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FRI0028 Redox mediated angiogenesis and inflammation in the arthritic joint
  1. M. Biniecka1,
  2. C. T. Ng1,
  3. E. Balogh1,
  4. D. J. Veale1,
  5. U. Fearon1
  1. 1Translation Research Group, Dublin Academic Medical Centre, St. Vincent’s University Hospital, Dublin, Ireland

Abstract

Background In the inflamed joint hypoxia is a powerful trigger of endothelial cell (EC) activation, proliferation and survival. Furthermore, hypoxia induces oxidative stress which arises from an imbalance between Reactive Oxygen Species (ROS) production and antioxidant defences. One of the major sources of ROS in vascular cells is Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase, also known as Nox enzyme. While Nox activity and excessive ROS are thought to actively regulate angiogenesis and inflammation, their role in RA remain to be defined.

Objectives To examine the role of Nox-derived ROS and Nox-dependent redox signaling events in regulating hypoxia-induced angiogenesis and inflammation in the arthritic joint.

Methods Patients with inflammatory arthritis (IA; n=48) underwent arthroscopy and synovial tissue oxygen (tpO2) measurements, synovial tissue biopsies and clinical assessment were obtained. Macroscopic synovitis/vascularity was measured by visual analogue scale. Nox2 mRNA expression in isolated IA peripheral blood mononuclear cells (PBMC) was measured by Real-time PCR. Synovial levels of Nox (isoform Nox2), cell-specific markers of inflammation (CD3, CD8, CD20, CD68), markers of angiogenesis (VEGF, Ang2, Factor VIII) and redox signaling factor (NF-κB) were quantified by immunohistology/immunofluorescence. Human microvascular endothelial cells (HMVEC) were stimulated with 4-HNE (Nox2 activator) and DPI (Nox2 inhibitor) under normoxia and 3% hypoxia. Secretion levels VEGF, IL-6 and IL-8 were measured by ELISA and tube formation was assessed using matrigel angiogenic assay.

Results Low in vivo pO2 levels in the inflamed joint (median [range] 26.6 [3.2–63] mm Hg) were significantly associated with synovial Nox2 expression (r=-0.43, p<0.01). Higher Nox2 mRNA expression was detected in PBMC from patients with tpO2<20mmHg compared to patients with tpO2>20mmHg (p<0.05). High synovial Nox2 expression correlated with macroscopic vascularity (r=0.46, p=0.005), synovitis (r=0.37, p=0.03), and with increased number of cell-specific markers of T cells (CD3 r=0.90, p<0.0001; CD8 r=0.72, p<0.002), B cells (CD20 r=0.82, p<0.003) and macrophages (CD68 r=0.44, p<0.01). There was a colocalisation and strong association between synovial Nox2 and expression of VEGF (p<0.005; r=0.52), Ang2 (p<0.05; r=0.32) and a number of blood vessels (p<0.004; r=0.50). In addition, synovial Nox2 expression was linked to redox activation of NF-κB signaling pathway. Functionally, 4-HNE significantly induced VEGF and IL-8 levels and promoted angiogenic tube formation (all p<0.05), an effect that was potentiated in 3% hypoxic conditions (all p<0.001). DPI significantly inhibited 4-HNE-induced VEGF, IL-6, IL-8 and tube formation under normoxia and 3% hypoxia (all p<0.05).

Conclusions NADPH oxidase is highly expressed in synovial tissue. NADPH-derived ROS promote angiogenic and proinflammatory processes in the inflamed joint, effects that may in part be mediated through hypoxic activation of downstream redox sensitive signaling events.

Disclosure of Interest M. Biniecka: None Declared, C. Ng: None Declared, E. Balogh: None Declared, D. Veale Grant/research support from: Abbott, Opsona, Pfizer, Roche, Consultant for: MSD, Pfizer, Roche, Speakers bureau: Janssen, MSD, Pfizer, UCB, U. Fearon: None Declared

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