title: Dysregulated bioenergetics: a key regulator of joint inflammation.
abstract: OBJECTIVE
  This study examines the relationship between synovial hypoxia and cellular bioenergetics with synovial inflammation.
  
  
  METHODS
  Primary rheumatoid arthritis synovial fibroblasts (RASF) were cultured with hypoxia, dimethyloxalylglycine (DMOG) or metabolic intermediates. Mitochondrial respiration, mitochondrial DNA mutations, cell invasion, cytokines, glucose and lactate were quantified using specific functional assays. RASF metabolism was assessed by the XF24-Flux Analyzer. Mitochondrial structural morphology was assessed by transmission electron microscopy (TEM). In vivo synovial tissue oxygen (tpO2mmHg) was measured in patients with inflammatory arthritis (n=42) at arthroscopy, and markers of glycolysis/oxidative phosphorylation (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), PKM2, GLUT1, ATP) were quantified by immunohistology. A subgroup of patients underwent contiguous MRI and positron emission tomography (PET)/CT imaging. RASF and human dermal microvascular endothelial cells (HMVEC) migration/angiogenesis, transcriptional activation (HIF1α, pSTAT3, Notch1-IC) and cytokines were examined in the presence of glycolytic inhibitor 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO).
  
  
  RESULTS
  DMOG significantly increased mtDNA mutations, mitochondrial membrane potential, mitochondrial mass, reactive oxygen species and glycolytic RASF activity with concomitant attenuation of mitochondrial respiration and ATP activity (all p<0.01). This was coupled with altered mitochondrial morphology. Hypoxia-induced lactate levels (p<0.01), which in turn induced basic fibroblast growth factor (bFGF) secretion and RASF invasiveness (all p<0.05). In vivo glycolytic markers were inversely associated with synovial tpO2levels <20 mm Hg, in contrast ATP was significantly reduced (all p<0.05). Decrease in GAPDH and GLUT1 was paralleled by an increase in in vivo tpO2in tumour necrosis factor alpha inhibitor (TNFi) responders. Novel PET/MRI hybrid imaging demonstrated close association between metabolic activity and inflammation. 3PO significantly inhibited RASF invasion/migration, angiogenic tube formation, secretion of proinflammatory mediators (all p<0.05), and activation of HIF1α, pSTAT3 and Notch-1IC under normoxic and hypoxic conditions.
  
  
  CONCLUSIONS
  Hypoxia alters cellular bioenergetics by inducing mitochondrial dysfunction and promoting a switch to glycolysis, supporting abnormal angiogenesis, cellular invasion and pannus formation.
authors.affiliation: Centre for Arthritis and Rheumatic Diseases, Dublin Academic Medical Centre, St. Vincent's University Hospital, Dublin, Ireland.
authors.forename: M
authors.initials: M
authors.lastname: Biniecka
authors.affiliation: Department of Molecular Rheumatology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland.
authors.forename: M
authors.initials: M
authors.lastname: Canavan
authors.affiliation: Department of Molecular Rheumatology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland.
authors.forename: T
authors.initials: T
authors.lastname: McGarry
authors.affiliation: Centre for Arthritis and Rheumatic Diseases, Dublin Academic Medical Centre, St. Vincent's University Hospital, Dublin, Ireland.
authors.forename: W
authors.initials: W
authors.lastname: Gao
authors.affiliation: Centre for Arthritis and Rheumatic Diseases, Dublin Academic Medical Centre, St. Vincent's University Hospital, Dublin, Ireland.
authors.forename: J
authors.initials: J
authors.lastname: McCormick
authors.affiliation: Centre for Arthritis and Rheumatic Diseases, Dublin Academic Medical Centre, St. Vincent's University Hospital, Dublin, Ireland.
authors.forename: S
authors.initials: S
authors.lastname: Cregan
authors.affiliation: Centre for Arthritis and Rheumatic Diseases, Dublin Academic Medical Centre, St. Vincent's University Hospital, Dublin, Ireland.
authors.forename: L
authors.initials: L
authors.lastname: Gallagher
authors.affiliation: Centre for Arthritis and Rheumatic Diseases, Dublin Academic Medical Centre, St. Vincent's University Hospital, Dublin, Ireland.
authors.forename: T
authors.initials: T
authors.lastname: Smith
authors.affiliation: Department of Surgery, Trinity College Dublin, St James's Hospital, Dublin, Ireland.
authors.forename: J J
authors.initials: JJ
authors.lastname: Phelan
authors.affiliation: Department of Radiology, School of Medicine and Biomedical Sciences, University College Dublin, Dublin, Ireland.
authors.forename: J
authors.initials: J
authors.lastname: Ryan
authors.affiliation: Department of Surgery, Trinity College Dublin, St James's Hospital, Dublin, Ireland.
authors.forename: J
authors.initials: J
authors.lastname: O'Sullivan
authors.affiliation: Department of Rheumatology and Immunology, Singapore General Hospital, Singapore, Singapore.
authors.forename: C T
authors.initials: CT
authors.lastname: Ng
authors.affiliation: Centre for Arthritis and Rheumatic Diseases, Dublin Academic Medical Centre, St. Vincent's University Hospital, Dublin, Ireland.
authors.forename: D J
authors.initials: DJ
authors.lastname: Veale
authors.affiliation: Department of Molecular Rheumatology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland.
authors.forename: U
authors.initials: U
authors.lastname: Fearon
mesh_terms: D000600:Amino Acids, Dicarboxylic / Q000378:metabolism; D001172:Arthritis, Rheumatoid / Q000503:physiopathology*; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D016207:Cytokines / Q000032:analysis; D004272:DNA, Mitochondrial / Q000378:metabolism; D004734:Energy Metabolism / Q000502:physiology*; D005347:Fibroblasts / Q000378:metabolism*; D005947:Glucose / Q000032:analysis; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D007596:Joints / Q000378:metabolism; D019344:Lactic Acid / Q000032:analysis; D008928:Mitochondria / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013583:Synovial Membrane / Q000166:cytology
chemical_list: D000600:Amino Acids, Dicarboxylic; D016207:Cytokines; D004272:DNA, Mitochondrial; D017382:Reactive Oxygen Species; D019344:Lactic Acid; D005947:Glucose; C040947:oxalylglycine
keywords: Fibroblasts; Inflammation; Rheumatoid Arthritis; Synovitis; TNF-alpha