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Skin microbiota plays an essential role in the development and function of the cutaneous immune system, in the maintenance of the skin barrier through the release of antimicrobial peptides, and in the metabolism of some natural products. With the aim of characterizing changes in the cutaneous microbiota specifically associated with wound healing in the diabetic condition, we performed a 16 S rRNA gene Next Generation Sequencing of skin swabs taken within the ulcer bed of ten diabetic patients before (t0) and after 20 days of therapy (t20) with a fluorescein-based galenic treatment. Considering the twenty most representative genera, we found at t20 an increase of Corynebacterium, Peptostreptococcus, and Streptococcus, and a decrease of Enterococcus, Finegoldia, and Peptoniphilus genera. However, differences were not significant due to the high variability among samples and the small patient cohort. S. aureus was the most abundant species at t0 and was reduced by therapy in four patients. Comparing the microbiome in the ulcer bed and in the perilesional tissue of the same patient at t0, no major differences were observed. Taken together, our data indicate that in the absence of antibiotic-based therapy the healing process of diabetic ulcers is accompanied by changes in the microbiome composition., Competing Interests: Declarations Competing interests Carlo Alessio Tenoglio is the legal representative of Blufarma S.r.l., the producer of the FluorexinTM galenic product used in clinical practice for diabetic ulcer treatment. All other authors declare no competing interests., (© 2024. The Author(s).)

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.

In the development of targeted oncology drugs, it is important to assess drug effectiveness in individual patients. We evaluated the possibility of reproducing in an ex-vivo system the biological effects observed in vitro and in vivo by the combined administration of two chemotherapeutic drugs, gemcitabine and a small inhibitor of Wee1. We found that modulation of both CDC2 phosphorylation and of a previously-identified gene signature was detectable in human skin equivalents obtained with primary keratinocytes from three individuals. Therefore, we suggest that human skin equivalents could represent a promising tool for the identification and validation of novel pharmacodynamic biomarkers.

Reduced microcirculation and diminished expression of growth factors contribute to wound healing impairment in diabetes. Placenta growth factor (PlGF), an angiogenic mediator promoting pathophysiological neovascularization, is expressed during cutaneous wound healing and improves wound closure by enhancing angiogenesis. By using streptozotocin-induced diabetic mice, we here demonstrate that PlGF induction is strongly reduced in diabetic wounds. Diabetic transgenic mice overexpressing PlGF in the skin displayed accelerated wound closure compared with diabetic wild-type littermates. Moreover, diabetic wound treatment with an adenovirus vector expressing the human PlGF gene (AdCMV.PlGF) significantly accelerated the healing process compared with wounds treated with a control vector. The analysis of treated wounds showed that PlGF gene transfer improved granulation tissue formation, maturation, and vascularization, as well as monocytes/macrophages local recruitment. Platelet-derived growth factor, fibroblast growth factor-2, and vascular endothelial growth factor mRNA levels were increased in AdCMV.PlGF-treated wounds, possibly enhancing PlGF-mediated effects. Finally, PlGF treatment stimulated cultured dermal fibroblast migration, pointing to a direct role of PlGF in accelerating granulation tissue maturation. In conclusion, our data indicate that reduced PlGF expression contributes to impaired wound healing in diabetes and that PlGF gene transfer to diabetic wounds exerts therapeutic activity by promoting different aspects of the repair process.

Summary Background Granulocyte/macrophage colony-stimulating factor (GM-CSF), a cytokine with pleiotropic functions, has been successfully employed in the treatment of chronic skin ulcers. The biological effects underlying GM-CSF action in impaired wound healing have been only partly clarified. Objectives To investigate the effects of GM-CSF treatment of chronic venous ulcers on lesion vascularization and on the local synthesis of the angiogenic factors vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). Methods Patients with nonhealing venous leg ulcers were treated with intradermal injection of recombinant human GM-CSF, and biopsies were taken at the ulcer margin before and 5 days after administration. Wound vascularization was analysed by immunohistochemistry using antiplatelet endothelial cell adhesion molecule-1/CD31 and anti-alpha-smooth muscle actin antibodies. VEGF and PlGF transcription was assessed by in situ hybridization. To identify the cell populations transcribing VEGF within the ulcer bed, the VEGF hybridization signal was correlated with the immunostaining for different cell type markers on serial sections. Direct induction of VEGF transcription by GM-CSF was investigated in GM-CSF-treated cultured macrophages and keratinocytes. Results Blood vessel density was significantly increased in the ulcer bed following GM-CSF treatment. VEGF transcripts were localized in keratinocytes at the ulcer margin both before and after GM-CSF treatment, whereas a VEGF hybridization signal was evident within the ulcer bed only following administration. PlGF mRNA was barely detectable in keratinocytes at the ulcer margin and was not visibly increased after treatment. Unlike VEGF, a specific PlGF hybridization signal could not be detected in cells within the ulcer following GM-CSF administration. Monocytes/macrophages were the main cell population transcribing VEGF after GM-CSF treatment. In vitro analysis demonstrated that VEGF transcription can be directly stimulated by GM-CSF in a differentiated monocytic cell line, but not in keratinocytes. Conclusions Our data show that increased vascularization is associated with GM-CSF treatment of chronic venous ulcers and indicate that inflammatory cell-derived VEGF may act as an angiogenic mediator of the healing effect of GM-CSF in chronic ulcers.

The placenta growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family that has been shown to play an important role in promoting adult pathological angiogenesis. Besides inducing its own signaling in endothelial cells, PlGF exerts its angiogenic action by synergising with VEGF. In the skin, PlGF expression is upregulated during wound healing and PlGF-deficient mice show delayed wound closure, indicating that this factor promotes angiogenesis during skin repair. Moreover, PlGF expression by melanoma cells has been linked to tumor growth. The analysis of a transgenic mouse model constitutively expressing high levels of PlGF in basal keratinocytes has shown that this factor has strong angiogenic properties in the skin during both embryonic and post-natal life. Furthermore, PlGF delivery to the skin via an adenoviral vector induces the formation of large and stable blood vessels, but contrary to VEGF application, does not affect lymphatic vessel functionality. Such evidence opens the possibility of employing PlGF for therapeutic modulation of skin angiogenesis.

Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family, comprising at least five cytokines specifically involved in the regulation of vascular and/or lymphatic endothelium differentiation. Several lines of evidence indicate a role for PlGF in monocyte chemotaxis and in potentiating the activity of VEGF, but the exact function of this cytokine is not fully understood. To define the biological role of PlGF in vivo, we have produced a transgenic mouse model overexpressing this factor in the skin by using a keratin 14 promoter cassette. Our data indicate that PlGF has strong angiogenic properties in both fetal and adult life. PlGF overexpression results in a substantial increase in the number, branching and size of dermal blood vessels as well as in enhanced vascular permeability. Indeed, intradermally injected recombinant PlGF was able to induce vessel permeability in wild-type mice. The analysis of vascular endothelial growth factor receptor 1/flt-1 and vascular endothelial growth factor receptor 2/flk-1 indicates that the two receptors are induced in the skin endothelium of transgenic mice suggesting that both are involved in mediating the effect of overexpressed PlGF.