomaterial influences EPC function with bioglass evoking a vascularizing response. In this study the influence of a composite biomaterial based on polylactic acid (PLA) and either 20 or 40% bioglass, BG20 and BG40, respectively, on the differentiation and survival of EPCs in vitro was investigated. Subsequently, the effect of the composite material on early vascularization in a rat calvarial critical size defect model with or without EPCs was evaluated. Human EPCs were cultured with β-TCP, PLA, BG20 or BG40, and seeding efficacy, cell viability, cell morphology and apoptosis were analysed in vitro. BG40 released the most calcium, and improved endothelial differentiation and vitality best. This effect was mimicked by adding an equivalent amount of calcium to the medium and was diminished in the presence of the calcium chelator, EGTA. To analyze the effect of BG40 and EPCs in vivo, a 6-mm diameter critical size calvarial defect was created in rats (n = 12). Controls (n = 6) received BG40 and the treatment group (n = 6) received BG40 seeded with 5×10⁵ rat EPCs. Vascularization after 1 week was significantly improved when EPCs were seeded onto BG40, compared to implanting BG40 alone. This indicates that Ca²⁺ release improves EPC differentiation and is useful for enhanced early vascularization in critical size bone defects.     

Application of platelet derived growth factor-BB and diabetic wound healing: the relationship with oxidative events

The reasons that cause delay in wound healing in diabetes are a decrease in the level of growth factors secretion, an increase in the destruction of growth factors and in oxidative stress. Platelet derived growth factor (PDGF) is one of the important growth factors that play a role in all phases of wound healing. This study investigates time-dependent effects of topically PDGF-BB administration on oxidative events on the healing of dorsolateral-excisional wounds in diabetic rats. Forty-two female Wistar-albino rats with streptozotocin-induced diabetes were divided into four groups: control group, untreated group, chitosan-treated group, chitosan?+?PDGF-BB-treated group. Two identical full-thickness excisional skin wounds were made under anaesthesia in all rats except for the control group. In the PDGF-BB-treated and chitosan-treated groups, the wounds were treated topically PDGF-BB (7?ng/mL, single daily dose) and blank chitosan gel (equal amount) after wounding, respectively. After these administrations, on day 3 and day 7 of wound healing, rats were sacrificed. Thiobarbituric acid reactive substances, glutathione, nitric oxide, ascorbic acid levels, and superoxide dismutase activity in wound tissues were spectrophotometrically measured. PDGF-BB administration significantly increased TBARS levels and non-enzymatic antioxidant levels in early phase of diabetic wound healing. PDGF-BB dramatically reduced NO_x levels on day 3 and sharply increased NO_x levels on day 7 of wound healing. Consequently, PDGF-BB administration can be effective on oxidative balance in the early phase of diabetic wound healing.     

Treatment of non-healing wounds with autologous bone marrow cells, platelets, fibrin glue and collagen matrix

Background aimsRecalcitrant diabetic wounds are not responsive to the most common treatments. Bone marrow-derived stem cell transplantation is used for the healing of chronic lower extremity wounds.MethodsWe report on the treatment of eight patients with aggressive, refractory diabetic wounds. The marrow-derived cells were injected/applied topically into the wound along with platelets, fibrin glue and bone marrow-impregnated collagen matrix.ResultsFour weeks after treatment, the wound was completely closed in three patients and significantly reduced in the remaining five patients.ConclusionsOur study suggests that the combination of the components mentioned can be used safely in order to synergize the effect of chronic wound healing.     

