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Background: Reactive oxygen species (ROS) are increased in diabetic conditions and play a causal role in diabetic foot ulcers (DFU). We previously showed that ROS up-regulate miR-200c expression, that in turns causes apoptosis, senescence, ROS upregulation and nitric oxide decrease, leading to endothelial disfunction. Methods: The aim of this study is to dissect miR-200c role in DFU and to explore the potential role of anti-miR-200c and antioxidant catalase (CAT) in promoting wound healing (WH). miR-200c inhibition and CAT treatment were performed either in immortalized keratinocytes (HaCaT) or in primary fibroblasts (FBs) and keratinocytes (KCs) deriving from diabetic patients (pts) undergoing amputations. Primary cells deriving from pts undergoing saphenectomies were used as controls. The miR-200c blockade was performed either via lentiviral particles bearing an anti-miR-200c sequence or locked nucleic acid (LNA) anti-miR-200c oligos. Equine CAT was administered on cell medium. The WH assay was performed in vivo on diabetic (db/db) mice by a topical treatment with CAT and LNA anti-miR-200c on wounds dissolved in a Pluronic gel mixture, administered every three days. Results: We found that miR-200c levels were increased by different stimuli known to induce ROS, such as ultraviolet radiation (UV), hydrogen peroxide (H2O2), and high glucose in HaCaT. miR-200c was also upregulated in skin biopsies, in FBs and KCs isolated from pts with DFU vs controls. Forced miR-200c expression induced ROS in both FBs and KCs, and CAT reduced it. miR-200c inhibition improved WH in HaCaT, both under basal conditions and after UV and H2O2 treatment, and the simultaneous treatment with CAT accelerated it. miR-200c inhibition accelerated WH in KCs of DFU pts, increasing its protein targets: sirtuin 1 (SIRT1), the transcription factors FOXO1 and ZEB1 and decreasing p66Shc phosphorylation at Ser-36, that is induced by ROS, and the co-treatment with CAT showed synergistic effects in reducing ROS and cytotoxicity. Interestingly, CAT treatment decreased miR-200c expression in FBs and KCs of DFU pts. Topical administration of anti-miR-200c and CAT in a WH model of diabetic mice accelerated closure. Conclusions: Anti-miR-200c and CAT could be considered a novel treatment for DFU and, possibly, for other types of non-diabetic skin ulcers. [ABSTRACT FROM AUTHOR]

UVB radiation induces DNA damage generating several thymine photo-adducts (TDPs), which can lead to mutations and cellular transformation. The DNA repair pathways preserve genomic stability by recognizing and removing photodamage. These DNA repair side products may affect cellular processes. We previously synthesized novel thymine biomimetic thymine dimers (BTDs) bearing different alkane spacers between nucleobases. Thus, the present study investigates whether novel BTDs and their TDPs can modulate DNA damage safeguard pathways of primary keratinocytes and fibroblasts using 2D and 3D models. We found that the p53/p21waf1 pathway is activated by BTDs and TDPs in primary cells similar to UVB exposure. Compound 1b can also induce the p53/p21waf1 pathway in a 3D skin model. However, BTDs and TDPs exhibit distinct effects on cell survival. They have a protective action in keratinocytes, which maintain their clonogenic ability following treatments. Conversely, compounds induce pro-apoptotic pathways in fibroblasts that exhibit reduced clonogenicity. Moreover, compounds induce inflammatory cytokines mainly in keratinocytes rather than fibroblasts. Matrix metalloproteinase 1 is up-regulated in both cell types after treatments. Therefore, BTDs and TDPs can act in the short term as safeguard mechanisms helping DNA damage response. Furthermore, they have distinct biological effects depending on photodamage form and cell type. [ABSTRACT FROM AUTHOR]

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 siblings with RDEB 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 (HDACi) 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 enzyme-linked immunosorbent assay (ELISA). The effects of givinostat and valproic acid (VPA) on RDEB fibroblast fibrotic behaviour were assessed by a collagen–gel contraction assay, Western blot and immunocytofluorescence for α-smooth muscle actin, and ELISA for released transforming growth factor (TGF)-β1. RNA sequencing 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 HDACi lowered fibrotic traits, including contractility, TGF-β1 release and proliferation. VPA administration to RDEB mice mitigated severe manifestations affecting the eyes and paws. These effects were associated with fibrosis inhibition. Proteomic analysis of mouse skin revealed that VPA almost normalized protein sets involved in protein synthesis and immune response, processes linked to the increased susceptibility to cancer and bacterial infections seen in people with RDEB. Conclusions Dysregulated histone acetylation contributes to RDEB pathogenesis by facilitating the progression of fibrosis. Repurposing of HDACi could be considered for disease-modifying treatments in RDEB. [ABSTRACT FROM AUTHOR]

Epidermal development and maintenance are finely regulated events requiring a strict balance between proliferation and differentiation. Alterations in these processes give rise to human disorders such as cancer or syndromes with skin and annexes defects, known as ectodermal dysplasias (EDs). Here, we studied the functional effects of two novel receptor-interacting protein kinase 4 (RIPK4) missense mutations identified in siblings with an autosomal recessive ED with cutaneous syndactyly, palmoplantar hyperkeratosis and orofacial synechiae. Clinical overlap with distinct EDs caused by mutations in transcription factors (i.e. p63 and interferon regulatory factor 6, IRF6) or nectin adhesion molecules was noticed. Impaired activity of the RIPK4 kinase resulted both in altered epithelial differentiation and defective cell adhesion. We showed that mutant RIPK4 resulted in loss of PVRL4/nectin-4 expression in patient epidermis and primary keratinocytes, and demonstrated that PVRL4 is transcriptionally regulated by IRF6, a RIPK4 phosphorylation target. In addition, defective RIPK4 altered desmosome morphology through modulation of plakophilin-1 and desmoplakin. In conclusion, this work implicates RIPK4 kinase function in the p63-IRF6 regulatory loop that controls the proliferation/differentiation switch and cell adhesion, with implications in ectodermal development and cancer., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)