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Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits
Published on: June 2, 2019
The developmental and acute phases of insulin-induced laminitis involve minimal metalloproteinase activity
M A de Laat1, M T Kyaw-Tanner, A R Nourian
1Australian Equine Laminitis Research Unit, School of Veterinary Science, The University of Queensland, Gatton, Queensland 4343, Australia. m.delaat@uq.edu.au
Abstract:
Metalloproteinases have been implicated in the pathogenesis of equine laminitis and other inflammatory conditions, through their role in the degradation and remodelling of the extracellular matrix environment. Matrix metalloproteinases (MMPs) and their inhibitors are present in normal equine lamellae, with increased secretion and activation of some metalloproteinases reported in horses with laminitis associated with systemic inflammation. It is unknown whether these enzymes are involved in insulin-induced laminitis, which occurs without overt systemic inflammation. In this study, gene expression of MMP-2, MMP-9, MT1-MMP, ADAMTS-4 and TIMP-3 was determined in the lamellar tissue of normal control horses (n=4) and horses that developed laminitis after 48 h of induced hyperinsulinaemia (n=4), using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Protein concentrations of MMP-2 and MMP-9 were also examined using gelatin zymography in horses subject to prolonged hyperinsulinaemia for 6h (n=4), 12h (n=4), 24h (n=4) and 48 h (n=4), and in normal control horses (n=4). The only change in gene expression observed was an upregulation of MMP-9 (p<0.05) in horses that developed insulin-induced laminitis (48 h). Zymographical analysis showed an increase (p<0.05) in pro MMP-9 during the acute phase of laminitis (48 h), whereas pro MMP-2 was present in similar concentration in the tissue of all horses. Thus, MMP-2, MT1-MMP, TIMP-3 and ADAMTS-4 do not appear to play a significant role in the pathogenesis of insulin-induced laminitis. The increased expression of MMP-9 may be associated with the infiltration of inflammatory leukocytes, or may be a direct result of hyperinsulinaemia. The exact role of MMP-9 in basement membrane degradation in laminitis is uncertain as it appears to be present largely in the inactive form.
Insights
Matrix metalloproteinase-9 (MMP-9) was upregulated in horses with insulin-induced laminitis. However, MMP-2 and other enzymes did not appear to play a significant role in this condition.
Area of Science:
- Equine pathology
- Biochemistry
- Molecular biology
Background:
- Matrix metalloproteinases (MMPs) are crucial in extracellular matrix remodeling and implicated in equine laminitis.
- While MMPs are involved in inflammatory laminitis, their role in insulin-induced laminitis is unclear.
- Insulin-induced laminitis occurs without overt systemic inflammation, suggesting different pathogenic mechanisms.
Purpose of the Study:
- To investigate the gene and protein expression of specific matrix metalloproteinases (MMPs) and their inhibitors in equine insulin-induced laminitis.
- To determine the involvement of MMP-2, MMP-9, MT1-MMP, ADAMTS-4, and TIMP-3 in the pathogenesis of insulin-induced laminitis.
Main Methods:
- Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was used to assess gene expression of MMP-2, MMP-9, MT1-MMP, ADAMTS-4, and TIMP-3 in lamellar tissue.
- Gelatin zymography was employed to measure protein concentrations of MMP-2 and MMP-9 in horses at various time points after induced hyperinsulinaemia.
- Control horses and horses with experimentally induced insulin-induced laminitis were used for comparative analysis.
Main Results:
- Upregulation of MMP-9 gene expression was observed in horses that developed insulin-induced laminitis (48 h).
- Zymographical analysis revealed an increase in pro MMP-9 during the acute phase (48 h) of laminitis.
- Pro MMP-2 concentrations remained similar across all groups, and no significant changes were noted for MMP-2, MT1-MMP, TIMP-3, and ADAMTS-4.
Conclusions:
- MMP-2, MT1-MMP, TIMP-3, and ADAMTS-4 do not appear to be significantly involved in the pathogenesis of insulin-induced laminitis.
- Increased MMP-9 expression may be linked to inflammatory cell infiltration or a direct effect of hyperinsulinaemia.
- The precise role of MMP-9 in basement membrane degradation in laminitis requires further investigation, as it was largely found in an inactive form.
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