Advances in the Canine Cranial Cruciate Ligament, 2nd edition

82 Etiopathogenesis of Cruciate Ligament Rupture

L-arginine

O 2 , NADPH

H 2 O, NADP+ iNOS, cNOS

N-hydroxy-L-arginine

iNOS, cNOS

O 2 , 1/2 equiv NADPH

H 2 O, 1/2 equiv NADP+

L-citrulline + NO

Figure 10.1 Production of nitric oxide via the oxidation
of L-arginine by a family of nitric oxide synthase (NOS)
enzymes.

suppresses different cellular protein synthesis
in various cell types, and produces marked
cytotoxicity by inhibiting mitochondrial respi-
ration or inducing DNA alterations. Reaction
of NO with the superoxide anion (O 2 .–),
another product of activated macrophages,
may yield peroxynitrite (ONOO–) which medi-
ates oxidative DNA damage and subsequently
programmed cell death described for several
cell types, including chondrocytes (Del Carlo &
Loeser 2002).
The list of diseases where iNOS is currently
thought to be involved is large and rapidly
increasing. In multiple organ failure following
septic shock, type I diabetes or ulcerative col-
itis, massive NO formation due to iNOS acti-
vation was characterized. Another chronic local
inflammatory process with iNOS participation
occurs in the arthritic joint (Del Carlo & Loeser
2008).

Nitric oxide activity in articular tissues

Apart from iNOS, the isoforms cNOS and
nNOS which produce lower amounts of NO,
have also been detected in different healthy and
osteoarthritic joint tissues (Aminet al. 1995; Pel-
letieret al. 2000; Di Mauroet al. 2006). However,
nNOS was more frequently expressed in nor-
mal than in osteoarthritis (OA) chondrocytes

(Rosaet al. 2008). Within joints, NO is mainly

produced by superficial cartilage chondrocytes

and by synoviocytes or macrophages in the

inflamed synovial membrane; all cells in which

iNOS expression is upregulated during OA

(Aminet al. 1995; Hauselmann ̈ et al. 1998).

The importance of iNOS expression and the

subsequent increase of NO in the pathogenesis

of OA is endorsed by experiments in vivo

demonstrating that specific inhibition of iNOS

results in decreased production of cytokines,

matrix metalloproteinases (MMPs) and per-

oxynitrite (Pelletieret al. 1999). Furthermore,

ex vivostudies demonstrated that the canine

cranial cruciate ligament (CrCL) actively pro-

duces NO mainly derived from the inducible

NOS pathway (Sprenget al. 2000), suggesting

that ligament fibroblasts are actively involved

in extracellular matrix (ECM) homeostasis.

Additionally, the activation of CrCL with a pro-

inflammatory mixture of lipopolysaccharide

(LPS)/IL-1/TNF leads to increased NO produc-

tion and iNOS activity, indicating that ligament

fibroblasts indeed produce substantial amounts

of NO under a specific stimulus (Louiset al.

2006).

Upon exposure to pro-inflammatory

cytokines, including IL-1β, IL-17, TNFα,

IFNγ, and LPS, as well as to shear stress, iNOS

expression is upregulated, resulting in an

increased NO production that perpetuates the

release of inflammatory cytokines and other

catabolic processes.

As assessment of the unstable NO is

extremely difficult, NO production must

be calculated by measuring its stable end

products nitrite or nitrate. In ligament tissue,

especially in partially or totally ruptured CrCL,

the assessment of NO production is difficult.

Specimens of naturally diseased CrCL are

usually retrieved from canine patients during

surgical repair, leading to the problem that

highly traumatized tissue is examined without

knowing if a measured production of NO and

NO metabolites is triggered by the original

CrCL pathology, or is only a consequence of

the mechanical trauma of ligament disruption.

Interestingly, normal CrCL produces more NO

compared to torn CrCL, normal cartilage or

normal medial collateral ligaments of the stifle

in dogs (Sprenget al. 2000)