Spadoni 2015 Abstract MiPschool London 2015

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Mitochondria contribution to oxidative stress in fibroblasts isolated from patients affected by systemic sclerosis.

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Tatiana S, Silvia S, Armando G (2015)

Event: MiPschool London 2015

Systemic Sclerosis or Scleroderma (SSc) is a chronic fibrotic disease which can affect skin and internal organs, resulting in significant morbidity. SSc fibroblasts isolated from lesional areas of patients 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 we provided evidence that 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 investigated whether mitochondria also play a role in ROS production in SSc fibroblasts. To this aim, SSc fibroblasts were incubated with the mitochondria-targeted antioxidant MitoQ, and then with dihydroethidium (DHE), used to assess all intracellular superoxide production, or MitoSOX™ Red to detect mitochondria superoxide. Treatment with MitoQ reduced MitoSOX™ Red staining, and partially decreased DHE fluorescence, suggesting that mitochondria contribute to the redox state in SSc fibroblasts. To confirm the real contribution of mitochondria in SSc redox state, we purified mitochondria from fibroblasts isolated from SSc patients or healthy subjects as controls and measured ROS production using Amplex Red Assay Kit in a microplate reader. SSc mitochondrial fractions generated significantly higher levels of ROS compared to controls. Mitochondrial and cytoplasmatic fractions from SSc and control samples were also analyzed by western blot.

In this study we demonstrated that mitochondria contribute to the abnormal redox state of SSc fibroblasts. 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.


Labels: MiParea: Patients  Pathology: Other  Stress:Oxidative stress;RONS 

Tissue;cell: Endothelial;epithelial;mesothelial cell, Fibroblast 





Affiliations

Dept Sc Cliniche e Molecolari, Univ Poliecnica delle Marche, Ancona, Italy. - t.spadoni@univpm.it

References

  1. Baroni SS, Santillo M, Bevilacqua F, Luchetti M, Spadoni T, Mancini M, Fraticelli P, Sambo P, Funaro A, Kazlauskas A, Avvedimento EV, Gabrielli A (2006) Stimulatory autoantibodies to the PDGF receptor in systemic sclerosis. N Engl J Med 354:2667-76.
  2. Svegliati S, Marrone G, Pezone A, Spadoni T, Grieco A1, Moroncini G, Grieco D, Vinciguerra M4, Agnese S, Jüngel A, Distler O, Musti AM, Gabrielli A, Avvedimento EV (2014) Oxidative DNA damage induces the ATM-mediated transcriptional suppression of the Wnt inhibitor WIF-1 in systemic sclerosis and fibrosis. Sc Signaling 7:ra84.
  3. Spadoni T, Svegliati Baroni S, Amico D, Albani L, Moroncini G, Avvedimento EV, Gabrielli A (2015) A reactive oxygen species-mediated loop maintains the increased expression of NOX2 and NOX4 in skin fibroblasts from patients with systemic sclerosis. Arthritis Rheum doi: 10.1002/art.39084
  4. Lee DY, Wauquier F, Eid AA, Roman LJ, Ghosh-Choudhury G, Khazim K, Block K, Gorin Y (2013) Nox4 NADPH oxidase mediates peroxynitrite-dependent uncoupling of endothelial nitric-oxide synthase and fibronectin expression in response to angiotensin II: role of mitochondrial reactive oxygen species. J Biol Chem 288:28668-86.