John F Tarlton1 and A J F Webster University of Bristol, Langford, Bristol, UK
A BIOCHEMICAL AND BIOMECHANICAL BASIS FOR THE
PATHOGENESIS OF CLAW HORN LESIONS// Proceedings of 12th
IINTERNATIIONAL CONFERENCE ON BOVIINE LAMENESS/Section 6
DISEASES OF THE CLAW & CLAW HORN, 2002.-Р.395-397
Abstract
The supportive capacity of the connective tissue suspending the pedal bone in the hooves of
heifers around the time of first calving was determined, and compared with age matched
maiden heifers. Increased laxity, reduced rigidity, and decreased load bearing capacity at
appropriate physiological extension, demonstrated a clear deterioration of structural integrity
in hooves of heifers around first calving, by comparison with the maiden heifers.
Furthermore, these changes progressed from 2wks pre-calving to 12 weeks post calving. This
would permit the pedal bone to drop, transmitting greater loads to other structures, in
particular the sole, leading to ulceration, haemorrhage, white line lesions, and lameness. The
biochemical mechanisms underlying these changes were explored. There was a marginal
increased expression of proMMP-2, and a highly significant increase in the levels of activated
MMP-2 in the calving heifers. A novel gelatinolytic protease of approximately 52kD
("hoofase"), resistant to inhibitors of serine, cystein, aspartate and metallo-proteinases, was
expressed at high levels in the hooves of calving heifers, but was absent in the maiden heifers.
There was a highly significant relationship between MMP-2 activation and expression of
"hoofase", which was found to be capable of activating MMP-2 in vitro. Levels of “hoofase”
were greatest at 2wks pre-calving, with levels declining at 4 and 12wks post calving,
suggesting a causal relationship with the loss in supportive capacity, rather than being a
consequence of such changes. The hooves of a cow diagnosed with laminitis were also found
to express high levels of "hoofase", further demonstrating its role in the pathogenesis of cattle
lameness.
Introduction
The most common and intractable cause of lameness in dairy cattle is claw horn lesions
(CHL). Early pathogenesis of CHL appears to involve sinkage of the distal phalanx,
increasing pressure on the sole and the risk of sole ulceration and white line disease. We
hypothesise that the primary causal event involves increased laxity of the connective tissue
suspending the pedal bone within the hoof, linked to metabolic changes associated with
parturition.
Materials and methods
Hooves were removed from heifers aged 2-2.5 years killed two weeks before first calving (C2), and 4 and 12 weeks post-calving (C+4 and C+12), and from age-matched maiden heifers.
Segments were dissected from the anterior walls of lateral hind claws, to include horn, corium
and bone, and the biomechanical properties measured on an Instron 6200 mechanical test
frame. Corium was removed from the hoof segments, pulverised, lyophilised and extracted in
20mM triethanolamine/0.1% Brij 35 at 20μl per mg of tissue. Levels of gelatinolytic
proteases were determined using gelatin gel zymography. In brief, extracts were
electrophoresed on polyacrylamide gels with gelatin substrate incorporated, incubated overnight
in proteolysis buffer, and stained with coomassie blue. Densitometric scanning was
used to quantify levels of proteases. For inhibition studies, inhibitors for matrix
metalloproteinases (EDTA, 10mM; 1,10 phenanthroline 10mM; hydroxamate, 1mM), serine
proteases (AEBSF, 2mM; soybean trypsin inhibitor, 50μg/ml; leupeptin, 25μg/ml), cystein
proteases (E64, 10μM; leupeptin, 25μg/ml), or aspartic proteases (pepstatin, 10μM), were
added to the proteolysis buffer. Pooled extracts expressing high levels of "hoofase" were
subjected to cation exchange chromatography on a FPLC, and fractions containing partially
purified "hoofase" (determined by zymography) were used in activation experiments on
proMMP-2 prepared from human acute wound fluid.
Results
There was a reduced capacity for support in the hooves of calving (versus maiden) heifers,
and this progressed from C-2 to C+12. Overall capacity to resist loading, i.e. the stress-strain
gradient to failure, decreased in the calving heifers, reaching minimum levels at C+12 (Fig
1A, p=0.034). Greater laxity in the supportive connective tissue was identified, with increased
displacement experienced to initiate support (Fig 1B, p<0.0005), and to reach maximum
support (Fig 1C, p=0.012), in the calving heifers, again progressing to C+12. At a
displacement of 2mm, judged to be of a scale experienced during normal physiological
loading, there was a marked decrease in the support provided by the hoof connective tissue of
calving heifers (Fig 1D, p=0.001).
A previously unidentified ~52kD gelatinolytic protease, "hoofase”, was detected in all those
specimens derived from calving heifers, but was absent in the maiden heifers (fig 2).
Hoofase was not inhibited by leupeptin, soybean trypsin inhibitor, AEBSF, E64 or pepstatin, and
was therefore not typical of a serine, cystein, or aspartic protease. It was only partially
inhibited by EDTA and 1.10-phenanthroline, and unaffected by hydroxamate, and is therefore
not a matrix metalloproteinase (MMP). Hoofase was also found in hooves of a heifer
suffering from clinical laminitis, and therefore may be specific to calving and laminitic cows.
Highest levels of hoofase were seen in the heifers 2wks before calving (Fig 3A, p<0.0005).
There was a marginal increase in levels of proMMP-2, a connective tissue protease of great
importance in both physiological and pathological remodelling (Fig 3B, p=0.056 at C-2), and
a highly significant increase in activated MMP-2 (Fig 3C, p<0.0005 at C-2) consistent with
elevated matrix remodelling. Little or no MMP-9 was detected in maiden, calving or laminitic
specimens.
Discussion
Reduction in the supportive capacity of the hoof wall connective tissue around calving is
consistent with a role in the early pathogenesis of CHL. The increased stress transmitted to
other supportive structures predisposes to sole ulceration and white line separation. Early
clinical signs of lameness have been demonstrated to occur around first calving, particularly
when combined with external trauma. The absence of MMP-9 indicates that inflammation is
not a major feature in the pathogenesis of hoof pathology around calving. The appearance of
the 52kD hoofase prior to calving, may be significant in the pathogenesis of CHL. As well as
degrading matrix components, hoofase is able to activate MMP-2, an important mediator of
collagen remodelling. The peak in hoofase activity is in advance of the maximum
biomechanical and clinical signs of disease, implying a causal role in pathology. The link with
clinical lameness was confirmed by the presence of hoofase in laminitis, and this protease
may represent a therapeutic target. The identity of hoofase is not known. Inhibition studies
indicate that it is not a typical serine, cystein, aspartic or metallo-protease, but may be a
member of the ADAM family of proteases.
References
A complete list of references may be provided upon request of the authors.