Identification of the Critical Therapeutic Entity in Secreted Hsp90α That Promotes Wound Healing in Newly Re-Standardized Healthy and Diabetic Pig Models

Chronic and non-healing skin wounds represent a significant clinical, economic and social problem worldwide. Currently, there are few effective treatments. Lack of well-defined animal models to investigate wound healing mechanisms and furthermore to identify new and more effective therapeutic agents still remains a major challenge. Pig skin wound healing is close to humans. However, standardized pig wound healing models with demonstrated validity for testing new wound healing candidates are unavailable. Here we report a systematic evaluation and establishment of both acute and diabetic wound healing models in pigs, including wound-creating pattern for drug treatment versus control, measurements of diabetic parameters and the time for detecting delayed wound healing. We find that treatment and control wounds should be on the opposite and corresponding sides of a pig. We demonstrate a strong correlation between duration of diabetic conditions and the length of delay in wound closure. Using these new models, we narrow down the minimum therapeutic entity of secreted Hsp90α to a 27-amino acid peptide, called fragment-8 (F-8). In addition, results of histochemistry and immunohistochemistry analyses reveal more organized epidermis and dermis in Hsp90α-healed wounds than the control. Finally, Hsp90α uses a similar signaling mechanism to promote migration of isolated pig and human keratinocytes and dermal fibroblasts. This is the first report that shows standardized pig models for acute and diabetic wound healing studies and proves its usefulness with both an approved drug and a new therapeutic agent.     

Expression of TGF-β3 in isolated fibroblasts from foreskin

Background:

The multifunctional transforming growth factor beta (TGF-β) is a glycoprotein that exists in three isoforms. TGF-β3 expression increases in fetal wound healing and reduces fibronectin and collagen I and III deposition, and also improves the architecture of the neodermis which is a combination of blood vessels and connective tissue during wound healing. Fibroblasts are key cells in the wound healing process. TGF-β3 plays a critical role in scar-free wound healing and fibroblast actions in the wound healing process. The aim of this study was to express the TGF-β3 gene (tgf-b3) in human foreskin fibroblasts (HFF’s).

Methods:

We obtained HFF’s from a newborn and a primary fibroblast culture was prepared. The cells were transfected with TGF-β3-pCMV6-XL5 plasmid DNA by both lipofection and electroporation. Expression of TGF-β3 was measured by enzyme-linked immunosorbent assay (ELISA).

Results:

The highest TGF-β3 expression (8.3-fold greater than control) was obtained by lipofection after 72 hours using 3 µl of transfection reagent. Expression was 1.4-fold greater than control by electroporation.

Conclusions:

In this study, we successfully increased TGF-β3 expression in primary fibroblast cells. In the future, grafting these transfected fibroblasts onto wounds can help the healing process without scarring.Key Words: Fibroblasts, Gene expression, TGF-β3     

Application of decellularized human reticular allograft dermal matrix promotes rapid re-epithelialization in a diabetic murine excisional wound model

Background aimsThe treatment and care of human wounds represent an enormous burden on the medical system and patients alike. Chronic or delayed healing wounds are characterized by the inability to form proper granulation tissue, followed by deficiencies in keratinocyte migration and wound re-epithelialization, leading to increased likelihood of infection and poor wound outcomes. Human reticular acellular dermal matrix (HR-ADM) is one type of tissue graft developed to enhance closure of delayed healing wounds that has demonstrated clinical utility through accelerating closure of lower extremity diabetic ulcers, but the mechanisms underlying this clinical success are not well understood.MethodsThe authors utilized a diabetic murine splinted excisional wound model to investigate the effects of HR-ADM application on wound closure.ResultsThe authors demonstrate that application of HR-ADM served as a dermal scaffold and promoted rapid re-epithelialization and keratinocyte proliferation, resulting in accelerated wound closure while minimizing granulation tissue formation. HR-ADM-applied wounds also demonstrated evidence of cellular infiltration, neovascularization and collagen remodeling by the host organism.ConclusionsThese data suggest that HR-ADM supports epidermal closure in delayed healing wounds and remodeling of the matrix into host tissue, lending further support to the clinical success of HR-ADM described in clinical reports.     

