title: Aging enhances the sensitivity of endothelial cells toward apoptotic stimuli: important role of nitric oxide.
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-alpha-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.
authors.affiliation: Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Frankfurt, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Hoffmann
authors.forename: J
authors.initials: J
authors.lastname: Haendeler
authors.forename: A
authors.initials: A
authors.lastname: Aicher
authors.forename: L
authors.initials: L
authors.lastname: Rössig
authors.forename: M
authors.initials: M
authors.lastname: Vasa
authors.forename: A M
authors.initials: AM
authors.lastname: Zeiher
authors.forename: S
authors.initials: S
authors.lastname: Dimmeler
mesh_terms: D000375:Aging / Q000187:drug effects / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D001011:Aorta; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D008077:Lipoproteins, LDL / Q000494:pharmacology; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D009603:Nitroso Compounds / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D013314:Stress, Mechanical; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D008077:Lipoproteins, LDL; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D011518:Proto-Oncogene Proteins; D013438:Sulfhydryl Compounds; D014409:Tumor Necrosis Factor-alpha; C099252:oxidized low density lipoprotein; D009569:Nitric Oxide; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases