The development and validation of novel biomarkers to assess the skin barrier function

Background: The uppermost skin layer, stratum corneum (SC), is a physical and biochemical barrier against environmental insults. The SC resembles a wall with terminally differentiated keratinocytes, corneocytes, as “bricks” that are embedded in lamellar layers as “mortar”. The corneocytes gain in maturity by transglutaminase enhancing rigidity and hydrophobicity. External conditions act on the skin homeostasis and thus understanding these effects on a molecular level might provide new targets for the cosmetic industry. Objectives: Tape stripping is an established approach to collect successive layers of the SC. These tapes were used to identify biomarkers for the maturation of corneocytes by measuring enzyme activities and protein expression in the photo exposed (PE) cheek and photoprotected (PP) post auricular sites. Methods: The SC integrity, cohesion and thickness was determined as a reference measurement. Immunostaining of structural proteins and enzymes was used to characterise the corneocyte from the first and ninth tape strip of both anatomical sites. The relative corneocyte envelope maturity assay was developed by determining the rigidity and hydrophobicity of corneocytes that to correlate to the SC properties. In addition, ex vivo maturation was tested at a range of relative humidities (RH) to determine the optimal RH. The corneocyte maturation and enzyme activities were investigated at low, optimal and high RH in the presence of protease inhibitors. Results: The SC of the PE cheek site is thinner with a lower integrity and higher cohesion compared to the PP post auricular site. The corneocytes from the PE cheek site are less mature than from the PP post auricular site. The corneocytes from the PE cheek site were able to increase in rigidity but not in hydrophobicity. Humidity has an impact on proteases which in turn are able to deactivate transglutaminase activity and thus influence corneocyte maturation. Conclusions: Various methods were tested and correlated with the SC integrity in human subjects. The determination of protein expression was suggested for a set of biomarkers involved in lipid processing enzymes (12R-lipoxygenase and epidermal lipoxygenase 3), structural CE proteins (involucrin and skin-specific protein 32), proteases (cathepsin D and V) and transglutaminase. The impaired ex vivo maturation of samples from the PE cheek point towards pre-mature desquamation and thus provide new pharmaceutical targets for moisturising skin care products.