NADPH Oxidase Activation and Endothelial Nitric Oxide Synthase Uncoupling Contribute to Endothelial Dysfunction in Antiphospholipid Syndrome
Authors: Wu Meifang , Kundu Suman , McCrae Keith R. 
Published: October 2019

Abstract
Introduction: Antiphospholipid syndrome (APS) is characterized by thrombosis and/or recurrent fetal loss in the presence of persistently elevated antiphospholipid antibodies (APLA). The majority of pathologic APLA are directed against β2-glycoprotein I (β2GPI). APLA cause endothelial dysfunction, though the underlying mechanisms have not been clearly delineated.
Methods: Endothelial cells (EC) were incubated with β2GPI and either control antibodies or anti-β2GPI antibodies affinity-purified from sera of patients with APS, in the absence or presence of diapocynin, an NADPH oxidase (NOX) inhibitor, or siRNA against NOX1, NOX2, or NOX4. Generation of reactive oxygen species (ROS) in EC and conditioned medium were measured using fluorescent dyes (CM-H2DCFDA and CellROX Deep Red) or chemiluminescent substrate. NOX1, NOX2, NOX4, 3-nitrotyrosine, and thioredoxin reductase 1 (TrxR1) expression were analyzed by western blot. eNOS monomer and dimer were detected using cold (4°C) SDS-PAGE and immunoblot. Immunoprecipitation of TrxR1 was performed using protein A/G agarose. EC activation was assessed by measuring expression of E-selectin.
Results: Incubation of EC with β2GPI and anti-β2GPI antibodies stimulated ROS generation in EC, as well as the release of ROS into conditioned medium. Expression of NOX2, but not NOX1 or NOX4, was significantly increased in EC exposed to anti-β2GPI antibodies, but not control IgG. Preteatment of endothelial cells with diapocynin, a NOX inhibitor, or siRNA against NOX2 and NOX4, but not NOX1, inhibited endothelial cell activation by anti-β2GPI antibodies. Furthermore, anti-β2GPI antibody-treated EC generated more peroxynitrite, as determined by 3-nitrotyrosine expression. Treatment of EC with anti-β2GPI antibodies increased eNOS monomer/dimer ratio, suggesting eNOS uncoupling. Compared to control human IgG, the TrxR1 immunoprecipitate from EC treated with β2GPI and anti-β2GPI antibodies contained more 3-nitrotyrosine level, suggesting TrxR1 tyrosine residue modification by nitration and possible loss of function.
Conclusions: β2GPI and anti-β2GPI antibodies stimulate ROS generation in EC, with release into the conditioned medium. The impairment of EC activation by diapocynin, NOX2 siRNA, or NOX4 siRNA suggests that NOX mediate EC activation by anti-β2GPI antibodies. Anti-β2GPI antibodies induce nitrative stress in EC, which might be explained by eNOS uncoupling induced by these antibodies. The nitration of TrxR1, an enzyme that prevents eNOS uncoupling, may result in the inactivation of TrxR1 and contribute to eNOS uncoupling. Taken together, these studies provide preliminary evidence of the contribution of NOX activation and eNOS uncoupling in mediating oxidative and nitrative stress and induction of EC dysfunction by APLA.

Disclosures
McCrae: Janssen: Membership on an entity's Board of Directors or advisory committees; Syntimmune: Consultancy; Momenta: Consultancy; Halozyme: Membership on an entity's Board of Directors or advisory committees.