FRI0242 Role of Mitochondria- and Nadph Oxidase-Derived ROS in Fibroblasts Isolated from Patients Affected by Systemic Sclerosis

Background

Oxidative stress plays an important role in the development of fibrosis under various pathological conditions. Fibroblasts isolated from patients with Systemic Sclerosis (SSc) overproduce reactive oxygen species (ROS), overexpress type I collagen and α-smooth muscle actin (α-SMA) and show DNA damage and activation of checkpoint kinase ATM (1, 2). NADPH oxidase (NOX) is an important source of ROS, and its overactivity or overexpression are often associated with chronic diseases, characterized by tissue damage and fibrosis. Recently, we have demonstrated that NOX2 and NOX4 are critical components of NADPH oxidase complex in SSc fibroblasts and ROS generated by NOX play a primary role in the pathological activation of dermal fibroblasts (3). Since a recent study reported that NOX4 localizes to membranes and mitochondria and contributes to the generation of mitochondrial ROS (4), we wondered whether mitochondria also play a role in ROS production in SSc fibroblasts.

Objectives

The aim of this study was to investigate the contribution of mitochondrial ROS to oxidative stress in SSc.

Methods

ROS production was analysed by confocal microscopy using dihydroethidium (DHE, to assess all intracellular superoxide), or MitoSOX™ Red (to detect mitochondria superoxide), or using a microplate reader following Amplex® Red kit protocol (to measure H₂O₂). Mitochondria were purified using a mitochondria isolation kit. For protein expression, mitochondrial and cytosolic fractions were subjected to western blot with specific antibodies.

Results

SSc fibroblasts incubated with the mitochondria-targeted antioxidant MitoQ showed a reduced MitoSOX™ Red staining, and a partially decreased DHE fluorescence, suggesting that mitochondria contribute to the redox state in SSc fibroblasts. Mitochondria purified from SSc fibroblasts generated significantly higher levels of ROS compared to controls. Incubation of normal cells with PDGF, a profibrotic cytokine able to activate a SSc-like phenotype, led to a significant increase of total and mitochondrial ROS levels compared to unstimulated cells. Mitochondrial and cytosolic fractions from SSc and activated normal fibroblasts were also analyzed to evaluate different protein expression patterns.

Conclusions

In this study we demonstrated that mitochondria contribute to the abnormal redox state of SSc fibroblasts and activated normal cells. Further studies may clarify whether mitochondrial ROS are generated by a mitochondrial NOX isoform or are the result of the interplay between mitochondria and NOX enzymes located outside the organelles.

References

Baroni S et al, N Engl J Med 2006; 354:2667–76; Svegliati S et al, Science Signaling 2014; 7: ra84; Spadoni T et al, Arthritis Rheum 2015; 67:1611–1622; Lee DY et al, J Biol Chem 2013;288:28668–28686.

Disclosure of Interest

None declared