482 EQUINE VETERINARY JOURNAL Equine vet. J. (2008) 40 (5) 482-487 doi: 10.2746/042516408X270353 Summary Reasons for performing study: Enzymatic separation at the hoof lamellar dermal-epidermal interface may play a role in the development of laminitis and characterising and locating matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of MMPs or TIMPs) in lamellar tissues may further understanding of pathogenesis. Objectives: To clone and sequence the cDNA encoding lamellar MMP-14 and TIMP-2, and quantify their transcription in normal and laminitic tissue; and to develop antibody to locate MMP-14 in lamellar tissues. Methods: Tissue samples were obtained from an oligofructose induced model of laminitis. Total RNA was isolated, amplified by RT-PCR, cloned into a vector and sequenced. Real-time PCR was used to quantify MMP-14 and TIMP-2 expression. Rabbit anti-equine MMP-14 antibody was developed to analyse MMP-14 proteins from hoof tissues. Results: Immunohistochemistry detected MMP-14 in the cytoplasm of normal lamellar basal and parabasal cells in close proximity to the lamellar basement membrane. In laminitis affected tissue MMP-14 immunostaining was depleted in lamellar basal cells. Quantitative real-time PCR showed MMP-14 and TIMP-2 expression significantly (P<0.05) elevated and lowered respectively in laminitis affected tissues. Conclusion: MMP-14, located in the cytoplasm of normal lamellar basal cells, disappears during laminitis development. The pathology of laminitis is associated with increased and lowered transcription of MMP-14 and TIMP-2, respectively. Potential relevance: Enzymes have a role in laminitis pathology and inhibition of their activity may prevent laminitis. Introduction Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases involved in physiological and pathological degradation of the extracellular matrix (ECM) (Woessner 1991; Mignatti and Rifkin 1993). The activities of MMPs are controlled by natural inhibitors known as tissue inhibitors of metalloproteinases (TIMPs). MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are capable of degrading type IV collagen, a major constituent of the hoof lamellar basement membrane. MMP-2 is secreted as an inactive proenzyme and requires proteolytic removal of its N-terminal profragment to be activated. A transmembrane MMP named membrane type-1 MMP (MMP-14) has been identified on the surfaces of cells, and was shown to activate pro-MMP-2 specifically (Sato et al. 1994). Correlations between MMP-14 expression and pro-MMP-2 activation have been reported in various human tumours (Nomura et al. 1995; Tokuraku et al. 1995; Fishman et al. 1996; Gilles et al. 1996; Yamamoto et al. 1996; Ueno et al. 1997; Yoshizaki et al. 1997; Kitagawa et al. 1998; Jiang et al. 2006), suggesting the involvement of MMP-14 in pro-MMP-2 activation in such tumours. Laminitis is a disease involving the separation of epidermal basal cells in the inner hoof wall from their underlying basement membrane. The separation process involves lysis and dysadhesion of the lamellar basement membrane (Pollitt 1996; Pollitt and Daradka 1998) and cleavage of the laminin 5 filaments that attach basal cells to their underlying basement membrane (French and Pollitt 2004). The clinical consequence of these ultrastructural and molecular perturbations is a crippling and painful disease that affects horses. A laminitis induction model, based on alimentary dosing with the nonstructural carbohydrate oligofructose, which reproduces the lamellar lesions and clinical signs of natural cases of laminitis, was used in this study (van Eps and Pollitt 2006). It has been shown that the activity of MMP-2 in the hoof lamellae of horses with laminitis increases (Johnson et al. 1998; Pollitt et al. 2003; Kyaw-Tanner and Pollitt 2004; van Eps and Pollitt 2004). The present study investigated the expression of MMP-14 and TIMP-2 in hoof tissues of both normal and laminitic horses. The objective was to provide insight into the pathology of laminitis at the molecular level in the hope of developing more efficacious preventive and treatment strategies. Materials and methods Hoof tissue samples The project was approved by a University of Queensland Animal Ethics Committee (AEC) that monitors compliance with the Animal Welfare Act (2001) and The Code of Practice for the care Equine laminitis: Membrane type matrix metalloproteinase-1 (MMP-14) is involved in acute phase onset M. T. KYAW-TANNER, O. WATTLE † , A. W. VAN EPS and C. C. POLLITT* Australian Equine Laminitis Research Unit, School of Veterinary Science, Faculty of Natural Resources Agriculture and Veterinary Science, The University of Queensland, Brisbane, Queensland 4072, Australia; and † Department of Clinical Sciences, University of Agricultural Sciences, Uppsala, Sweden. Keywords: horse; RT-PCR; TIMPs; matrix metalloproteinases; laminitis *Author to whom correspondence should be addressed. [Paper received for publication 26.04.06; Accepted 12.12.07]