Expression and Correlation of HER2/P53/VEGF in Marjolin's Ulcer.

Marjolin's ulcer is described as malignant lesions developed in the injured skin, which can cause several kinds of malignancies. Our results showed that no HER2 but p53 was detected in Majorlin's ulcer samples. Meanwhile, by statistical analysis, we found that the positive rate of p53 in Majorlin's ulcer samples was associated with the pathological type of ulcer canceration and degree of tumor differentiation. The positive expression rate of vascular endothelial growth factor (VEGF) was 62.5% in poorly differentiated squamous cell carcinoma (SCC), 39.4% in moderately differentiated SCC, and 66.7% in well-differentiated SCC, respectively. Furthermore, some cases of Majorlin's ulcer with positive P53 were negative for VEGF, while some cases with positive VEGF were negative for P53. Image superposition showed that VEGF expression was absent or minimal in p53-positive cases. However, P53 was not expressed or rarely expressed in VEGF-positive cases. Our results of this study will suggest that P53 can be used as the mark of Marjolin's ulcer differentiation, and there may be some interaction between P53 and VEGF in Marjolin's ulcer. The regulation of microenvironment in the oncogenesis, progression, and differentiation of Marjolin's ulcer is complex and needs further study.

Sympathetic System in Wound Healing: Multistage Control in Normal and Diabetic Skin.

In this review, we discuss sympathetic regulation in normal and diabetic wound healing. Experimental denervation studies have confirmed that sympathetic nerve endings in skin have an important and complex role in wound healing. Vasoconstrictor neurons secrete norepinephrine (NE) and neuropeptide Y (NPY). Both mediators decrease blood flow and interact with inflammatory cells and keratinocytes. NE acts in an ambiguous way depending on receptor type. Beta2-adrenoceptors could be activated near sympathetic endings; they suppress inflammation and re-epithelialization. Alpha1- and alpha2-adrenoceptors induce inflammation and activate keratinocytes. Sudomotor neurons secrete acetylcholine (ACh) and vasoactive intestinal peptide (VIP). Both induce vasodilatation, angiogenesis, inflammation, keratinocytes proliferation and migration. In healthy skin, all effects are important for successful healing. In treatment of diabetic ulcers, mediator balance could be shifted in different ways. Beta2-adrenoceptors blockade and nicotinic ACh receptors activation are the most promising directions in treatment of diabetic ulcers with neuropathy, but they require further research.

Rhein promotes the proliferation of keratinocytes by targeting oestrogen receptors for skin ulcer treatment.

BACKGROUND: The Sheng-ji Hua-yu (SJHY) formula is a quite effective Traditional Chinese Medicines (TCM) in the treatment of delayed diabetic wounds. Previous research has shown that the SJHY formula has significant anti-inflammatory and wound-healing effects, but the precise mechanism remains unknown. The purpose of this study was to evaluate the effects of rhein, a compound extracted from SJHY formula, in keratinocytes and to investigate the underlying mechanisms. METHODS: Microscale thermophoresis (MST) technology was used to confirm that rhein binds directly to oestrogen receptors (ERs). Rhein was then used to treat keratinocytes in vitro. Cell cycle and proliferation analysis, Real-time polymerase chain reaction (RT-PCR) and Western-blot were conducted. RESULTS: Rhein increased the proportion of cells in the S phase of the cell cycle and promoted keratinocyte proliferation. ICI 182,780, an ER inhibitor, was also used to treat keratinocytes. The expression of c-myc mRNA and protein induced by rhein was antagonized by ICI 182,780, indicating that this induction is ER dependent. Intervention with ICI 182,780 had no effect on the upregulation of FosB and JunD, indicating that activator protein 1 (AP-1) members (FosB and JunD) are involved in rhein-induced c-myc mRNA and protein expression but does not require the ER. CONCLUSION: The present study found that rhein stimulates keratinocyte proliferation by activating the oestrogen signalling pathway via the oestrogen receptor, which induces the expression of c-myc in collaboration with FosB and JunD, thereby accelerating the process of re-epithelialization.

Dietary prenylated flavonoid xanthohumol alleviates oxidative damage and accelerates diabetic wound healing via Nrf2 activation.

Diabetic skin ulcer is one of the most common complications in patients suffering diabetes mellitus. Xanthohumol (XN), a hop-derived prenylated dietary flavonoid, has multiple health beneficial bioactivities. In the present study, we reported XN alleviates oxidative damage and accelerates diabetic wound healing via Nrf2 activation. In vitro, XN attenuated hydrogen peroxide (H2O2)-induced cytotoxicity, ROS production, cell apoptosis, as well as high glucose-induced cell damage. Mechanistic studies further demonstrated that XN could stabilize nuclear factor erythroid 2-related factor 2 (Nrf2) and promote its nuclear translocation, which was associated with AMPKα activation and covalent modification of Keap1 by XN. In vivo, XN increased Nrf2 expression and accelerated diabetic wound healing. Our study revealed a novel function of XN in diabetic wound healing as well as the underlying molecular mechanisms, suggesting XN is a promising lead compound and a potential food and/or drug candidate for the treatment of diabetic skin ulcers.

Angiogenic efficacy of ASC spheroids filtrated on porous nanosheets for the treatment of a diabetic skin ulcer.

Stem cell transplantation is expected to be an effective treatment for intractable skin ulcers by promoting angiogenesis; however, it is challenging to quickly realize a sufficient bloodstream for the ulcers. For this treatment, sheet-like materials with monolayer cells such as cell sheets have been investigated. However, they have a limitation of cell number that can be transplanted at one time due to the two-dimensional, monolayer cell structure, and sufficient secretion of growth factors cannot be expected. In this regard, cellular aggregates, such as spheroids, can reproduce three-dimensional cell-cell interactions that cause biological functions of living tissues more representative than monolayer cells, which is important to achieving efficient secretion of growth factors. In this study, we focused on free-standing porous polymer ultrathin films ("porous nanosheets") comprising poly(d,l-lactic acid) (PDLLA) and succeeded in developing a spheroid-covered nanosheet, on which more than 1000 spheroids from adipose-tissue derived stem cells (ASCs) were loaded. The porous structure with an average pore diameter of 4 µm allowed for facile filtration and carrying spheroids on the nanosheet, as well as sufficient oxygen and nutrients inflow to the cells. The spheroid-covered nanosheet achieved homogeneous transference of spheroids to a whole skin defect in diabetic model mice. Given the continuous release of vascular endothelial growth factor (VEGF) from the spheroids, the transplanted spheroids promoted healing with more accelerated angiogenesis than a nanosheet with a monolayer of cells. The spheroid-covered nanosheet may be a new regenerative material for promoting intractable skin ulcer healing.

CD21low B cells are predictive markers of new digital ulcers in systemic sclerosis.

The objective of this study was to evaluate the predictive role of CD21low B cells as markers of new digital ulcers in systemic sclerosis patients. Peripheral blood B cell subpopulations and clinical assessments have been evaluated in 74 systemic sclerosis patients at baseline and after a 12-month follow-up. After a 12-month follow-up, 23 (31.1%) systemic sclerosis patients developed new digital ulcers. The median percentage of CD21low B cells was significantly higher in patients with than without new digital ulcers [10.1 (4.3-13.6) versus 4.8 (3.5-7.4); p < 0.01]. The 10% cut-off shows good diagnostic accuracy [area under the curve (AUC) = 0.732, confidence interval (CI) = 0.587-0.878; P = 0.01]. Kaplan-Meier curves show a significantly reduced free survival from new digital ulcers in systemic sclerosis patients with CD21low B cells ≥ 10% (p < 0.0001). In multivariate analysis, CD21low B cells ≥ 10%, modified Rodnan skin score (mRSS) and systolic pulmonary arterial pressure (sPAP) are associated with the development of new digital ulcers. We hypothesize that CD21low B cells are a predictive marker of new digital ulcers in systemic sclerosis patients.

Epstein-Barr Virus-Positive Mucocutaneous Ulcer: A Unique and Curious Disease Entity.

Epstein-Barr virus (EBV)-positive mucocutaneous ulcer (EBVMCU) was first described as a lymphoproliferative disorder in 2010. EBVMCU is a unifocal mucosal or cutaneous ulcer that often occurs after local trauma in patients with immunosuppression; the patients generally have a good prognosis. It is histologically characterized by proliferating EBV-positive atypical B cells accompanied by ulcers. On the basis of conventional pathologic criteria, EBVMCU may be misdiagnosed as EBV-positive diffuse large B-cell lymphoma or other lymphomas. However, its prognosis differs from that of EBV-associated lymphomas, in that patients with EBVMCU frequently show spontaneous regression or complete remission without chemotherapy. Therefore, EBVMCU is now recognized as a low-grade malignancy or a pseudo-malignant lesion. Avoiding unnecessary chemotherapy by distinguishing EBVMCU from other EBV-associated lymphomas will reduce the burden and unnecessary harm on patients. On the basis of these facts, EBVMCU was first described as a new clinicopathological entity by the World Health Organization in 2017. In this review, we discuss the clinicopathological characteristics of previously reported EBVMCU cases, while focusing on up-to-date clinical, pathological, and genetic aspects.

BET bromodomain inhibitors regulate keratinocyte plasticity.

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.

Functional Analysis of Estrogen Receptor 1 in Diabetic Wound Healing: A Knockdown Cell-Based and Bioinformatic Study.

BACKGROUND Diabetic wound (DW) treatment is a serious challenge for clinicians, and the underlying mechanisms of DWs remain elusive. We sought to identify the critical genes in the development of DWs and provide potential targets for DW therapies. MATERIAL AND METHODS Datasets of GSE38396 from the Gene Expression Omnibus (GEO) database were reviewed. Pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology term analyses were carried out, and Cytoscape software (Cytoscape 3.7.2) was used to construct the protein interaction network. Serum samples from patients with diabetes and control participants were collected, and the expression of estrogen receptor 1 (ESR1) was measured by quantitative reverse-transcription polymerase chain reaction. In addition, the function of ESR1 in human skin fibroblasts was investigated in vitro. RESULTS Eight samples were analyzed using the Morpheus online tool, which identified 637 upregulated and 448 downregulated differentially expressed genes. The top 5 KEGG pathways of upregulated differentially expressed genes were associated with sphingolipid metabolism, estrogen signaling, ECM-receptor interaction, MAPK signaling, and PI3K-Akt signaling. The hub genes for DWs were JUN, ESR1, CD44, SMARCA4, MMP2, BMP4, GSK3B, WDR5, PTK2, and PTGS2. JUN, MMP2, and ESR1 were the upregulated hub genes, and ESR1 was found to be consistently enriched in DW patients. Inhibition of ESR1 had a stimulative role in human skin fibroblasts. CONCLUSIONS ESR1 was identified as a crucial gene in the development of DWs, which suggests potential therapeutic targets for DW healing.

Melatonin loaded lecithin-chitosan nanoparticles improved the wound healing in diabetic rats.

Wound healing in diabetic patients remains a worldwide problem that can cause amputations and even lead to death. This work aimed to produce lecithin-chitosan nanoparticles loaded with melatonin (MEL-NP) incorporated in a topical formulation to be evaluated for healing in the in vivo animal model for diabetes. To produce nanoparticles, an ethanolic solution containing soybean lecithin and melatonin was added dropwise to an aqueous solution of chitosan under sonication. The nanoparticles were physicochemical characterized and evaluated in vivo for toxicity using the Galleria mellonella model and its potential for wound healing in diabetic rats. The MEL-NPs presented a particle size of 160 nm and a zeta potential of 25 mV. The melatonin entrapment efficiency was 27%. Our results indicated that treatment with MEL-NP improved wound healing demonstrated by wound closure earlier than the other treatments evaluated. A desired therapeutic effect was achieved by MEL-NP in the induction of fibroblast and angiogenic proliferation. In addition, it was accompanied by an expressive collagen deposition. Considering the observed data, the MEL-NP developed could be used as a proof of concept to develop a promising strategy for the healing of diabetic wound.

