The Journal of Experimental Medicine
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© The Rockefeller University Press, 0022-1007/1997/2/601/ $5.00
The Journal of Experimental Medicine, Volume 185, Number 4, February 17, 1997 601-608

Articles

Suppression of Apoptosis by Nitric Oxide via Inhibition of Interleukin-1β–converting Enzyme (ICE)-like and Cysteine Protease Protein (CPP)-32–like Proteases

Stefanie Dimmeler, Judith Haendeler, Michael Nehls, and Andreas M. Zeiher

From the Molecular Cardiology Group, Department of Internal Medicine IV, University of Frankfurt, Germany

Physiological levels of shear stress alter the genetic programm 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-1β–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.

Address correspondence to Andreas M. Zeiher, Department of Internal Medicine IV, Division of Cardiology, University of Frankfurt, Theodor Stern-Kai 7, 60590 Frankfurt, Germany.

1 Abbreviations used in this paper: AFC, 7-amino-4-trifluoro-methylcoumarin; AMC, 7-amino-4-coumarin; CCP, cysteine protease protein; DTNB, 5,5'dithiobis (2-nitrobenzoate); eNOS, endothelial NO-synthase; HUVEC, human umbilical venous endothelial cells; ICE, interleukin-1β-converting enzyme; LDH, lactate-dehydrogenase; LNMA, NG-monomethyl-L-arginine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NO, nitric oxide; SNP, sodium nitroprusside; SNAP, S-nitrosopenicillamine.


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