Histone deacetylase inhibition mitigates fibrosis-driven disease progression in recessive dystrophic epidermolysis bullosa.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a blistering disease caused by mutations in the gene encoding type VII collagen (C7). RDEB is associated with fibrosis, which is responsible for severe complications. The phenotypic variability observed in RDEB siblings suggests that epigenetic modifications contribute to disease severity. Identifying epigenetic changes may help to uncover molecular mechanisms underlying RDEB pathogenesis and new therapeutic targets. OBJECTIVES: To investigate histone acetylation in RDEB skin and to explore histone deacetylase inhibitors (HDACis) as therapeutic molecules capable of counteracting fibrosis and disease progression in RDEB mice. METHODS: Acetylated histone levels were detected in human skin by immunofluorescence and in RDEB fibroblasts by ELISA. The effects of Givinostat and valproic acid (VPA) on RDEB fibroblast fibrotic behaviour were assessed by collagen-gel contraction assay, Western blot and immunocytofluorescence for α-smooth muscle actin, ELISA for released transforming growth factor-ß1 (TGF-ß1). RNA-seq was performed in HDACi- and vehicle-treated RDEB fibroblasts. VPA was systemically administered to RDEB mice, and effects on overt phenotype were monitored. Fibrosis was investigated in the skin using histological and immunofluorescence analyses. Eye and tongue defects were examined microscopically. Mass spectrometry proteomics was performed on skin protein extracts from VPA-treated RDEB and control mice. RESULTS: Histone acetylation decreases in RDEB skin and primary fibroblasts. RDEB fibroblasts treated with HDACis lowered fibrotic traits including contractility, TGF-ß1 release, and proliferation. VPA administration to RDEB mice mitigated severe manifestations affecting eyes and paws. These effects were associated with fibrosis inhibition. Proteomic analysis of mouse skin revealed that VPA almost normalised protein sets involved in protein synthesis and immune response, processes linked to the increased susceptibility to cancer and bacterial infections observed in RDEB patients. CONCLUSIONS: Dysregulated histone acetylation contributes to RDEB pathogenesis by facilitating the progression of fibrosis. Repurposing of HDACi could be considered for disease-modifying treatments of RDEB.

Gamma-Secretase Inhibitors Downregulate the Profibrotic NOTCH Signaling Pathway in Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare skin fragility disorder caused by mutations in COL7A1. RDEB is hallmarked by trauma-induced unremitting blistering, chronic wounds with inflammation, and progressive fibrosis, leading to severe disease complications. There is currently no cure for RDEB-associated fibrosis. Our previous studies and increasing evidence highlighted the profibrotic role of NOTCH pathway in different skin disorders, including RDEB. In this study, we further investigated the role of NOTCH signaling in RDEB pathogenesis and explored the effects of its inhibition by γ-secretase inhibitors DAPT and PF-03084014 (nirogacestat). Our analyses demonstrated that JAG1 and cleaved NOTCH1 are upregulated in primary RDEB fibroblasts (ie, RDEB-derived fibroblasts) compared with controls, and their protein levels are further increased by TGF-ß1 stimulation. Functional assays unveiled the involvement of JAG1/NOTCH1 axis in RDEB fibrosis and demonstrated that its blockade counteracts a variety of fibrotic traits. In particular, RDEB-derived fibroblasts treated with PF-03084014 showed (i) a significant reduction of contractility, (ii) a diminished secretion of TGF-ß1 and collagens, and (iii) the downregulation of several fibrotic proteins. Although less marked than PF-03084014-treated cells, RDEB-derived fibroblasts exhibited a reduction of fibrotic traits also upon DAPT treatment. This study provides potential therapeutic strategies to antagonize RDEB fibrosis onset and progression.

Is Melanoma Progression Affected by Thyroid Diseases?

Clinical and epidemiological evidence indicate a relationship between thyroid diseases and melanoma. In particular, the hypothyroidism condition appears to promote melanoma spread, which suggests a protective role of thyroid hormones against disease progression. In addition, experimental data suggest that, in addition to thyroid hormones, other hormonal players of the hypothalamic-pituitary-thyroid (HPT) axis, namely the thyrotropin releasing hormone and the thyrotropin, are likely to affect melanoma cells behavior. This information warrants further clinical and experimental studies in order to build a precise pattern of action of the HPT hormones on melanoma cells. An improved knowledge of the involved molecular mechanism(s) could lead to a better and possibly personalized clinical management of these patients.

