Newly Defined ATP-Binding Cassette Subfamily B Member 5 Positive Dermal Mesenchymal Stem Cells Promote Healing of Chronic Iron-Overload Wounds via Secretion of Interleukin-1 Receptor Antagonist.

In this study, we report the beneficial effects of a newly identified dermal cell subpopulation expressing the ATP-binding cassette subfamily B member 5 (ABCB5) for the therapy of nonhealing wounds. Local administration of dermal ABCB5+ -derived mesenchymal stem cells (MSCs) attenuated macrophage-dominated inflammation and thereby accelerated healing of full-thickness excisional wounds in the iron-overload mouse model mimicking the nonhealing state of human venous leg ulcers. The observed beneficial effects were due to interleukin-1 receptor antagonist (IL-1RA) secreted by ABCB5+ -derived MSCs, which dampened inflammation and shifted the prevalence of unrestrained proinflammatory M1 macrophages toward repair promoting anti-inflammatory M2 macrophages at the wound site. The beneficial anti-inflammatory effect of IL-1RA released from ABCB5+ -derived MSCs on human wound macrophages was conserved in humanized NOD-scid IL2rγ null mice. In conclusion, human dermal ABCB5+ cells represent a novel, easily accessible, and marker-enriched source of MSCs, which holds substantial promise to successfully treat chronic nonhealing wounds in humans. Stem Cells 2019;37:1057-1074.

NLRP3 activation induced by neutrophil extracellular traps sustains inflammatory response in the diabetic wound.

Persistent inflammatory response in the diabetic wound impairs the healing process, resulting in significant morbidity and mortality. Mounting evidence indicate that the activation of Nod-like receptor protein (NLRP) 3 inflammasome in macrophages (MΦ) contributes to the sustained inflammatory response and impaired wound healing associated with diabetes. However, the main trigger of NLRP3 inflammasome in the wounds is not known. Neutrophils, as sentinels of the innate immune system and key stimulators of MΦ, are immune cells that play the main role in the early phase of healing. Neutrophils release extracellular traps (NETs) as defense against pathogens. On the other hand, NETs induce tissue damage. NETs have been detected in the diabetic wound and implicated in the impaired healing process, but the mechanism of NETs suspend wound healing and its role in fostering inflammatory dysregulation are elusive. Here, we report that NLRP3 and NETs production are elevated in human and rat diabetic wounds. NETs overproduced in the diabetic wounds triggered NLRP3 inflammasome activation and IL-1ß release in MΦ. Furthermore, NETs up-regulated NLRP3 and pro-IL-1ß levels via the TLR-4/TLR-9/NF-κB signaling pathway. They also elicited the generation of reactive oxygen species, which facilitated the association between NLRP3 and thioredoxin-interacting protein, and activated the NLRP3 inflammasome. In addition, NET digestion by DNase I alleviated the activation of NLRP3 inflammasome, regulated the immune cell infiltration, and accelerated wound healing in diabetic rat model. These findings illustrate a new mechanism by which NETs contribute to the activation of NLRP3 inflammasome and sustained inflammatory response in the diabetic wound.

Potential correlation of wound bed score and biomarkers in chronic lower leg wounds: an exploratory study.

OBJECTIVE: The wound bed score is a validated tool to monitor wound healing in chronic wounds, and depends on visual examination by trained personnel. This study describes the feasibility of adding some biochemical and immunohistochemical parameters to increase the objectivity and specificity of the wound bed score Method: Patients with chronic wounds on the lower leg with different durations were enrolled to assess the correlation between the wound bed score and specific wound-related biomarkers, namely MMP-9, MMP-2, NGAL, albumin, integrin α2/ß1, and other histochemical (CD68, PK1, CD32, fractalkine, periostin) and immunocytochemical markers from biopsies and smears taken from wound edges and bed. RESULTS: The study examined samples from 10 patients. Patients with an unfavourable wound bed score had a low expression of periostin and fractalkine in the wound bed tissue. CD68 PK1 showed a low or negative expression in the majority of the samples. Patients negative for CD68 PK1 were also negative for CD32. Principal component analysis revealed that the albumin level and the amount of proteins were associated with a high wound bed score. Two different subsets of patients could be discriminated either by integrin α2/ß1 and albumin percentages or the MMP-9 and MMP-2 activities Conclusion: These preliminary results pave the way towards an improved wound status diagnosis and an advanced quality of wound care and management. These findings need confirming with a large number of patients and at different time points.

