Aging Enhances the Sensitivity of Endothelial Cells Toward Apoptotic Stimuli

• DOI: 10.1161/hh2001.097796
• Publication Date: 2007-09-28T18:32:49Z
• Authors: Jörg Hoffmann; Judith Haendeler; Alexandra Aicher; Lothar Rössig; Mariuca Vasa; Andreas M. Zeiher; Stefanie Dimmeler
• Keywords: Male; Mice, Knockout; Aging; 0303 health sciences; Nitric Oxide Synthase Type III; Caspase 3; Nitric Oxide Synthase Type II; Apoptosis; Nitric Oxide; Enzyme Activation; Lipoproteins, LDL; Mice; 03 medical and health sciences; Amino Acid Substitution; Caspases; Animals; Humans; Nitric Oxide Donors; Endothelium, Vascular; Nitric Oxide Synthase; Aorta; Cells, Cultured

Abstract—

Advanced aging leads to impaired endothelial NO synthesis and enhanced endothelial cell apoptosis; therefore, we investigated the sensitivity of aged endothelial cells toward apoptotic stimuli and determined the role of NO. Human umbilical vein endothelial cells (HUVECs) were cultured until 14th passage. In aged cells, oxLDL and tumor necrosis factor-α–induced apoptosis and caspase-3–like activity were significantly enhanced more than 3-fold compared with young cells (passage 3). Because NO contributes to protection against endothelial cell death via S-nitrosylation of caspases, we determined endothelial NO synthase (eNOS) protein expression and the content of S-nitrosylated proteins. Aged HUVECs showed significantly reduced eNOS expression (35±10%) and a decrease in the overall S-NO content (33±3%), suggesting that eNOS downregulation may be involved in age-dependent increase of apoptosis sensitivity. Indeed, eNOS knockout endothelial cells showed a significantly enhanced apoptosis induction. Exogenous NO donors abolished increased apoptosis and caspase-3–like activity. In contrast, the application of shear stress, which exerts a profound apoptosis inhibitory effect via upregulation of NO synthesis in young cells, failed to inhibit apoptosis in aged cells. Moreover, no upregulation of eNOS protein expression and S-NO content in response to shear stress was detected in aged cells. Overexpression of wild-type eNOS completely restored the antiapoptotic effect of shear stress, whereas only a partial inhibitory effect was detected under steady conditions. Strikingly, transfection of constitutively active phosphomimetic eNOS (S1177D) further abrogated apoptosis in aged HUVECs. Thus, aging of endothelial cells is associated with decreased NO synthesis and concomitantly increased sensitivity of apoptosis, which may contribute to functional impairment of the endothelial monolayer.