Equine laminitis: increased transcription of matrix metalloproteinase-2 (MMP-2) occurs during the developmental phase.
BACKGROUND
The dysadhesion and destruction of lamellar basement membrane of laminitis may be due to increased lamellar metalloproteinase activity. Characterising lamellar metalloproteinase-2 (MMP-2) and locating it in lamellar tissues may help determine if laminitis pathology is associated with increased MMP-2 transcription.


OBJECTIVE
To clone and sequence the cDNA encoding lamellar MMP-2, develop antibody and in situ hybridisation probes to locate lamellar MMP-2 and quantitate MMP-2 transcription in normal and laminitis tissue.


METHODS
Total RNA was isolated, fragmented by RT-PCR, cloned into vector and sequenced. Rabbit anti-equine MMP-2 and labelled MMP-2 riboprobe were developed to analyse and quantitate MMP-2 expression.


RESULTS
Western immunoblotting with anti-MMP-2 detected 72 kDa MMP-2 in hoof tissue homogenates and cross-reacted with human MMP-2. Immunohistochemistry and in situ hybridisation detected MMP-2 in the cytoplasm of basal and parabasal cells in close proximity to the lamellar basement membrane. Northern analysis and quantitative real-time PCR showed MMP-2 expression significantly (P < 0.01) elevated in laminitis affected tissues.


CONCLUSIONS
The lamellar pathology of laminitis is associated with increased transcription of MMP-2.


CONCLUSIONS
Real-time PCR analysis of lamellar MMP-2 accurately monitors laminitis development at the molecular level and can be used diagnostically and for testing preventive strategies. Controlling increased MMP-2 transcription may ameliorate or prevent laminitis in high risk clinical situations. Our findings represent a warning to clinicians that the basement membrane lesion of laminitis is insidious and well under way before clinical signs are apparent.
Kyaw-Tanner
M
M
Australian Equine Laminitis Research Unit, School of Veterinary Science, Faculty of Natural Resources Agriculture and Veterinary Science, The University of Queensland, Brisbane, Queensland 4072, Australia.
Pollitt
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005534:Foot Diseases / Q000201:enzymology / Q000662:veterinary*; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D006724:Hoof and Claw / Q000201:enzymology / Q000473:pathology; D006734:Horse Diseases / Q000201:enzymology*; D006736:Horses; D007150:Immunohistochemistry / Q000662:veterinary; D017403:In Situ Hybridization / Q000662:veterinary; D007249:Inflammation / Q000201:enzymology / Q000662:veterinary; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry / Q000235:genetics; D011897:Random Allocation; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D014158:Transcription, Genetic
D018076:DNA, Complementary; D012313:RNA; D020778:Matrix Metalloproteinase 2