Investigation of proteolysis of the basement membrane during the development of equine laminitis

It is well established that failure of the lamellar basement membrane (BM) occurs during the development of equine laminitis. This is due to loss of the crucial BM components; laminins and collagens along with loss of attachment complex, the hemidesmosome, of the basal cell to the underlying BM. Previous studies have suggested that Ln-332 may be the primary protein involved in lamellar failure. However, the details of the progression and mechanism involved in this pathology are not currently fully known. This thesis aimed to refine the proteolytic processes and mechanisms occurring during the development of oligofructose induced laminitis. Through the use of novel temporal lamellar biopsies obtained during the development of laminitis induction, it was determined that loss of both Ln-332 and collagen type IV occurs as early as 12 hours post induction. This loss of reactivity initially occurred in a focal pattern with increasing loss as the disease progressed in severity. At the later stages of laminitis, separation of the basal epithelial cell from the dermal tissue was also observed, however at these points the BM still appeared intact. This suggests that more than one mechanism may be involved in disease pathology; one resulting in fragmentation of the BM while a second results in loss of the cell attachment allowing the intact BM to slip away. Immunohistochemical analysis of lamellar tissue revealed a unique pattern of reactivity for the Ln-332 γ2 antibody D4B5, in which no reactivity was observed in normal lamellar tissue, yet the epitope recognized by this antibody becomes apparent during disease development. This initially led to the hypothesis that cleavage of the γ2 subunit and the release of biologically active fragments may occur. However, at the molecular level, no γ2 fragments were detected by western blotting. In vitro cleavage of partially purified equine Ln-332 revealed that both MMP-2 and MT1-MMP were able to process the molecule to produce fragments corresponding to the biologically active counterparts. This suggests that the change in reactivity with this antibody may be due to other mechanisms such as decreased interaction of Ln-332 with other BM components resulting in loss of structural stability of the BM allowing for a change in the orientation of Ln-332. Increased MMP-2, MMP-9 and MT1-MMP expression has been demonstrated in laminitis and this was assumed to be the causative agent resulting in tissue destruction and failure. However, work in this thesis found no increase in gene expression of MMP- 2 and MT1-MMP, as well as no activation of pro MT1-MMP. Increased pro MMP-9 gene and protein expression was observed early in the disease progression yet no MMP- 9 activation occurred. Additionally, activation of MMP-2 was found to occur late in laminitis progression at least 12 hours following BM degradation, thus MMP-2 activation is a secondary effect of laminitis development. Thus, other proteases are expected to result in BM processing. Gene expression of the metalloprotease ADAMTS-4, was observed to increase early during laminitis development, suggesting this is a putative factor involved in intensifying the degradation of the lamellar BM. Work in this thesis also revealed that both Ln-332 and collagen type IV are widely distributed throughout organs in the equine body and localized primarily to BM structures. A novel finding of this thesis is that not only does BM degradation occur in the lamellar BM, it also occurs in organs remote from the hoof. At both the onset of lameness and the acute phase of laminitis, fragmentation of both Ln-332 and collagen type IV also occurs in both the skin and stomach. Recent studies have indicated that both leukocyte emigration and increased cytokine expression occurs in the lamellar tissue during laminitis. Work in this thesis added to this knowledge as leukocyte infiltration into the lamellar tissue occurs early during oligofructose laminitis induction as does increased IL-6 gene expression. Overall, work conducted in this thesis has added to the knowledge of the events occurring during laminitis development. Even though the complete mechanism of tissue destruction and lamellar failure was not established, the progression of events is now more clear in that BM degradation is one of the first events to occur, while MMP-2 activation occurs secondarily. Thus, other mechanisms must be at work early during laminitis development and discovering what they are must remain a research priority for the realization of effective therapeutic strategies.