The Influence of Chronic Wound Extracts on Inflammatory Cytokine and Histatin Stability.

Chronic ulcers represent a major health burden in our society. Despite many available therapies, a large number of ulcers do not heal. Protein based therapies fail in part due to proteolytic activity in the chronic wound bed. The aim of this in vitro study was to determine whether typical inflammatory cytokines and human salivary histatins remain stable when incubated with chronic wound extracts. Furthermore we determined whether a short exposure of histatins or cytokines was sufficient to exert long term effects on fibroblast migration. Stability of human recombinant cytokines IL-6 and CXCL8, and histatin variants (Hst1, Hst2, cyclic Hst1, minimal active domain of Hst1) in the presence of chronic wound extracts isolated from non-healing ulcers, was monitored by capillary zone electrophoresis. Migration-stimulating activity was assessed using a dermal fibroblast wound healing scratch assay. Histatins and cytokines stayed stable in saline for > 24 h at 37°C, making them ideal as an off-the-shelf product. However, incubation with chronic wound extracts resulted in serious breakdown of Hst1 and Hst2 (~50% in 8 h) and to lesser extent cyclic Hst1 and the minimal active domain of Hst1 (~20% in 8 h). The cytokines IL-6 and CXCL8 were more stable in chronic wound extracts (~40% degradation in 96 h). An initial 8-hour pulse of histatins or cytokines during a 96-hour study period was sufficient to stimulate fibroblast migration equally well as a continuous 96-hour exposure, indicating that they may possibly be used as novel bioactive therapeutics, exerting their activity for up to four days after a single exposure.

Clinical Safety and Preliminary Efficacy of Plasmid pUDK-HGF Expressing Human Hepatocyte Growth Factor (HGF) in Patients with Critical Limb Ischemia.

OBJECTIVE: Critical limb ischemia (CLI) is the most severe form of peripheral arterial disease and a major unmet public health care need. This phase I clinical study was performed to assess the safety and preliminary efficacy of naked plasmid DNA (pUDK-HGF) expressing human hepatocyte growth factor (HGF) in patients with critical limb ischemia (CLI). DESIGN: Twenty-one patients with CLI were enrolled and randomly divided into four dose groups (4-16 mg) to receive local injection of pUDK-HGF into ischemic calf and/or thigh muscles twice on days 1 and 15. Safety, including adverse events and physiological parameters, and preliminary efficacy, including pain severity score (VAS), ulcer size, transcutaneous oxygen pressure (TcPO2), and ankle brachial index (ABI), were evaluated throughout a 3 month follow up period. RESULTS: All doses of pUDK-HGF were well tolerated by the patients. None of the adverse effects was considered to be related to pUDK-HGF injection. Two significant clinical results were observed after pUDK-HGF administration. The mean VAS value of all patients decreased from 4.52 at baseline to 0.30 (p < .01), and pain had disappeared in 14 out of 17 evaluable patients by day 91. Two of four ulcers had completely healed, with the other two patients having more than 25% ulcer size reduction in the long axis diameter. Of five patients with gangrene, one gangrenous wound had healed completely and two patients showed marked size reduction by day 91. The mean hemodynamic parameters (ABI, TcPO2) were also improved. CONCLUSION: Intramuscular injection of pUDK-HGF is safe, and may provide symptomatic relief for CLI patients. A larger, randomized, double blinded phase II trial will provide more information on safety and efficacy.

Macrophage PPARγ and impaired wound healing in type 2 diabetes.

