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Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases.

Dimmeler S, Haendeler J, Nehls M, Zeiher AM - J. Exp. Med. (1997)

Bottom Line: Physiological levels of shear stress alter the genetic program of cultured endothelial cells and are associated with reduced cellular turnover rates and formation of atherosclerotic lesions in vivo.This anti-apoptotic activity of shear stress decreased after pharmacological inhibition of endogenous nitric oxide (NO)-synthase by NG-monomethyl-L-arginine and was completely reproduced by exogenous NO-donors.Endothelial-derived nitric oxide (NO) as well as exogenous NO donors inhibited TNF-alpha-induced cysteine protease activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine IV, University of Frankfurt, Germany.

ABSTRACT
Physiological levels of shear stress alter the genetic program of cultured endothelial cells and are associated with reduced cellular turnover rates and formation of atherosclerotic lesions in vivo. To test the hypothesis that shear stress (15 dynes/cm2) interferes with programmed cell death, apoptosis was induced in human umbilical venous cells (HUVEC) by tumor necrosis factor-alpha (TNF-alpha). Apoptosis was quantified by ELISA specific for histone-associated DNA-fragments and confirmed by demonstrating the specific pattern of internucleosomal DNA-fragmentation. TNF-alpha (300 U/ml) mediated increase of DNA-fragmentation was completely abrogated by shear stress (446 +/- 121% versus 57 +/- 11%, P <0.05). This anti-apoptotic activity of shear stress decreased after pharmacological inhibition of endogenous nitric oxide (NO)-synthase by NG-monomethyl-L-arginine and was completely reproduced by exogenous NO-donors. The activation of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like cysteine proteases was required to mediate TNF-alpha-induced apoptosis of HUVEC. Endothelial-derived nitric oxide (NO) as well as exogenous NO donors inhibited TNF-alpha-induced cysteine protease activation. Inhibition of CPP-32 enzyme activity was due to specific S-nitrosylation of Cys 163, a functionally essential amino acid conserved among ICE/CPP-32-like proteases. Thus, we propose that shear stress-mediated NO formation interferes with cell death signal transduction and may contribute to endothelial cell integrity by inhibition of apoptosis.

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(A) CPP-32–like activity in HUVEC treated with NO donors  (10 μM) or Ac-DEVD (100 μM) and TNF-α (300 U/ml) for 18 h. (B)  Northern blot analysis of CPP32. RNA from HUVEC was prepared after  6 h of incubation with TNF-α (300 U/ml), SNP and SNAP (10 μM) as indicated. 10 μg of total RNA was resolved, blotted and sequentially hybridized to full-length human cDNA of CPP-32 and GAPDH.
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Figure 2: (A) CPP-32–like activity in HUVEC treated with NO donors (10 μM) or Ac-DEVD (100 μM) and TNF-α (300 U/ml) for 18 h. (B) Northern blot analysis of CPP32. RNA from HUVEC was prepared after 6 h of incubation with TNF-α (300 U/ml), SNP and SNAP (10 μM) as indicated. 10 μg of total RNA was resolved, blotted and sequentially hybridized to full-length human cDNA of CPP-32 and GAPDH.

Mentions: Next, we measured ICE/CPP-32–like proteases activity in HUVEC incubated for 18 h in the presence of TNF-α (300 U/ml) and SNP or SNAP (10 μM). ICE- and CPP32–like protease activities were directly determined in the homogenate. Exogenous NO completely suppressed the increase in CPP-32–like protease activity induced by TNF-α (Fig. 2 A). Similarly, endogenous NO, induced by shear stress, also inhibited TNF-α stimulation of both proteases (P = 0.04), an effect completely reversed by addition of the NO-synthase inhibitor LNMA (99 ± 3% of TNF-induced CPP-32–like activity).


Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases.

Dimmeler S, Haendeler J, Nehls M, Zeiher AM - J. Exp. Med. (1997)

(A) CPP-32–like activity in HUVEC treated with NO donors  (10 μM) or Ac-DEVD (100 μM) and TNF-α (300 U/ml) for 18 h. (B)  Northern blot analysis of CPP32. RNA from HUVEC was prepared after  6 h of incubation with TNF-α (300 U/ml), SNP and SNAP (10 μM) as indicated. 10 μg of total RNA was resolved, blotted and sequentially hybridized to full-length human cDNA of CPP-32 and GAPDH.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196141&req=5

Figure 2: (A) CPP-32–like activity in HUVEC treated with NO donors (10 μM) or Ac-DEVD (100 μM) and TNF-α (300 U/ml) for 18 h. (B) Northern blot analysis of CPP32. RNA from HUVEC was prepared after 6 h of incubation with TNF-α (300 U/ml), SNP and SNAP (10 μM) as indicated. 10 μg of total RNA was resolved, blotted and sequentially hybridized to full-length human cDNA of CPP-32 and GAPDH.
Mentions: Next, we measured ICE/CPP-32–like proteases activity in HUVEC incubated for 18 h in the presence of TNF-α (300 U/ml) and SNP or SNAP (10 μM). ICE- and CPP32–like protease activities were directly determined in the homogenate. Exogenous NO completely suppressed the increase in CPP-32–like protease activity induced by TNF-α (Fig. 2 A). Similarly, endogenous NO, induced by shear stress, also inhibited TNF-α stimulation of both proteases (P = 0.04), an effect completely reversed by addition of the NO-synthase inhibitor LNMA (99 ± 3% of TNF-induced CPP-32–like activity).

Bottom Line: Physiological levels of shear stress alter the genetic program of cultured endothelial cells and are associated with reduced cellular turnover rates and formation of atherosclerotic lesions in vivo.This anti-apoptotic activity of shear stress decreased after pharmacological inhibition of endogenous nitric oxide (NO)-synthase by NG-monomethyl-L-arginine and was completely reproduced by exogenous NO-donors.Endothelial-derived nitric oxide (NO) as well as exogenous NO donors inhibited TNF-alpha-induced cysteine protease activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine IV, University of Frankfurt, Germany.

ABSTRACT
Physiological levels of shear stress alter the genetic program of cultured endothelial cells and are associated with reduced cellular turnover rates and formation of atherosclerotic lesions in vivo. To test the hypothesis that shear stress (15 dynes/cm2) interferes with programmed cell death, apoptosis was induced in human umbilical venous cells (HUVEC) by tumor necrosis factor-alpha (TNF-alpha). Apoptosis was quantified by ELISA specific for histone-associated DNA-fragments and confirmed by demonstrating the specific pattern of internucleosomal DNA-fragmentation. TNF-alpha (300 U/ml) mediated increase of DNA-fragmentation was completely abrogated by shear stress (446 +/- 121% versus 57 +/- 11%, P <0.05). This anti-apoptotic activity of shear stress decreased after pharmacological inhibition of endogenous nitric oxide (NO)-synthase by NG-monomethyl-L-arginine and was completely reproduced by exogenous NO-donors. The activation of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like cysteine proteases was required to mediate TNF-alpha-induced apoptosis of HUVEC. Endothelial-derived nitric oxide (NO) as well as exogenous NO donors inhibited TNF-alpha-induced cysteine protease activation. Inhibition of CPP-32 enzyme activity was due to specific S-nitrosylation of Cys 163, a functionally essential amino acid conserved among ICE/CPP-32-like proteases. Thus, we propose that shear stress-mediated NO formation interferes with cell death signal transduction and may contribute to endothelial cell integrity by inhibition of apoptosis.

Show MeSH
Related in: MedlinePlus