Understanding the mechanisms of epidermal barrier damage and repair using 3D skin equivalent models

The epidermis of the skin provides an essential barrier between our bodies and the external environment through the formation of a dense cornified layer of cross-linked proteins and lipids at the surface of the skin. Chemical insult or impaired lipid synthesis can damage the cornified layer and lead to barrier dysfunction; however the fundamental relationships between lipid content and barrier biophysical properties remains poorly defined. In addition, there is a need for improved human-based experimental models for investigating lipid function and barrier repair in vitro, and for the evaluation of new therapeutic compounds.
The aim of this PhD studentship is to develop a 3D organotypic skin model of impaired lipid synthesis in order to dissect the functional relationships between lipid synthesis, barrier mechanics, and tissue repair. In addition, this platform will be used to evaluate lipid mimetic compounds developed by the GSK Skin Health group. The project will employ shRNA to stably knockdown key enzymes involved in lipid biosynthesis and advanced biophysical methods to characterise the effects of impaired lipid synthesis and the response to lipid-mimetic treatments on skin barrier function. The project will consist of three major objectives.
1. Build and characterise 3D skin models of impaired lipid synthesis (Months 1-18). The primary lipid components of the epidermis include long chain fatty acids, cholesterol, and sphingolipids. Human keratinocyte cell lines with stable knockdown of key enzymes for each major pathway will be generated using lentiviral transduction of shRNA constructs. We will target fatty acid synthase (fatty acids), HMG-CoA reductase (cholesterol), and serine palmitoyl transferase (sphingolipids) as they are essential enzymes within the lipid synthesis pathways and have all been shown to be upregulated during epidermal barrier recovery (Feingold KR, J Lipid Res, 2009). Keratinocyte cell lines with stable knockdown for each enzyme will be used to construct 3D organotypic models, and the effects on tissue biophysics will be assessed by atomic form microscopy (AFM) methods available within our group, while terminal differentiation and barrier function will be examined by immunohistochemistry and permeability assays, respectively.
2. Develop quantitative relationships between lipid content and epidermal mechanics (Months 19-33).
Lipidomic profiling of human skin equivalents lacking different components of the lipid biosynthesis pathways will be performed by mass spectrometry through a commercially available service (Lipotype GmbH). These lipid profiles will then be correlated with biomechanical data to develop quantitative and predictive models of the relationship between lipid content and skin barrier function.