326 Two methods of keratinocyte extraction: Do they involve two different types of keratinocyte behavior?

Abstract

We recently showed that the activities of the serine proteases of the plasminogen system (plasmin and urokinase) are elevated in facial stratum corneum (SC) compared to forearm SC and correlated with TEWL. We also demonstrated that plasmin activity is increased on sunexposed SC. Moreover, elevated plasmin activity was associated with reduced corneocyte maturation. The protease/protease-inhibitor balance of the plasminogen system may be key for the SC maturation processes and optimal barrier function. Thus, our aim was to develop a specific dual plasmin and urokinase inhibitor for topical application of barrier-impaired skin. Conventional synthetic, characterization and modelling methods were used in the development of the dual competitive inhibitor benzylsulfonyl-D-Ser-homoPhe-(4-amidino-benzylamide (BSFA) with Ki values 46 and 25 nM for plasmin and urokinase, respectively. The catalytic domain of both proteases is highly conserved and in both models BSFA showed a similar binding mode in silico. In a study on normal human keratinocytes stimulated with inflammatory cytokines (IL-1b, TNFa) the addition of BSFA (100mM) led to a down-regulation of both plasmin (40%) and urokinase (97%) activities. Additionally, an increased gene expression (4.5x) of transglutaminase 1 was induced by BSFA in the cytokine-stimulated keratinocytes. Finally, in a four week, vehicle-controlled study on 44 female Caucasian subjects the effect of a topically applied BSFA hydrogel formulation (10 ppm) was evaluated for basal function and recovery of the barrier of facial skin that was previously challenged by tape stripping. Compared to the vehicle treated group the BSFA treated group showed significantly improved SC barrier integrity, chemo-sensation to a lactic acid challenge and facial skin condition. Our data reveal that the topical application of an inhibitor of the plasminogen system is a novel approach for the treatment of barrier impaired skin and premature desquamation. Gaining a better understanding of the different processes and their regulations occurring during skin aging is fundamental to improve solutions to fight the signs of skin aging. The emergence of miRNA studies and their roles in post-transcriptional inhibition of gene expression highlights the importance of these small RNAs in the skin aging process. miRNA maturation is divided into two major steps: the primary miRNA is processed by Drosha to generate a pre-miRNA; and after exportation in the cytoplasm, Dicer allows the final maturation of miRNA. On senescent lung fibroblasts, it is proved that the expression of these two key markers decreases, involving the dysregulation of 20% of miRNAs. Among them, miRNA-19b expression was shown to decrease with aging except for the centenarians, who keep a high level of this miRNA, suggesting its association with longevity. Firstly, we evaluated the expression of Drosha and Dicer by qPCR in in vitro skin fibroblasts that were aged by replicative senescence. We observed the decrease in the expression of these two key markers, as we...