Preserving vessel function during ischemic disease: new possibilities of inorganic nitrite therapy

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in the USA and throughout the industrialized world. Moreover, the incidence of CVD is on the rise globally and continues to place a significant economic and sociological burden on humanity as a whole. There is no doubt that continued research into the pathophysiological mechanisms of CVD has made excellent strides in clinical diagnosis and management. However, significant limitations still exist with several aspects of CVD pathology, namely our ability to protect and restore tissue function in response to acute or chronic ischemic events. Acute or chronic vessel occlusion contributes to nearly all forms of CVD and the ability to thwart these events along with restoration of ‘healthy’ vascular function is still lacking. What is more, a common underlying feature of cardiovascular disease is defective bioavailability of nitric oxide (NO), which has broad physiological roles ranging from regulation of vascular reactivity and growth, inflammation and thrombosis. Indeed, effective NO therapy has been considered by many to be the ‘holy grail’ for CVD intervention, yet realization of this goal has remained elusive. Recent work from our laboratory and others has begun to identify straightforward yet novel approaches for selective NO therapy that involve chemical reduction of the inorganic nitrite anion (NO₂^-) back into bioavailable NO. While this may be a seemingly simplistic approach, inorganic nitrite therapy may hold significant potential as an effective and selective NO donor for acute and chronic ischemia secondary to cardiovascular disease.

Acute & chronic ischemic cardiovascular disease

The insidious nature of atherosclerotic vessel disease is a common underlying mediator of multiple aspects of CVD. Specifically, vascular atherosclerotic lesions may progress to frank rupture, typically resulting in thrombosis and acute tissue ischemia (e.g., myocardial infarction or stroke) or lesions may continually evolve without thrombosis and compromise luminal diameter, vascular reactivity and subsequent blood flow, resulting in chronic tissue ischemia, such as peripheral artery disease (PAD). The time course of the ischemic event clearly impacts tissue function, with acute events typically being more deleterious than chronic ones. However, the primary clinical challenge for ischemic disorders is to minimize tissue damage and restore vascular function and blood flow to the affected area as quickly as possible. Significant progress has been made with regard to prevention of ischemic disorders through pharmacological regulation of cholesterol levels and hypertension, along with lifestyle modifications aimed at reducing risk factors such as smoking and tight control of diabetes. Clinical management of both acute and chronic ischemic disorders is evolving, with therapeutic strategies focusing on pharmacological regulation of lipid levels (statins), regulating metabolic demand of affected tissue coupled with maintenance of tissue perfusion (antidiabetic and antihypertension agents), as well as antiplatelet therapy (clopidogrel and cilostazol). However, the overall efficacy of these agents for chronic ischemia disorders, such as PAD, intermittent claudication and critical limb ischemic disease, remains low, highlighting the need for additional therapeutic approaches.

Several experimental studies have suggested that stimulation of therapeutic angiogenesis and arteriogenesis may be very beneficial for chronic ischemic tissue disorders, thereby re-establishing tissue perfusion and preventing further tissue damage [1,2]. Many different agents, including growth factors, autocoids, endocrine hormones, transcription factors and others, have been reported to stimulate vascular growth; however, few of these agents have been successful in large multicenter, randomized, double-blind, controlled clinical trials for PAD [1,2]. This may be due to the relatively narrow target effect of such agents as they typically do not affect a multiplicity of factors, such as defects in vascular reactivity, enhanced thrombogenic and inflammatory activity, and increased serum lipids. However, the gaseous free radical NO holds significant promise for ischemic tissue therapy as the biological effects of this molecule involve anti-inflammatory, antithrombogenic, proangiogenic, proarteriogenic and enhanced cytoprotection features [3,4]. Unfortunately, studies have shown that the clinical utility of gaseous NO or chemical NO donors may be limited by systemic pressor effects, potential cytotoxic effects owing to a lack of tissue specificity and difficulties in drug delivery [5,6]. In spite of all of these issues, hope may still exist for meaningful NO therapy in clinical settings. Recent work from many laboratories demonstrates that nitrite, the metabolic by-product of NO oxidation, is a surprisingly effective NO donor that may bypass many of the current problems with NO therapy for ischemic diseases.