NMR-based metabolomic analysis for the effects of Huiyang Shengji extract on rat diabetic skin ulcers.

ETHNOPHARMACOLOGICAL RELEVANCE: Huiyang Shengji formula (HSF) is a compound Chinese herbal medicine prescription, and has long been used for treating chronic non-healing wounds. AIM OF THE STUDY: The purpose of this study was to provide new insight into molecular mechanisms of healing effects of the HSF treatments. MATERIALS AND METHODS: We established a rat diabetic skin ulcer (DSU) model, and assessed healing effects of four HSF treatments on DSUs by calculating wound healing rates and immunohistochemical detection of the expressions of angiogenesis-related factors in the model rats (Mod) relative to normal rats (Nor), including Huiyang extract (HE), Shengji extract (SE), Huiyang Shengji extract (HSE) and HSE associated with acupuncture (Ac-HSE). We then performed NMR-based metabolomic analyses on skin tissues of the Nor, Mod, HSE-treated, Ac-HSE-treated rats to address metabolic mechanisms underlying these effects. RESULTS: These treatments up-regulated expressions of two angiogenesis-related factors VEGF and CD31, and improved efficacy of healing DSUs, in which HSE and Ac-HSE exhibited the most significant effects. Compared with Mod, HSE and Ac-HSE groups shared four characteristic metabolites (lactate, histidine, succinate and acetate) and four significantly altered metabolic pathways with Nor. Both HSE and Ac-HSE treatments could partly reverse the metabolically disordered pathological state of DSUs to the normal state. They might improve wound healing through promoting glucose metabolism, BCAAs metabolism, and enhancing antioxidant capacity and angiogenesis in DSU tissues. Ac-HSE significantly enhanced wound healing rates compared to HSE, potentially owing to significant capacities of enhancing anti-oxidation and angiogenesis and interfering three more metabolic pathways. CONCLUSIONS: This work provides a mechanistic understanding of the healing effects of the HSE and Ac-HSE treatments on DSUs, is of benefit to improvements of the HSF treatments for clinically healing chronic non-healing wounds.

Understanding Transcriptional Networks Regulating Initiation of Cutaneous Wound Healing.

The epidermis has an essential function in creating a barrier against the external environment to retain proper fluid balance and block the entry of pathogens. When damage occurs to this barrier, the wound must quickly be sealed to avoid fluid loss, cleared of invading pathogens, and then keratinocytes must re-form an intact barrier. This requires complex integration of temporally and spatially distinct signals to execute orderly closure of the wound, and failure of this process can lead to chronic ulceration. Transcription factors serve as a key integration point for the myriad of information coming from the external environment, allowing for an orderly process of re-epithelialization. Importantly, transcription factors engage with and alter the chromatin structure around key target genes through association with different chromatin-modifying complexes. In this review, we will discuss the current understanding of how transcription is regulated during the initiation of re-epithelialization, and the exciting technological advances that will allow for a more refined mechanistic understanding of the re-epithelialization process.

Transcriptomic Analysis of a Diabetic Skin-Humanized Mouse Model Dissects Molecular Pathways Underlying the Delayed Wound Healing Response.

Defective healing leading to cutaneous ulcer formation is one of the most feared complications of diabetes due to its consequences on patients' quality of life and on the healthcare system. A more in-depth analysis of the underlying molecular pathophysiology is required to develop effective healing-promoting therapies for those patients. Major architectural and functional differences with human epidermis limit extrapolation of results coming from rodents and other small mammal-healing models. Therefore, the search for reliable humanized models has become mandatory. Previously, we developed a diabetes-induced delayed humanized wound healing model that faithfully recapitulated the major histological features of such skin repair-deficient condition. Herein, we present the results of a transcriptomic and functional enrichment analysis followed by a mechanistic analysis performed in such humanized wound healing model. The deregulation of genes implicated in functions such as angiogenesis, apoptosis, and inflammatory signaling processes were evidenced, confirming published data in diabetic patients that in fact might also underlie some of the histological features previously reported in the delayed skin-humanized healing model. Altogether, these molecular findings support the utility of such preclinical model as a valuable tool to gain insight into the molecular basis of the delayed diabetic healing with potential impact in the translational medicine field.

