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The results of the study showed that LPS exerted its well-described proinflammatory
effects 31] in isolated equine synoviocytes by increasing expression of SAA, IL-6 and MCP-1.
Moreover, LPS induced alteration of expression of factors affecting the fibrinolytic
potential of the synoviocytes (up-regulation of uPA and down-regulating PAI-1) and
caused a decrease in mRNA expression of TF. Thrombin treatment also induced inflammatory
changes by increased expression of SAA, IL-6 and MCP-1, but decreased fibrinolysis
by an early increase in PAI-1. Fibrinogen exposure also caused an increase in expression
of inflammatory markers (IL-6 and MCP-1). In contrast to LPS, fibrinogen induced increased
mRNA expression of TF and PAI-1 in the synoviocytes—changes that have the potential
to increase extravascular coagulation (TF) and inhibit fibrinolysis (PAI-1). All three
treatments decreased the mRNA expression of PAR-1.

A common characteristic of several joint conditions is inflammation, and studies have
shown its importance in development and healing of disease processes 15], 17], 18]. Lately haemostatic reactions have obtained interest, since upregulation of factors
involved in coagulation and fibrinolytic pathways has been shown to occur intra-articularly
in mice with antigen-induced arthritis 19] and in humans with naturally occurring arthritis 15], 23]. It thus seems, just as in systemic reactions, that both inflammatory and haemostatic
pathways are activated and likely interlinked in the joint cavity 17], 18]. One link between inflammation and haemostasis is fibrinogen, which is an acute phase
protein upregulated during inflammation, but also serves a crucial role in haemostasis.
Fibrinogen has been shown to serve as inducer and regulator of inflammatory reactions
in joint diseases in humans and rodents 17], 18], 24], 32]. Thrombin is another link between inflammation and haemostasis. While thrombin has
traditionally been thought of as a procoagulant through its induction of fibrin formation,
it has also been shown to be a potent inducer of production of inflammatory biomolecules
such as MCP-1 and IL-6 and activator of leukocytes 14], 33]. In horses, inflammatory changes in joints with spontaneous or experimentally-induced
arthritis are well-described 20], 34], 35]. In contrast, only few studies have attempted to assess haemostatic factors intra-articularly
in horses. Two recent studies have described increases in d-dimer in synovial fluid
of horses with joint disease 28], 36], indicating that fibrinolysis is active in the intra-articular compartment of horses
with joint inflammation. It is thus possible that the cross-talk between inflammation
and haemostasis suggested as a hallmark of human joint disease 37], 38] may also be at play in horses.

LPS is a well-known inflammatory inducer, and it was therefore not surprising that
it caused an up-regulation of mRNA expression of the inflammatory markers SAA and
IL-6 starting at 6 h and continuing till the end of the study (48 h). Similar results
have been found in other in vitro studies showing LPS-induced increases in SAA mRNA
expression in chicken synovial fibroblast 30] and in IL-6 mRNA expression in equine and human synoviocytes 14], 16]. LPS exposure also caused the equine synoviocytes to express MCP-1 mRNA. While this
to our knowledge is the first time that MCP-1 is described in horses, similar results
have been found in IL-1?-treated human synoviocytes 39] and in mice with haemophilic synovitis 40]. MCP-1 serves to recruit mononuclear cells to the joint compartment 39]–41], and MCP-1 has been suggested to correlate with the degree of inflammatory changes
in human synovial membrane 42].

Thrombin stimulation caused significant increases in mRNA expressions of the three
inflammatory markers of this study (SAA, IL-6 and MCP-1) in the synoviocytes, resembling
the response observed after LPS stimulation. Thrombin-induced IL-6 production has
previously been shown in human synovial fibroblasts 14], and MCP-1 induction by thrombin has been shown in endothelial cells 43] and MCP-1 was found elevated in synovial fluid from haemophilic mice 40]. Proinflammatory effects of thrombin have thus been demonstrated across species and
tissues.

The inflammation-inducing potential of fibrinogen is less well described than that
of LPS. In the present study, fibrinogen induced a significant increase in mRNA expression
of IL-6 and MCP-1 after 48 and 6 h of stimulation, respectively. In species other
than the horse, fibrinogen exposure has been described to cause inflammatory changes:
increased IL-6 concentrations were found in culture supernatant from fibrinogen-treated
human pancreatic stellate cells 25], and fibrinogen induced increases in MCP-1 expression was shown in endothelial cells
of humans and pigs 44], 45]. In articular tissue, fibrinogen exposure has been shown to exert proinflammatory
functions. Human synovial fibroblasts cultured in vitro in the presence of fibrinogen
had increased expression of intercellular adhesion molecules (ICAM) and interleukin-8
(IL-8) 24]. By increasing expression of ICAM, IL-8, and MCP-1 fibrinogen may serve to attract
and retain leukocytes intra-articularly, but at this stage the exact effects of fibrinogen
in the intra-articular compartment are unknown.

Factors involved in haemostasis pathways were induced in equine synoviocytes after
LPS, thrombin and fibrinogen exposure. Fibrinogen-treated synoviocytes showed increased
mRNA expression of TF at 6 h and thereafter a gradual decline. In contrast, LPS-treatment
decreased the mRNA expression of TF at 48 h. This is surprising and interesting, since
LPS stimulation has been found to increase TF in equine peritoneal macrophages 46]. In vivo, a gradual increase in TF mRNA expression over 7 days has been demonstrated
in mice with antigen-induced arthritis 19] and increased TF activities along with increased fibrinogen concentrations have been
found in inflamed synovial fluid from human arthritis 23]. TF initiates cell based haemostasis 47] resulting in thrombin formation 48], but TF is also described as a link between inflammation and haemostasis 23]. TF has been suggested to play an active role in attraction of monocytes to the synovial
membrane 49], and a significant correlation between synovial fluid TF concentrations and leukocyte
counts has been demonstrated 15]. To further characterize and understand the synovial TF response in horses, the in
vitro results presented here need to be corroborated by in vivo data.