Ameliorative effects of clindamycin - nanoceria conjugate: A ROS responsive smart drug delivery system for diabetic wound healing study

BackgroundIncreased incidence of antibiotic-resistant species calls for development of new types of nano-medicine that can be used for healing of bacteria-caused wounds, such as diabetic foot ulcer. As diabetic patients have inefficient defense mechanism against reactive oxygen species (ROS) produced in our body as a by-product of oxygen reduction, the process of wound healing takes longer epithelialisation period. Ceria nanoparticles (CNPs) are well-known for their antibacterial and ROS-scavenging nature. Yet till now no significant effort has been made to conjugate ceria nanoparticles with drugs to treat diabetic wounds.MethodsIn this experiment, CNPs were synthesized in-house and clindamycin hydrochloride was loaded onto it by physical adsorption method for reactive oxygen species responsive drug delivery. Various physico-chemical characterisations such as Transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, Energy dispersive X-ray, Thermogravimetric study etc. were performed to affirm the formation of both nanoceria along with drug encapsulated nanoceria.ResultsBoth of these as-prepared formulations inhibited the growth of Gram-positive as well as Gram-negative bacteria confirmed by Disk diffusion study; exhibiting their antibacterial effect. In-vitro drug release study was carried out in physiological environment both in absence and presence of hydrogen peroxide solution to test the reactive ROS-responsiveness of the drug loaded nanocomposites. It also exhibited faster wound healing in diabetes-induced rats. Therefore, it could successfully lower the amount of serum glucose level, inflammation cytokines, hepatotoxic and oxidative stress markers in diabetic rats as confirmed by various ex vivo tests conducted.ConclusionThus, drug loaded ceria nanoparticles have the potential to heal diabetic wounds successfully and can be considered to be useful for the fabrication of appropriate medicated suppositories beneficial for diabetic foot ulcer treatment in future.     

mESC衍生内皮祖细胞定向分化为血管内皮细胞促伤口愈合

缺血性功能障碍是重要的全球健康问题。血管内皮细胞 (vascular endothelial cell, VEC) 在血管生成和创面修复中发挥关键作用,血管重建不足可导致慢性不愈合伤口。因此,了解有效的血管内皮细胞生成策略有助于受损组织中的血管再生。胚胎干细胞 (embryonic stem cell, ESC) 在组织的内皮化研究中应用广泛。内皮祖细胞 (endothelial progenitor cell, EPC) 是血管内皮细胞发育中不可或缺的部分。本研究目的在于找到一种小鼠胚胎干细胞 (mouse embryonic stem cell, mESC) 衍生为内皮祖细胞的快速、易筛选且高重复性的方法,并从内皮祖细胞定向分化中获得存活率高和功能性好的血管内皮细胞。结果表明,胚胎干细胞通过10 ng/mL VEGF和5 ng/mL bFGF定向诱导分化为增殖能力强的“铺路石”样祖细胞。同时,差异贴壁法有助于EPC的筛选。而EPC可诱导3 d的祖细胞高表达CD133和CD34(相对表达量分别为0.88 ± 0.04和2.12 ± 0.02);采用acctuse酶消化祖细胞,并在50 ng/mL VEGF和25 ng/mL bFGF的条件下诱导7 d分化为血管内皮样细胞,该细胞不仅高表达内皮细胞标志基因CD31、CD144、LAMA5、Tek、KDR和vWF,高表达标志蛋白CD31、CD144、LAMA5(相对表达量分别为1.07 ± 0.03、0.60 ± 0.02和0.70 ± 0.02),而且具有良好的迁移、成管和Weibel Palade (W-P) 小体形成能力。随后,将PBS、EPC和VEC分别应用于大小相同的创面治疗,EPC和VEC均能加快组织愈合程度(相对愈合率分别为78.93 ± 75.35%、95.57 ± 83.73%和100.00 ± 0.00%),VEC明显增强了伤口的血管生成能力和炎症反应。该研究初步证实,mESC衍生的EPC定向诱导7 d后可分化为血管内皮细胞。此内皮细胞具有较好的组织修复功能,干细胞促进血管生成的生理途径有望成为组织重塑的新靶点。     

