Advances in the Canine Cranial Cruciate Ligament, 2nd edition

Role of Nitric Oxide Production and Matrix Protease Activity in Cruciate Ligament Degeneration 85

2005; Doomet al. 2008). However, some MMPs
have opposite roles in the development of spe-
cific diseases. For example, MMP-2 and MMP-
9 (Gelatinase A and B) have very similar sub-
strate specificities for matrix proteins, but have
opposite non-matrix-related actions: MMP-2 is
important for the degradation of inflammatory
factors, whereas MMP-9 is an important prod-
uct of macrophages and neutrophils. Experi-
mentally, MMP-2 deficiency leads to exacer-
bated arthritis, whereas a deficiency of MMP-9
reduces arthritis (Itohet al. 2002).
Various studies have detected MMPs in CrCL
tissue during aging, degeneration, or in torn
CrCLs. Results of a study on cellular and ECM
changes in the human anterior cruciate lig-
ament (ACL) showed that constitutive MMP
expression (MMP-1, -3, and -13) declines with
aging, while a marked upregulation occurs in
degenerated ACL, mainly in chondrocyte-like
cells but not in fibroblast-like cells (Hasegawa
et al. 2013). It was suggested that decreased
MMP expression with aging may reflect a
reduced capacity to remodel and maintain the
tissue, whereas an increase in ACLs from OA
knees may be caused by phenotypic changes
and contribute to degeneration. These find-
ings are supported by examination of MMP
gene expression in ruptured and intact CrCLs
from dogs separated into different age groups.
MMP-13 and both gelatinases were significantly
increased in ruptured CrCLs only, whereas
the collagen I degrading enzymes MMP-1 and
MMP-3 were present in torn and in intact aged
CrCLs (Muiret al. 2005). The similar pattern of
metalloprotease expression in torn and aging
human and canine cruciate ligaments suggests
that changes in collagenolytic enzymes influ-
ence the development of subtle joint laxity over
time, and subsequently induces the progression
of CR (Fooset al. 2001; Muiret al. 2005).
Degradative enzymes have the potential to
diffuse from the ligament into the synovial
fluid, particularly if there are defects in the syn-
ovial lining and exposure of ligament to the
joint environment. Thus, many studies have
evaluated MMP activity in synovial fluid of
dogs with CR. In the synovial fluid of dogs with
acute CR, MMP-2 and MMP-9 activities are sig-
nificantly elevated compared to synovial fluid
of unaffected or contralateral joints (Rabillard
et al. 2012; Bolandet al. 2014; Murakamiet al.

2016). The activity of MMP-13, however, was

similar and MMP-3 was not detected in syn-

ovial fluid in either healthy or affected joints.

Moreover, activity levels were not influenced by

surgical stabilization and postoperative doxy-

cycline treatment (Rabillardet al. 2012).

MMP measurement in synovial fluid is only

of limited value in understanding pathophys-

iologic pathways because it is not possible to

differentiate the origin of the MMP production

when measured in synovial fluid (Fujitaet al.

2006; Salinardiet al. 2006). Beside CrCL tissue,

elevated MMPs originate from other joint

tissues, particularly inflamed synovium or

cartilage.

Influence of NO and MMPs on CrCL

structure

The pathogenesis of canine CR involves

the complex processes that change a normal,

healthy ligament into an altered, nonfunctional,

and potentially torn ligament. Alterations have

been evaluated in both the ligament fibrob-

lasts and the ECM, and characterized from the

molecular to the macroscopic level, yet the exact

role still remains to be verified. One of the main

reasons for this is the difficulty in studying the

early phase of the disease process that leads

to CR.

There is good evidence that NO and MMPs

modulate the consequences of CR, for exam-

ple OA (Abramson 2008). On the other hand,

almost all scientific information on NO and

MMP involvement in the early and subclinical

stages of the CR condition is only descriptive

and fragmented, and does not allow any defi-

nite conclusions to be made on the exact path-

way that leads to CrCL fiber rupture. Some of

these data concerning direct actions of NO and

MMP on the ligament are described below.

External stimuli induce NO and MMP in

ligaments

Recent findings of a study using a novel canine

model revealed that synovial debridement

of the CrCL has detrimental effects on clini-

cal signs such as pain, and also on effusion,

function, and histology (Bozynskiet al. 2015).