Metabolic Profiling Reveals Changes in Serum Predictive of Venous Ulcer Healing.

OBJECTIVE: The aim of this study was to identify potential biomarkers predictive of healing or failure to heal in a population with venous leg ulceration. SUMMARY BACKGROUND DATA: Venous leg ulceration presents important physical, psychological, social and financial burdens. Compression therapy is the main treatment, but it can be painful and time-consuming, with significant recurrence rates. The identification of a reliable biochemical signature with the ability to identify nonhealing ulcers has important translational applications for disease prognostication, personalized health care and the development of novel therapies. METHODS: Twenty-eight patients were assessed at baseline and at 20 weeks. Untargeted metabolic profiling was performed on urine, serum, and ulcer fluid, using mass spectrometry and nuclear magnetic resonance spectroscopy. RESULTS: A differential metabolic phenotype was identified in healing (n = 15) compared to nonhealing (n = 13) venous leg ulcer patients. Analysis of the assigned metabolites found ceramide and carnitine metabolism to be relevant pathways. In this pilot study, only serum biofluids could differentiate between healing and nonhealing patients. The ratio of carnitine to ceramide was able to differentiate between healing phenotypes with 100% sensitivity, 79% specificity, and 91% accuracy. CONCLUSIONS: This study reports a metabolic signature predictive of healing in venous leg ulceration and presents potential translational applications for disease prognostication and development of targeted therapies.

Topical Aspirin Administration Improves Cutaneous Wound Healing in Diabetic Mice Through a Phenotypic Switch of Wound Macrophages Toward an Anti-inflammatory and Proresolutive Profile Characterized by LXA4 Release.

Patients with diabetes present a persistent inflammatory process, leading to impaired wound healing. Since nonhealing diabetic wound management shows limited results, the introduction of advanced therapies targeting and correcting the inflammatory status of macrophages in chronic wounds could be an effective therapeutic strategy to stop the sustained inflammation and to return to a healing state. In an excisional skin injury in a diet-induced diabetic murine model, we demonstrate that topical administration of low-dose aspirin (36 µg/wound/day) improves cutaneous wound healing by increasing wound closure through the promotion of the inflammation resolution program of macrophages. This treatment increased efferocytosis of wound macrophages from aspirin-treated diabetic mice compared with untreated diabetic mice. We also show that aspirin treatment of high-fat-fed mice oriented the phenotype of wound macrophages toward an anti-inflammatory and proresolutive profile characterized by a decrease of LTB4 production. The use of diabetic mice deficient for 5-LOX or 12/15-LOX demonstrated that these two enzymes of acid arachidonic metabolism are essential for the beneficial effect of aspirin on wound healing. Thus, aspirin treatment modified the balance between pro- and anti-inflammatory eicosanoids by promoting the synthesis of proresolving LXA4 through 5-LOX, LTA4, 12/15-LOX signaling. In conclusion, the restoration of an anti-inflammatory and proresolutive phenotype of wound macrophages by the topical administration of low-dose aspirin represents a promising therapeutic approach in chronic wounds.

Clinical Grade Human Pluripotent Stem Cell-Derived Engineered Skin Substitutes Promote Keratinocytes Wound Closure In Vitro.

Chronic wounds, such as leg ulcers associated with sickle cell disease, occur as a consequence of a prolonged inflammatory phase during the healing process. They are extremely hard to heal and persist as a significant health care problem due to the absence of effective treatment and the uprising number of patients. Indeed, there is a critical need to develop novel cell- and tissue-based therapies to treat these chronic wounds. Development in skin engineering leads to a small catalogue of available substitutes manufactured in Good Manufacturing Practices compliant (GMPc) conditions. Those substitutes are produced using primary cells that could limit their use due to restricted sourcing. Here, we propose GMPc protocols to produce functional populations of keratinocytes and fibroblasts derived from pluripotent stem cells to reconstruct the associated dermo-epidermal substitute with plasma-based fibrin matrix. In addition, this manufactured composite skin is biologically active and enhances in vitro wounding of keratinocytes. The proposed composite skin opens new perspectives for skin replacement using allogeneic substitute.

FBN2 Silencing Recapitulates Hypoxic Conditions and Induces Elastic Fiber Impairment in Human Dermal Fibroblasts.

