Oxidative stress impairs energy metabolism in primary cells and synovial tissue of patients with rheumatoid arthritis

Emese Balogh,Ursula Fearon,Trudy Mcgarry,Chin-Teck Ng,Carl Orr,Monika Biniecka,Zoltan Szekanecz,Douglas J Veale

doi:10.1186/s13075-018-1592-1

Abstract

BackgroundIn this study, we examined the effect of oxidative stress on cellular energy metabolism and pro-angiogenic/pro-inflammatory mechanisms of primary rheumatoid arthritis synovial fibroblast cells (RASFC) and human umbilical vein endothelial cells (HUVEC).MethodsPrimary RASFC and HUVEC were cultured with the oxidative stress inducer 4-hydroxy-2-nonenal (4-HNE), and extracellular acidification rate, oxygen consumption rate, mitochondrial function and pro-angiogenic/pro-inflammatory mechanisms were assessed using the Seahorse analyser, complex I–V activity assays, random mutation mitochondrial capture assays, enzyme-linked immunosorbent assays and functional assays, including angiogenic tube formation, migration and invasion. Expression of angiogenic growth factors in synovial tissue (ST) was assessed by IHC in patients with rheumatoid arthritis (RA) undergoing arthroscopy before and after administration of tumour necrosis factor inhibitors (TNFi).ResultsIn RASFC and HUVEC, 4-HNE-induced oxidative stress reprogrammed energy metabolism by inhibiting mitochondrial basal, maximal and adenosine triphosphate-linked respiration and reserve capacity, coupled with the reduced enzymatic activity of oxidative phosphorylation complexes III and IV. In contrast, 4-HNE elevated basal glycolysis, glycolytic capacity and glycolytic reserve, paralleled by an increase in mitochondrial DNA mutations and reactive oxygen species. 4-HNE activated pro-angiogenic responses of RASFC, which subsequently altered HUVEC invasion and migration, angiogenic tube formation and the release of pro-angiogenic mediators. In vivo markers of angiogenesis (vascular endothelial growth factor, angiopoietin 2 [Ang2], tyrosine kinase receptor [Tie2]) were significantly associated with oxidative damage and oxygen metabolism in the inflamed synovium. Significant reduction in ST vascularity and Ang2/Tie2 expression was demonstrated in patients with RA before and after administration of TNFi.ConclusionsOxidative stress promotes metabolism in favour of glycolysis, an effect that may contribute to acceleration of inflammatory mechanisms and subsequent dysfunctional angiogenesis in RA.

Highlights

In this study, we examined the effect of oxidative stress on cellular energy metabolism and proangiogenic/pro-inflammatory mechanisms of primary rheumatoid arthritis synovial fibroblast cells (RASFC) and human umbilical vein endothelial cells (HUVEC)
For the first time to our knowledge, that inhibition of Oxygen consumption rate (OCR) following 4-HNE-induced oxidative stress was associated with a shift in RASFC metabolism towards glycolysis. 4HNE reduced basal mitochondrial respiration (p < 0.05), paralleled by a reduction in maximal mitochondrial respiration (p < 0.001), adenosine triphosphate (ATP) synthesis (p = 0.1) and reserve capacity (p < 0.01) (Fig. 1b)
We observed increases in reactive oxygen species (ROS) production and Mitochondrial DNA (mtDNA) point mutations in RASFC in the presence of 4-HNE compared with basal cells (p < 0.001 and p = 0.06, respectively) (Fig. 3a). 4-HNE protein adduction may alter protein activity; we examined the activity of the individual proteins of mitochondrial oxidative phosphorylation (OXPHOS) complexes I–V. 4-HNE significantly reduced the activity of complex III by 8% and complex IV by 70% compared with basal values

Summary

Introduction

We examined the effect of oxidative stress on cellular energy metabolism and proangiogenic/pro-inflammatory mechanisms of primary rheumatoid arthritis synovial fibroblast cells (RASFC) and human umbilical vein endothelial cells (HUVEC). An increase in the metabolic state towards glycolysis has been shown in primary rheumatoid arthritis synovial fibroblasts (RASFC), CD4 T cells, T-helper type 17 (TH17) cells, macrophages and dendritic cells [7,8,9,10]. This is paralleled by elevated lactate levels and diminished glucose in RA synovial fluids as well as by increased activity of key glycolytic enzymes in the RA synovium, indicating that anaerobic glycolysis is favoured in this hypoxic environment [11,12,13]. In vitro studies by our group have shown that hypoxia and Toll-like receptor 2 (TLR2)-induced inflammation promoted mitochondrial dysfunction and oxidative stress and reprogrammed the nature of cellular respiration in RA synovial cells [14, 15]

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