Macrophages undergo a transition from pro-inflammatory to healing-associated phenotypes that is critical for efficient wound healing. However, the regulation of this transition during normal and impaired healing remains to be elucidated. In our studies, the switch in macrophage phenotypes during skin wound healing was associated with up-regulation of the peroxisome proliferator-activated receptor (PPAR)γ and its downstream targets, along with increased mitochondrial content. In the setting of diabetes, up-regulation of PPARγ activity was impaired by sustained expression of IL-1ß in both mouse and human wounds. In addition, experiments with myeloid-specific PPARγ knockout mice indicated that loss of PPARγ in macrophages is sufficient to prolong wound inflammation and delay healing. Furthermore, PPARγ agonists promoted a healing-associated macrophage phenotype both in vitro and in vivo, even in the diabetic wound environment. Importantly, topical administration of PPARγ agonists improved healing in diabetic mice, suggesting an appealing strategy for down-regulating inflammation and improving the healing of chronic wounds.

Enhanced deposition of cartilage oligomeric matrix protein is a common feature in fibrotic skin pathologies.

Skin fibrosis is characterized by activated fibroblasts and an altered architecture of the extracellular matrix. Excessive deposition of extracellular matrix proteins and altered cytokine levels in the dermal collagen matrix are common to several pathological situations such as localized scleroderma and systemic sclerosis, keloids, dermatosclerosis associated with venous ulcers and the fibroproliferative tissue surrounding invasively growing tumors. Which factors contribute to altered organization of dermal collagen matrix in skin fibrosis is not well understood. We recently demonstrated that cartilage oligomeric matrix protein (COMP) functions as organizer of the dermal collagen I network in healthy human skin (Agarwal et al., 2012). Here we show that COMP deposition is enhanced in the dermis in various fibrotic conditions. COMP levels were significantly increased in fibrotic lesions derived from patients with localized scleroderma, in wound tissue and exudates of patients with venous leg ulcers and in the fibrotic stroma of biopsies from patients with basal cell carcinoma. We postulate enhanced deposition of COMP as one of the common factors altering the supramolecular architecture of collagen matrix in fibrotic skin pathologies. Interestingly, COMP remained nearly undetectable in normally healing wounds where myofibroblasts transiently accumulate in the granulation tissue. We conclude that COMP expression is restricted to a fibroblast differentiation state not identical to myofibroblasts which is induced by TGFß and biomechanical forces.

Involvement of IL-1 and oncostatin M in acanthosis associated with hypertensive leg ulcer.

Hypertensive leg ulcer (HLU) is an inflammatory disease characterized by intense pain, alteration of vascularization, and skin necrosis. The optimal treatment relies on surgical removal of necrotic tissues covered by a split-skin graft. We studied the histomorphology of the lesions and investigated the involvement of inflammatory cells and cytokines to further define the physiopathology of HLU. We report epidermis acanthosis and a preferential occlusion of the precapillary arterioles with infiltration of neutrophils, macrophages, and T lymphocytes in the dermis. OSM, IL-1ß, and IL-6 were overexpressed in the ulcer, whereas the Th17-derived cytokines were not. In vitro, the addition of IL-1ß and OSM promoted acanthosis and destructuring of reconstructed epidermis. Exogenous IL-1ß and OSM synergistically induced epidermal acanthosis in mice. These data show that OSM and IL-1ß are not only a biological characteristic signature of HLU, but these cytokines reflect a specific inflammatory state, directly involved in the pathogenesis. We suggest that anti-cytokine biotherapies could be an alternative strategy to surgery to treat HLU.

A prospective pilot study of ultrasound therapy effectiveness in refractory venous leg ulcers.

