Article Text
Abstract
Objectives 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 (tpO2 mmHg) 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 tpO2 levels <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 tpO2 in 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.
- Inflammation
- Fibroblasts
- Rheumatoid Arthritis
- TNF-alpha
- Synovitis
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Footnotes
Handling editor Tore K Kvien
MB and MC are joint first authors.
Twitter Follow Mary Canavan at @mary_canavan
Contributors MB, MC, TMcG, WG, JM, SC, LG, TS, CTN, UF, JJP, UF conducted experiments, analysis of data and manuscript preparation. UF, DJV, MB, MC, JOS participated in the data analysis and interpretation and manuscript preparation. UF, DJV, MB, MC and JOS participated in the study design. UF and DJV supervised the research. DJV and CTN recruited all patients, performed the arthroscopies, measured the in vivo pO2 levels and provided all clinical information. JR created MRI/PET hybrid images and designed the imaging programme. DJV, UF, CTN and JR interpreted MRI and PET image data. All authors read and approved the final manuscript.
Funding This work was supported by the Health Research Board of Ireland, the Programme for Research in Third Level Institutions (PRTLI), Ireland and the EU-IMI programme ‘BeThe Cure’ (grant number 4379-2422-R13798).
Competing interests None declared.
Ethics approval St. Vincent's Healthcare Group Medical Research and Ethics Committee.
Provenance and peer review Not commissioned; externally peer reviewed.