Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of TNF-α–induced apoptosis by shear stress, nitric  oxide and ICE-like and CPP-32–like protease inhibitors in human endothelial cells. (A) HUVEC were incubated with LNMA (1 mM) and/or  TNF-α (300 U/ml) for 18 h with or without additional shear stress (ss)  and DNA fragmentation was determined. Results are means ± SE, with  * P <0.05 versus TNF-α; ** P <0.05 versus TNF-α + shear stress. (B) Dosedependent inhibition of TNF-α–induced apoptosis by the NO donors  SNP and SNAP. HUVEC were incubated with SNP and SNAP with or  without TNF-α (300 U/ml) for 18 h and DNA fragmentation was measured. (C) Effect of protease inhibitors or NO donors on TNF-α induced  apoptosis after 18 h of incubation. Substances were added just before  TNF-α addition in the following concentrations: SNP and SNAP (10 μM);  ICE-like protease inhibitor Ac-YVAD-CHO and CPP-32–like inhibitor  Ac-DEVD-CHO (100 μM); 8-bromo-cGMP (1 mM). Results are means  ± SE, with * P <0.05 versus TNF-α.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196141&req=5

Figure 1: Inhibition of TNF-α–induced apoptosis by shear stress, nitric oxide and ICE-like and CPP-32–like protease inhibitors in human endothelial cells. (A) HUVEC were incubated with LNMA (1 mM) and/or TNF-α (300 U/ml) for 18 h with or without additional shear stress (ss) and DNA fragmentation was determined. Results are means ± SE, with * P <0.05 versus TNF-α; ** P <0.05 versus TNF-α + shear stress. (B) Dosedependent inhibition of TNF-α–induced apoptosis by the NO donors SNP and SNAP. HUVEC were incubated with SNP and SNAP with or without TNF-α (300 U/ml) for 18 h and DNA fragmentation was measured. (C) Effect of protease inhibitors or NO donors on TNF-α induced apoptosis after 18 h of incubation. Substances were added just before TNF-α addition in the following concentrations: SNP and SNAP (10 μM); ICE-like protease inhibitor Ac-YVAD-CHO and CPP-32–like inhibitor Ac-DEVD-CHO (100 μM); 8-bromo-cGMP (1 mM). Results are means ± SE, with * P <0.05 versus TNF-α.

Mentions: TNF-α triggered apoptosis in HUVEC in a time-dependent fashion with maximum effects after 18 h exposure (Fig. 1 A). There was no increase in lactate dehydrogenase activity by TNF-α treatment excluding the induction of cell necrosis (data not shown). Exposure of HUVEC to physiological levels (15 dynes/cm2) of laminar shear stress dramatically reduced basal and TNF-α–triggered apoptosis. Shear stress results in an immediate increase in endothelial NO production and further chronically enhances NO synthesis by increasing endothelial NO-synthase expression (12, 21). In our experimental setting, NO synthesis measured by cGMP levels increased more than twofold 5 min after the onset of shear stress. Furthermore, increased protein levels of endothelial NO-synthase (eNOS) were sustained for 18 h of shear stress exposure up to 150 ± 10% compared with controls. Although TNF-α slightly reduced eNOS protein levels at baseline, shear stress increased eNOS protein to a similar extent in the presence of TNF-α (132 ± 4%). Inhibition of NO formation by NG-monomethyl- l-arginine (LNMA) significantly inhibited the effect of shear stress on the reduction of TNF-α–mediated cell death and completely restored basal apoptosis (Fig. 1 A). Thus, physiological concentrations of endogenous NO appear to be capable of suppressing TNF-α–triggered apoptosis of HUVEC. To demonstrate the antiapoptotic potential of NO, we investigated the effect of exogenous NO donors. Coincubation with SNP or S-nitrosopenicillamine (SNAP) dosedependently decreased TNF-α–triggered apoptosis (Fig. 1 B). NO donors did not affect apoptosis in the absence of TNF-α (Fig. 1 B). Low concentrations up to 50 μM of NO-donors were shown to be protective, whereas higher concentrations (>300 μM) revealed the known proapoptotic effect (22) (data not shown). TNF-α–induced apoptosis correlated with a decrease of cell viability by 22 ± 6%, which was completely prevented by NO donors. The inhibition of TNF-α–induced apoptosis by NO appeared to be independent of elevated cGMP levels, since the cGMP analogue 8-bromo-cGMP did not affect DNA fragmentation (Fig. 1 C).