Six cases of perforating pilomatricoma: Anetodermic changes with expression of matrix metalloproteinases.

Perforating pilomatricoma (PP) is a rare clinical variant of pilomatricoma presenting as a crusted or ulcerated nodule. Previous reports have suggested that the tumor cells perforate the epidermis through a process of transepithelial elimination. Here, we report six cases of PP and examine the mechanism of transepithelial elimination in PP. Histologically, the dermis above or around the tumor nest exhibited edema, dilated vascular spaces, sparse collagen bundles and absence of elastic fibers, suggesting anetodermic changes in all cases. Immunohistochemistry demonstrated many CD68-positive macrophages around the tumor nests. Matrix metallopeptidase (MMP)-9 and MMP-12 were expressed in the inflammatory cells and tumor cells, and were also present in the epidermis and fibroblasts in all cases. We speculate that in PP anetodermic change caused by MMP and elastases including MMP-9 and MMP-12 may precede elimination of the tumor.

Autonomic nerve dysfunction and impaired diabetic wound healing: The role of neuropeptides.

Lower extremity ulcerations represent a major complication in diabetes mellitus and involve multiple physiological factors that lead to impairment of wound healing. Neuropeptides are neuromodulators implicated in various processes including diabetic wound healing. Diabetes causes autonomic and small sensory nerve fibers neuropathy as well as inflammatory dysregulation, which manifest with decreased neuropeptide expression and a disproportion in pro- and anti- inflammatory cytokine response. Therefore to fully understand the contribution of autonomic nerve dysfunction in diabetic wound healing it is crucial to explore the implication of neuropeptides. Here, we will discuss recent studies elucidating the role of specific neuropeptides in wound healing.

The Effect of Shear Force on Skin Viability in Patients with Type 2 Diabetes.

BACKGROUND: Shear is a major risk factor in the development of diabetic foot ulcers, but its effect on the skin of patients with type 2 diabetes mellitus (DM) remains to be elucidated. The aim was to determine skin responses to shear in DM patients with and without diabetic polyneuropathy (DNP). METHODS: The forearm skin was loaded with 14.5 N shear (+2.4 kPa pressure) and with 3.5 kPa pressure for 30 minutes in 10 type 2 DM patients without DNP, 10 type 2 DM patients with DNP, and 10 healthy participants. A Sebutape collected IL-1α (measure of tissue damage). A laser Doppler flowmeter measured cutaneous blood cell flux (CBF) as a measure of the reactive hyperaemic skin response. FINDINGS: Reactive hyperaemia and IL-1α release was significantly increased after shear loading in all three groups and was higher compared to the responses to pressure loading. The reactive hyperaemic response after shear loading was impaired in patients with type 2 DM compared to healthy participants but did not differ between patients with and without DNP. The reactive hyperaemic response was negatively correlated with the blood glucose level but did not correlate with the DNP severity score. INTERPRETATION: Shear is important in the development of tissue damage, but the reparative responses to shear are impaired in patients with type 2 DM. DNP was not associated with altered skin responses, suggesting that the loss of protective sensation to sense shear to skin remains a key factor in the development of diabetic foot ulcers in patients with DNP.

Th1- and Th17-Related Cytokines in Venous and Arterial Blood of Sclerodermic Patients with and without Digital Ulcers.

