Interpreting antioxidant responses to angiotensin AT1 receptor antagonists: pharmacology or chemistry?

The efficacy of targeting the renin–angiotensin system (RAS) in the treatment of hypertension is well defined and has been the subject of numerous clinical trials [1–3]. Recently, interest has been generated by the hypothesis that activation of vascular NAD(P)H oxidase by angiotensin II is important in the pathophysiology of hypertension [4–7]. There is also evidence that angiotensin II can activate reactive oxygen species (ROS) generation in cardiomyocytes [8,9]. In this issue of the journal, Guo et al. [10] report data that support this hypothesis and extends it to show that ROS, possibly generated by NAD(P)H oxidase, contributes to diastolic dysfunction in Dahl salt-sensitive (DSS) hypertensive rats. One of the important features of DSS hypertensive rats, as used by Guo et al. [10], is that circulating angiotensin II levels are not elevated in comparison to non-hypertensive controls [11]. In addition hypertension appears to be independent of the RAS because interventions such as angiotensin-converting enzyme inhibition or AT1 receptor antagonism are either ineffective [11–15] or, at best, partially effective [16,17] in controlling blood pressure. However, left-ventricular tissue levels of angiotensin II are significantly elevated in hypertensive DSS rats [16,18] and untreated rats fed a high-salt diet suffer from left-ventricular remodelling and eventual heart failure [13]. In their study, Guo et al. [10] compared the effects of the angiotensin II AT1 receptor antagonist, candesartan cilexetil, with the superoxide dismutase mimetic, tempol, in DSS rats fed an 8% NaCl, high-salt diet. In agreement with previous studies [11,15], candesartan did not reduce systolic blood pressure, although diastolic heart failure was reduced and levels of reactive oxygen species (ROS), together with NAD(P)H oxidase activity and expression of NAD(P)H oxidase components, were inhibited. Similar results were obtained with tempol but, in contrast, systolic blood pressure was significantly reduced. The results obtained with tempol are consistent with those obtained with other antioxidants [19–25]. The finding that tempol reduced left-ventricular expression of NAD(P)H oxidase components [10] indicates that ROS can upregulate the expression of NAD(P)H oxidase. As reported by Guo et al. [10], there is other evidence to support this view in the literature [14,26]. It is evident from the data reported by Guo et al. [10] that diastolic dysfunction in DSS rats fed a high-salt diet can be ameliorated by interventions targeted against ROS generation to an equal degree to that observed with an AT1 receptor antagonist. These data raise the possibility that candesartan, and by implication other AT1 receptor antagonists, might mediate at least part of their beneficial effects on diastolic heart failure in the DSS rat model of hypertension by quenching free radicals. Support for the idea that angiotensin II receptor antagonists might quench free radicals is provided in a recent report by Miyata et al. [27] who used a cell-free in-vitro system to assess the ability of various AT1 receptor antagonists to inhibit the formation of advanced glycation end products. As part of their study, Miyata et al. [27] examined the ability of the angiotensin II AT1 receptor antagonist, olmesartan, to scavenge free radicals. They found that olmesartan significantly quenched a number of different types of free radical. Of particular note was the ability of olmesartan to quench hydroxyl radicals. At a concentration of 0.8 mmol/l, olmesartan reduced the in-vitro generation of hydroxyl radicals by approximately 80%. Unfortunately, the ability of lower concentrations of olmesartan, or other AT1 receptor antagonists, to reduce hydroxyl radical generation was not tested. However, as Miyata et al. [27] point out, AT1 receptor antagonists share a common chemical core. Therefore, it is possible that the ability to quench hydroxyl free radicals is a common feature of other AT1 receptor antagonists that contain the core chemical structure, 5-(4′-methylbiphenyl-2-yl)-1H-tetrazol. Only one difference between candesartan and tempol was identified by Guo et al. [10] in that tempol significantly reduced systolic blood pressure whereas candesartan had no significant effect. As previously discussed, interventions targeted against the RAS are ineffective [11–15] or, at best, partially effective [16,17] at controlling systolic blood pressure in DSS rats fed a high-salt diet. However, there are numerous reports of antioxidants reducing elevated blood pressure in the salt-induced hypertension of DSS rats [19–25], emphasizing the role of ROS in the hypertensive phenotype of this model. It is equally evident that activation of NAD(P)H oxidase is not involved in the salt-induced hypertension of DSS rats because treatment with an inhibitor of NAD(P)H oxidase, apocynin [12], or an inhibitor of NAD(P)H oxidase assembly, gp91ds-tat [14], failed to reduce elevated blood pressure in hypertensive DSS rats. Therefore, the differential effect of tempol and candesartan on blood pressure could indicate that the actions of candesartan in this model are not due to scavenging of free radicals because, if this were so, candesartan would be expected to reduce