The long-term goal of this proposal is gain a better understanding of the biomolecular mechanisms underlying keloid pathogenesis. Keloids are the result of pathologic over-healing and represent a worldwide biomedical burden with approximately two billion people at risk. Keloids are not only aesthetically unattractive, but can result in functional impairments and physical deformity with many attendant fiscal, social, and psychological sequelae. In general, we propose to study the molecular biology of keloid pathogenesis using a four-pronged approach. First, we intend to employ a novel co-culture system to investigate the interactions of two important cell-types (keratinocytes and fibroblasts) believed to be critical to the formation of keloids in vivo. We will utilize mix and match combinations of normal skin-derived human keratinocytes (NHKs) or keloid-derived human keratinocytes (KHKs) with normal skin-derived human fibroblasts (NHFs) or keloid-derived human fibroblasts (KHFs). This step will allow us to isolate and focus on these four cell types and their specific interactions. Second, because keloids have a propensity to develop in areas of the body where skin stretch and movement are prevalent, we will examine the effect of equibiaxial strain on these four cell types and determine its effect on gene expression and protein synthesis. Third, we intend to make use of the strong environment for microarray analysis here at Stanford University to study the gene expression of NHKs, KHKs, NHFs, and KHFs. This powerful technology, will allow us to analyze differential gene expression on a genome-wide basis. We can, therefore, use microarray analysis to characterize the large-scale gene expression phenotypes of these four cell types in isolation as well as in coculture. Finally, we intend to use an innovative grafting technique to place cutaneous keratinocyte/acellular dermal grafts onto nude mice. We will pre-seed these grafts with either NHKs or KHKs and eventually NHFs and KHFs in mix and match combinations. In this way, we hope to create an in vivo analog to our in vitro coculture model. The central hypothesis to be tested in this proposal is that KHKs interact with KHFs in promoting keloid pathogenesis
