Background/Purpose: To examine the relationship between synovial hypoxia, cellular bioenergetics and mitochondrial dysfunction with synovial inflammation.

Methods: Primary RASFC were cultured with 3% hypoxia, DMOG (stabilizes HIF1α) or lactic acid.  Reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and mitochondrial mass (MM) were quantified using fluorescent probes, RASFC invasion by Transwell Matrigel^TM chambers, and VEGF, bFGF by ELISA. Alterations in the synovial mitochondrial genome were assessed by Random Mutation Capture assay. RASFC metabolism was assessed by the XF24 Flux analyser (Seahorse) which quantifies real-time aerobic and anaerobic bioenergetic profiles. To examine if altered metabolism is also observed in vivo, 44 patients with active inflammatory arthritis underwent arthroscopy, clinical assessment and synovial tissue oxygen (tpO₂) measurements. Synovial microscopic levels of glycolysis and oxidative phosphorylation (GAPDH, PKM2, GLUT1, ATP), inflammation (CD3, CD68) and angiogenesis (FactorVIII/αSMA) were quantified by immunohistology. A subgroup of patients underwent contiguous MRI and PET/CT imaging.  Finally we examined the effect by blocking glycolysis using a small molecule, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) (which blocks PFKFB3) in RASFC, human microvascular endothelial cells (HMVEC) and RA synovial explant cultures. Migration, angiogenesis, HIF1α activation and pro-inflammatory mediators were assessed using specific functional assays, western blot and ELISA.  

Results: In RASFC, hypoxia induced increased mitochondrial DNA mutations, MM, MMP, ROS and invasion but inhibited ATP indicating altered cellular energy metabolism (all p<0.01). Hypoxia increased the rate of glycolysis with concomitant attenuation of mitochondrial respiration (p<0.01), decreased basal and maximal respiration (p<0.01) with a simultaneous induction in glycolytic cell activity (p<0.001). Hypoxia inhibited RASFC glucose secretion (p<0.05) and increased lactate levels (p<0.05). Lactic acid in turn, induced RASFC invasion and secretion of bFGF and VEGF. Elevated RASFC glycolysis was concurrent with in vivo assessments, with synovial expression of GAPDH, PKM2 and GLUT1 significantly higher in patients with tpO2 levels in vivo<20mmHg (all p<0.05), in contrast to ATP which was significantly lower (p<0.05). Glycolytic marker expression also correlated with increased macroscopic/microscopic vascularity and synovitis (all p<0.05). PET/MRI hybrid images demonstrated close association between metabolic turnover and site of inflammation with tpO2 levels in vivo. Finally 3PO significantly inhibited RASFC migration, HIF1α activation, angiogenesis and secretion of IL-6, IL-8, MCP-1 and ICAM-1 (all p<0.05).

Conclusion: Hypoxia alters cellular bioenergetics by down-regulating mitochondrial respiration and promoting a switch to glycolysis in the inflamed joint. This may enable synovial cells to generate sufficient ATP to support enhanced synovial proliferation, angiogenesis and pannus formation. Blockade of glycolysis may represent a potential therapeutic strategy for RA and other autoimmune diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/metabolic-reprogramming-in-the-inflamed-joint-inhibits-pro-inflammatory-mechanisms/