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Nicotinamide is an essential nutrient, a precursor of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate, known to reverse the symptoms of pellagra (Pellagra Preventive vitamin—vitamin PP, also known as vitamin B3). More recently a number of other biological activities of nicotinamide have been described, including its cytoprotective effects on neural and vascular tissues (Chong et al., 2002; Maiese and Chong, 2003) as well as its anti-inflammatory activity (Ungerstedt et al., 2003).
Nicotinamide is metabolized in the liver by cytochrome P450 to nicotinamide-N-oxide (N-Ox) but mainly by nicotinamide N-methyltransferase (NNMT) to 1-methylnicotinamide (MNA) that is further metabolized to 1-methyl-2-pyridone-5-carboxamide (2-PYR) or 1-methyl-4-pyridone-5-carboxamide by aldehyde oxidase (Aoyama et al., 2000) (Figure 1). Despite the early discovery of nicotinamide and its metabolites, NNMT, a major liver enzyme in nicotinamide metabolism, has only recently been genetically characterized in mouse and human (Yan et al., 1999).
Over the years, nicotinamide metabolites have been frequently measured in rodents and humans. The level of MNA in plasma and urine was analysed initially to diagnose niacin deficiency (Vivian et al., 1958) and then to monitor renal tubular excretion (Maiza et al., 1992) or peroxisome proliferation in the liver (Delaney et al., 2005). An increased concentration of urine MNA was found for example in patients with Parkinson's disease (Aoyama et al., 2000) or liver cirrhosis (Pumpo et al., 2001). In all these studies, MNA was considered as an inactive biomarker. Other nicotinamide metabolites were also considered as inactive.
In light of the above, it was quite surprising that MNA applied topically proved to be effective in alleviating the inflammatory responses of various skin diseases (Gebicki et al., 2003; Wozniacka et al., 2005). These pilot studies provided the first evidence for the biological activity of MNA in vivo, although the mechanism of MNA activity was not defined. The present study was undertaken to characterize the profile of cardiovascular action of MNA and its in vivo anti-thrombotic activity, relative to those of structurally related compounds. We have demonstrated that MNA is a unique anti-thrombotic agent as it limits platelet-dependent experimental thrombosis by a mechanism dependent on prostacyclin (PGI2) synthesized by the inducible isoform of cyclooxygenase (COX-2).
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In the present work, we showed that MNA exhibited anti-thrombotic activity in vivo that was mediated by PGI2 derived from vascular COX-2, while it was devoid of direct antiplatelet and vasodilator activity.
The anti-thrombotic effects of MNA were analysed in two complementary experimental models of platelet-dependent thrombosis. The first one involved extracorporeal thrombus formation, in which aggregates of platelets, formed on the collagen strip superfused with blood were dissipated by MNA. This response was considered as a thrombolytic response. The second one concerned intravascular thrombus formation in response to vascular injury, whereby MNA prevented the thrombus formation. Both models have been well-characterized in previous studies (Gryglewski et al., 2001; Wojewodzka-Zelezniakowicz et al., 2006). Importantly, in both models of arterial thrombosis platelets make a major contribution to the development of thrombosis, as the inhibition of platelet activation by ASA or stimulation of endothelial release of PGI2 by angiotensin-converting enzyme inhibitors afforded pronounced anti-thrombotic effects (Schumacher et al., 1993; Gryglewski et al., 2001; Wojewodzka-Zelezniakowicz et al., 2006). In contrast, in venous thrombosis induced by the occlusion of the vena cava, platelets play a minor role and the stimulation of endothelial release of NO, rather then that of PGI2, seems to provide the pronounced anti-thrombotic effect (Cylwik et al., 2004). Thus, our present findings that MNA with its PGI2 releasing properties, while ineffective in venous thrombosis, displays the ability to limit platelet-dependent thrombosis is fully consistent with the nature of these experimental models and highlights the specificity of MNA and PGI2-triggered mechanisms towards arterial, platelet-dependent, thrombosis. It would be still worthwhile to examine MNA activity in a different animal model of platelet-dependent thrombosis, sensitive to agents that modulate the COX pathways (Hennan et al., 2002) to confirm our conclusion.
We provide the following evidence for the involvement of the COX-2/PGI2 pathway in the MNA-induced response. In vitro, MNA (up to high millimolar concentrations) did not affect platelet aggregation—hence mechanisms of the anti-thrombotic action of MNA could not be explained by a direct antiplatelet action of MNA. On the other hand, the thrombolytic response to MNA was associated with a release of PGI2 (assayed as 6-keto-PGF1α) into arterial plasma, while neither TXB2 nor PGE2 levels changed in response to MNA. Moreover, in the presence of a non-selective COX inhibitor (indomethacin) or a selective COX-2 inhibitor (rofecoxib) the thrombolytic response to MNA and concomitant 6-keto-PGF1α release were abolished. Also in the arterial thrombosis model, the MNA-induced effect was markedly inhibited in the presence of indomethacin or rofecoxib, supporting our conclusion that the anti-thrombotic activity of MNA in vivo in both experimental systems was mediated by PGI2 formed via COX-2.
Although in vitro, PGI2 and NO seem to be released from the endothelium in a coupled manner (Gryglewski et al., 1986), PGI2-dependent thrombolysis induced by MNA in vivo, was not associated with NO-dependent hypotension. Indeed MNA did not change either mean blood pressure or carotid blood flow when applied intravenously. Moreover, the NOS inhibitor L-NAME did not modify the MNA-induced thrombolytic response. In line with this result, in aortic rings or mesenteric arteries in vitro, MNA was devoid of NO-dependent vasorelaxant activity. Accordingly, MNA appears to be a relatively selective releaser of vascular PGI2 from COX-2 in vivo.