Innovative Cell and Platelet Rich Plasma Therapies for Diabetic Foot Ulcer Treatment: The Allogeneic Approach.

Cutaneous chronic wounds are a major global health burden in continuous growth, because of population aging and the higher incidence of chronic diseases, such as diabetes. Different treatments have been proposed: biological, surgical, and physical. However, most of these treatments are palliative and none of them can be considered fully satisfactory. During a spontaneous wound healing, endogenous regeneration mechanisms and resident cell activity are triggered by the released platelet content. Activated stem and progenitor cells are key factors for ulcer healing, and they can be either recruited to the wound site from the tissue itself (resident cells) or from elsewhere. Transplant of skin substitutes, and of stem cells derived from tissues such as bone marrow or adipose tissue, together with platelet-rich plasma (PRP) treatments have been proposed as therapeutic options, and they represent the today most promising tools to promote ulcer healing in diabetes. Although stem cells can directly participate to skin repair, they primarily contribute to the tissue remodeling by releasing biomolecules and microvesicles able to stimulate the endogenous regeneration mechanisms. Stem cells and PRP can be obtained from patients as autologous preparations. However, in the diabetic condition, poor cell number, reduced cell activity or impaired PRP efficacy may limit their use. Administration of allogeneic preparations from healthy and/or younger donors is regarded with increasing interest to overcome such limitation. This review summarizes the results obtained when these innovative treatments were adopted in preclinical animal models of diabetes and in diabetic patients, with a focus on allogeneic preparations.

Dysregulation of microRNA expression in diabetic skin.

BACKGROUND: Clinical skin manifestations are common in diabetes; however, molecular mechanisms underlying such defects are largely unknown. Several findings indicate a role for microRNAs (miRNAs) in skin homeostasis. OBJECTIVE: To investigate whether miRNA expression is altered in diabetic skin. METHODS: Type 1 and 2 mouse models of diabetes were used. MiRNA profiling was performed on RNA extracted from the skin of type 1 diabetic mice and non-diabetic controls. Expression levels of pri-miRNAs and of miRNA-biogenesis genes were also analyzed. Biogenesis gene expression analysis was performed in human dermal fibroblasts cultured in hyperglycemic, hypoxic or oxidative stress conditions. RESULTS: Several miRNAs were differentially expressed in diabetic skin with a general down-modulation as compared to controls. Bioinformatics analysis of signature-miRNA target genes showed the enrichment in pathways involved in skin homeostasis, such as TGF-ß and Wnt. MiRNA alteration in diabetic skin associated with reduced expression levels of DROSHA, DGCR8, XPO5, DICER1, AGO2, both as mRNA and protein. Reduced biogenesis gene expression did not correlate with accumulation of pri-miRNAs, which displayed differences in expression levels similar to those found for their mature miRNAs. Experiments with cultured fibroblasts showed that hypoxia and oxidative stress induced the down-regulation of miRNA-biogenesis genes in this skin cell type. CONCLUSION: A general down-regulation of differentially expressed miRNAs was found in diabetic skin. This alteration is part of and is dependent from a wider transcriptional defect also affecting the expression of pri-miRNAs and of genes responsible for miRNA biogenesis. Such an alteration is likely contributing to diabetic skin manifestations.

Non-animal models in dermatological research.

Despite widely used for basic and preclinical studies in dermatology, available animal models only partly recapitulate human skin features often leading to disappointing outputs when preclinical results are translated to the clinic. Therefore, the need to develop alternative, non-animal models is widely recognized to more closely recapitulate human skin pathophysiology and to address the pressing ethical demand of reducing the number of animals used for research purposes, following the globally accepted 3Rs principle (Replacement, Reduction and Refinement). Skin is the outermost organ of the body, and, as such, easily accessible. Different skin cell types can be propagated in vitro and skin can be reconstructed for therapeutic transplantation as well as for in vitro modeling of physiopathological conditions. Bioengineered skin substitutes have been developed and evolved from elementary to complex systems, more and more closely resembling complete skin architecture and biological responses. In silico analyses take advantage from the huge amount of data already available from human studies for identifying and modeling molecular pathways involved in skin pathophysiology without further animal testing. The present review recapitulates the available non-animal models for dermatological research and sheds lights on their prospective technological evolution.

