Hypoxia and oxidative stress are involved in mediating angiogenic processes in the inflamed synovial tissue. In this study we examine the expression of angiogenic markers in inflammatory arthritis and the effects of tumour necrosis factor alpha inhibitors (TNFi) on angiogenesis in relationship to synovial hypoxia in vivo and oxidative stress. In vitro oxidative stress-induced angiogenic responses are also investigated.Materials and methods
44 IA patients pre/post-TNFi underwent knee joint arthroscopy and in vivo oxygen levels (tpO2) were measured. Expression of angiogenic growth factors in paired serum, synovial fluid and synovial tissue was accessed by ELISA and immunohistochemistry. Pro-angiogenic processes, mitochondrial function and metabolism were assessed in RA Synovial Fibroblast Cells (RASFCs) and Human Microvascular Endothelial Cells (HMVECs) under normoxia or 3% hypoxia in the presence of oxidative stress inducer - 4-HNE using the tube formation and invasion assays and the XFe Extracellular Flux Seahorse Analyzer.Results
VEGF and Ang2 were expressed in the joint synovial fluid and Ang1 and PDGF-B were expressed at a systemic level. The highest synovial tissue (ST) VEGF, Ang2 and Tie2 positivity was observed in vascular region compared to lining and sublining layers (all p<0.05). Significant reduction in synovial vascularity and ST Ang2/Tie2 expression, paralleled by an increase in in vivo tpO2 was found after TNFi (all p<0.05). ST oxidative stress expression (8-oxo-dG and 4-HNE) was colocalized with VEGF, Ang2 and Tie2 and showed an association pre/post anti TNF-α therapy. In vitro 4-HNE promoted RASFCs secretion of pro-angiogenic mediators, ROS production, and cell proliferation (all p<0.05). In HMVECs 4-HNE elevated angiogenic tube-formation and cell invasiveness (all p<0.05), effects that were further potentiated by 3% hypoxia. Furthermore, 4-HNE significantly increases glycolytic activity of RASFC and HMVECs with concomitant attenuation of mitochondrial respiration.Conclusions
Differential local and systemic expression of angiogenic factors may indicate a constant remodelling of synovial vasculature. Reduced levels of Ang2 and Tie2 following TNFi demonstrate a relationship between angiogenesis, hypoxia and TNFα pathway. Redox signalling can alter angiogenic responses of synovial cells. Oxidative stress alters cellular bioenergetics by promoting switch to glycolysis mitochondrial respiration supporting cellular invasion and dysregulated angiogenesis.