Vascular endothelial cells synthesize vasodilators such as NO, prostacyclin, hydrogen peroxide, carbon monoxide and epoxyeicosatrienoic acids, as well as vasoconstrictors, like thromboxane A2, prostaglandin H2 and endothelin-1. These substances play key roles in the local regulation of vascular tone and BP. Our results indicate that Trp metabolism to Kyn via the action of IDO is a novel endothelium-dependent pathway contributing to vascular relaxation and the regulation of BP in systemic inflammation.
Several lines of evidence support our contention that IDO can contribute to BP regulation. First, IDO was expressed in endothelial cells of resistance vessels of mice hypotensive as a result of systemic inflammation, and in these mice inhibition of IDO using 1-Me-Trp restored normal BP. Second, 1-Me-Trp had no effect on BP in animals in the absence of inflammation, or in animals suffering from inflammation but that were deficient in IDO or IFNγ and hence did not express IDO19
. Third, administration of the IDO product, Kyn, decreased arterial BP in hypertensive rats, relaxed pre-constricted arteries and attenuated vessel constriction. Fourth, provision of the IDO substrate, Trp, to arteries expressing active IDO caused relaxation that was inhibited by 1-Me-Trp.
Our findings show that endothelial expression of IDO and the associated conversion of Trp to Kyn contribute to vascular tone regulation. Indeed, in the models of systemic inflammation used, IDO induction is predominantly in vascular endothelial cells, indicating that they represent a major source of IDO activity in vivo
. Circulating concentrations of key pro-inflammatory cytokines, including IFNγ, increase in systemic inflammation18
. IFNγ is a principal inducer of IDO in various cells including those of the blood vessel wall16
. Consistent with this report, we found that IFNγ treatment caused a time- and dose-dependent induction of IDO in endothelial cells of different species, and this was associated with the conversion of Trp to Kyn. Therefore, many inflammatorys condition might provide an environment in which IDO-mediated formation of Kyn contributes to the regulation of vascular tone.
Our findings that administered Kyn lowered BP in hypertensive rats and activated isolated sGC suggest that Kyn itself is the vasoactive metabolite of Trp. The levels of Kyn required to achieve these biological activities are in the high micromolar-to-low millimolar range. Such concentrations accumulate in the lumen of both IFNγ-treated porcine coronary arteries and brain blood vessels of mice infected with PbA26
, the latter calculated from the tissue Kyn concentration, based on the exclusive localization of IDO in endothelial cells14,15,19
, and the knowledge that endothelial cells account for ≤1% of brain mass15
. Nevertheless, we cannot exclude the possibility that a Kyn derivative or trace contaminant present in Kyn was responsible for some of the activities observed, although when examined by NMR and mass spectrometry, the commercial Kyn preparations used for the present studies were pure (data not shown). Being a heme ligand32
, we also considered the possibility that formate, a co-product of IDO action on Trp, contributes to vessel relaxation. In contrast to Kyn, however, formate (≤ 5 mM) did not relax pre-constricted aortic rings (not shown). Therefore, formate is not a participant in the regulation of vascular tone, similar to the Kyn metabolites, 3-hydroxykynurenine, kynurenic acid, 3-hydroxyanthranilate and quinolinic acid.
Hypotension is a common complication of sepsis and cerebral malaria in humans2,20
. We observed pharmacological inhibition of IDO to restore normal BP in endotoxemic and infected wild type mice, and genetic deletion of IDO to attenuate the drop in BP in mice challenged with LPS. Of potential clinical relevance, attenuation of hypotension was achieved with IDO inhibition commencing as late as 8 h after induction of endotoxemia. Also, a recent study reported pharmacological and genetic blockade of IDO to decrease mortality induced by LPS33
, although that study did not investigate a role of IDO in the regulation of BP. Notwithstanding this, however, hypotension was still observed in Ido-/-
mice, in both cerebral malaria and endotoxemia. This may reflect redundancy in the regulation of BP. For example, mice deficient in heme oxygenase-1 (that generates carbon monoxide) or soluble epoxide hydrolase (that inactivates epoxyeicosatrienoic acids) show normal basal BP34,35
. Interestingly however, both types of mice reveal an attenuated BP response when challenged with LPS35,36
, similar to the situation observed in the present study with Ido-/-
mice (). In the case of heme oxygenase-1-deficient mice, increased expression of endothelin-1 was suggested as a compensatory mechanism36
, whereas our studies (Supplementary Fig. 3
online) suggest an increase in the contribution of iNOS to hypotension seen in Ido-/-
mice. The latter appears to contrast with a recent report indicating decreased bioavailability of NO in PbA infection37
. However, that study37
examined blood-related parameters of NO bioavailability, whereas our measure of NO activity was limited to the vessel wall. Also, we observed an increased contribution only in the case of Ido-/-
mice, which were not studied by Gramaglia and co-workers37
. Compared with iNOS, the contribution of IDO to the drop in BP in endotoxemic mice is smaller (~30 versus ~10 mmHg, respectively). However, IDO may be relatively more important in humans, where iNOS plays a lesser role while IDO activity is more pronounced11
A common thread of NO-modulated processes in the vasculature is their dependence on cGMP production by the heme-containing sGC. Activation of sGC occurs when NO binds to heme. We provide direct evidence that Kyn can activate all facets of this pathway, resulting in blood vessel relaxation. Surprisingly, Kyn also activated oxidized/heme-free sGC, in sharp contrast to NO, which requires reduced heme to function. Although Kyn is not a strong activator of oxidized/heme-free sGC, it would be expected to cause biologically relevant increases in cGMP. Our findings reveal Kyn as the first natural substance capable of activating oxidized/heme-free sGC, raising the intriguing possibility that IDO-mediated conversion of Trp to Kyn may represent a previously unknown endogenous ‘back-up’ system contributing to vessel relaxation in situations where sGC becomes oxidized/heme-depleted and hence refractory to activation by NO. Oxidative stress associated with inflammation and vascular disease states leads to a form of sGC that is indistinguishable from the in vitro
oxidized/heme-free enzyme, such that the physiological existence of oxidized and finally heme-free sGC can no longer be rejected29
In conclusion, our findings provide novel insights into the regulation of BP that have potentially far-reaching implications. Our work provides a potential novel therapeutic strategy for the regulation of vascular tone in hypertension, through manipulating the tissue activity of IDO and/or concentration of Trp and Kyn. We hypothesize that Trp metabolism to Kyn could be important in systemic inflammation, perhaps particularly in conditions where NO bioactivity is impaired due to dysfunctional sGC.