Folic acid enlarges the armamentarium for the treatment of coronary vasospasm

Abstract

Qipshidze et al. (2010) described in the Journal of Cell Physiology that folic acid ameliorates acetylcholine-induced vasoconstriction in hyperhomocysteinemic cystathionine beta synthase heterozygote (CBS /þ) mice, and that this effect can be explained by an increase in endothelial nitric oxide (eNOS) expression and nitric oxide (NO) bioavailability in the coronary artery. The authors have investigated the role of eNOS in acetylcholine-induced vasoconstriction in CBS /þ mice by measuring total protein (by immunolabelling) and mRNA levels (by RT-PCR). However, they did not perform eNOS uncoupling assessment, which is of essential importance to evaluate the active or inactive state of eNOS. Qipshidze et al. (2010) assessed only the NO-pathway using diaminofluorescein-2-diacetat staining. However, it should be considered that compartmentalization of diaminofluorescein-2 around elastic fibers limits its potential to characterize the site of NO production at the subcellular level. Further, reaction of diaminofluorescein-2 with reagents commonly used in NO research, such as ferricyanide, N-ethylmaleimide, and tempol, may also interfere with diaminofluorescein-2 fluorescence that can be misinterpreted as NO signals. Importantly, in cell-free systems, the diaminofluorescein-2 dependent signal is enhanced in the presence of superoxide, suggesting that diaminofluorescein-2 is indeed more sensitive to the degradation product of NO, peroxynitrite, rather than to NO itself (Roychowdhury et al., 2002). This would imply that the detection of higher signals in the presence of diaminofluorescein-2 do not necessarily indicate higher levels of bioavailable NO but can rather indicate the reverse, higher levels of peroxynitrite and NO scavenging. In any case, even if diaminofluorescein-2 were specific for NO or more sensitive for NO than for peroxynitrite, higher steady state levels of NO must not necessarily indicate an effect of eNOS and higher production of NO but can also indicate reduced oxidative stress or a direct anti-oxidant effect of folate. Thus, the demonstration of recoupling of eNOS from its monomeric to its dimeric state would have been essential to justify the authors’ conclusion. In addition, UV illumination can lead to formation of high levels of nitrosating species that interferewith NO detection coming from enzymatic sources (Rodriguez et al., 2005). Therefore, assessment of NO alone by diaminofluorescein-2-diacetate cannot conclude whether or not this molecule is generated by eNOS. Other methods, such as eNOSmonomer/dimer blot, dichlorofluorescin staining, and NOS-dependent ROS production using lucigenin-enhanced chemiluminescence should also be considered. Unfortunately, the authors also did not investigate the role of folic acid-induced changes in high-energy phosphate metabolism. It has been described that folic acid, besides its well-known homocysteine-lowering and antioxidant effect, preserves the high-energy phosphate metabolism during myocardial ischemia with subsequent less myocardial necrosis, better myocardial function, and less arrhythmias (Moens et al., 2008). Title et al. (2000) have demonstrated that endothelial function, as assessed by flow-mediated dilation (FMD) in patients with coronary artery diseases, is ameliorated by reducing the level of plasma homocysteine. This is in contrast with a study ofDoshi et al. (2001), which showed no correlation between FMD improvement and plasma homocysteine level in patients with coronary artery diseases. This result is supported by Moens et al. (2007) demonstrating in patients with acute myocardial infarction that the beneficial effect of folic acid on endothelial dysfunction is independent of baseline homocysteine-level. Chambers et al. (2000) have shown that improved FMD correlates with the reduction in free (unbound)-reduced homocysteine in patients with coronary heart diseases. Unfortunately, Qipshidze et al. (2010) did not assess any of the different forms (reduced, mixed disulphides, and total) of homocysteine. It has been described that smoothmuscle constriction of the coronary artery, the so-called coronary vasospasm, is associated with increased markers of oxidative stress (Motoyama et al., 1998) and inflammation (Li et al., 2008). Interestingly, certain behavioral traits including type A personality, panic disorder, and severe anxiety, have been described to be associated with coronary artery vasospasm (Rasmussen et al., 1986; Vidovich et al., 2009). However, no data are available regarding whether these clinical situations are associated with changes in endothelial NOS. Translation of these pre-clinical data to humans, and especially to patientswith these behavioral conditions is therefore well encouraged. The data provided in the paper of Qipshidze et al. (2010) are intriguing since they open theraupetic possibilities for patient with coronary vasospasms. Nitrates and calcium channel