Subject(s)
Cartilage, Articular/metabolism , Gene Expression Regulation/physiology , Laminin/physiology , Matrix Metalloproteinase 3/genetics , Osteoarthritis/metabolism , DNA Primers/chemistry , Humans , Immunohistochemistry , Matrix Metalloproteinase 3/metabolism , Real-Time Polymerase Chain ReactionABSTRACT
OBJECTIVE: Sphingosine-1-phosphate (S1P) is a messenger molecule, with important functions in inflammation and wound healing. The present study was performed to elucidate a possible role of S1P signaling in articular chondrocytes. METHODS: Human and bovine primary chondrocytes were cultured in monolayer. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect S1P receptor mRNA. Proliferation of S1P stimulated chondrocytes was measured by 3H-thymidine uptake. Supernatants of cultured bovine chondrocytes stimulated with S1P alone or in combination with interleukin-1beta (IL-1beta) were tested for nitric oxide (NO) formation and expression of inducible nitric oxide synthase (iNOS). Matrixmetalloprotease-13 (MMP-13) and aggrecanase-1 (ADAMTS-4) were evaluated using real-time PCR. Glycosaminoglycan (GAG) loss from bovine cartilage explants was evaluated using the dimethylene blue method. RESULTS: S1P1, S1P2 and S1P3 but not S1P4 and S1P5 receptor mRNA were detected in human and bovine chondrocytes. S1P dose dependently induced proliferation in bovine and human chondrocytes. S1P significantly reduced NO formation and iNOS mRNA and protein expression, both in un-stimulated and IL-1beta stimulated bovine chondrocytes. Furthermore, S1P dose dependently inhibited IL-1beta induced expression of ADAMTS-4 and MMP-13 and diminished IL-1beta mediated GAG depletion from cartilage explants. CONCLUSION: These results suggest that S1P provides an anti-catabolic signal in articular chondrocytes.
Subject(s)
Cell Proliferation/drug effects , Chondrocytes/chemistry , Chondrocytes/drug effects , Lysophospholipids/physiology , Nitric Oxide/antagonists & inhibitors , Receptors, Lysosphingolipid/analysis , Sphingosine/analogs & derivatives , ADAM Proteins/antagonists & inhibitors , ADAM Proteins/metabolism , ADAMTS4 Protein , Animals , Cattle , Cells, Cultured , Dose-Response Relationship, Drug , Glycosaminoglycans/metabolism , Humans , In Vitro Techniques , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/physiology , Matrix Metalloproteinase 13/metabolism , Matrix Metalloproteinase Inhibitors , Nitric Oxide/biosynthesis , Nitric Oxide Synthase Type II/antagonists & inhibitors , Nitric Oxide Synthase Type II/metabolism , Polymerase Chain Reaction/methods , Procollagen N-Endopeptidase/antagonists & inhibitors , Procollagen N-Endopeptidase/metabolism , Sphingosine/physiologyABSTRACT
Measurements of pulmonary capillary blood flow by a nitrous oxide rebreathing technique (QN2O) were performed in 21 anesthetized and artificially ventilated minipigs with normal lungs and in nine minipigs with thrombin-induced (75-150 U kg-1 h-1) lung pathology. QN2O was calculated with the Hook-Meyer-formula and compared to cardiac output measurements (thermodilution, QT, or direct Fick's principle, QFick). The coefficient of variation in double QN2O measurements was 0.05. If the tidal volume to dead space ration (VD/VT) is normal, the nitrous oxide method works well, but when the efficacy of ventilation worsens, this gas uptake method fails to detect the circulation of the poorly ventilated parts of the lung. The mean ratio QN2O/QFick in pigs with normal lungs (58 measurements) was 1.00 +/- 0.10 (mean +/- s.d.). During thrombin infusion, the mean ratio QN2O/QT was 0.84 +/- 0.17 (n = 49). After corrections for shunt perfusion (Qs), the mean ration QN2O/(QT-Qs) was 0.89 +/- 0.17 (n = 49). QN2O/QT-Qs) decreased with increasing VD/VT. In measurements during thrombin infusion with VD/VT less than 0.33, the mean ratio QN2O/(QT-Qs) was 0.97 +/- 0.11 (n = 21), with a VD/VT between 0.33 and 0.44, the mean ratio QN2O (QT-QS) was 0.90 +/- 0.08 (n = 20), and with a VD/VT greater than or equal to 0.45, this ratio was 0.62 +/- 0.18 (n = 8). In the presence of only moderate functional inhomogeneities, this noninvasive rebreathing method will offer reliable data on pulmonary perfusion.