Nitric oxide metabolism in health & disease

It is now well accepted that NO plays a central and critical role in maintaining cardiovascular health and that its bioavailability is reduced during cardiovascular disease [7–9]. The primary enzyme responsible for NO production in the cardiovascular system is endothelial nitric oxide synthase (eNOS), which is regulated by numerous molecules and signaling pathways [10,11]. Importantly, eNOS activity is also largely responsible for systemic NO production, as the amount of enzyme expression is often directly proportional to NO metabolite levels [12,13]. NO readily diffuses across lipid bilayers and its biological fate is dictated predominately by reactions with metalloproteins and other free radical species; the classic example being activation of the heme enzyme soluble guanylate cyclase, which initiates a signal cascade leading to vessel dilation and platelet inhibition [14]. In addition, NO may also be oxidized through various mechanisms resulting in the formation of nitrite that can be further oxidized to nitrate (NO₃^-) [14,15]. Traditionally, nitrite and nitrate have been viewed as inert biological end products of NO metabolism [12]. However, new studies are directly challenging this notion and clearly suggest that both nitrite and nitrate may have important biological functions in regulating production of NO from NOS-independent pathways.

Previous opinions regarding the biological effects of nitrite were grim due to their potential to form carcinogenic nitrosoamines, which has led to tight regulations for human consumption, even though unequivocal data supporting a role for nitrite in human carcinogenesis are not available [16]. These concerns aside, substantial levels of nitrite and nitrate are found in green leafy vegetables present in diets that are more usually associated with cardiovascular health, and are used as food preservatives [17,18]. Moreover, recent elegant reviews have addressed the historical importance of inorganic nitrite and nitrate in medicine and biology, which show that these compounds may have previously been useful for disease conditions of the cardiovascular system in antiquity [19,20].

Today, there is a newfound appreciation for various metabolic pathways and uses of inorganic nitrate and nitrite that were previously unknown. Studies within the past few years have revealed that inorganic nitrite can undergo a one-electron reduction back to NO through various mechanisms, including but not limited to, deoxyhemoglobin, deoxymyoglobin, xanthine oxidoreductase, eNOS, acidic disproportionation and members of the mitochondrial electron transport chain [21–25]. The ability of nitrite to be reduced back to NO classifes it as a unique NO donor under permissive biological conditions such as tissue ischemia that entails many of these potential reducing agents. Importantly, nitrite reduction to NO by these factors selectively occurs only in tissue compartments that are ischemic, thereby resulting in targeted NO production where it is most needed. Therefore, inorganic nitrite treatment during tissue ischemia represents a novel type of NO donor therapy that, so far, has no discernable effect on normoxic tissue.

Beneficial effects of nitrite therapy

The recent appreciation for biological mechanisms that result in NO formation from nitrite have led to numerous studies showing that inorganic nitrite affects several biological processes through a NO-dependent pathway. The majority of investigations have centered on the ability of deoxyhemoglobin to mediate a one-electron reduction of nitrite back to NO, which mediates vasodilation and increased tissue blood flow. Cosby et al. demonstrated that, near physiological concentrations of sodium nitrite, human forearm blood flow was significantly increased as measured by wire plethysmography during exercise [26]. Consistent with these results, Crawford et al. reported that deoxyhemoglobin increases nitrite-mediated relaxation of isolated vessel ring segments through a NO-dependent pathway involving increased soluble guanylate cyclase activation and cGMP formation [27]. Importantly, these and other studies have shown that progressive oxygen desaturation of hemoglobin, as seen in the arterial to venous gradient of blood flow, results in increased nitrite reduction to NO [28,29]. Data from our laboratory also demonstrate that during profound tissue ischemia, very low doses of nitrite specifically increase ischemic tissue blood flow through an NO-dependent manner [30]. Other studies have also shown inorganic nitrite therapy to enhance tissue blood flow in other ischemic disorders, such as sickle cell disease, hypoxic pulmonary hypertension, and subarachnoid hemorrhage-induced cerebral vasospasm [31–33]. Despite the evidence mentioned earlier and other recent studies regarding nitrite-dependent vasodilation, it is still not exactly clear how much deoxyhemoglobin-mediated nitrite reduction to NO contributes to hypoxic vasodilation in vivo compared with other known hypoxic vasodilatory mechanisms [34]. This issue aside, it is abundantly clear that nitrite reduction by deoxyhemoglobin represents a potent salvage pathway for bioavailable NO during pathological and physiological states of tissue ischemia.

