Abstract 10099: Loss of CD70 Impairs Nitric Oxide Homeostasis and Augments Intracellular Oxidative Stress in Endothelial Cells

Abstract

Introduction: TNF superfamily ligands exert multiple effects on endothelial cell (EC) function, metabolism, and redox status. CD70 is a TNF superfamily member classically expressed on antigen-presenting cells and drives T memory cell polarization. Despite evidence of expression by ECs, the role of CD70 in endothelial biology has not been defined. We hypothesized that CD70 affects EC function through regulation of nitric oxide (NO) generation and cellular oxidative stress. Methods: Human aortic and pulmonary artery ECs (HAECs and HPAECs, respectively) were transfected with control and CD70-directed siRNA (siCD70). NO levels of individual cells were monitored in real-time after stimulation with ATP using the intracellular fluorescent biosensor c-geNOp. Similarly, intracellular hydrogen peroxide was assayed in real-time using the intracellular fluorescent biosensor Hyper7 following stimulation by auranofin and histamine. For both studies, 70-100 individual cells, averaged over 4 experiments, were analyzed using confocal microscopy. Control and siCD70-treated cell lysates (n = 4-6) were analyzed by Western blot and quantitative RT-PCR, as well as for glutathione peroxidase (GPx) enzyme activity. Results: CD70 knockdown was associated with a reduction in agonist-stimulated NO levels in HAECs and HPAECs by 30% and 40%, respectively. Protein levels of eNOS decreased after CD70 knockdown, with a reduction of 50% in HAECs and 75% in HPAECs. Phosphorylation at Ser1177 of eNOS in response to EC stimulation with ATP was reduced after siCD70 treatment, and expression of the key eNOS-stimulatory protein Hsp90 was decreased. Intracellular hydrogen peroxide was significantly increased (20% in HAECs and 80% in HPAECs) following ROS stimulation in siCD70-treated versus control cells. This change in redox status was associated with a decrease in mitochondrial superoxide dismutase protein and an increase in GPx-1 expression and enzyme activity. Conclusions: Loss of CD70 in ECs is associated with reduced eNOS expression and bioactivity as well as increased susceptibility to intracellular oxidative stress, potentially from a mitochondrial source. These results suggest an important homeostatic role for CD70 in endothelial function and redox status.