The balance between uPA and PAI-1 determines the fibrinolytic activity in the joint
50], 51]. The LPS-treated equine synoviocytes in this study showed a significant increase
in uPA mRNA expression at 6 h followed by a steady decline, and a gradual decrease
in PAI-1 mRNA expression throughout the 48-h study period, which would increase fibrinolysis.
In contrast, fibrinogen- and thrombin-treated synoviocytes did not show changes in
uPA mRNA expression, while mRNA expression of PAI-1 increased significantly at 24
and 6 h, respectively. Taken together, this suggests that an inflammatory stimulus
increases the fibrinolytic capacity of equine synoviocytes first by an uPA increase
and then a PAI-1 decrease, whereas the haemostatic proteins fibrinogen and thrombin
appear to inhibit the fibrinolytic capacity of equine synoviocytes by upregulation
of PAI-1. In human synovial tissue obtained from patients with OA and RA both uPA
and PAI-1 mRNA expression was increased 32], and in mice with antigen-induce arthritis uPA mRNA expression in synovial tissue
quickly increased and peaked around 4 h, while PAI-1 was increased in the first 3 days
after induction of arthritis 19]. These findings seemingly suggest that inflamed joints develop a pro- or hyper-coagulable
state with tendency to produce and clot more fibrinogen 19]. If the same is true for the horse, the results of this study indicate that fibrinolytic
pathways are intact in initial inflammatory arthritis, and when fibrinogen and thrombin
accumulation occurs in the joint cavity, coagulation increases and fibrinolysis decreases,
making the joint potentially vulnerable to fibrin deposition and its deleterious effects.

PAR-1, a trans-membrane receptor for thrombin and other proteinases 52], showed significantly lower expression at 24 and 48 h after all three treatments
compared to the non-treated controls. LPS caused the most down-regulation of PAR-1
expression. PAR-1 knockout mice have shown less clinical symptoms in antigen-induced
arthritis compared to wild type mice 53], and PAR-1 has also been demonstrated in human RA synovial fibroblasts 54], 55]. These results indicate that PAR-1 plays a role in joint disease processes 22]. Increased synovial fibroblast proliferation 54] and increased mRNA expression in synovial fibroblast of inflammatory cytokines IL-6,
IL-8 and TNF-? have been shown to be mediated through PAR-1 14], 56]. Whether inflammation up- or down-regulates expression of PAR-1 in articular tissues
is not currently clear. Opposite to our results, PAR-1 has been found in increased
levels in inflamed human muscle cells 57] and infected murine smooth muscle cell 58], and thrombin and LPS have been shown to induce PAR-1 mRNA expression in endothelial
cells 59] and in rat astrocytes 60]. In accordance with the findings of our study, decreasing levels of PAR-1 mRNA has
been found in ischemic rat brain 61] and after TNF-? stimulation of human endothelial cells 62]. Decreased PAR-1 expression in response to inflammatory and haemostatic stimuli might
indicate a down-regulation of inflammatory pathways in equine synovial fibroblasts.

VEGF was the only marker investigated, whose expression pattern was not changed in
response to any of the treatments. VEGF was initially expected to show increased expression
pattern in the stimulated synoviocytes, as it has been demonstrated to have an angiogenic
role in inflammatory joint diseases 63] and has been found in increased concentrations in synovial fluid of human OA and
RA patients 21], 64]. The angiogenic effects and pro-inflammatory properties of VEGF in synovial inflammation
can potentially lead to hyperplasia of the synovial membrane, pannus formation and
development of osteoarthritis 65], 66]. Several cell types such as rat lung pericytes, human pancreatic stellate cells,
human fibroblast, human and bovine endothelial cells have shown up-regulation of VEGF
mRNA after LPS, thrombin or fibrinogen treatment 25], 67]–69]. While studies in humans demonstrated increased VEGF concentrations in synovial fluid
from RA patients 21], 64], a study by Salvi et al. 19] detected no change in VEGF expression in synovial tissue from antigen-induced arthritis
in mice. Whether VEGF is consistently up-regulated in joint inflammation is thus not
clear at this stage and warrants further study.

This is the first study evaluating the relationship between inflammation and haemostasis
in equine fibroblast-like synoviocytes. Obviously, the single cell type with exposure
to one inducing factor at a time cannot reflect the complexity of events taking place
in an inflamed joint, and further studies are warranted before firm conclusions regarding
the interplay between inflammatory and haemostatic pathways in equine joint disease
may be drawn. However, the findings of our study help elucidate specific interactions
between individual reactants, which are otherwise impossible to tease out of the complex
microenvironment of the diseased joint.

In the present study, passage-4 equine synovial fibroblasts were used. It is well
documented that at this passage the cell population is homogenous, because the culture
environment does not favour the macrophage-like synoviocytes 29], 30]. It is clear from the results of the present study that it is important to include
non-treated cell cultures at all investigated time points to control for culture-induced
effects on mRNA expression of the chosen markers unrelated to the tested treatments.
The non-treated cultures showed significant increases in mRNA expression of SAA, MCP-1,
TF, VEGF and PAR-1 over time, showing that the culture environment affected the synoviocytes.