Decorin counteracts disease progression in mice with recessive dystrophic epidermolysis bullosa.

Loss-of-function mutations in the gene encoding type VII collagen underlie recessive dystrophic epidermolysis bullosa (RDEB), a disease characterized by skin and mucosal blistering, impaired wound healing, and diffuse dermal inflammation and fibrosis. Transforming growth factor-ß signaling plays a crucial role in determining RDEB fibrotic microenvironment that leads to the development of disabling secondary disease manifestations, including hand and foot deformities. Experimental findings indicate that expression levels of decorin, a small leucine-rich proteoglycan and an endogenous TGF-ß inhibitor, can modulate RDEB disease phenotype by contrasting dermal fibroblast fibrotic behavior. In this study, the ability of decorin to modify RDEB course was investigated by systemically treating RDEB mice with a lentivirus expressing human decorin. Overexpressed decorin was able to enhance survival, and to limit digit contraction and the development of paw deformities. These effects were associated with decreased TGF-ß1 levels and TGF-ß signaling activation. Fibrotic traits were strongly reduced in paw skin and also attenuated in the non-chronically injured back skin. However, the expression of pro-inflammatory proteins was not decreased in both paw and back skin. Our findings confirm TGF-ß role in promoting fibrosis and disease progression in RDEB, and show that decorin counteracts disease manifestations by inhibiting TGF-ß activation. More generally, our data indicate that modifying extracellular matrix composition is an option to improve RDEB disease course.

Thyroid diseases and skin autoimmunity.

The skin is the largest organ of the body, at the boundary with the outside environment. Primarily, it provides a physical and chemical barrier against external insults, but it can act also as immune organ because it contains a whole host of immune-competent cells of both the innate and the adaptive immune systems, which cooperate in eliminating invading pathogens following tissue injury. On the other hand, improper skin immune responses lead to autoimmune skin diseases (AISD), such as pemphigus, bullous pemphigoid, vitiligo, and alopecia. Although the interplay among genetic, epigenetic, and environmental factors has been shown to play a major role in AISD etiology and progression, the molecular mechanisms underlying disease development are far from being fully elucidated. In this context, epidemiological studies aimed at defining the association of different AISD with other autoimmune pathologies revealed possible shared molecular mechanism(s) responsible for disease progression. In particular, over the last decades, a number of reports have highlighted a significant association between thyroid diseases (TD), mainly autoimmune ones (AITD), and AISD. Here, we will recapitulate the epidemiology, clinical manifestations, and pathogenesis of the main AISD, and we will summarize the epidemiological evidence showing the associations with TD as well as possible molecular mechanism(s) underlying TD and AISD pathological manifestations.

Vitiligo and Autoimmune Thyroid Disorders.

Vitiligo represents the most common cause of acquired skin, hair, and oral depigmentation, affecting 0.5-1% of the population worldwide. It is clinically characterized by the appearance of disfiguring circumscribed skin macules following melanocyte destruction by autoreactive cytotoxic T lymphocytes. Patients affected by vitiligo usually show a poorer quality of life and are more likely to suffer from depressive symptoms, particularly evident in dark-skinned individuals. Although vitiligo is a non-fatal disease, exposure of affected skin to UV light increases the chance of skin irritation and predisposes to skin cancer. In addition, vitiligo has been associated with other rare systemic disorders due to the presence of melanocytes in other body districts, such as in eyes, auditory, nervous, and cardiac tissues, where melanocytes are thought to have roles different from that played in the skin. Several pathogenetic models have been proposed to explain vitiligo onset and progression, but clinical and experimental findings point mainly to the autoimmune hypothesis as the most qualified one. In this context, it is of relevance the strong association of vitiligo with other autoimmune diseases, in particular with autoimmune thyroid disorders, such as Hashimoto thyroiditis and Graves' disease. In this review, after a brief overview of vitiligo and its pathogenesis, we will describe the clinical association between vitiligo and autoimmune thyroid disorders and discuss the possible underlying molecular mechanism(s).