Venous insufficiency is the most common cause of leg ulcers in the United States. Venous leg ulcers cost the health care system billions of dollars annually, and healing rates are less than 70% with standard of care; therefore, new therapies are needed to increase healing times and minimize associated costs. Non contact ultrasound therapy has been used to treat a variety of chronic wounds including venous leg ulcers, and it is thought that ultrasound has an effect on decreasing the bacterial count in wounds, although the exact mechanism of action of ultrasound is yet to be determined. We conducted an open labelled pilot study of 10 refractory venous ulcers of large size to determine the effect of non contact ultrasound on wound closure, bacterial counts, expression of inflammatory cytokines and pain reduction. We lacked a sham control group but we compared the baseline and end of treatment assessments and noted the differences. We found a significant reduction in wound area (P = 0·0039) over the 4-week treatment period. We also found a decline in individual and total bacterial counts; however, these differences were not significant. For all patients, there was also a trend toward reduced inflammatory cytokine expression compared with baseline levels; however, this reduction did not reach statistical significance. Interestingly, there was a correlation between healing and change in cytokine expression, which showed statistically significance for tumour necrosis factor (TNF)-αP = 0·0395, IL-1a P = 0·0351, IL-6 P = 0·0508, IL-8 P = 0·0990. Pain as measured by the visual analogue scale (VAS) was reduced from 4 at the baseline to 2·7 by the end of the study. In conclusion, we found that patients treated with ultrasound therapy and compression therapy show clinical improvement over the course of 4 weeks and had a decrease in inflammatory cytokines, bacterial counts and pain.

Selective release of cytokines, chemokines, and growth factors by minced skin in vitro supports the effectiveness of autologous minced micrografts technique for chronic ulcer repair.

A new effective surgical procedure to repair chronic ulcers called minced micrografts technique has been recently reported. The technique consists in spreading a finely minced skin sample upon the wound bed. In this study, we investigate the in vitro release of cytokines (interleukin-6, tumor necrosis factor-α, interleukin-1α, and granulocyte-colony stimulating factor), chemokines (monocyte chemoattractant protein-1 and growth-related oncogene-α), and growth factors (platelet-derived growth factor, basic fibroblast growth factor, vascular endothelial growth factor, hepatocyte growth factor, and nerve growth factor) by minced (referred to as the minced sample) vs. not minced (referred to as the whole sample) human skin biopsy samples from the same donor. Factor release in the culture medium at different time points was detected using a multiplexed protein assay. The minced sample, which could behave like the skin fragments used in vivo in the autologous minced micrografts technique, expressed higher levels of tumor necrosis factor-α, interleukin-1α, platelet-derived growth factor, and basic fibroblast growth factor, and lower levels of interleukin-6, monocyte chemoattractant protein-1, growth related oncogene-α, and vascular endothelial growth factor compared with the whole sample. In conclusion, mincing of healthy skin may allow appropriate regulation of the inflammatory phase of wound healing and could induce overexpression of some growth factors, which facilitates the proliferative phase of healing.

Elevated uric acid correlates with wound severity.

Chronic venous leg ulcers are a major health issue and represent an often overlooked area of biomedical research. Nevertheless, it is becoming increasingly evident that new approaches to enhance healing outcomes may arise through better understanding the processes involved in the formation of chronic wounds. We have for the first time shown that the terminal purine catabolite uric acid (UA) is elevated in wound fluid (WF) from chronic venous leg ulcers with relative concentrations correlating with wound chronicity. We have also shown a corresponding depletion in UA precursors, including adenosine, with increased wound severity. Further, we have shown that xanthine oxidase, the only enzyme in humans that catalyses the production of UA in conjunction with a burst of free radicals, is active in chronic WF. Taken together, this provides compelling evidence that xanthine oxidase may play a critical role in the formation of chronic wounds by prolonging the inflammatory process.

Skin iron deposition characterises lipodermatosclerosis and leg ulcer.

BACKGROUND: It is commonly reported that chronic venous disease (CVD) increases the skin iron content in which the excess is stored as haemosiderin. Despite increasing interest in the role of haemosiderin in venous ulceration, no study has systematically evaluated the occurrence of iron overload in the limbs of patients with CVD. PURPOSE: To evaluate skin haemosiderin deposition in relation to the presence and severity of skin changes in CVD legs designated according to the clinical, etiologic, anatomic and pathophysiologic (CEAP) classification. METHODS: A total of 85 skin biopsies were taken from the medial aspect of 49 limbs with CVD of CEAP clinical stages C2, C3, C4 and C6. The content of ferric ions was assessed by Perl's Prussian Blue (PPB) stain. RESULTS: No haemosiderin deposition was found in normal skin of C2, C3 and C4A legs, in less severe regions of pigmentation and in some parts of more severely affected limbs. Haemosiderin was always present in lipodermatosclerotic skin and ulcers. Occasionally, haemosiderin was found in the apparently normal perilesional skin of C4b and C6 legs. The regenerating dermis at the base of healing ulcers showed none or light haemosiderin deposition. CONCLUSION: Iron overload is not present in the less severe stages of skin damage due to CVD but lipodermatosclerosis and leg ulcers are always accompanied by haemosiderin deposition. In fact, no severe skin changes occur in CVD legs until iron overload occurs. Our results are in agreement with previous reports suggesting that a genetic inability to counteract skin iron overload is present in these patients. A more detailed analysis of disordered iron metabolism should be undertaken in CVD patients.

