Hypertrophic scars in deep dermal burns comprise a devastating physical disfigurement for which there is no reliable treatment or prevention. Patients suffer from chronic discomfort due to raised, red, contracted scars. Chronic neuropathic pain constitutes one common complaint; itching impedes ability to sleep and work and negatively impacts quality of life. The Duroc porcine fibroproliferative model offers an opportunity for pre- clinical testing of therapeutic interventions, but is expensive and involves multiple surgeries on large vertebrate animals. Observations that cultured Duroc and Yorkshire fibroblasts maintain a fibroproliferative genotype and phenotype suggest a potential for mechanistic hypothesis development without involving large numbers of pigs. A barrier to understanding hypertrophic scar pathophysiology has been limited ability to define fibroproliferative signaling. Our plan to use RNA sequencing (RNA-Seq) to assess Duroc and Yorkshire fibroblast genomic responses will identify unprecedented expression patterns. Our repository of human DNA with a prospectively collected clinical database allows us to correlate genetic results with severity of scarring. This proposal marries cutting edge methodologies with unique repositories to determine cellular events leading to impaired healing and to identify therapeutic interventions for wound repair. Our novel hypothesis is that Duroc porcine fibroproliferation represents a reproducible biological model of human cutaneous hypertrophic scar formation. We propose 3 specific aims:
Aim 1 : To define the differential transcriptome expression induced by the fibroproliferative mediator transforming growth factor ?1 (TGF?1) in Duroc and Yorkshire fibroblasts. Our hypothesis for Aim 1 is that cultured Duroc fibroblasts have an intrinsic TGF?1-resistent fibroproliferative genotype that represents a reproducible in vitro model that can be used to study cutaneous scarring. We will use RNA Seq to identify novel differentially expressed genes in Yorkshire and Duroc fibroblasts and validate our results by assessing gene expression and protein localization in porcine wounds.
Aim 2 : To determine whether interactions between dorsal root ganglion fibers and dermal fibroblasts modulate Duroc cutaneous responses to injury. Our hypotheses for Aim 2 are that 1) genomic differences in Duroc fibroblast responses to neuroinflammatory signaling cause a fibroproliferative phenotype and 2) Duroc fibroblast-derived signals lead to increased neurogenesis in Duroc dorsal root ganglion cells. We will use the same approach as in Aim 1.
Aim 3 : To determine whether differentially expressed Duroc fibroproliferative genes correlate with human hypertrophic scarring. Our hypothesis for Aim 3 is that differentially expressed genes identified in Aims 1 and 2 have altered expression patterns in human hypertrophic scars and/or have associated single nucleotide polymorphisms that correlate with severity of human scarring. We will validate porcine RNA-Seq data by assessing gene expression and protein localization in human hypertrophic scars. We will determine whether variant alleles in genes identified in Aims 1 and 2 correlate with severity of scarring or itching.
In this new project, we propose that we can utilize cultured porcine fibroblasts rather than live animals to identify previously unrecognized fibrogenic mediators that can be targeted as novel therapeutic interventions to prevent scarring and relieve patient suffering. We will confirm that the novel molecules are differentially expressed in Duroc swine fibroproliferative scars and human hypertrophic scars. We will also determine whether mutations in the identified genes are associated with scar severity in humans.
|Carter, Damien W; Sood, Ravi F; Seaton, Max E et al. (2018) MC1R gene polymorphisms are associated with dysfunctional immune responses and wound infection after burn injury. J Surg Res 231:448-452|