EGF and TGF-alpha in wound healing and repair

Wound healing is a localized process which involves inflammation, wound cell migration and mitosis, neovascularization, and regeneration of the extracellular matrix. Recent data suggest the actions of wound cells may be regulated by local production of peptide growth factors which influence wound cells through autocrine and paracrine mechanisms. Two peptide growth factors which may play important roles in normal wound healing in tissues such as skin, cornea, and gastrointestinal tract are the structurally related peptides epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). EGF/TGF-alpha receptors are expressed by many types of cells including skin keratinocytes, fibroblasts, vascular endothelial cells, and epithelial cells of the GI tract. In addition, EGF or TGF-alpha are synthesized by several cells involved in wound healing including platelets, keratinocytes, and activated macrophages. Healing of a variety of wounds in animals and patients was enhanced by treatment with EGF or TGF-alpha. Epidermal regeneration of partial thickness burns on pigs or dermatome wounds on patients was accelerated with topical application of EGF or TGF-alpha, and EGF treatment accelerated healing of gastroduodenal ulcers. EGF also increased tensile strength of skin incisions in rats and corneal incisions in rabbits, cats, and primates. Additional research is needed to better define the roles of EGF, TGF-alpha and their receptor in normal wound healing, to determine if alterations have occurred in the EGF/TGF-alpha system in chronic wounds, and optimize vehicles for effective delivery of peptide growth factors to wounds.     

Adiponectin promotes endothelial progenitor cell number and function

Obesity-linked diseases are associated with suppressed endothelial progenitor cell (EPC) function. Adiponectin is an adipose-derived protein that is downregulated in obese and diabetic subjects. Here, we investigated the effects of adiponectin on EPCs. EPC levels did not increase in adiponectin deficient (APN-KO) in response to hindlimb ischemia. Adenovirus-mediated delivery of adiponectin increased EPC levels in both WT and APN-KO mice. Incubation of human peripheral blood mononuclear cells with adiponectin led to an increase of the number of EPCs. Adiponectin induced EPC differentiation into network structures and served as a chemoattractant in EPC migration assays. These data suggest that hypoadiponectinemia may contribute to the depression of EPC levels that are observed in patients with obesity-related cardiovascular disorders.     

Curcumin differentially regulates TGF-beta1, its receptors and nitric oxide synthase during impaired wound healing

Wound healing is a highly ordered process, requiring complex and coordinated interactions involving peptide growth factors of which transforming growth factor-beta (TGF-beta) is one of the most important. Nitric oxide is also an important factor in healing and its production is regulated by inducible nitric oxide synthase (iNOS). We have earlier shown that curcumin (diferuloylmethane), a natural product obtained from the plant Curcuma longa, enhances cutaneous wound healing in normal and diabetic rats. In this study, we have investigated the effect of curcumin treatment by topical application in dexamethasone-impaired cutaneous healing in a full thickness punch wound model in rats. We assessed healing in terms of histology, morphometry, and collagenization on the fourth and seventh days post-wounding and analyzed the regulation of TGF-beta1, its receptors type I (tIrc) and type II (tIIrc) and iNOS. Curcumin significantly accelerated healing of wounds with or without dexamethasone treatment as revealed by a reduction in the wound width and gap length compared to controls. Curcumin treatment resulted in the enhanced expression of TGF-beta1 and TGF-beta tIIrc in both normal and impaired healing wounds as revealed by immunohistochemistry. Macrophages in the wound bed showed an enhanced expression of TGF-beta1 mRNA in curcumin treated wounds as evidenced by in situ hybridization. However, enhanced expression of TGF-beta tIrc by curcumin treatment observed only in dexamethasone-impaired wounds at the 7th day post-wounding. iNOS levels were increased following curcumin treatment in unimpaired wounds, but not so in the dexamethasone-impaired wounds. The study indicates an enhancement in dexamethasone impaired wound repair by topical curcumin and its differential regulatory effect on TGF-beta1, it's receptors and iNOS in this cutaneous wound-healing model.