Degradation of lamellar basement membrane components occurs prior to changes in MMP expression and activation.
Introduction
Failure of lamellar basement membrane (BM) integrity and cell dysadhesion occurs during the development of equine laminitis. Ln-332 may be involved due to its purported cleavage by MMPs. For this to be true, Ln-332 cleavage and MMP activation must occur together. We conducted temporal studies on the progression of BM and protease events during laminitis.
Materials and Methods
Laminitis was experimentally induced in normal Standardbred horses (n = 5) by alimentary oligofructose dosing. Sequential lamellar biopsies were collected post dosing. Tissue samples were analyzed using immunohistochemisty (Ln-332 and collagen type IV), western blotting (MT1-MMP), gelatin zymography (MMP-2, MMP-9) and real-time PCR (MMP-2, 9, MT1-MMP, ADAMTS-4).
Results
Loss of Ln-332 and collagen type IV occurred 12 hours post dosing. No significant increase in gene expression of MMP-2 or MT1-MMP, or activation of MT1-MMP occurred. Activation of MMP-2 occurred late in laminitis progression. Increased MMP-9 gene and protein expression was observed early but without activation. Increased gene expression of ADAMTS-4 coincided with BM degradation.
Discussion
Loss of Ln-332 and collagen type IV in lamellar BM, 12 hours post induction indicates early BM damage. MMP-2 activation occurred too late for primary involvement in laminitis development. However, expression of ADAMTS-4 coincided with BM degradation suggesting a role for this enzyme.
Clinical Relevance
Damage to the lamellar BM occurs well before clinical signs of lameness.
Conclusion
Control of enzymatic degradation of lamellar BM should remain a focus of laminitis preventive strategy.
Australian Equine Laminitis Research Unit, School of Veterinary Science, The University of Queensland, Brisbane, Australia
FULL TEXT