Topical treatment with the vitamin D analogue calcipotriol enhances the upregulation of the antimicrobial protein hCAP18/LL-37 during wounding in human skin in vivo.

Cathelicidin antimicrobial protein, hCAP18, is the sole cathelin protein in human. Its C-terminal peptide, which is released enzymatically from the holoprotein, has broad antimicrobial activity but also has effects on eukaryotic cells. hCAP18 is present in leukocytes and is produced at epithelial interfaces as part of the innate immune system. In normal intact skin, there is low constitutive expression of hCAP18, which is rapidly upregulated upon injury. Accumulating evidence indicates that hCAP18/LL-37 may serve a key role in protecting the integrity of the epithelium and also actively promote re-epithelialization and tissue repair. Molecular mechanisms responsible for controlling hCAP18 gene expression in vivo are only partly understood. Vitamin D(3) and its analogue calcipotriol were recently found to directly induce transcription of the hCAP18 gene via functional vitamin D responsive elements in the hCAP18 gene promoter. Skin is the major source for vitamin D(3) in human, where its production is dependent on ultraviolet B (UVB) radiation. We have shown that exposure to UVB, sufficient to produce vitamin D(3), upregulates hCAP18 in human skin in vivo. In the present study, we demonstrate that the upregulation of hCAP18/LL-37 following acute skin injury is further enhanced, at both hCAP18 mRNA and protein levels, after topical treatment with the vitamin D(3) analogue calcipotriol. In chronic ulcers, calcipotriol treatment upregulated hCAP18 mRNA, whereas no consistent upregulation of hCAP18 protein was detected. Our results further support the role of vitamin D(3) as a key physiologic regulator of hCAP18/LL-37 in human skin.

Fibronectin fragmentation is a feature of periodontal disease sites and diabetic foot and leg wounds and modifies cell behavior.

BACKGROUND: Fibronectin (FN) undergoes fragmentation in periodontal disease sites and in poorly healing diabetic wounds. The biologic effects of FN fragments on wound healing remain unresolved. This study characterized the pattern of FN fragmentation and its effects on cellular behavior compared to intact FN. METHODS: Polyclonal antibodies were raised against FN and three defined recombinant segments of FN and used to analyze gingival crevicular fluid from periodontal disease sites in systemically healthy subjects and in subjects with diabetes, as well as chronic leg and foot wound exudates from subjects with diabetes. Subsequently, the behavior of human gingival fibroblasts (hGFs) and HT1080 reference cells were analyzed by measuring cell attachment, migration, and chemotaxis in the presence of intact FN or recombinant FN fragments. RESULTS: FN fragmentation was evident in fluids from periodontal disease sites and diabetic leg and foot wounds. However, no fragmentation pattern distinguished systemically healthy subjects from subjects with diabetes. hGFs and HT1080 cells required significantly higher concentrations of FN fragments to achieve attachment comparable to intact FN. Cells cultured on FN fragments also were morphologically different from cells cultured on full-length FN. Migration was reduced for hGFs cultured on FN fragments relative to full-length FN. In contrast, FN fragments increased HT1080 fibrosarcoma cell migration over intact FN. CONCLUSIONS: FN fragmentation is a prominent feature of periodontal and chronic leg and foot wounds in diabetes. Furthermore, cell culture assays confirmed the hypothesis that exposure to defined FN fragments significantly alters cell behavior.

Tissue and urinary haemosiderin in chronic leg ulcers.

