title: The role and control of matrix metalloproteinases (MMPs) in equine cutaneous wounds: a focus on both endogenous and exogenous proteases
Creators: name: McCarty, Sara
Publisher: name: University of Liverpool Repository
Year: 2015

description: Cutaneous wound healing is orchestrated by a number of physiological pathways which ultimately lead to re-formation of skin integrity and the production of functional scar tissue. Matrix metalloproteinases (MMPs) act to control the remodelling of the extracellular matrix (ECM). Regulation of MMPs is imperative for wound healing as excessive levels of MMPs can lead to disproportionate destruction of the wound ECM compared to ECM deposition. In addition to human MMPs, bacterial proteases have been found to be influential in tissue breakdown and as such have a role to play in the healing of infected wounds. Of particular interest in this thesis is Pseudomonas aeruginosa elastase, which has been reported in previous studies to induce degradation of host proteins including proteoglycans in chronic wounds and has also been shown to degrade host immune cell mediators. This evidence, along with recent data demonstrating the presence of P. aeruginosa biofilms in equine wounds, led to the hypothesis that P. aeruginosa proteases play a role in the non-healing of equine wounds and that the wound environment has an influence on the pathogenicity of P. aeruginosa in terms of protease expression. In this thesis I identify that isolates of P. aeruginosa obtained from equine wounds display a highly variable protease expression and biofilm forming potential (BFP). Furthermore, environmental changes made to P. aeruginosa cultures including changes to the initial culture pH (i.e. changes to the initial pH of the media in a non-buffered system) and depletion of oxygen in vitro were shown to alter protease expression. Further experimentation has shown that the biofilm mode of growth causes changes in the proteolytic profiles of these isolates. A further study was conducted to investigate the effects of exoproducts derived from P. aeruginosa in addition to purified P. aeruginosa elastase on the viability, growth and MMP/TIMP mRNA expression of equine dermal fibroblasts in vitro. Preliminary findings demonstrated that P. aeruginosa exoproducts, including purified elastase exert deleterious effects on both normal skin fibroblasts and granulation tissue fibroblasts in a concentration-dependent manner. Immunohistochemistry analysis of wound tissue revealed the presence of bacterial clusters disseminated throughout the wound bed and this bacterial staining occurred in conjunction with moderate staining of MMPs -2, -3 and -13 throughout the tissue. Furthermore, whilst data is preliminary, QRT-PCR analysis showed that mRNA of each of the MMPs and TIMPs of interest were expressed in normal skin fibroblasts and granulation tissue fibroblasts to a higher level than GapDH, with cells from different equine donors responding differently in terms of MMP/TIMP mRNA expression when treated with P. aeruginosa conditioned media and P. aeruginosa elastase. My results also identified the potential MMP modulating capacity of a novel foam dressing which incorporates polyphosphate. The dressing was effectively able to reduce the levels of MMP-2 and MMP-9 in both their active and latent forms and the dressing demonstrated its potential in the regulation of P. aeruginosa derived proteases. In light of the results from these investigations, it is proposed that in future work the proteolytic phenotype and BFP of infecting bacteria should be considered when investigating the clinical nature of chronically infected wounds.