It was quite surprising that, among the various structurally modified MNA analogues, we have not found a compound with better thrombolytic activity than MNA itself. Only the replacement of the amide group at the three-position of the pyridine ring by an acetyl group, as in the case of MAP, resulted in the retention of sustained dose-dependent thrombolysis (3–30 mg kg−1) with a concomitant rise in 6-keto-PGF1α in blood. Other tested compounds were inactive or much weaker thrombolytic agents. For example, 6-amino nicotinamide—an inhibitor of pentose phosphate pathway (PPP) (Gupte et al., 2003) and 1-ribosylnicotinamide—a newly discovered precursor of NAD+ (Bieganowski and Brenner, 2004)—were virtually inactive as thrombolytics. Thus the mechanism of MNA-induced thrombolysis would seem independent of PPP activity or of intracellular NAD+. Nicotinic acid and trigonelline were also ineffective thrombolytically, even though nicotinic acid, at very high doses, has previously been shown to induce a remarkable thrombolytic response in cats that was attributed to the release of PGI2 (Swies and Dabrowski, 1984). In turn, the nicotinamide-induced response was substantially weaker than that of MNA. The observed weak anti-thrombotic activity of nicotinamide may be explained by the fact that only a minor part of nicotinamide is transformed to MNA by liver nicotinamide-N-methyltransferase upon direct intravascular administration, while the different magnitude of the response to nicotinic acid in cats vs rats may underline species differences known to mark the selective biological response to nicotinic acid (Declercq et al., 2005).
It is of note that in patients with peripheral artery disease the drugs with the nicotinic acid moiety such as β-pyridylcarbinol (Ronicol) or xanthinol nicotinate (Sadamin) exert their antiplatelet actions through the release of endothelial PGI2 (Dembinska-Kiec et al., 1983; Bieron et al., 1998), while nicotinic acid-induced flushing is mediated by a stimulation of the GPR109A receptor and the subsequent release of PGD2 and PGE2 from COX-1 (Benyo et al., 2005; Pike, 2005). It remains to be tested whether the MNA-induced release of PGI2 from COX-2 involves this vascular nicotinic acid-like receptor or other mechanisms.
There is overwhelming evidence that COX-2 derived PGI2 affords vasculoprotective, cardioprotective and anti-atherogenic activity (Gryglewski, 1980; Dowd et al., 2001; Grosser et al., 2006). The biological importance of the vascular COX-2/PGI2 pathway has recently been emphasized by the increased risk of myocardial infarction and stroke reported in patients treated with selective COX-2 inhibitors (Grosser et al., 2006). It is clear today that the long-term use of drugs known to inhibit COX and subsequently to depress PGI2 production proved to be harmful, while pharmacological stimulation of PGI2in vivo with the use of MNA might be beneficial in vascular diseases. Indeed MNA, being a stable and non-toxic molecule, seems to be a good candidate for a drug to boost the endogenous COX-2/PGI2 pathway. So far, PGI2 or its stable analogues have been widely used in the treatment of pulmonary hypertension (Wise and Jones, 1996) and have been proven effective in cases of peripheral arterial disease (Gryglewski, 1980) or liver injury (Ohta et al., 2005). It will be important to test the therapeutic effectiveness of MNA.
It is important to note that in our experiments, MNA afforded anti-thrombotic action, not only in normotensive rats, but also in rats with renovascular hypertension (2K-1C hypertension). Hypertension is one of the most important risk factors of arterial thrombosis and its clinical consequences such as acute coronary syndrome or ischaemic stroke. Therefore studying thrombosis in hypertensive rats more closely resembles a clinically relevant situation. In various cardiovascular pathologies, including hypertension, endothelial dysfunction develops that is characterized by an impaired production of NO, an impairment of basal PGI2 production (Gryglewski, 1980; Frein et al., 2005) and a compensatory increase in PGI2 formation by COX-2 (FitzGerald et al., 2000). Our results suggest that in the setting of impaired NO-dependent function, the COX-2/PGI2 pathway is able to be stimulated pharmacologically with MNA. These findings have important therapeutic implications.
Finally, the demonstration of biological activity of MNA may bring a new understanding of the mechanism of the pharmacological activities of nicotinamide. Indeed, the anti-diabetic, neuroprotective (Satoh et al., 1999; Gosteli, 2005) as well as anti-inflammatory action of nicotinamide, at least in part, may be mediated by a MNA-COX-2/PGI2 pathway, as outlined here.
Summing up, we demonstrate here—to our knowledge for the first time—the novel biological activity of MNA in vivo that greatly surpasses that of closely related compounds. MNA appears as an anti-thrombotic agent that limits platelet-dependent experimental thrombosis by a mechanism dependent on the COX-2/PGI2 pathway. Although our study focused on exogenously applied MNA, our results could imply that endogenous MNA formed in the liver by nicotinamide N-methyltrasferase is an endogenous activator of the COX-2/PGI2 pathway and may play an important regulatory role in limiting thrombosis, as well as inflammatory processes in the cardiovascular system. Our findings of novel in vivo biological activity of MNA may have potentially important physiological, biochemical as well as therapeutic implications and warrant further studies.