OBJECTIVE: The aim of this study was to assess the relationship between urinary and tissue haemosiderin in chronic leg ulcers, and its value as a diagnostic test for venous ulceration. METHODS: 45 patients with chronic leg ulcers were recruited to the study (24 venous, 6 ischaemic, 6 lymphoedematous, 5 rheumatoid and 4 sickle cell). Punch biopsy of the ulcer edge was taken and early morning urine samples were collected. Positive Prussian-blue urinary haemosiderin granules were measured with a haemocytometer following Perls' staining. The percentage area of histological section staining positively with Perls' was measured using image analysis. RESULTS: 84 urine samples and 46 ulcer biopsies were collected. Urinary haemosiderin was present in 92% of venous ulcer patients, but was absent in the ischaemic ulcer patients (p<0.0001). Significantly more urinary haemosiderin granules were detected in venous ulcer patients compared with patients who had lymphoedema (p<0.05). Tissue haemosiderin was detected in all ulcer types investigated. No correlation was found between the amounts of haemosiderin deposited in the tissue and the amount found in urine (r(2)=0.06). CONCLUSIONS: Haemosiderin is present in the urine of most patients with venous ulcers but not in ischaemia ulcers.

[Jingjielianqiao Decoction promotes leg ulcer healing in rabbits].

OBJECTIVE: To study the effect of Jingjielianqiao Decoction in promoting leg ulcer in rabbits. METHODS: Nine adult male New Zealand albino rabbits with chronic leg ulcers were randomized into 3 groups, namely group A treated with Jingjielianqiao Decoction, group B with Shengjiyuhong Decoction, and group C with normal saline. Gross observation of the wounds was carried out regularly for evaluating the changes in the ulcerous area, depth and wound surface excretion. After 3 weeks of treatment, the tissues on the edge of the ulcer were sampled and prepared for routine pathological examination, electron microscopy and immunohistochemistry for vascular endothelial growth factor (VEGF) and CD34. The number of blood vessels and their areas were also recorded. RESULTS: The wounds showed no significant differences between the 3 groups by gross observation during the treatment, but after completion of the 3-week treatment, routine pathological examination and electron microscopy revealed significant differences between the groups. Immunohistochemistry for VEGF and CD34 yielded comparable results between groups A and B (positive control), but showed significant differences between group C and the other two groups (P<0.01). CONCLUSION: Jingjielianqiao Decoction and Shengjiyuhong Decoction can obviously promote the healing of leg ulcer in rabbits.

Expression of growth factors and growth factor receptor in non-healing and healing ischaemic ulceration.

OBJECTIVES: To characterise the histological and cytokinetic characteristics of purely ischaemic ulcers and the processes that underpin healing following successful revascularisation. DESIGN: Prospective observational study. MATERIALS AND METHODS: Biopsies were taken immediately pre- and 6 weeks following successful revascularisation of solely ischaemic ulceration. They were evaluated for morphological differences using H&E staining for the platelet derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), TGFbeta receptorIII (TGFbetaRIII), transforming growth factor beta 1 and 3 (TGFbeta1 and TGFbeta3) and von Willebrand factor (vWF) expression using immunohistochemistry. Localisation and quantification of these growth factors and receptors was assessed systematically by three independent investigators who were blinded to the timing of biopsy. RESULTS: Pre-operatively, small vessel vasculitis, necrosis and infection with a profuse neutrophil and macrophage infiltrate was observed in all samples. Post-operative biopsies revealed a proliferation of new capillaries in and around the ulcer edge and base. vWF staining confirmed an endothelial layer within these new vessels. Following successful revascularisation there was less infection and inflammation with minimal vasculitis. These newly formed capillaries had increased staining for TGFbeta3, PDGFR and TGFbetaRIII with staining for PDGFR also localised to dermal fibroblasts which were larger and more numerous. Accelerated epithelial cell proliferation was observed with detachment from the underlying dermis. CONCLUSIONS: Healing of purely ischaemic ulcers is characterised by vasculogenesis associated with increased presence of the proangiogenic cytokines PDGF and TGFbeta3. These findings show promise for the use of growth factor manipulation to aid healing in ischaemic ulcers.