Most chronic wounds are characterized by varying degrees of hypoxia and low partial pressures of O2 that may favor the development of the wound and/or delay healing. However, most studies regarding extracellular matrix remodeling in wound healing are conducted under normoxic conditions. Here, we investigated the consequences of hypoxia on elastic network formation, both in a mouse model of pressure-induced hypoxic ulcer and in human primary fibroblasts cultured under hypoxic conditions. In vitro, hypoxia inhibited elastic fiber synthesis with a reduction in fibrillin-2 expression at the mRNA and protein levels. Lysyl oxidase maturation was reduced, concomitant with lower enzymatic activity. Fibrillin-2 and lysyl oxidase could interact directly, whereas the downregulation of fibrillin-2 was associated with deficient lysyl oxidase maturation. Elastic fibers were not synthesized in the hypoxic inflammatory tissues resulting from in vivo pressure-induced ulcer. Tropoelastin and fibrillin-2 were expressed sparsely in hypoxic tissues stained with carbonic anhydrase IX. Different hypoxic conditions in culture resulted in the arrest of elastic fiber synthesis. The present study demonstrated the involvement of FBN2 in regulating elastin deposition in adult skin models and described the specific impact of hypoxia on the elastin network without consequences on collagen and fibronectin networks.

Biphasic Effects of Blue Light Irradiation on Human Umbilical Vein Endothelial Cells.

Blue light regulates biological function in various cells, such as proliferation, oxidative stress, and cell death. We employed blue light illumination on human umbilical vein endothelial cells utilizing a LED device at 453 nm wavelength and revealed a novel biphasic response on human umbilical vein endothelial cells (HUVECs). The results showed that low fluence blue light irradiation promoted the fundamental cell activities, including cell viability, migration and angiogenesis by activating the angiogenic pathways such as the VEGF signaling pathway. In contrast, high fluence illumination caused the opposite effect on those activities by upregulating pro-apoptotic signaling cascades like ferroptosis, necroptosis and the p53 signaling pathways. Our results provide an underlying insight into photobiomodulation by blue light and may help to implement potential treatment strategies for treating angiogenesis-dependent diseases.

A novel conjunctive microenvironment derived from human subcutaneous adipose tissue contributes to physiology of its superficial layer.

BACKGROUND: In human subcutaneous adipose tissue, the superficial fascia distinguishes superficial and deep microenvironments showing extensions called retinacula cutis. The superficial subcutaneous adipose tissue has been described as hyperplastic and the deep subcutaneous adipose tissue as inflammatory. However, few studies have described stromal-vascular fraction (SVF) content and adipose-derived stromal/stem cells (ASCs) behavior derived from superficial and deep subcutaneous adipose tissue. In this study, we analyzed a third conjunctive microenvironment: the retinacula cutis superficialis derived from superficial subcutaneous adipose tissue. METHODS: The samples of abdominal human subcutaneous adipose tissue were obtained during plastic aesthetic surgery in France (Declaration DC-2008-162) and Brazil (Protocol 145/09). RESULTS: The SVF content was characterized in situ by immunofluorescence and ex vivo by flow cytometry revealing a high content of pre-adipocytes rather in superficial subcutaneous adipose tissue microenvironment. Adipogenic assays revealed higher percentage of lipid accumulation area in ASCs from superficial subcutaneous adipose tissue compared with retinacula cutis superficialis (p < 0.0001) and deep subcutaneous adipose tissue (p < 0.0001). The high adipogenic potential of superficial subcutaneous adipose tissue was corroborated by an up-regulation of adipocyte fatty acid-binding protein (FABP4) compared with retinacula cutis superficialis (p < 0.0001) and deep subcutaneous adipose tissue (p < 0.0001) and of C/EBPα (CCAAT/enhancer-binding protein alpha) compared with retinacula cutis superficialis (p < 0.0001) and deep subcutaneous adipose tissue (p < 0.0001) microenvironments. Curiously, ASCs from retinacula cutis superficialis showed a higher level of adiponectin receptor gene compared with superficial subcutaneous adipose tissue (p = 0.0409), widely known as an anti-inflammatory hormone. Non-induced ASCs from retinacula cutis superficialis showed higher secretion of human vascular endothelial growth factor (VEGF), compared with superficial (p = 0.0485) and deep (p = 0.0112) subcutaneous adipose tissue and with adipogenic-induced ASCs from superficial (p = 0.0175) and deep (p = 0.0328) subcutaneous adipose tissue. Furthermore, ASCs from retinacula cutis superficialis showed higher secretion of Chemokine (C-C motif) ligand 5 (CCL5) compared with non-induced (p = 0.0029) and induced (p = 0.0089) superficial subcutaneous adipose tissue. CONCLUSIONS: This study highlights the contribution to ASCs from retinacula cutis superficialis in their angiogenic property previously described for the whole superficial subcutaneous adipose tissue besides supporting its adipogenic potential for superficial subcutaneous adipose tissue.

