Deficiency of seven in absentia homologue (Siah) and its effect on wound healing

Wound healing in the skin is a complex and well orchestrated process involving the interaction of numerous cell types as healing progresses through phases involving inflammation, proliferation and remodelling. Impairment to the wound repair process can lead to delayed function in scarring tissue, the difficulty in healing chronic wounds and high cost of wound care, there is considerable interest in the factors involved in the regulation of normal healing. Following injury, vascular damage often occurs resulting in low oxygen tension (hypoxia). Slight changes in systemic and cellular oxygen concentrations induce tightly regulated response conditions. Most of these responses occur through the induction of the transcription factor hypoxia inducible factor-1 (HIF-1) which regulates many processes needed to carry out tissue repair processes during wound ischaemia. For this reason HIF-1 is viewed as a positive regulator of wound healing and a potential regulator of tissue fibrosis. Seven in absentia homologue proteins (Siah) are a family of E3 ubiquitin ligases that tag HIF-hydroxylases for degradation during hypoxia and thus increase HIF-1α availability. In this context, Siah deficient cells are unable to adequately stabilise HIF-1α under hypoxic conditions. We hypothesized that Siah would have a regulatory role in early wound healing where hypoxic microenvironments are common and that Siah would be important for cell functionality in hypoxia. Thus, this thesis aims to address the role of Siah2 in dermal wound healing in vivo by examining the effect of Siah2 deficiency using Siah2 knockout (transgenic) mice and in vitro through the study of cultured dermal fibroblasts (murine Siah2-deficient fibroblasts and human fibroblasts). The results indicate that in the absence of Siah2, cutaneous wound repair is significantly impaired, albeit transiently. Specifically, macrophage invasion, re-epithelialisation, matrix deposition, fibroblast-myofibroblast transition, angiogenesis and wound contraction are delayed or reduced in Siah2 deficient wound repair. Furthermore, Siah2 knockout fibroblasts showed impaired functionality under hypoxic conditions (specifically collagen synthesis, αSMA induction) and an impaired response to exogenous TGFβ (delayed cell migration and reduced collagen synthesis). Moreover, transient inhibition of Siah in human dermal fibroblasts using a transfected Siah1/2 inhibitor (phyllopod, a Drosophila protein) reduced the functionality (VEGF release, collagen induction, cell migration and TGF beta receptor type 1 induction) of otherwise healthy dermal cells. The results suggest for the first time, a role for Siah proteins as positive regulators of early wound healing through their regulation of HIF-1α fate and possibly through HIF-1 independent mechanisms. Aberrant wound healing is a concern of epidemic promotions and current strategies are far from ideal. In conclusion, this thesis demonstrates that Siah is a positive regulator of wound healing and cell functions related to healing and we propose that these findings may have important clinical implications for both impaired and over-elaborate forms of healing that result in scarring.