Leukotrienes are potent pro-inflammatory lipid mediators and over the past years several studies have implicated LTs, in particular LTB4
, in the inflammatory component underlying vascular inflammation, atherosclerosis, and atherothrombosis 
. The role of LTs in diseases related to the myocardium, however, remains unclear. Thus, in previous studies with preclinical models, hearts of otherwise healthy animals (rodents and dogs) have been subjected to ischemic damage and effects of genetic or pharmacological intervention of the leukotriene pathway have been studied with variable effects 
. In recent elegant studies, the second major receptor for cys-LTs, viz. CysLT2 has been implicated in vascular responses to cys-LT and ischemia-reperfusion injury 
. Here, a transgenic model was employed with artificial overexpression of CysLT2 in endothelial cells and, as in most previous investigations, the potential impact of pre-existing vascular disease or atherosclerosis was not addressed. Furthermore, is important to distinguish between models of ischemia/reperfusion with infarction and models of hypoxia without infarction. Accordingly, although studies have addressed the potential role of LTs in the setting of myocardial infarction and ischemia-reperfusion injury with influx of inflammatory cells (for review see 
) understanding of the coupling between a hypoxic myocardial environment and induction of LT signaling is lacking. In particular, it is unclear whether and/or which of the molecular components of the LT system is expressed in the myocardium and if production of LTs occur. In addition, it is not known whether endogenous LT signaling elicits adverse effects in response to bouts of hypoxia. We therefore constructed an experimental system to test the hypothesis that cys-LT signaling may compromise the myocardial oxygen supply in chronic ischemic heart disease during bouts of hypoxia.
In the present study we chose the Apoe−/−
mouse as a model of atherosclerotic heart disease and assessed the gene expression profile of the LT cascade. The Apoe−/−
mouse develops hypercholesterolemia and extensive atherosclerotic lesions throughout the arterial tree as well as coronary atherosclerosis 
. In addition, this mouse model exhibits cardiac hypertrophy and interstitial remodeling 
, findings that we were able to reproduce in this investigation (). We found that in the Apoe−/−
heart, levels of LTC4
S and CysLT1 were specifically and significantly upregulated as compared to control C57BL/6J mice. Since LTC4
S catalyzes the committed step in cys-LT formation and the CysLT1 receptor is known to mediate classical pro-inflammatory actions of cys-LT 
, this expression profile is consistent with increased cys-LT signaling in the heart in atherosclerotic disease.
Previous clinical investigations have demonstrated increased levels of urinary LTE4
, a stable and bioactive metabolite of LTC4
, in patients with sleep apnea 
and acute coronary syndromes 
. Moreover, treatment with a FLAP inhibitor suppressed biomarkers of infarction in a clinical trial of patients with a genetic variant of FLAP that confers increased risk of myocardial infarction 
. However, these studies have measured systemic levels of LTs and none of them have presented direct evidence for a localized myocardial production of these mediators.
Furthermore, it is not previously known whether an acute hypoxic stimulus increases the expression of genes involved in LT production and signaling, which in turn would point to a possible link between hypoxia and inflammation in the heart. It was therefore of interest to assess the effects of a bout of hypoxic stress on LT-pathway gene expression. In our model, there was a pronounced increase in the expression of LTC4S and CysLT1 in response to hypoxic stress. In addition, we could detect a tendency towards increased expression of 5-LO, the upstream enzyme in LT biosynthesis. One could argue that increased expression of cys-LT system components are due to an increased influx of inflammatory cells in chronic hypoxic myocardium or following hypoxic stress. Although a contribution from recruited leukocytes cannot be entirely excluded it does not appear a likely explanation to our data, because we had no evidence of myocardial infarction by histology or troponin measurements and we counted inflammatory cells and found no differences in numbers of CD68+ cells.
CysLT1 protein was localized by immunohistochemistry to vascular (possibly endothelial) cells in heart tissue of Apoe−/− mice. In the absence of a specific antiserum for LTC4S, we used a biochemical approach to demonstrate the presence of active enzyme in the mouse myocardium. Thus, incubation of heart membrane preparations with intact LTA4, followed by analysis with HPLC coupled to EIA, revealed significant formation of LTC4, which increased in heart tissue from Apoe−/− mice, particularly following hypoxic stress. Because there is only one membrane enzyme with significant ability to conjugate LTA4 with GSH to form LTC4, i.e., LTC4S, we conclude that this enzyme is up-regulated in hypoxic heart tissue from Apoe−/− mice.
To test whether pharmacological interruption of the cys-LT signaling pathway could have an impact on the hypoxic load of the myocardium, Apoe−/−
mice were subjected to hypoxia in the presence or absence of montelukast, a selective antagonist of CysLT1. Administration of this drug reduced the hypoxic load of the myocardium to levels equal to those observed in hearts of Apoe−/−
mice under normoxic conditions. Since expression of LTC4S was also increased in the heart of Apoe−/−
mice, an alternative pharmacological approach to block cys-LT signaling could be inhibition of LTC4
. This strategy may in fact be even more effective since recent data have shown that there are multiple, cross-talking, and cross-regulatory receptors for cys-LT 
. However, to date no selective LTC4
S inhibitors have been developed. The choice of the classical CysLT1 antagonist montelukast in our study was guided by the selective upregulation of CysLT1 and the fact that montelukast is a widely prescribed asthma drug. Since both the endogenous ligands and the synthetic antagonists are known to display off-target actions, it had been desirable to study the effects of, e.g. modulatory GPR17 signaling, a selective LTC4S inhibitor, or additional CysLT1 antagonists. Due to the lack of sufficiently selective pharmacological tools and extended time needed for this mouse model, we focused on montelukast.
In studies of the pathological role of LTs, animal disease models have been of variable value. For instance, few, if any, mouse models reproduce human LT-driven asthma and for atherosclerosis, the Apoe−/−
model has provided ambiguous results 
. Therefore, it was of particular interest to compare our data from the Apoe−/−
model with human tissues. Needless to say, samples of human tissues that perfectly match the mouse model, or vice versa, are not available. However, we used a unique collection of human heart biopsies from both ischemic and non-ischemic parts of the same heart and could demonstrate similar changes in the expression levels of LTC4
S and CysLT1 as those observed in the Apoe−/−
model (). These data suggest that the chronic ischemic human heart is also susceptible to increased cys-LT signaling and that blocking of cys-LT signaling may exert similar effects in human heart tissue under hypoxic stress. In this context it is interesting to note that a recent report demonstrates that males using montelukast have a significantly reduced risk of MI, supporting the clinical relevance of our work 
In conclusion, we have demonstrated that LTC4S and CysLT1 receptor gene expression is up-regulated in the heart and can be further enhanced by bouts of hypoxic stress in a mouse model of atherosclerotic heart disease. Blocking cys-LT signaling by montelukast abolishes the aggravated hypoxic load in response to hypoxia indicating that cys-LTs compromise oxygen supply, presumably via actions on the myocardial microcirculation. We also demonstrate a highly similar gene-expression pattern in the chronic ischemic human heart suggesting that similar mechanisms are operating in humans. These findings link myocardial hypoxia to inflammatory cys-LT signaling, vasoconstriction and acute hypoxic events, and prompts for testing of anti-leukotriene drugs in humans at risk for decreased blood oxygen content (e.g. sleep apnea) and with obstructive coronary artery disease.