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• Research Paper Volume 4, Issue 10 pp 664-673

  Cystathionine beta synthase modulates senescence of human endothelial cells

  Relevance score: 16.829777

  Eva Albertini, Rafał Kozieł, Angela Dürr, Michael Neuhaus, Pidder Jansen-Dürr

  Keywords: cystathionine beta synthase, senescence, molecular biology of aging, transsulfuration

  Published in Aging on October 18, 2012

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  Availability of methionine is known to modulate the rate of aging in model organisms, best illustrated by the observation that dietary methionine restriction extends the lifespan of rodents. However, the underlying mechanisms are incompletely understood. In eukaryotic cells, methionine can be converted to cysteine through the reverse transsulfuration pathway thereby modulating intracellular methionine availability. Whereas previous results obtained in yeast and fruit flies suggest that alterations in the reverse transsulfuration pathway modulate the rate of aging, it is not known whether this function is conserved in evolution. Here we show that depletion of cystathionine beta synthase (CBS), a rate limiting enzyme in the reverse transsulfuration pathway, induces premature senescence in human endothelial cells. We found that CBS depletion induces mild mitochondrial dysfunction and increases the sensitivity of endothelial cells to homocysteine, a known inducer of endothelial cell senescence and an established risk factor for vascular disease. Our finding that CBS deficiency induces endothelial cell senescence in vitro, involving both mitochondrial dysfunction and increased susceptibility of the cells to homocysteine, suggests a new mechanism linking CBS deficiency to vascular aging and disease.

  

  (A) Protein was isolated from HUVEC and HDF in passage 6 (young) and subsequent passages as indicated; HUVEC reached senescence at passage 25, whereas HDF reached senescence at passage 27. CBS levels were determined by Western blot analysis, GAPDH served as loading control. (B) Left panels: CBS gene expression was inactivated by lentiviral shRNA vectors in early-passage HUVEC and HDF, as indicated. Cumulative population doublings (cPDL) were calculated, beginning 12 days after infection. Growth curves represent three independent experiments in each case. Right panels: CBS knockdown was verified by Western blot. (C) Bar diagram displaying relative percentage of BrdU positive (= proliferating) HUVEC 42 days after infection with lentiviral shRNA vectors. The percentage of BrdU positive cells was determined using flow cytometry; data are represented as mean ± SE (n=3). ***: p<0.001. (D) Bar diagram displaying relative percentage of annexin V positive (= apoptotic) HUVEC 42 days after infection with lentiviral shRNA vectors. The percentage of apoptotic cells was determined using flow cytometry; data are represented as mean ± SE (n=5). n.s: not significant

  
  
  

  

  CBS gene expression was inactivated by lentiviral shRNA vectors in early-passage HUVEC, HAEC and HDF, as indicated. (A) Left panel: Cumulative population doublings (cPDL) of CBS-depleted HAEC were calculated beginning 12 days after infection. Growth curve represents three independent experiments. Right panel: CBS knockdown was verified by Western blot. (B) The percentage of SA-ß-gal positive cells was determined 20 days after infection; data are represented as mean ± SE (n=3). ***: p<0.001, n.s: not significant. Micrographs show representative pictures of HUVEC and HAEC stained for SA-ß-gal. (C) Protein was isolated from control and CBS-depleted HUVEC and HAEC, as indicated. Protein levels were determined by Western blot, GAPDH served as loading control.

  
  
  

  

  (A) Left panel: CBS-depleted HUVEC were subjected to high resolution respirometry. The experimental regime started with routine respiration, followed by addition of oligomycin, and stepwise titration of FCCP. Finally, respiration was inhibited by sequential addition of rotenone and antimycin A (Ant.A). Respirometry results were normalized to citrate synthase activity; data are represented as mean ± SE (n=6 and 7 for CBS knockdown and control, respectively). Right panel: Respiratory control ratio was determined as the ratio of uncoupled respiration to oligomycin-inhibited respiration. (B) The electric potential of the inner mitochondrial membrane was measured in situ using flow cytometry in intact cells stained with the JC-1 fluorescent probe. The ratio of cells with high to cells with low mitochondrial membrane potential was calculated as described in the methods. Data are represented as mean ± SE (n=3).

  
  
  

  

  (A) Left panel: Cumulative population doublings of CBS overexpressing (OE) and control HUVEC were calculated, beginning 11 days after infection. Growth curves represent three independent experiments in each case. Right panel: CBS Western blot after lentiviral infection with the CBS overexpression and control construct. (B) Left panel: Bar diagram displaying relative percentage of SA-ß-gal positive cells 46 days after lentiviral infection with the CBS overexpression and control construct. Data are represented as mean ± SE (n=3). ***: p<0.001 Right panel: Representative micrographs of cells stained for SA-ß-gal activity.

  
  
  

  

  CBS gene expression was inactivated by lentiviral shRNA vectors in early-passage HUVEC and HDF, as indicated, and homocysteine (Hcy) was added. Cells were counted after 72 h (panel A), and the percentage of SA-ß-gal positive cells was determined after 84 h (panel B). Data are represented as mean ± SE (n=3). Micrographs show representative pictures of cells stained for SA-ß-gal. ***: p<0.001, **: p<0.01, n.s.: not significant.