Nitrite therapy has also been studied in several experimental models of acute ischemia–reperfusion (I–R) injury. Seminal work by Duranski et al. showed that low-dose sodium nitrite therapy was highly effective in preventing I–R tissue injury in both the murine heart and liver, and identified the importance of NO formation in mediating cytoprotection [35]. The beneficial effect of nitrite therapy for myocardial I–R injury has also been corroborated in a canine model, which revealed that nitrite dosing as late as 5 mins before reperfusion still confers significant tissue protection [36]. Conversely, the beneficial effects of nitrite therapy for I–R injury may not be as clear in other organs. Jung et al. have shown that sodium nitrite therapy is protective in an experimental model of stroke involving middle cerebral artery occlusion in mice [37]. However, Schatlo et al. reported that nitrite adjuvant therapy coupled with thrombolysis did not confer additional protection in a middle cerebral artery occlusion delayed reperfusion model in rats. This may have been due to differences in duration of tissue ischemia, the nitrite dosing regimen, species and age of animals, and the coadministration of recombinant tissue plasminogen activator [38]. A similar issue also exists regarding whether nitrite therapy is protective in experimental models of renal I–R injury. Basireddy et al. reported that sodium nitrite therapy was not protective in left renal I–R injury in rats; conversely, Tripatara et al. showed that nitrite therapy confers protection in bilateral renal I–R in rats [39,40]. Interestingly, whereas both studies showed that intravenous or intraperitoneal administration of nitrite failed to confer protection, direct topical application of nitrite to the kidney did mediate protection, demonstrating that different therapeutic regimens may need to be employed to evaluate nitrite therapy in different organ systems. Together, these reports show that low-dose sodium nitrite therapy is protective against acute I–R injury, but more work is needed to precisely understand the pathological conditions where NO therapy confers the greatest benefit, the metabolic fate of administered nitrite and the protective mechanisms involved with nitrite therapy.