systolic blood pressure in a similar manner to that seen with tempol. However, there is at least one other study where an AT1 receptor antagonist (losartan) significantly lowered blood pressure in salt-induced hypertensive DSS rats [16]. As already discussed, a feature of this model is that hypertension is not associated with elevated circulating levels of angiotensin II [11], nor is it associated with activation of NAD(P)H oxidase [12,14], a major mechanism implicated in the aetiology of angiotensin II-dependent hypertension [28–34]. Therefore, it appears unlikely that the partial amelioration of hypertension observed with losartan [16] is due to blockade of AT1 receptors resulting in reduced NAD(P)H oxidase activation, and might instead be due to an antioxidant property of losartan based on its chemical structure. Differences between losartan and other AT1 receptor antagonists, such as candesartan, might then be due to differences in antioxidant efficacy rather than differences in the degree of AT1 receptor antagonism. Experimentally, there is a large body of evidence to support the view that, in renin-dependent hypertension, the mechanism of hypertension involves the generation of free radicals via activation of vascular NAD(P)H oxidase [4,5]. However, given that expression of NAD(P)H oxidase components can be inhibited by an antioxidant [19], a free radical scavenging mechanism of action for AT1 receptor antagonists cannot be ruled out. Differences between individual angiotensin II AT1 receptor antagonists in the same model have been noted. Using stroke-prone spontaneously hypertensive rats, Takai et al. [35] investigated the effects of two different angiotensin II AT1 receptor antagonists, losartan and telmisartan, that have similar binding affinities for the AT1 receptor [36]. Both antagonists reduced blood pressure to a similar degree, but expression of the NAD(P)H oxidase component, p22phox, was lower in telmisartan-treated rats than in those receiving losartan. The differential effect was ascribed to differences in lipophilicity, with telmisartan being more lipophilic than losartan. However, if telmisartan is a better free radical scavenger than losartan, this could also explain the results obtained. Unfortunately, no data are available with respect to the antioxidant properties of telmisartan. Numerous studies have demonstrated that infusion of angiotensin II causes hypertension associated with NAD(P)H oxidase activation [32,37–41]. However, these findings are not conclusive evidence that angiotensin II infusion induces hypertension by activating NAD(P)H oxidase. There are a large number of stimuli that have been shown to activate NAD(P)H oxidase including, but not limited to, endothelin [42], vascular endothelial growth factor [43], cytokines [44], leukotrienes [45], shear stress [6], cardiac ischaemia-reperfusion [46], vascular hypoxia-reoxygenation [47], hypercholesterolaemia [48], low-density lipoproteins [49] and oxidized low density lipoproteins [50]. Given the large number of triggers for NAD(P)H oxidase activation, it is possible that its activation after angiotensin II infusion is indirect. Indeed, taking just one example, it has been shown that angiotensin II activates NAD(P)H oxidase in rat vascular smooth muscle cells by stimulating the release of leukotriene B4 [51]. The problems associated with using AT1 receptor antagonists to demonstrate that angiotensin II-infusion activates NAD(P)H oxidase via stimulation of AT1 receptors has been discussed previously. In brief, hypertension induced by angiotensin II infusion can be inhibited by scavengers of free radicals [37,38,41] and, because AT1 antagonists might be associated with antioxidant properties due to their chemical structure [27], their efficacy cannot be taken as conclusive evidence of AT1 receptor activity in a mechanism involving ROS generation. The results reported by Guo et al. [10] indicate that, although an AT1 receptor antagonist had no significant impact on blood pressure in a RAS-independent model of hypertension, diastolic dysfunction was relieved. Important caveats about the interpretation of the data were raised by Guo et al. [10], as expanded upon in this editorial. In essence, caution needs to be exercised when interpreting the results obtained with AT1 receptor antagonists where ROS generation might be involved in the mechanism. Although it is not clear as to what degree the chemical structure of angiotensin II AT1 receptor antagonists (as opposed to their pharmacology) contributes to their well-publicized antioxidant activities, on the basis of the current state of knowledge, it would be unwise to ignore this potential mechanism. Indeed, it is surprising, given the widespread evidence of a relationship between the RAS and ROS, that a comprehensive quantitative investigation, using a cell-free in-vitro system similar to that used by Miyata et al. [27], has not been undertaken to assess the ability of different AT1 receptor antagonists to scavenge free radicals. Indeed, the identification of an AT1 receptor antagonist without, or with a very limited ability to quench free radicals in a cell-free assay would provide the tool required to clarify whether in-vivo antioxidant responses induced by AT1 receptor antagonists are mediated by virtue of their chemical structure or pharmacology.