Laminitis is a painful and often life-threatening disease of the lamellar tissue that connects the axial skeleton of a horse with the inner surface of the hoof capsule. Depending on the underlying cause, the disease can be categorized as SIRS-induced laminitis, endocrinopathic laminitis, and supporting limb laminitis.1,2 A characteristic feature of SIRS-induced laminitis is damage of the lamellar dermoepidermal junction, which is believed to result from changes in the basal epidermal cells and degradation and dysadhesion of the adjacent basement membrane.3–5 This basement membrane is attached to the underlying dermal stroma via numerous anchoring fibrils mainly composed of type VII collagen.6,7 In experimentally induced SIRS-related laminitis, basement membrane damage is associated with loss of type VII collagen,3 and therefore, it appears plausible that degradation of this collagen type contributes to the lamellar failure characteristic of SIRS-induced laminitis in horses.
Like most constituents of the basement membrane, type VII collagen of the anchoring fibrils can be degraded by MMPs, which play a major role in the remodeling of the extracellular matrix in several physiologic and pathological processes.8,9 Of all known MMPs, only 2 collagenases (MMP-1 and MMP-8) and the 2 gelatinases (MMP-2 and MMP-9) are capable of cleaving type VII collagen9; therefore, these 4 MMPs may be relevant to the pathogenesis of SIRS-induced laminitis in horses.
Matrix metalloproteinase-1 (collagenase-1) is produced by several lamellar cell types, including fibroblasts, keratinocytes, and endothelial cells, with increased production on demand (eg, by inflammatory stimulation).8 This protein is capable of degrading type VII collagen of the anchoring fibrils and also other components of the basement membrane,8,9 and it is therefore of particular interest in laminitis research.5,10 The amount of gene expression and cellular production of MMP-1 was increased in horses with versus without CHO-induced laminitis (an often-used in vivo model of SIRS-related laminitis) in a previous study,5 but the contribution of MMP-1 to the pathological remodelling of the lamellar tissue remained doubtful because only the inactive form of MMP-1 was detected.
Matrix metalloproteinase-8 (collagenase-2) is predominantly associated with neutrophils, which store this protein in their specific granules and release it instantly after neutrophil activation,8 but MMP-8 can also be produced at lower amounts by other inflammatory cells as well as by keratinocytes, fibroblasts, smooth muscle cells, and endothelial cells.11,12 The MMP-8 can cleave type VII collagen, but it can also degrade other collagens of the adjacent dermal stroma.8,9 To the authors’ knowledge, the relevance of MMP-8 in the pathogenesis of laminitis has not yet been investigated, but owing to its association with activated neutrophils, one could reasonably anticipate that it may play a role in the pathogenesis of SIRS-induced laminitis.
Matrix metalloproteinase-2 (gelatinase A) is produced by a variety of lamellar cell types, including keratinocytes, fibroblasts, and endothelial cells.8 Matrix metalloproteinase-9 (gelatinase B) is constitutively produced in neutrophils, stored in their tertiary granules, and released from the granules on neutrophil activation, but its expression is also inducible in other cell types, including keratinocytes and endothelial cells with increased expression due to stimulation by inflammatory mediators or by hypoxia.8,13,14 Both MMPs can degrade type VII collagen and other components of the basement membrane.8,9,15 The role of MMP-2 and MMP-9 in the pathogenesis of SIRS-induced laminitis remains unclear; both enzymes have long been associated with degradation of the basement membrane in the course of laminitis, but their ability to initiate the typical lamellar basement membrane damage has been questioned given findings of in vivo laminitis experiments.4,16,17
An established risk factor for SIRS-induced laminitis is endotoxemia, which develops when LPS from gram-negative bacteria enter the circulation, which can happen as a result of various inflammatory diseases, such as gastrointestinal diseases, pleuropneumonia, retained placenta, or metritis.18–21 Direct exposure of the lamellar tissue to LPS through extracorporeal perfusion of isolated equine limbs has been shown to induce pathological changes consistent with those of CHO-induced and naturally acquired laminitis.22,23
The purpose of the study reported here was to use extracorporeal perfusion of isolated equine limbs with LPS-supplemented perfusate as an ex vivo model of SIRS-induced laminitis to investigate the effect of LPS on the expression of MMPs in the lamellar tissue that are capable of cleaving type VII collagen of the anchoring fibrils. Our hypothesis was that the perfusion of the isolated limbs with a clinically relevant concentration of LPS would result in a significant increase in lamellar presence of at least one of these MMPs.
Supported by a research grant from the Vienna University of Veterinary Medicine and an award for the development of alternatives to animal experiments from the International Federation Against Animal Experiments (Internationaler Bund der Tierversuchsgegner).
The authors declare that there were no conflicts of interest.
The authors thank Magdalena Helmreich and Claudia Höchsmann for technical assistance and Dr. Alexander Tichy for help with the statistical analyses.
Interquartile (25th to 75th percentile) range
Systemic inflammatory response syndrome
Sigma-Aldrich Handels GmbH, Vienna, Austria.
Santa Cruz Biotechnology Inc, Santa Cruz, Calif.
Abcam, Cambridge, England.
Abnova/EMBLEM, Heidelberg, Germany.
AbDSerotec, Oxford, England.
BrightVision poly-HRP-anti-rabbit antibody, ImmunoLogic, Duiven, Netherlands.
BrightVision poly-HRP-anti-mouse antibody, ImmunoLogic, Duiven, Netherlands.
IBM SPSS Statistics, version 24, IBM Corp, Armonk, NY.