1. The NADPH oxidase Nox4 restricts the replicative lifespan of human endothelial cells
- Author
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Dietmar Herndler-Brandstetter, Rafal Koziel, Martin Hermann, Ruth Greussing, Barbara Lener, Haymo Pircher, Hermann Unterluggauer, Eveline Hütter, and Pidder Jansen-Dürr
- Subjects
HBSS, Hanks balanced salt solution ,DPI, diphenyliodinium ,FISH, fluorescence in situ hybridization ,Biochemistry ,HDF, human diploid fibroblast ,BrdU, bromodeoxyuridine ,chemistry.chemical_compound ,replicative senescence ,Superoxides ,HUVEC, human umbilical vein endothelial cell ,oxidative stress ,ERK1/2, extracellular-signal-regulated kinase 1/2 ,Cells, Cultured ,Cellular Senescence ,chemistry.chemical_classification ,NADPH oxidase ,Membrane Glycoproteins ,8-oxo-dG, 8-oxodeoxyguanosine ,Superoxide ,Nox, NADPH oxidase ,qPCR, quantitative real-time PCR ,PBGD, porphobilinogen deaminase ,NOX4 ,RLU, relative light units ,Telomere ,NGS, normal goat serum ,Cell biology ,PDL, population doubling ,Mitochondrial respiratory chain ,NADPH Oxidase 4 ,Gene Knockdown Techniques ,shRNA, small-hairpin RNA ,NADPH Oxidase 2 ,JNK, c-Jun N-terminal kinase ,cardiovascular system ,Cell aging ,Oxidation-Reduction ,Research Article ,Senescence ,MAP Kinase Signaling System ,Biology ,human umbilical vein endothelial cell (HUVEC) ,Nox4 ,RT, reverse-transcription ,ROS, reactive oxygen species ,SOD, superoxide dismutase ,Humans ,Molecular Biology ,Free-radical theory of aging ,Reactive oxygen species ,PNA, peptide nucleic acid ,urogenital system ,Endothelial Cells ,NADPH Oxidases ,Cell Biology ,telomere attrition ,chemistry ,biology.protein ,DNA damage ,SA-β-gal, senescence-associated β-galactosidase ,MAPK, mitogen-activated protein kinase - Abstract
The free radical theory of aging proposes that ROS (reactive oxygen species) are major driving forces of aging, and are also critically involved in cellular senescence. Besides the mitochondrial respiratory chain, alternative sources of ROS have been described that might contribute to cellular senescence. Noxs (NADPH oxidases) are well-known sources of superoxide, which contribute to the antimicrobial capabilities of macrophages, a process involving the prototypical member of the family referred to as Nox2. However, in recent years non-phagocytic homologues of Nox2 have been identified that are involved in processes other than the host defence. Superoxide anions produced by these enzymes are believed to play a major role in signalling by MAPKs (mitogen-activated protein kinases) and stress-activated kinases, but could also contribute to cellular senescence, which is known to involve oxygen radicals. In HUVECs (human umbilical vein endothelial cells), Nox4 is predominantly expressed, but its role in replicative senescence of HUVECs remains to be elucidated. Using shRNA (small-hairpin RNA)-mediated knockdown of Nox4, implicating lentiviral vectors, we addressed the question of whether lifelong depletion of Nox4 in HUVECs would influence the senescent phenotype. We found a significant extension of the replicative lifespan of HUVECs upon knockdown of Nox4. Surprisingly, mean telomere length was significantly reduced in Nox4-depleted cells. Nox4 depletion had no discernable influence on the activity of MAPKs and stress-activated kinases, but reduced the degree of oxidative DNA damage. These results suggest that Nox4 activity increases oxidative damage in HUVECs, leading to loss of replicative potential, which is at least partly independent of telomere attrition.
- Published
- 2009