Pathomechanisms of Altered Wound Healing in Recessive Dystrophic Epidermolysis Bullosa.

Individuals with recessive dystrophic epidermolysis bullosa (RDEB), a rare genetic skin disease, carry mutations in the COL7A1 gene that codes for type VII collagen, an extracellular matrix component of the basement membrane zone forming the anchoring fibrils. As a consequence, RDEB individuals manifest unremitting skin blistering that evolves into chronic wounds, inflammation, and fibrosis. These features play a central role in the development of more severe disease complications, such as mitten deformities of hands and feet and aggressive epithelial cancers. Despite being recognized as a central clinical issue for RDEB, wound healing impairment has been only marginally investigated. Recently, studies with disease mouse models started to shed light on the molecular mechanisms underlying the altered healing response of RDEB. In turn, alterations found in RDEB skin cell behavior fostered the understanding of mechanisms that may be responsible for defective skin repair. This review summarizes findings related to healing impairment in RDEB, and highlights therapeutic strategies for ameliorating healing.

Stromal microenvironment in type VII collagen-deficient skin: The ground for squamous cell carcinoma development.

Recessive dystrophic epidermolysis bullosa (RDEB) is a skin fragility disease caused by mutations that affect the function and/or the amount of type VII collagen (C7), the major component of anchoring fibrils. Hallmarks of RDEB are unremitting blistering and chronic wounds leading to tissue fibrosis and scarring. Nearly all patients with severe RDEB develop highly metastatic squamous cell carcinomas (SCC) which are the main cause of death. Accumulating evidence from a murine RDEB model and human RDEB cells demonstrates that lack of C7 also directly alters the wound healing process. Non-healing RDEB wounds are characterized by increased inflammation, high transforming growth factor-ß1 (TGF-ß1) levels and activity, and are heavily populated by myofibroblasts responsible for enhanced fibrogenesis and matrix stiffness. These changes make the RDEB stroma a microenvironment prone to cancer initiation, where cells with features of cancer-associated fibroblasts are found. Here, we discuss recent knowledge on microenvironment alterations in RDEB, highlighting possible therapeutic targets to prevent and/or delay fibrosis and SCC development.

Epigenetic Control of Skin Re-Epithelialization: the NF-kB/JMJD3 Connection.

Major changes in gene expression must occur at wound site to establish the cellular responses required for rapid healing. Although epigenetic remodeling plays a role in gene modulation, the mechanisms responsible for epigenetic regulation during healing are largely unknown. The study from Na et al. sheds light on the role of histone demethylase Jumonji domain-containing protein D3 in promoting keratinocyte function after injury, and it links Jumonji domain-containing protein D3-dependent gene expression to NK-kB activity.

The E3 ligase Itch knockout mice show hyperproliferation and wound healing alteration.

The HECT-type E3 ubiquitin ligase Itch is absent in the non-agouti-lethal 18H or Itchy mice, which develop a severe immunological disease. Several of the known Itch substrates are relevant for epidermal development and homeostasis, such as p63, Notch, c-Jun and JunB. By analysing Itchy mice before the onset of immunological alterations, we investigated the contribution of Itch in skin development and wound healing. Itchy newborn mice manifested hyperplastic epidermis, which is not present in adulthood. Itch(-/-) cultured keratinocytes showed overexpression of proliferating markers and increased capability to proliferate, migrate and to repair a scratch injury in vitro. These data correlated with improved in vivo wound healing in Itchy mice, at late time points of the repair process when Itch is physiologically upregulated. Despite healing acceleration, epidermal remodelling was delayed in the scars of Itch(-/-) mice, as indicated by enhanced epidermal thickening, keratinocyte proliferation and keratin 6 expression, and retarded keratin 14 polarization to the basal layer. Itch(-/-) keratinocyte prolonged activation was not associated with increased immune cell persistence in the scars. Our in vitro and in vivo results indicate that Itch plays a role in epidermal homeostasis and remodelling and this feature does not seem to depend on immunological alterations.

Interleukin-22 Promotes Wound Repair in Diabetes by Improving Keratinocyte Pro-Healing Functions.

Impaired re-epithelialization, imbalanced expression of cytokines and growth factors, and vascular disease contribute to healing impairment in diabetes. IL-22, a pro-inflammatory cytokine mediating a cross-talk between immune system and epithelial cells, has been shown to have a role in repair processes. In this study we aimed to investigate IL-22 regenerative potential in the poor healing context of diabetic wounds. By using streptozotocin-induced diabetic mice, we demonstrated that IL-22 wound treatment significantly accelerated the healing process, by promoting re-epithelialization, granulation tissue formation, and vascularization. Improved re-epithelialization was associated with increased keratinocyte proliferation and signal transducer and activator of transcription 3 (STAT3) activation. We showed that endogenous IL-22 content was reduced at both mRNA and protein level during the inflammatory phase of diabetic wounds, with fewer IL-22-positive cells infiltrating the granulation tissue. We demonstrated that IL-22 treatment promoted proliferation and injury repair of hyperglycemic keratinocytes and induced activation of STAT3 and extracellular signal-regulated kinase transduction pathways in keratinocytes grown in hyperglycemic condition or isolated from diabetic patients. Finally, we demonstrated that IL-22 treatment was able to inhibit diabetic keratinocyte differentiation while promoting vascular endothelial growth factor release. Our data indicate a pro-healing role of IL-22 in diabetic wounds, suggesting a therapeutic potential for this cytokine in diabetic ulcer management.

Monozygotic twins discordant for recessive dystrophic epidermolysis bullosa phenotype highlight the role of TGF-ß signalling in modifying disease severity.

Recessive dystrophic epidermolysis bullosa (RDEB) is a genodermatosis characterized by fragile skin forming blisters that heal invariably with scars. It is due to mutations in the COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils connecting the cutaneous basement membrane to the dermis. Identical COL7A1 mutations often result in inter- and intra-familial disease variability, suggesting that additional modifiers contribute to RDEB course. Here, we studied a monozygotic twin pair with RDEB presenting markedly different phenotypic manifestations, while expressing similar amounts of collagen VII. Genome-wide expression analysis in twins' fibroblasts showed differential expression of genes associated with TGF-ß pathway inhibition. In particular, decorin, a skin matrix component with anti-fibrotic properties, was found to be more expressed in the less affected twin. Accordingly, fibroblasts from the more affected sibling manifested a profibrotic and contractile phenotype characterized by enhanced α-smooth muscle actin and plasminogen activator inhibitor 1 expression, collagen I release and collagen lattice contraction. These cells also produced increased amounts of proinflammatory cytokines interleukin 6 and monocyte chemoattractant protein-1. Both TGF-ß canonical (Smads) and non-canonical (MAPKs) pathways were basally more activated in the fibroblasts of the more affected twin. The profibrotic behaviour of these fibroblasts was suppressed by decorin delivery to cells. Our data show that the amount of type VII collagen is not the only determinant of RDEB clinical severity, and indicate an involvement of TGF-ß pathways in modulating disease variability. Moreover, our findings identify decorin as a possible anti-fibrotic/inflammatory agent for RDEB therapeutic intervention.

Diabetes impairs adipose tissue-derived stem cell function and efficiency in promoting wound healing.

Adipose tissue-derived stem cells (ASCs) are gaining increasing consideration in tissue repair therapeutic application. Recent evidence indicates that ASCs enhance skin repair in animal models of impaired wound healing. To assess the therapeutic activity of autologous vs. allogeneic ASCs in the treatment of diabetic ulcers, we functionally characterized diabetic ASCs and investigated their potential to promote wound healing with respect to nondiabetic ones. Adipose tissue-derived cells from streptozotocin-induced type 1 diabetic mice were analyzed either freshly isolated as stromal vascular fraction (SVF), or following a single passage of culture (ASCs). Diabetic ASCs showed decreased proliferative potential and migration. Expression of surface markers was altered in diabetic SVF and cultured ASCs, with a reduction in stem cell marker-positive cells. ASCs from diabetic mice released lower amounts of hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, and insulin-like growth factor-1, growth factors playing important roles in skin repair. Accordingly, the supernatant of diabetic ASCs manifested reduced capability to promote keratinocyte and fibroblast proliferation and migration. Therapeutic potential of diabetic SVF administered to wounds of diabetic mice was blunted as compared with cells isolated from nondiabetic mice. Our data indicate that diabetes alters ASC intrinsic properties and impairs their function, thus affecting therapeutic potential in the autologous treatment for diabetic ulcers.

