Expression and activity of metalloproteinases in the digital laminae of horses with starch gruel-induced laminitis

s / Journal of Equine Veterinary Science 31 (2011) 562-609 566 Scientific – Inflammation Part 2 Expression and activity of metalloproteinases in the digital laminae of horses with starch gruel-induced laminitis Le Wang , Erica A. Pawlak , Philip J. Johnson , James K. Belknap , Dominique Alfandari , and Samuel J. Black 1 University of Massachusetts, Amherst, MA, University of Missouri, College of Veterinary Medicine, Columbia, MO, 3 The Ohio State University, College of Veterinary Medicine, Columbus, OH Take-Home Message: Over-expression of metalloproteinases (ADAMTS-4,MMP-1,MMP-12, andMMP-13) in the laminaeofhorses withstarchgruel-induced laminitismayberesponsible for structural failure of the tissue. An important component of the pathology is ADAMTS-4 cleavage of versican and accompanying physiologic remodeling of basal epithelial cells, as evidenced by suppressed expression of genes encoding versican, b catenin, and integrin b4. Introduction: Starch gruel-induced laminitis replicates disease parameters occurring in horses with naturally acquired acute laminitis. Thus, this model is used to study pathological processes leading to structural failure of the digital laminae. In this study it was used to investigate the role(s) of metalloproteinases in the loss of laminar integrity during acute laminitis. Materials and Methods: Acute laminitis was induced in horses by the nasogastric administration of a starch gruel. Metalloproteinase gene expression was determined using cDNA prepared from laminar RNA and real-time quantitative polymerase chain reaction. Protein expression and processing were determined by sodium dodecylsulfate polyacrylamide gel electrophoresis of laminar extracts and quantitativeWestern blotting. Protein distribution and abundance were determined by quantitative immunofluorescent staining. Results: Expression of genes encoding ADAMTS-4, MMP-1, MMP-12, and MMP-13 was elevated in the laminae of horses with starch gruel-induced laminitis. There were temporal differences in gene expression as follows: MMP-12 and ADAMTS-4 gene expression was increased w100-fold at the onset of fever, and was sustained thereafter; MMP-1 gene expression was increasedw25 fold at the onset of Obel grade 1 lameness (OG-1) and increased thereafter; MMP-13 gene expression was increased w100 fold at the onset of Obel grade 3 lameness (OG-3). There was no treatment-induced increase in the expression of genes encoding MMP-2, MMP-3, MMP-9, MMP-14, MMP-15, MMP-16, or MMP-25. Elevated expression of the gene encoding ADAMTS-4 was accompanied by elevated ADAMTS-4 protein expression and activity. This upregulation resulted in increased cleavage of its substrates, aggrecan and versican, with which it co-localizes in basal epithelial cells. Increased cleavage of versican by ADAMTS-4 was accompanied by suppressed expression of the genes encoding versican, b catenin, and integrin b4; a reduction in the presence of versican and attachment proteins; and detachment of some epithelial cells from the basement membrane, thus compromising the epidermal-dermal junction. Discussion: The timing of elevated expression of genes encoding ADAMTS-4, MMP-1, MMP-12, andMMP-13 indicates independent regulation, but it does not preclude a cascade effect. Elevated metalloelastase (MMP-12) and collagenase (MMP-1, MMP-13) gene expression, if accompanied by elevated enzyme activity, would lead to reduced resistance of the laminae to stretch deformation and loss of tensile strength in the laminae. Loss of versican from basal epithelial cells correlates with profound changes in their physiology, including suppressed expression of genes encoding b catenin and integrin b4, an effect consistent with epithelial-to-mesenchymal transition as described by Sheng et al. (Mol Biol Cell 2006; 17:2009–2020). The protein b catenin is a component of the adherens junction, which sustains epithelial cell-cell association, while integrin b4 is a component of the hemidesmosome, which sustains epithelial cell-basement membrane attachment. Conclusions and Clinical Relevance: Over-expression of MMP1, MMP-12, MMP-13, and ADAMTS-4 in the digital laminae may result in degradation of extracellular matrix components required to maintain the shape and tensile strength of the laminae. It may also reduce production of epithelial cell proteins required to sustain the integrity of the epidermal-dermal junction. Inflammatory gene expression in the liver, lung, and kidney in the carbohydrate overload model of equine laminitis B.S. Leise ,M.Watts , P.J. Johnson , S.J. Black , and J.K. Belknap 2 1 Colorado State University College of Veterinary Medicine and Biomedical Sciences, Fort Collins, CO, 2 The Ohio State University College of Veterinary Medicine, Columbus, OH, 3 University of Missouri College of Veterinary Medicine, Columbia, MO, University of Massachusetts, Amherst, MA Take-Home Message: Differences in the magnitude and type of inflammatory response exist between the laminae and othermajor organs of the horse after carbohydrate overload. Although inflammation is present in the liver, lung, and kidney, it is not sufficient to result in organ failure as is commonly seen in the laminae. Introduction: The carbohydrate (CHO) overload model is used to study the development of laminitis in horses. Administration of CHO causes changes in the gastrointestinal microflora which result in systemic inflammatory response syndrome (SIRS), with subsequent injury and failure of the laminae. Human patients with sepsis/SIRS often undergo multiple organ injury and failure. In the adult horse, the most common “organ” to fail secondary to SIRS/sepsis is the foot. Organs known to be commonly injured in human sepsis, such as the liver, lung, and kidney, seem much less likely to be injured in the horse following CHO overload; however, such injury has not been investigated. The purpose of this study was to determine how other target organs of sepsis respond in the horse after administration of CHO overload. Materials and Methods: Samples of liver, lung, and kidney were obtained from 18 horses administered either water (control group, n1⁄46, euthanized 24 h after water administration) or 17.6 g of starch/kg body weight (CHO group, n1⁄412) via nasogastric tube. Horses administered CHO were euthanized after showing a 2degree increase in rectal temperature (DEV group, n1⁄46) or at the onset of Obel grade 1 lameness (OG-1 group, n1⁄46). Real timequantitative polymerase chain reaction for the cytokines IL-1b, IL6, CXCL8, IL-10, TNFa, COX-1, and COX-2 was performed using