Whereas most of the studies mentioned earlier assessed nitrite therapy in an acute ischemic setting, no information exists on the possibility of nitrite therapy for chronic ischemic diseases, which characterizes PAD. To address this, our laboratory used a murine model of critical hind limb ischemia by permanent femoral artery occlusion and observed that continuous low-dose sodium nitrite therapy was highly effective in re-establishing ischemic tissue blood flow by significantly increasing both angiogenesis and arteriogenesis in the ischemic hind limb [30]. Interestingly, lower nanomolar doses (165 µg/kg) of sodium nitrite were much more therapeutically effective than higher micromolar doses (3.3 mg/kg), and repeated nitrite administration did not alter nonischemic tissue perfusion or vascular growth. These findings are noteworthy as they demonstrate that effective nitrite doses are far below maximal recommended US FDA levels and that continuous nitrite therapy provides selective benefit only to the tissues in jeopardy. Moreover, results from our work are in agreement with Dejam et al. who have reported that continuous inorganic nitrite therapy does not induce tolerance (unlike organic nitrites) or have adverse effects; however, micromolar nitrite concentrations can decrease mean arterial blood pressure, which will require close scrutiny during clinical trials [41]. Results from our study also revealed that sodium nitrite therapy selectively stimulates endothelial cell proliferation in ischemic tissues in a NO-dependent manner that does not involve alteration of tissue cGMP levels. These data are surprising as nitrite therapy has been shown to increase vascular cGMP in single-bolus dose studies [42]. These differences are probably due to the doses of nitrite used, the duration of ischemia and subsequent metabolic changes of the tissues under prolonged oxygen deprivation, which all require further examination. Last, our study also revealed interesting differences in nitrite metabolism, as tissue nitrite levels were quickly elevated within 3 days only in ischemic tissues, but significantly declined by day 7, while tissue levels of nitrosothiols and iron-nitrosyl proteins were only elevated at day 7. The reason for these differences is presently unclear; however, given the fact that nitroso-modified species often represent a NO storage reservoir, it is possible that greater metabolic demand for NO exists early during chronic ischemia, but diminishes over time owing to restoration of tissue perfusion, thereby allowing restoration of tissue NO reserves [43]. Studies are underway to address these and other questions, including whether nitrite therapy facilitates chronic ischemic tolerance. Nonetheless, we and others agree that inorganic nitrite therapy may hold significant promise in effectively treating chronic ischemia disorders, such as PAD, critical limb ischemia and vasculitis, by stimulating therapeutic angiogenesis and arteriogenesis as well as conferring ischemic tissue cytoprotection [44].

Future considerations

As with any new therapeutic approach, one must consider possible downsides to such great potential. The possible side effect of methemoglobinemia has not been problematic in human and nonhuman primate studies thus far; however, continued monitoring of this and changes in blood pressure will be required during clinical trials, as discussed previously [41]. It is quite likely that hemodynamic issues will be minimal as low nanomolar doses of sodium nitrite, which robustly increase ischemic tissue vascular growth and collateral vessel perfusion, do not significantly alter mean arterial pressure, thus affording a wide therapeutic dosing window. Another possible concern with sodium nitrite for therapeutic angiogenesis is the possibility of enhanced tumorigenesis or malignancy. This has been a universal worry for all agents (VEGF, bFGF and others) previously used to induce therapeutic angiogenesis. However, no evidence of increased malignancy has presented during therapeutic angiogenesis trials for PAD or critical limb ischemic disease [1,45]. In fact, one could envisage that sodium nitrite therapy may be useful in preventing tumor progression as NO can be directly cytotoxic to tumor cells and might also normalize tumor blood flow, thereby enhancing chemotherapy or radiotherapy efficacy. Last, micromolar concentrations of inorganic nitrite and nitrate could alter steroid hormone synthesis in vitro and in vivo; however, the effective nitrite doses useful for angiogenic and arteriogenic stimulation in the hind limb ischemia model are in the low nanomolar range [30]. Additional studies will be needed to better understand the full ramifications of clinical inorganic nitrite therapy.

Another important consideration is the route of administration for effective clinical use of inorganic nitrite. Many studies, including ours, have either employed injections of sodium nitrite directly into the peritoneal cavity where it is rapidly absorbed into the mesenteric circulation or have utilized direct intravenous injection. While these approaches are effective, an easier route of administration is clearly desirable. Recent studies have shown that dietary nitrite is an effective means by which to increase circulating nitrite levels that affords protection against acute myocardial I–R injury in mice [46,47]. Thus it appears that orally available sodium nitrite dosing is possible; however, additional work is necessary to determine whether this route of administration is beneficial for all the disease conditions discussed previously.

In summary, inorganic nitrite therapy represents a potent and novel treatment modality for ischemic tissue disorders due to its selective reduction to NO that activates several endogenous protective pathways. It is possible that low dose nitrite may be the long awaited ‘ideal’ NO donor for cardiovascular disease while also reinforcing the importance of eating your vegetables.

Financial & competing interests disclosure

The authors are inventors of pending patent applications related to the use of nitrite salts for cardiovascular and ischemic tissue injury conditions. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.