Glycation by glyoxal leads to profound changes in the behavior of dermal fibroblasts.

INTRODUCTION: Diabetes is a worldwide health problem that is associated with severe complications. Advanced Glycation End products (AGEs) such as Nε-(carboxymethyl)lysine, which result from chronic hyperglycemia, accumulate in the skin of patients with diabetes. The effect of AGEs on fibroblast functionality and their impact on wound healing are still poorly understood. RESEARCH DESIGN AND METHODS: To investigate this, we treated cultured human fibroblasts with 0.6 mM glyoxal to induce acute glycation. The behavior of fibroblasts was analyzed by time-lapse monolayer wounding healing assay, seahorse technology and atomic force microscopy. Production of extracellular matrix was studied by transmission electronic microscopy and western blot. Lipid metabolism was investigated by staining of lipid droplets (LDs) with BODIPY 493/503. RESULTS: We found that the proliferative and migratory capacities of the cells were greatly reduced by glycation, which could be explained by an increase in fibroblast tensile strength. Measurement of the cellular energy balance did not indicate that there was a change in the rate of oxygen consumption of the fibroblasts. Assessment of collagen I revealed that glyoxal did not influence type I collagen secretion although it did disrupt collagen I maturation and it prevented its deposition in the extracellular matrix. We noted a pronounced increase in the number of LDs after glyoxal treatment. AMPK phosphorylation was reduced by glyoxal treatment but it was not responsible for the accumulation of LDs. CONCLUSION: Glyoxal promotes a change in fibroblast behavior in favor of lipogenic activity that could be involved in delaying wound healing.

Master regulators in primary skin fibroblast fate reprogramming in a human ex vivo model of chronic wounds.

Fibroblasts are important players in regulating tissue homeostasis. In the dermis, they are involved in wound healing where they differentiate into contractile myofibroblasts leading to wound closure. In nonhealing chronic wounds, fibroblasts fail to undertake differentiation. We established and used a human ex vivo model of chronic wounds where fibroblasts can undergo normal myofibroblast differentiation, or take on a nondifferentiable pathological state. At the whole genome scale, we identified the genes that are differentially regulated in these two cell fates. By coupling the search of evolutionary conserved regulatory elements with global gene network expression changes, we identified transcription factors (TF) potentially involved in myofibroblast differentiation, and constructed a network of relationship between these key factors. Among these, we found that TCF4, SOX9, EGR2, and FOXS1 are major regulators of fibroblast to myofibroblast differentiation. Conversely, down-regulation of MEOX2, SIX2, and MAF causes reprogramming of fibroblasts to myofibroblasts even in absence of TGF-ß, the natural inducer of myofibroblast differentiation. These results provide insight into the fibroblast differentiation program and reveal a TF network essential for cellular reprogramming. They could lead to the development of new therapeutics to treat fibroblast-related human pathologies.

Effects of TLC-Ag dressings on skin inflammation.