Expression of vascular endothelial growth factor-C in primary cutaneous melanoma predicts sentinel lymph node positivity.

BACKGROUND: Vascular endothelial growth factor-C (VEGF-C), a lymphatic vessel growth factor, has been involved in the formation of lymph nodal metastases in different tumor types. Early evidences indicate that VEGF-C expression in human primary melanoma could be predictive of lymph nodal metastases, whereas the role of lymphangiogenesis is still controversial. METHODS: By immunohistochemical analysis, we investigated VEGF-C or CC chemokine receptor 7 expression, together with the lymphatic and blood vessel network, in 36 patients with primary skin melanomas and metastases at the sentinel lymph node biopsy (SLN-positive), and 26 melanoma patients with negative SLN biopsy (SLN-negative). RESULTS: We found that VEGF-C expression in primary melanoma specimens was significantly associated with SLN-positive (p < 0.001), particularly in thin melanomas. An association between augmented peritumoral lymphatic vessel area and SLN-positive (p < 0.02) was also seen. Conversely, no association between either expression of the CC chemokine receptor 7 in the primary tumor, or intratumoral lymphatic vessel or peritumoral and intratumoral blood vessel area, and SLN-positive was found. CONCLUSIONS: Our results, taking into account the expression of either VEGF-C or related histopathological markers, indicated the possibility to use VEGF-C immunohistochemistry as a marker of metastatic progression, especially in thin cutaneous melanomas.

The atypical retinoid E-3-(3'-Adamantan-1-yl-4'-methoxybiphenyl-4-yl)-2-propenoic acid (ST1898) displays comedolytic activity in the rhino mouse model.

Retinoids represent the first-line therapy for the treatment of acne vulgaris. Their effect is comedolytic and anti-comedogenic, and associates with hyperplasia and deregulated differentiation of the epidermis, and decreased inflammation. We here tested the comedolytic effect of the novel atypical retinoid E-3-(3'-Adamantan-1-yl-4'-methoxybiphenyl-4-yl)-2-propenoic acid (ST1898) in the rhino mouse, as a model of comedogenic acne, and compared this effect to that of adapalene (Differin® gel), as reference compound. Topical administration of 0.1% ST1898 for three weeks exerted a comedolytic effect comparable to that of adapalene 0.1%. In ST1898-treated mice, epidermal hyperplasia was significantly reduced and the expression of keratinocyte differentiation markers was less perturbed compared to adapalene-treated animals. Moreover, keratin 6, which stains activated keratinocytes, was strongly and uniformly induced in interfollicular epidermis of adapalene-treated mice, while only faintly and focally expressed in ST1898-treated ones. Our data indicate that ST1898 has strong comedolytic activity but modest topical side effects.

p63-microRNA feedback in keratinocyte senescence.

We investigated the expression of microRNAs (miRNAs) associated with replicative senescence in human primary keratinocytes. A cohort of miRNAs up-regulated in senescence was identified by genome-wide miRNA profiling, and their change in expression was validated in proliferative versus senescent cells. Among these, miRNA (miR)-138, -181a, -181b, and -130b expression increased with serial passages. miR-138, -181a, and -181b, but not miR-130b, overexpression in proliferating cells was sufficient per se to induce senescence, as evaluated by inhibition of BrdU incorporation and quantification of senescence-activated ß-galactosidase staining. We identified Sirt1 as a direct target of miR-138, -181a, and -181b, whereas ΔNp63 expression was inhibited by miR-130b. We also found that ΔNp63α inhibits miR-138, -181a, -181b, and -130b expression by binding directly to p63-responsive elements located in close proximity to the genomic loci of these miRNAs in primary keratinocytes. These findings suggest that changes in miRNA expression, by modulating the levels of regulatory proteins such as p63 and Sirt1, strongly contribute to induction of senescence in primary human keratinocytes, thus linking these two proteins. Our data also indicate that suppression of miR-138, -181a, -181b, and -130b expression is part of a growth-promoting strategy of ΔNp63α in epidermal proliferating cells.