Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Role of Reactive Oxigen Species and ROS-Dependent Downstream Signalling in the Prothrombotic State Elicited by Antiphospholipid Antibodies from Primary Antiphospholipid Syndrome Patients.
Lopez-Pedrera1, Chary, Ruiz-Limon1, Patricia, Aguirre1, Maria Angeles, Barbarroja1, Nuria, Rodriguez-Ariza1, Antonio, Perez-Sanchez1, Carlos, Collantes-Estevez1, Eduardo
The role of antiphospholipid antibodies (aPL) in the induction of a procoagulant state is clearly established; yet, the precise intracellular mechanisms are poorly understood. Besides their role in tissue vascular injury, reactive oxygen species (ROS) may act as important signalling molecules, activating redox-sensitive signalling cascades that potentially link the activation of receptors by their agonists to gene expression.
We investigated the relevance of ROS in the prothrombotic/proinflammatory state elicited by aPL and studied ROS-dependent downstream signalling pathways leading to monocyte activation.
Design and Methods:
Monocytes from healthy individuals were treated with affinity purified IgG from 7 APS patients (aPL-IgG), or IgG normal human serum (IgG-NHS) from 7 healthy donors. Then, time and dose-dependent ROS production was analyzed. Inhibitory studies on ROS production were performed by preincubation of the treated monocytes 1h with antioxidants N-acetyl-L-cysteine (NAC), and vitamin C (vit C), as well as with the mitochondrial inhibitor, rotenone. Peroxides, peroxynitrite generation and mitochondrial membrane potential (MMP), were analysed by flow cytometry using specific fluorescent probes. As procoagulant/proinflammatory markers, cell surface TF, VEGF and Flt1 expression were evaluated by flow cytometry and western blot respectively. Nitric oxide (NO) levels and Total Antioxidant Capacity (TAC) were measured in the supernatant of the cell cultures. N-Tyr, iNOS expression, p38MAPK activation and IkappaB degradation were evaluated by Western blot. NFkB activity was quantified by EMSA.
Independent incubation of monocytes with aPL-IgG from 7 patients increased cellular ROS production and mitochondria depolarization, which were prevented by the antioxidants Vit C and NAC. Inhibition by rotenone further indicated an involvement of the mitochondrial transport chain as a source of ROS. The reduction of supernatant TAC after aPL-IgG treatment was abolished by ROS inhibitors. Moreover, aPL-induced iNOS, N-Tyr, and NO expression levels were reduced by treatment with ROS inhibitors. ROS produced by aPL-IgG treatment activated p38 MAPK and its subsequent target, the nuclear factor kappa B (NFkB), and controlled the up-regulation of TF, VEGF, and Flt1 in monocytes.
Our data indicate that the binding of aPL-IgG to the monocyte membrane elicited a redox-sensitive signalling pathway that controls the procoagulant phenotype of that cells in the setting of APS. Thus, oxidative stress by aPL-IgG represents a new pathway potentially contributing to the thrombotic complications of APS. Supported by JA0042/2007, JA0246/2009, P08CVI04234 and PS09/01809.
To cite this abstract, please use the following information:
Lopez-Pedrera, Chary, Ruiz-Limon, Patricia, Aguirre, Maria Angeles, Barbarroja, Nuria, Rodriguez-Ariza, Antonio, Perez-Sanchez, Carlos, et al; Role of Reactive Oxigen Species and ROS-Dependent Downstream Signalling in the Prothrombotic State Elicited by Antiphospholipid Antibodies from Primary Antiphospholipid Syndrome Patients. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1357