mRNA expression of genes involved in inflammation and haemostasis in equine fibroblast-like synoviocytes following exposure to lipopolysaccharide, fibrinogen and thrombin

Abstract

BackgroundStudies in humans have shown that haemostatic and inflammatory pathways both play important roles in the pathogenesis of joint disease. The aim of this study was to assess mRNA expression of haemostatic and inflammatory factors in cultured equine fibroblast-like synoviocytes exposed to lipopolysaccharide (LPS), fibrinogen and thrombin. Synovial membranes were collected from metacarpo-phalangeal joints of 6 skeletally mature horses euthanized for non-orthopaedic reasons. Passage 4 fibroblast-like synoviocytes were left non-treated or treated with either 0.1 μg/ml LPS, 5 mg/ml fibrinogen or 5 U/ml thrombin and harvested at time points 0, 6, 24 and 48 h. mRNA expression of serum amyloid A (SAA), interleukin-6 (IL-6), monocyte chemotactic protein 1 (MCP-1), tissue factor (TF), plasminogen activator inhibitor 1 (PAI-1), urokinase plasminogen activator (uPA), vascular endothelial growth factor (VEGF) and protease activator receptor 1 (PAR-1) was assessed using quantitative real time reverse transcriptase PCR.ResultsLPS caused a significant increase in mRNA expression of SAA, IL-6, MCP-1 and uPA, and a decrease in TF, PAI-1 and PAR-1 when compared to non-treated cells. Treatment with thrombin resulted in increased mRNA expression of SAA, IL-6, MCP-1 and PAI-1, and a decreased PAR-1 expression compared to non-treated cells. The fibrinogen-treated synoviocytes showed significantly increased mRNA expression of IL-6, MCP-1, TF and PAI-1, and decreased PAR-1 expression compared to non-treated cells.ConclusionLPS, fibrinogen and thrombin induced an increased gene expression of inflammatory markers in isolated equine fibroblast-like synoviocytes. LPS caused changes in gene expression promoting increased fibrinolysis, while fibrinogen and thrombin changed the gene expression resulting potentially in reduced fibrinolysis. Overall, it appeared that both inflammatory and haemostatic stimuli affected expression of genes involved in inflammatory and haemostatic pathways, supporting their importance in equine joint diseases.

Changes in concentrations of haemostatic and inflammatory biomarkers in synovial fluid after intra-articular injection of lipopolysaccharide in horses

“…Pannus formation in equine infected synovial structures has been related to reduced prognosis and poor athletic outcome after septic arthritis [ 38 , 39 ]. The haemostatic functions of fibrinogen are well-known [ 43 ], but fibrinogen has also been shown to possess proinflammatory functions [ 16 ], as increased expression of inflammatory biomolecules were detected in cultured synoviocytes after fibrinogen stimulation [ 20 , 21 ]. The balance between fibrin formation and dissolution is thought to be important for resolution of IA inflammatory disease [ 23 , 24 ].…”

“…Fibrinogen is a key protein in haemostasis, interacting with platelets to form a fibrin-platelet clot to control haemorrhage or exudation from the circulation. Fibrin/fibrinogen has also been shown to induce inflammatory reactions in human and equine synoviocytes [ 20 , 21 ], and fibrinogen may thus contribute to both haemostasis and inflammation in the joint compartment. Fibrinogen is also an acute phase reactant, and similar to other acute phase reactants, such as SAA, haptoglobin and iron, plasma concentrations of fibrinogen will change in response to inflammatory and infectious conditions [ 22 ].…”

Acute lower limb ischemia secondary to diffuse peritonitis

“…In case of severe endogenous intoxication, the cardiovascular system plays a critical role since the further course of the disease is determined by hemodynamic changes. 13,14 Peritonitis, as a pathological process, can trigger thrombus formation due to the activation of the processes of hypovolemia, stress, pain, and adynamia. 15 In acute diffuse peritonitis, 4-9 liters of fl uid are lost that result in the development of hypovolemic or septic shock.…”

Regulatory effect of anti-gp130 functional mAb on IL-6 mediated RANKL and Wnt5a expression through JAK-STAT3 signaling pathway in FLS

“…Then, the synoviocytes were passaged using 0.25% trypsin/1 mM EDTA (Invitrogen). The cells were cryopreserved between passage 3–8, and cells from these passages were used for all experiments [ 21 ].…”

“…Then, the cells were fixed with 4% buffered paraformaldehyde after washing with PBS, and then permeabilized with 0.5% Triton X-100 for 10 min at room temperature. The subsequent steps were performed as described previously [ 21 ]. The samples were incubated overnight at 4°C with antibodies against WNT5A (1:100, Abcam, Cambridge, MA, USA) and RANKL (1:100, R&D Systems).…”