Background: Endothelial injury is regarded as a key early event in the development of atherosclerosis. High level of glycemic excursion has been reported to play an important role in the pathogenesis of atherosclerosis. Recently, proteinase-activated receptor-2 (PAR2), activated by factor (F) Xa, has been shown to play a pivotal part in diabetes-induced endothelial dysfunction. In the present study, we have investigated the effect of rivaroxaban, a direct FXa-inhibitor by analyzing cellular biological function of human umbilical vein endothelial cells (HUVECs) in a glycemic excursion-model.
Methods: HUVECs were starved in 1% fetal calf serum media with 0.5% glucose for 4 hours and treated with or without rivaroxaban 0.1μg/mL, followed by stimulation of glucose in concentration of 0.5 mM, 5mM, 15mM and 30 mM. Cell viability was measured after 3 hours of stimulation by glucose using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell without rivaroxaban treatment and then stimulated with 0.5mM glucose was used as control. The high glucose-induced cell apoptosis was also evaluated with or without pre-treatment of rivaroxaban based on TUNEL method, using fluorescein-dUTP to label DNA strand breaks. All TUNEL-positive HUVECs in whole mount slide were counted. The derivatives of reactive oxygen metabolites (dROMs) were measured 45 minutes after the glucose stimulation to evaluate the production of reactive oxygen species (ROS).
Results: In rivaroxaban treated group, there was a significant increase in cell viability in high-glucose condition (113% in 0.5mM, 103% in 5 mM, 125% in 15 mM compared with cells without rivaroxaban pre-treatment, respectively). There was a significant increase in TUNEL positive cells in a glucose concentration dependent manner without rivaroxaban treatment; however, rivaroxaban pre-treatment significantly inhibited cell apoptosis (75% decrease in 15 mM glucose-stimulated-cells). There was a significant increase in ROS production in glucose-stimulated HUVECs without rivaroxabantreatment, which was also inhibited when pre-treated with rivaroxaban (30% decrease compared without rivaroxaban-pretreatment) (p< 0.05).
Conclusions: Rivaroxaban increases endothelial cell viability and inhibits apoptosis induced by high level of glycemic excursion, possibility through ROS signaling. These results suggest that rivaroxaban may be effective in the prevention of atherosclerosis.