title: mRNA expression of genes involved in inflammation and haemostasis in equine fibroblast-like synoviocytes following exposure to lipopolysaccharide, fibrinogen and thrombin.
abstract: BACKGROUND
  Studies 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.
  
  
  RESULTS
  LPS 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.
  
  
  CONCLUSIONS
  LPS, 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.
authors.affiliation: Department of Large Animal Sciences, Medicine and Surgery group, University of Copenhagen, Højbakkegård allé 5, DK-2630, Tåstrup, Denmark. andreassenstine@hotmail.com.
authors.forename: Stine Mandrup
authors.initials: SM
authors.lastname: Andreassen
authors.affiliation: Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Dyrlægevej 16, DK-1870, Frederiksberg C, Denmark. lcb@sund.ku.dk.
authors.forename: Lise C
authors.initials: LC
authors.lastname: Berg
authors.affiliation: Department of Large Animal Sciences, University of Copenhagen, Grønnegårdsvej 8, DK-1870, Frederiksberg C, Denmark. saxmose@sund.ku.dk.
authors.forename: Søren Saxmose
authors.initials: SS
authors.lastname: Nielsen
authors.affiliation: Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Dyrlægevej 16, DK-1870, Frederiksberg C, Denmark. atk@sund.ku.dk.
authors.forename: Annemarie T
authors.initials: AT
authors.lastname: Kristensen
authors.affiliation: Department of Large Animal Sciences, Medicine and Surgery group, University of Copenhagen, Højbakkegård allé 5, DK-2630, Tåstrup, Denmark. stj@sund.ku.dk.
authors.forename: Stine
authors.initials: S
authors.lastname: Jacobsen
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005340:Fibrinogen / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006487:Hemostasis / Q000187:drug effects / Q000502:physiology; D006736:Horses; D007249:Inflammation / Q000378:metabolism; D008070:Lipopolysaccharides / Q000633:toxicity*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D013583:Synovial Membrane / Q000166:cytology*; D013917:Thrombin / Q000494:pharmacology*
chemical_list: D008070:Lipopolysaccharides; D012333:RNA, Messenger; D005340:Fibrinogen; D013917:Thrombin