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)

Inhibition of TNF-α–induced apoptosis by shear stress, nitric  oxide and ICE-like and CPP-32–like protease inhibitors in human endothelial cells. (A) HUVEC were incubated with LNMA (1 mM) and/or  TNF-α (300 U/ml) for 18 h with or without additional shear stress (ss)  and DNA fragmentation was determined. Results are means ± SE, with  * P <0.05 versus TNF-α; ** P <0.05 versus TNF-α + shear stress. (B) Dosedependent inhibition of TNF-α–induced apoptosis by the NO donors  SNP and SNAP. HUVEC were incubated with SNP and SNAP with or  without TNF-α (300 U/ml) for 18 h and DNA fragmentation was measured. (C) Effect of protease inhibitors or NO donors on TNF-α induced  apoptosis after 18 h of incubation. Substances were added just before  TNF-α addition in the following concentrations: SNP and SNAP (10 μM);  ICE-like protease inhibitor Ac-YVAD-CHO and CPP-32–like inhibitor  Ac-DEVD-CHO (100 μM); 8-bromo-cGMP (1 mM). Results are means  ± SE, with * P <0.05 versus TNF-α.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196141&req=5

Figure 1: Inhibition of TNF-α–induced apoptosis by shear stress, nitric oxide and ICE-like and CPP-32–like protease inhibitors in human endothelial cells. (A) HUVEC were incubated with LNMA (1 mM) and/or TNF-α (300 U/ml) for 18 h with or without additional shear stress (ss) and DNA fragmentation was determined. Results are means ± SE, with * P <0.05 versus TNF-α; ** P <0.05 versus TNF-α + shear stress. (B) Dosedependent inhibition of TNF-α–induced apoptosis by the NO donors SNP and SNAP. HUVEC were incubated with SNP and SNAP with or without TNF-α (300 U/ml) for 18 h and DNA fragmentation was measured. (C) Effect of protease inhibitors or NO donors on TNF-α induced apoptosis after 18 h of incubation. Substances were added just before TNF-α addition in the following concentrations: SNP and SNAP (10 μM); ICE-like protease inhibitor Ac-YVAD-CHO and CPP-32–like inhibitor Ac-DEVD-CHO (100 μM); 8-bromo-cGMP (1 mM). Results are means ± SE, with * P <0.05 versus TNF-α.
Mentions: TNF-α triggered apoptosis in HUVEC in a time-dependent fashion with maximum effects after 18 h exposure (Fig. 1 A). There was no increase in lactate dehydrogenase activity by TNF-α treatment excluding the induction of cell necrosis (data not shown). Exposure of HUVEC to physiological levels (15 dynes/cm2) of laminar shear stress dramatically reduced basal and TNF-α–triggered apoptosis. Shear stress results in an immediate increase in endothelial NO production and further chronically enhances NO synthesis by increasing endothelial NO-synthase expression (12, 21). In our experimental setting, NO synthesis measured by cGMP levels increased more than twofold 5 min after the onset of shear stress. Furthermore, increased protein levels of endothelial NO-synthase (eNOS) were sustained for 18 h of shear stress exposure up to 150 ± 10% compared with controls. Although TNF-α slightly reduced eNOS protein levels at baseline, shear stress increased eNOS protein to a similar extent in the presence of TNF-α (132 ± 4%). Inhibition of NO formation by NG-monomethyl- l-arginine (LNMA) significantly inhibited the effect of shear stress on the reduction of TNF-α–mediated cell death and completely restored basal apoptosis (Fig. 1 A). Thus, physiological concentrations of endogenous NO appear to be capable of suppressing TNF-α–triggered apoptosis of HUVEC. To demonstrate the antiapoptotic potential of NO, we investigated the effect of exogenous NO donors. Coincubation with SNP or S-nitrosopenicillamine (SNAP) dosedependently decreased TNF-α–triggered apoptosis (Fig. 1 B). NO donors did not affect apoptosis in the absence of TNF-α (Fig. 1 B). Low concentrations up to 50 μM of NO-donors were shown to be protective, whereas higher concentrations (>300 μM) revealed the known proapoptotic effect (22) (data not shown). TNF-α–induced apoptosis correlated with a decrease of cell viability by 22 ± 6%, which was completely prevented by NO donors. The inhibition of TNF-α–induced apoptosis by NO appeared to be independent of elevated cGMP levels, since the cGMP analogue 8-bromo-cGMP did not affect DNA fragmentation (Fig. 1 C).

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