Letter to the Editor

Department of Internal and Experimental Medicine (DMCS), University of Padova, Padova, Italy

To the Editor:

The -786C allele in the promoter, but not the Asp298 (894T) variant in exon 7, of the endothelial nitric oxide synthase (NOS3) gene has been recently associated with a blunted increase in blood flow average peak velocity induced by acetylcholine in coronary or mammary artery after a 4-week exercise training program.1 By providing a potential explanation for the remarkable individual heterogeneity of responses achieved with exercise in coronary artery disease (CAD) patients, these results extend previous landmark findings from this group.2

The authors carefully excluded patients with arterial hypertension, insulin-dependent diabetes mellitus, smoking, and hypercholesterolemia, eg, conditions that notoriously induce endothelial dysfunction. However, the study sample size was small; therefore, one wonders if its statistical power, of which no calculation was provided, was adequate and if a selection bias occurred. The latter is suggested by the lack of -786C homozygous, which occurred in 18% to 22% of patients in larger studies,3,4 and lack of a frequency of -786C allele (31.6%) and 894T allele (43.1%), which differed from those observed in larger studies.3,4

The average peak velocity increase induced by acetylcholine baseline was blunted in patients with the 894T polymorphism;1 furthermore, the 3 patients homozygous for this allele exhibited vasoconstriction at baseline with acetylcholine and an impaired average peak velocity increase after exercise training. Therefore, Erbs et al suggested that the presence of either the promoter or the exon 7 polymorphism attenuates endothelium-dependent vasodilatation in CAD patients. To explain the effect of the 894T variant, the authors quoted a study claiming this variant to be more vulnerable to intracellular cleavage, thus implying a decreased in vivo NO generation.5 However, this contention was subsequently disputed by a painstaking study that clearly demonstrated that the intracellular cleavage found in cells harboring the Asp298 substitution was an in vitro artifact6 caused by the acidic pH used. Indeed, as Erbs et al correctly acknowledged, the Asp298 replacement does not affect NOS3 biological activity.5,6

We reported not only that the T-786C substitution was in linkage disequilibrium with the 894T variant3 but also that only the -786C allele implied a blunted forearm blood flow response to acetylcholine in essential hypertensive patients.4 These findings strongly implicated this allele as a determinant of endothelial dysfunction, in keeping with findings in Japanese patients.7 The relevance of the -786C, but not of the 894T, variant was further confirmed by the GENICA study, a large investigation of white CAD patients, which showed an association of the -786C allele with multi-vessel CAD, particularly in patients with multiple risk factors.3 Therefore, we support the conclusion of Erbs et al regarding the relevance of the -786C allele as a functional determinant of endothelial dysfunction in CAD patients,1 a contention that accords well with the demonstration that this allele binds a replication protein A1 acting as a potent repressor of NOS3 transcription.8

Although evidence is accumulating on the functional relevance of the NOS3 promoter polymorphism, it remains altogether controversial whether the 894T variant implies blunted NOS3 activity. Consequently, caution should be used regarding attributing a blunted NO generation to these polymorphisms tested, because of the small number of observations that are prone to serendipity.

References

Erbs S, Baither Y, Linke A, Adams V, Shu Y, Lenk K, Gielen S, Dilz R, Schuler G, Hambrecht R. Promoter but not Exon 7 Polymorphism of Endothelial Nitric Oxide Synthase Affects Training-Induced Correction of Endothelial Dysfunction. Arterioscler Thromb Vasc Biol. 2003; 23: 1814–1819.

Hambrecht R, Wolf A, Gielen S, Linke A, Hofer J, Erbs S, Schoene N, Schuler G. Effect of Exercise on Coronary Endothelial Function in Patients with Coronary Artery Disease. N Engl J Med. 2000; 342: 454–460.

Rossi GP, Cesari M, Zanchetta M, Colonna S, Maiolino G, Pedon L, Cavallin M, Maiolino P, Pessina AC. The T-786C endothelial nitric oxide synthase genotype is a novel risk factor for coronary artery disease in Caucasian patients of the GENICA study. J Am Coll Cardiol. 2003; 41: 930–937.

Rossi GP, Taddei S, Virdis A, Cavallin M, Ghiadoni L, Favilla S, Versari D, Sudano I, Pessina AC, Salvetti A. The T-786C and Glu298Asp polymorphisms of the endothelial nitric oxide gene affect the forearm blood flow responses of Caucasian hypertensive patients. J Am Coll Cardiol. 2003; 41: 938–945.

Tesauro M, Thompson WC, Rogliani P, Qi L, Chaudhary PP, Moss J. Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: cleavage of proteins with aspartate vs. glutamate at position 298. Proc Natl Acad Sci U S A. 2000; 97: 2832–2835.

Fairchild TA, Fulton D, Fontana JT, Gratton JP, McCabe TJ, Sessa WC. Acidic hydrolysis as a mechanism for the cleavage of the Glu(298)–>Asp variant of human endothelial nitric-oxide synthase. J Biol Chem. 2001; 276: 26674–26679.

Nakayama M, Yasue H, Yoshimura M, Shimasaki Y, Kugiyama K, Ogawa H, Motoyama T, Saito Y, Ogawa Y, Miyamoto Y, Nakao K. T-786–>C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation. 1999; 99: 2864–2870.

Miyamoto Y, Saito Y, Nakayama M, Shimasaki Y, Yoshimura T, Yoshimura M, Harada M, Kajiyama N, Kishimoto I, Kuwahara K, Hino J, Ogawa E, Hamanaka I, Kamitani S, Takahashi N, Kawakami R, Kangawa K, Yasue H, Nakao K. Replication protein A1 reduces transcription of the endothelial nitric oxide synthase gene containing a -786T–>C mutation associated with coronary spastic angina. Hum Mol Genet. 2000; 9: 2629–2637.