Glycated Reconstructed Human Skin as a Platform to Study the Pathogenesis of Skin Aging

Abstract

The advanced glycation end products (AGEs) of proteins are common factors in the pathophysiology of a number of disorders related to aging. The skin generation of AGEs occurs mainly through nonenzymatic glycation reactions of extracellular matrix (ECM) proteins in the dermis. The AGEs have been touted as one of the factors responsible for healing impairment and loss of elasticity of healing skin, affecting growth, differentiation, and cellular motility, as well as cytokines response, metalloproteinases expression, and vascular hemostasis. In this study, we generated an in vitro full-thickness reconstructed skin based on a glycated collagen matrix dermal compartment to evaluate the effects of glycation on dermal ECM and ultimately on the epidermis. Epidermal differentiation and stratification patterns and the glycation-induced ECM changes were evaluated by histology, immunohistochemistry, and mRNA levels. In this study, we reported for the first time that changes in the dermal matrix caused by collagen I in vitro glycation processes also affect the epidermal compartment. We demonstrated that glycation of collagen induces expression of carboxymethyllysine in dermal and epidermal compartments and, consequently, an aging phenotype consisting of poor stratification of epidermal layers and vacuolization of keratinocyte cytoplasm. Increased expression of cell-cell adhesion markers, such as desmoglein and E-cadherin in glycated skins, is observed in the stratum spinosum, as well as an increased compression of dermal collagen matrix. We also submitted our 3D model of reconstructed glycated skin to screening of anti-AGE molecules, such as aminoguanidine, which prevented the glycated morphological status. Controlled human studies investigating the effects of anti-AGE strategies against skin aging are largely missing. In this context, we proposed the use of skin equivalents as an efficient model to investigate cellular interactions and ECM changes in the aging skin, and to elucidate the role of anti-AGEs molecules in this process.

Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells

“…Cells were isolated and cultivated as described previously by our group. 60, 79 Fibroblasts and melanocytes were maintained in a humidified incubator at 37 °C containing 5% of CO 2 , whereas keratinocytes at 7.5% CO 2 .…”

“…After polymerization, 25 × 10 4 human keratinocytes, 0.83 × 10 4 human melanocytes and 8.3 × 10 4 melanoma cells (SK-MEL-28-naïve or vemurafenib-resistant) were seeded on top of each lattice and the skins were kept submerged in the culture medium for 24 h. Subsequently, the culture was raised and maintained at the air–liquid interface for 12 days to allow complete keratinocytes stratification and differentiation. 64, 79 The RHS containing resistant cells were treated with vemurafenib every 72 h and the RHS containing naïve or resistant cells were treated with Gant61 in the last 72 h of culture period.…”

“…Measurement of melanoma area thickness was carried out using three images with five independent measurements of the melanoma areas thickness from three different experimental conditions. 79 …”

Molecular effects of 1-naphthyl-methylcarbamate and solar radiation exposures on human melanocytes

“…Experiments were performed with human primary skin melanocytes, extracted from the foreskin of two unrelated Caucasian donors (4- and 6-years of age) as described by Pennacchi et al (2015). Melanocytes were cultured with 254CF medium supplemented with calcium chloride 0.2 M and Human Melanocyte Growth Supplement (HMGS, Gibco, Invitrogen Cell Culture, USA), with the addition of Ampicillin and Streptomycin 100mg/L.…”

Epigenetic signature of differentially methylated genes in cutaneous melanoma

“…Three primary cultures of melanocytes isolated from the foreskin of healthy donors [8,9] were used as controls. The primary cultures were obtained in collaboration with the School of Pharmaceutical Sciences from the University of Sao Paulo (University of Sao Paulo -CEP HU/USP 943/09), and DNA samples were extracted from early passages.…”