The TLC-Ag dressings, a combination of technology lipido-colloid and silver salts, are used to promote healing in wounds with risks or signs of local infection, thanks to the antimicrobial properties of the silver salts. Nanocrystalline silver dressings containing nanocrystalline silver, also used to improve wound healing, present both antimicrobial and anti-inflammatory effects. The aim of this study was to investigate the anti-inflammatory effects of TLC-Ag dressings in a model of chronic skin inflammation induced by repeated application of 12-O-tetradecanoylphorbol-13-acetate to the skin of hairless mice, in comparison with TLC dressing, Silcryst nanocrystalline dressing, desonide cream 0.05%, a corticoid cream used as positive control, and gauze. Daily treatments of the mice began 7 days after the start of induction of chronic skin inflammation and lasted for 7 days. A macroscopic score was performed daily during the treatment period until the mice killing on day 15 and skin samples were taken for histopathological analysis. TLC-Ag reduced significantly the macroscopic score of chronic skin inflammation from day 10 in comparison with gauze and TLC dressing, similarly to Silcryst nanocrystalline dressing and desonide cream, which presented the best anti-inflammatory effects. No significant differences were observed between TLC dressing and gauze. TLC-Ag reduced significantly the microscopic score of chronic skin inflammation in comparison with TLC dressing and gauze, similarly to Silcryst nanocrystalline dressing but significantly less than desonide cream. These results demonstrate that TLC-Ag dressings present significant anti-inflammatory effects on chronic skin inflammation. They can improve wound healing, due to both the antimicrobial and anti-inflammatory properties.

Early events in HIV transmission through a human reconstructed vaginal mucosa.

OBJECTIVE: The early steps of HIV entry into intact vaginal mucosa still need to be clarified. Here we investigated how HIV translocated across the vaginal pluristratified epithelium, either by transcytosis or by uptake in Langerhans cells. METHODS: Using human primary fibroblasts and vaginal epithelial cells, we developed an in-vitro model of vaginal mucosa in which Langerhans cells could also be integrated. Owing to the absence of T lymphocytes and macrophages, we specifically studied the role of Langerhans cells in HIV transmission and the transcytosis of cell-associated HIV. RESULTS: Our model has a normal mucosal tissue architecture and Langerhans cells were efficiently integrated within the pluristratified epithelium. In addition, tight junction proteins' expression, high transepithelium resistance and low fluorescein isothiocyanate-BSA passage confirmed the integrity and impermeability of the reconstruction. Furthermore, we showed that human Langerhans cells also expressed tight junction proteins. Then, we demonstrated that neither transcellular nor intercellular transport of free infectious virus released by R5-infected or X4-infected peripheral blood mononuclear cells inoculated apically occured in the vaginal mucosa, irrespective to the presence of Langerhans cells. CONCLUSION: For the first time, we documented that, within 4 h following contact with HIV-infected cells, translocation of free HIV particles across a pluristratified mucosa is not detectable and that, in this context, it seemed that Langerhans cells do not increase HIV transmission. Moreover, we provided a useful model for the development of strategies preventing HIV entry into the female genital tract, especially for testing the efficiency of various microbicides.

Human Langerhans cells express a specific TLR profile and differentially respond to viruses and Gram-positive bacteria.

Dendritic cells (DC) are APCs essential for the development of primary immune responses. In pluristratified epithelia, Langerhans cells (LC) are a critical subset of DC which take up Ags and migrate toward lymph nodes upon inflammatory stimuli. TLR allow detection of pathogen-associated molecular patterns (PAMP) by different DC subsets. The repertoire of TLR expressed by human LC is uncharacterized and their ability to directly respond to PAMP has not been systematically investigated. In this study, we show for the first time that freshly purified LC from human skin express mRNA encoding TLR1, TLR2, TLR3, TLR5, TLR6 and TLR10. In addition, keratinocytes ex vivo display TLR1-5, TLR7, and TLR10. Accordingly, highly enriched immature LC efficiently respond to TLR2 agonists peptidoglycan and lipoteichoic acid from Gram-positive bacteria, and to dsRNA which engages TLR3. In contrast, LC do not directly sense TLR7/8 ligands and LPS from Gram-negative bacteria, which signals through TLR4. TLR engagement also results in cytokine production, with marked differences depending on the PAMP detected. TLR2 and TLR3 ligands increase IL-6 and IL-8 production, while dsRNA alone stimulates TNF-alpha release. Strikingly, only peptidoglycan triggers IL-10 secretion, thereby suggesting a specific function in tolerance to commensal Gram-positive bacteria. However, LC do not produce IL-12p70 or type I IFNs. In conclusion, human LC are equipped with TLR that enable direct detection of PAMP from viruses and Gram-positive bacteria, subsequent phenotypic maturation, and differential cytokine production. This implies a significant role for LC in the control of skin immune responses.