Proposed scheme for pathways contributing to cellular oxidative stress and NF-κB activation in aged endothelial cells. In aged endothelial cells, increased levels of O₂^•− generated by the electron transport chain and NAD(P)H oxidases [stimulated by elevated TNF-α levels and/or by the activated local renin-angiotensin system (RAS) in the vascular wall] are dismutated to H₂O₂. Increased cytoplasmic H₂O₂ levels and activation of toll-like receptors (TLRs) each contribute to the activation and nuclear translocation of NF-κB, which results in a proinflammatory shift in the endothelial gene expression profile, endothelial activation, and increased monocyte adhesiveness to the endothelium. The transcriptional activity of NF-κB is regulated by nucleophosmin (NPM) and SIRT-1 (hatched arrows represent inhibition), and both pathways exhibit age-related alterations. In addition, poly(ADP-ribose) polymerase (PARP-1) activation also modulates transcriptional activity of NF-κB. Increased O₂^•− production and/or downregulation of endothelial nitric oxide synthase (eNOS) are mutually responsible for impaired bioavailability of NO and endothelial vasodilator dysfunction in aged arteries. The model predicts that upregulation of TNF-α and/or impaired NO bioavailability may also contribute to development of mitochondrial oxidative stress during aging. Increased TNF-α levels also promote endothelial apoptotic cell death, which, along with increased oxidative stress and vascular inflammation, increases the risk for coronary artery disease. iNOS, inducible nitric oxide synthase; SOD2, superoxide dismutase 2.

A: reporter gene assay demonstrates the knockdown effects of NPM [by small interfering RNA (siRNA)] on TNF-α (10 ng/ml)-induced NF-κB reporter activity in cultured primary human coronary arterial endothelial cells (HCAECs). The resulting overexpression of NPM with respect to endothelial NF-κB activation is also shown, as well as effects of resveratrol (10 μmol/l) on TNF-α (10 ng/ml)-induced NF-κB reporter activity. Cells were transiently cotransfected with NF-κB-driven firefly luciferase and CMV-driven Renilla luciferase constructs, followed by NPM knockdown, NPM overexpression, or TNF-α stimulation. Cells were then lysed and subjected to luciferase activity assay. Following normalization, relative luciferase activity was obtained from 6 independent transfections. Data are means ± SE. *P < 0.05 vs. control; #P < 0.05 vs. TNF-α only; &P < 0.05 vs. NPM overexpression. B: effect of knockdown of SIRT-1 (siRNA) and/or resveratrol (10 μmol/l) treatment on mRNA expression of NPM in cultured HCAECs. Analysis of mRNA expression was performed by real-time QRT-PCR. β-Actin was used for normalization. Data are means ± SE (n = 5 for each group). C: NPM mRNA expression in carotid arteries of ad libitum-fed young (3 mo old), ad libitum fed aged (28 mo old), and caloric-restricted aged (28 mo old) F344 rats. Analysis of mRNA expression was performed by real-time QRT-PCR. β-Actin was used for normalization. Data are means ± SE (n = 5 for each group). *P < 0.05. vs. young; #P < 0.05 vs. ad libitum-fed aged. All animal use protocols were approved by the Institutional Animal Care and Use Committee of the New York Medical College, Valhalla, NY.