86 Etiopathogenesis of Cruciate Ligament Rupture
Synoviectomy of the rabbit CrCL induces a sig-
nificant increase in collagenase activity within
the ligament, indicating that the synovial sheath
is a critically important structure for joint health
(Amielet al. 1990). The released collagenase
after synoviectomy may reflect the reaction of
the CrCL to synovial fluid mediators released
from inflamed synovium. Canine CrCL explant
cultures, as well as ligament fibroblast mono-
layer cultures activated with an inflammatory
stimulus, produce significantly more NO and
MMP compared to non-stimulated cultures
over a period of 48 hours (Riitanoet al. 2002).
The same stimulus induces a significantly
higher iNOS expression in CrCL compared to
medial collateral ligaments and femoral head
ligaments (Louiset al. 2006). Different patterns
of NO activity may reflect the different under-
lying abilities to react, and the different suscep-
tibilities to pathological processes, between the
ligaments.
High NO levels inhibit collagen
synthesis in ligament fibroblasts
CrCL has been shown to produce more NO
compared to other ligaments (Caoet al. 2000;
Sprenget al. 2000). Experiments performed by
Cao and colleagues showed that NO production
was increased due to iNOS induction, particu-
larly in CrCL cells, and inhibited normal colla-
gen synthesis. High NO concentrations could,
therefore, be a reason for the insufficient heal-
ing capacities of ruptured CrCL in humans
and in dogs. Moreover, high NO concentra-
tions in canine CrCL may contribute to matrix
homeostasis disturbance, leading to an intrinsic
weakness of the ligament.
NO combined with peroxynitrite induces
apoptosis in the ligament
In vitro experiments have demonstrated that
different NO donors induce apoptosis in CrCL
cells (Murakamiet al. 2005; Forterreet al. 2011).
In addition, fibroblasts from the CrCL were
more susceptible to NO-induced apoptosis than
cells from the caudal cruciate ligament or from
the medial collateral ligament. CrCL apop-
tosis is influenced by inhibition of caspase-
3(Murakamiet al. 2005), and is attenuated
by treatment with ROS scavengers or a tyro-
sine kinase inhibitor (Forterreet al. 2012). On
the other hand, oral treatment with L-NIL, a
specific iNOS inhibitor, did not have a direct
influence on the amount of apoptotic ligament
fibroblasts in dogs with CR (Hoferet al. 2009).
Consequently, NO alone is not responsible for
increased levels of apoptosis in the CrCL. This
corresponds to other reports on chondrocytes
indicating that only the combination of NO and
ROS induces apoptosis (Del Carlo & Loeser
2002). Increased NO production could be an
early step in development of CR, and may con-
tribute to the induction of cell death followed
by catabolic derangement of the homeostasis of
the ligament.Conclusions
All of the above described observations corre-
spond with the theory that an initial event leads
to inflammation of the cruciate ligament com-
plex (Hayashiet al. 2004). Ligament fibroblasts
develop a dysregulation of matrix homeosta-
sis as a reaction to the inflammatory stimulus,
including an upregulation of matrix-degrading
enzymes, a downregulation of matrix produc-
tion, and peroxynitrite-induced premature lig-
ament fibroblast death (see Figure 10.2). The
role of NO, however, is less simple. By extrapo-
lating data fromin vitrocartilage experiments
it seems that inhibition of NOS generation is
able to decrease inflammation, but alone has
little effect on protecting the joint from car-
tilage loss. On the other hand, peroxynitrite
inhibition reduces intra-articular inflammation
and loss of articular cartilage (Bezerraet al.
2004). Other studies, however, have shown the
opposite: OA dogs treated with a specific iNOS
inhibitor showed a reduction in osteophyte for-
mation, a decrease in the severity of histological
cartilage lesions, and a reduced levels of apop-
tosis in cartilage compared to untreated dogs
(Pelletieret al. 2000). Adding to the complex-
ity of the known catabolic effects of NO, the
latter has also been shown to promote carti-
lage deposition, thereby highlighting a protec-
tive role of NO in tenocytes and human chronic
OA (Xiaet al. 2006; Hancock & Riegger-Krugh
2008). These conflicting results suggest that the
role of NO in OAneeds to be further elucidated.