The earliest clinical manifestation of SSc is usually Raynaud's phenomenon, a small-arteries vasospasm driven by vascular tone dysregulation and microcirculatory abnormalities, resulting in digital ulcers (DU) in up to 50% of patients. Many cytokines as well as growth factors have been shown to play a role in promoting vascular smooth muscle cell proliferation and fibroblast activation, leading to ischemic damage as well as skin fibrosis. We aim to investigate a possible difference in venous and arterial blood levels of many cytokines (Th1- and Th17-related), GM-CSF, and endothelin-1 (ET1) in patients with and without DU. In the same patients, the correlations between capillary damage, evaluated by nailfold videocapillaroscopy (NVC), extension of skin fibrosis, calculated by modified Rodnan skin score (mRSS), and cytokines, ET-1, and GM-CSF levels were also measured. Patients with DU showed venous levels of IL-1ß (p=0.024), IL-6 (p=0.012), IL-22(p=0.006), and TGF-ß (p=0.046) significantly higher compared to arterial levels and arterial levels of GM-CSF and TNF-alpha significantly higher compared to venous levels (p < 0.001). NVC abnormalities were correlated with arterial TNFa and venous IL22, IL23, and IL17 levels and negatively correlated with venous ET-1 levels, whereas mRSS showed a negative correlation with IL-21(ρ = -0.427, p=0.050). The increased Th17-cytokine levels in venous compared to arterial blood of patients with DU suggest local cytokine production on ulcer site. The higher TNFa and GM-CSF levels in arterial blood of DU patients support the attempt to mitigate the hypoxic damage, and the correlation between Th17-cytokines, mRSS, NVC, and ET1 agrees with the potent profibrotic stimulus at the onset of the disease, which decreases as the SSc progresses.

Estimation of Biological Parameters of Cutaneous Ulcers Caused by Leishmaniasis in an Animal Model Using Diffuse Reflectance Spectroscopy.

Cutaneous leishmaniasis (CL) is a neglected tropical disease that requires novel tools for its understanding, diagnosis, and treatment follow-up. In the cases of other cutaneous pathologies, such as cancer or cutaneous ulcers due to diabetes, optical diffuse reflectance-based tools and methods are widely used for the investigation of those illnesses. These types of tools and methods offer the possibility to develop portable diagnosis and treatment follow-up systems. In this article, we propose the use of a three-layer diffuse reflectance model for the study of the formation of cutaneous ulcers caused by CL. The proposed model together with an inverse-modeling procedure were used in the evaluation of diffuse-reflectance spectral signatures acquired from cutaneous ulcers formed in the dorsal area of 21 golden hamsters inoculated with Leishmanisis braziliensis. As result, the quantification of the model's variables related to the main biological parameters of skin were obtained, such as: diameter and volumetric fraction of keratinocytes, collagen; volumetric fraction of hemoglobin, and oxygen saturation. Those parameters show statistically significant differences among the different stages of the CL ulcer formation. We found that these differences are coherent with histopathological manifestations reported in the literature for the main phases of CL formation.

Platelet-rich plasma, a powerful tool in dermatology.

Platelet-rich plasma (PRP), a platelet concentrate contained in a small volume of plasma, has become a promising option in the last decade to treat different diseases related to the skin due to its high concentration of growth factors. When it is of autologous origin, it decreases the probability of suffering adverse reactions and transfusion-transmitted infections, thus it is an optimal and safe therapy for the patient. PRP has been used in the treatment of several dermatological conditions such as acne, alopecia, and skin ulcers. Its use has also extended to other skin conditions such as melasma, hyperpigmentation, and burns, where it stimulates tissue repair and regeneration. The purpose of this article is to review the management and treatment of different dermatological alterations with PRP. Although there are a variety of studies that support the use of PRP, more research is needed to standardise the protocols for obtaining, processing, and applying it as well as understanding the biological and molecular bases of its functioning.

Activated Protein C in Cutaneous Wound Healing: From Bench to Bedside.

Independent of its well-known anticoagulation effects, activated protein C (APC) exhibits pleiotropic cytoprotective properties. These include anti-inflammatory actions, anti-apoptosis, and endothelial and epithelial barrier stabilisation. Such beneficial effects have made APC an attractive target of research in a plethora of physiological and pathophysiological processes. Of note, the past decade or so has seen the emergence of its roles in cutaneous wound healing-a complex process involving inflammation, proliferation and remodelling. This review will highlight APC's functions and mechanisms, and detail its pre-clinical and clinical studies on cutaneous wound healing.