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Drug discovery

A lifeline for suffocating tissues

Nature volume 453, pages 11941195 (26 June 2008) | Download Citation

When a blood vessel becomes blocked, the ideal treatment would be a drug that induces new vessel formation in the damaged tissue, without affecting healthy tissues. With the chemical nitrite, we might be on to a winner.

Ischaemia occurs, for instance, when a blood vessel becomes occluded by a clot. It affects hundreds of millions of people worldwide, and is often life-threatening. So there is an urgent need for molecular factors that could stimulate new vessel growth (angiogenesis) and so promote revascularization of ischaemic tissues. Progress so far has not been particularly noteworthy, and one major problem is that some potential angiogenic factors promote vascularization in healthy tissues as well as in the ischaemic tissue, causing undesirable side effects. Writing in Proceedings of the National Academy of Sciences, Kumar et al.1 revive hope that a drug might one day become available. They report that systemic administration of nitrite (NO2), which is reduced to nitric oxide (NO), for protracted periods restores blood flow in ischaemic tissue without stimulating angiogenesis in healthy tissues.

The idea of therapeutic angiogenesis is not new. Over the past decade, considerable efforts have gone into assessing whether the administration of angiogenic factors, such as growth factors, signalling molecules and gene transcription factors, would improve revascularization. But despite initial promise, these molecules have mostly failed in clinical trials, chilling the enthusiasm for the feasibility of therapeutic angiogenesis. Reasons for the disappointing clinical results include technical difficulties in efficiently administering the angiogenic factor locally to the ischaemic tissue over sufficiently long periods of time; the short metabolic half-life of these factors; and the requirement for multiple angiogenic factors to build a mature, stable, functional vasculature2,3. Moreover, the potential risk of side effects, such as deregulation of blood pressure and stimulation of dormant tumour growth, has precluded systemic administration of angiogenic factors, especially those that act indiscriminately on vessels in healthy and ischaemic tissues.

A large body of evidence4,5 implicates NO — which was initially discovered as a factor that relaxes blood vessels — in stimulating angiogenesis. This signalling molecule increases the expression of various angiogenic factors, including VEGF, which, together with other mediators, increases NO levels through positive feedback. As well as stimulating the growth of nascent and immature vessels consisting only of fragile endothelial cells, NO recruits perivascular mural cells, which stabilize vessels, allowing them to become fully functioning conduits. Moreover, NO improves blood perfusion by inducing vessel dilation and possibly by enhancing the formation of collateral vessels, which supply the bulk of the flow to ischaemic tissues.

Another attractive property of NO is that it can protect tissues against ischaemic damage by slowing down cellular respiration. It does this at least in part through the nitrosylation of complex I proteins in the electron-transfer chain. This reduces the production of toxic reactive-oxygen species, which often occurs under ischaemic conditions6. What's more, at high NO levels, which may occur in inflamed tissues, nitrosylation inactivates oxygen sensors of the PHD family of proteins7. Inhibition of the PHD1 sensor protects tissues against ischaemic damage by reprogramming the cell's metabolism and reducing oxidative stress8. Both of these nitrosylating activities of NO therefore induce 'ischaemic tolerance'. So it is not surprising that NO has been considered a candidate for the revascularization and protection of ischaemic tissues.

But the therapeutic potential of NO is both context dependent and dose dependent. Despite its beneficial actions, it could have toxic effects at high concentrations. Indeed, in addition to enhancing programmed cell death and decreasing cell proliferation, NO inactivates the oxygen carrier haemoglobin and inhibits the cytochrome c oxidase enzyme, thus impairing cellular respiration. Moreover, under certain conditions, it stimulates oxygen sensors and promotes the formation of reactive-oxygen species. Finally, at least when given systemically as an organic nitrate compound, NO acts indiscriminately in diseased and healthy tissues.

The duality of NO activity could explain why, paradoxically, both molecules that produce NO and inhibitors of the enzyme NO synthase, which catalyses NO production, protect cells against ischaemic injury5. And it raises questions about the 'safe window' of NO levels for therapy. So why did Kumar et al.1 consider using nitrite as an angiogenic factor? In their study, the authors took into account the fact5,9 that nitrite is reduced to NO under ischaemic conditions, whereas in well-oxygenated tissues it is oxidized to apparently harmless nitrate (NO3; Fig. 1). In other words, nitrite acts as a site-selective 'pro-drug' by preferentially generating NO in ischaemic tissues, where it stimulates revascularization and cell protection, while avoiding potentially harmful NO generation in healthy tissues.

Figure 1: Targeted effect.
Figure 1

Kumar et al.1 find that, when administered systemically in mice, (a) nitrite (NO2) is converted to nitrate (NO3) in tissues that contain normal perfused blood vessels. b, In ischaemic tissues, by contrast, nitrite is selectively converted to nitric oxide (NO), where it stimulates revascularization, vasodilation and ischaemic tolerance.

Such a safety profile would overcome the difficulty of administering an angiogenic drug locally at the ischaemic site, and instead might allow systemic — even oral — delivery over prolonged periods. Kumar et al. show that, in mice, sodium nitrite promotes revascularization of ischaemic tissues by stimulating the formation of mature, perfused vessels, whereas treatment with a NO scavenger abolishes this effect, thus supporting the idea that conversion of nitrite to NO underlies its beneficial effect in ischaemia.

Nonetheless, as is often the case, intriguing questions remain. Reactive-oxygen species that form during ischaemia interact with nitrite to generate highly reactive peroxynitrite, which can damage DNA and proteins4,9. Will such damage caused by oxidative stress occur with long-term nitrite treatment? Also, some of the beneficial effects of NO are mediated by its acute vasodilatory effects. Will the benefits of nitrite therapy persist after its withdrawal?

Kumar et al. suggest that nitrite accumulation in ischaemic muscle stimulates revascularization in a NO-dependent manner three days after the start of ischaemia; but they also observe that products of NO metabolism are detectable only after seven days. These puzzling observations remain to be reconciled. Moreover, the extent to which the protection nitrite offers against ischaemic tissue damage relates to ischaemic tolerance rather than revascularization remains to be explored.

Another recent study10 documents cardioprotective effects of nitrite just one day after reperfusion of ischaemic heart tissue — too short a time-frame for angiogenesis to occur after permanent blockage of an artery. If it is ischaemic tolerance that precedes revascularization, how rapidly can nitrite induce it? Also, unlike chronic ischaemia, a severe acute ischaemic event is characterized by rapid cell death, and nitrite-mediated ischaemic tolerance might not suffice as a beneficial adaptation. So, will starting treatment after the acute event still be effective?

Finally, the dual activities of NO warrant a careful choice of the nitrite dose administered, ensuring that the optimal therapeutic dose of this drug won't cause a drop in systemic blood pressure. NO inhalation could be a promising delivery route that might induce less fluctuation in blood pressure. Furthermore, this delivery route selectively increases NO in ischaemic tissues, with cytoprotective results11. Kumar and colleagues' promising findings1 certainly warrant further study of the therapeutic potential of this molecule.

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  1. Massimiliano Mazzone and Peter Carmeliet are in the Vesalius Research Center, University of Leuven, Flanders, Institute for Biotechnology (VIB), B-3000 Leuven, Belgium.  peter.carmeliet@med.kuleuven.be

    • Massimiliano Mazzone
    •  & Peter Carmeliet

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