Activated oxygen metabolites

Angiotensin II, originally described as a potent vasoconstrictor, is now well recognized as having pleitropic actions in multiple organ systems. In the vasculature, angiotensin II, through the AT1 receptor, induces contraction, cell growth, migration, differentiation and secretion [1]. It is also pro-inflammatory and pro-fibrotic and plays a major role in maintaining vascular integrity [2]. At the subcellular level, these processes are highly regulated by complex networks of interacting signalling pathways [1]. Among the numerous signalling molecules involved in angiotensin II-mediated vascular actions, reactive oxygen species appear to play a pivotal role [3,4]. Angiotensin II stimulates production of reactive oxygen species in all vascular cell types, primarily through activation of cell membrane-associated NAD(P)H oxidase [5]. Although xanthine oxidase has also been implicated in vascular generation of free radicals, the contribution of this enzyme appears to be minor. Vascular NAD(P)H oxidase is a neutrophil-like NAD(P)H oxidase comprising, to varying degrees, the major phagocytic NADPH oxidase subunits; cell-membrane-associated p22phox and gp91phox (also known as nox2) or its homologues nox1 or nox4, and cytosolic-associated p47phox, p67phox and p40phox [6,7]. Angiotensin II-activated NAD(P)H oxidase, in the presence of molecular O2 and the substrate NAD(P), generates superoxide anion (•O2–), which is spontaneously or enzymatically [by superoxide dismutase (SOD)] dismutated to hydrogen peroxide (H2O2). Unlike phagocytic NAD(P)H oxidase, which generates •O2– extracellulary in a burst-like pattern, vascular NAD(P)H oxidase-mediated production is constitutive and inducible and is generated intracellularly in a slow and sustained manner [5]. Whereas •O2– is highly unstable and cell membrane impermeable, H2O2 is more stable, has a longer half-life and easily crosses cell membranes. In addition to the mitogenic, proinflammatory and profibrotic characteristics of angiotensin II-generated reactive oxygen species, emerging evidence indicates that oxygen-derived molecules regulate vascular reactivity. Studies performed as early as the 1980s reported that H2O2 is a potent vasodilator in cerebral arteries [8]. More recent studies suggest that H2O2 induces vasoconstriction in various vascular beds including rat mesenteric arteries [9], rat aorta [10], porcine pulmonary arteries [11] and canine cerebral arteries [12]. Superoxide has also been shown to induce vasoconstriction in certain vascular beds and to reduce relaxation to endothelium-dependent vasodilators [13]. This may be either the result of nitric oxide (NO) scavenging or the result of a direct contractile effect on vascular smooth muscle [14]. However, not all reports are in agreement with these findings. Indeed, studies of the effects of oxygen free radicals on vascular tone often report contradictory results, as demonstrated by Schuijt et al. [15] in the present issue of the journal. In their study, the role of endogenous reactive oxygen species in contractile responses to angiotensin II was questioned. Studies were performed in isolated human and porcine coronary or femoral arteries in organ baths and the bioavailability of reactive oxygen species was modulated using various pharmacological tools. Under these experimental conditions •O2– did not play a role in angiotensin II-induced vasoconstriction and, if anything, it resulted in vasodilation through H2O2 formation. These results support those previously found in cerebral arteries [16], coronary arteries [17] and pulmonary arteries [18], but are in contrast to those of Torrecillas et al. [19], who demonstrated that H2O2 is a critical intracellular mediator in vascular contractile responses to angiotensin II. How then do reactive oxygen species mediate their dual contractile/dilatory actions? (Fig. 1). Numerous studies have documented that oxygen metabolites produce vasoconstriction and/or vasodilation in various in vitro and in vivo preparations. At the subcellular level, it appears that •O2– enhances agonist-stimulated Ca2+ signalling in vascular smooth muscle cells, by stimulating Ca2+ influx and Ca2+ mobilization, resulting in increased intracellular free Ca2+ concentration ([Ca2+]i), actin–myosin complex formation and consequent increased contraction [20]. In the endothelium, •O2– also increases [Ca2+]i, which might stimulate Ca2+-dependent nitric oxide synthase activation resulting in increased NO production and consequent vasodilation [21]. It is also possible that elevated endothelial •O2– could quench NO leading to peroxynitrite formation, which has been described to be a weak vasodilator [22]. Thus, •O2– could have both vasoconstrictory and vasodilatory actions depending on the primary location and the concentration of the free radical. Similarly, H2O2 has a dual vasoactive effect. However, its mechanisms of action appear to be different to those of •O2–. Hydrogen peroxide induces vasodilation via stimulation of prostaglandins or through activation of Ca2+-dependent K+ channels [23,24] and vasoconstriction by stimulating Ca2+ influx and increasing vascular smooth muscle cell [Ca2+]i [25]. In the endothelium, H2O2 has been considered to be an important endothelium-derived hyperpolarizing factor [26,27]. Hydrogen peroxide activates calcium-activated K+ (KCa) channels (KCa) by direct modulation of the channels or by a cGMP or lipoxygenase-mediated mechanism. Endothelium-dependent H2O2-induced vasorelaxation has been demonstrated in human coronary and mesenteric arteries, cat cerebral arterioles, piglet pial arterioles, mouse mesenteric arteries and rat cerebral arteries [26–30]. The role of H2O2 as an endothelium-derived hyperpolarizing factor in human radial arteries remains unclear [31]. In vascular smooth muscle cells, cGMP is directly activated by nanomolar concentrations of NO or H2O2, leading to vasodilation. These cGMP-mediated mechanisms of relaxation are inhibited by SOD [24]. On the other hand, vascular smooth muscle cell H2O2 stimulates Ca2+ influx and mobilization, leading to increased [Ca2+]i and consequent vasoconstriction. H2O2-mediated vasoconstriction has been demonstrated in canine cerebral arteries [12], rat mesenteric arteries [9] and rat aorta [10], and is enhanced in arteries from spontaneously hypertensive rats [9].Fig. 1: Schematic demonstrating putative mechanisms whereby superoxide (•O2 −) and hydrogen peroxide (H2O2) mediate constriction and/or dilation in vessels. Increased bioavailability of •O2 − and H2O2 in vascular smooth muscle cells induces Ca2+ influx through activated Ca2+ channels and enhanced Ca2+ mobilization from sarcoplasmic reticular (SR) stores, resulting in increased concentrations of intracellular free Ca2+ ([Ca2+]i) and consequent contraction. Increased bioavailability of reactive oxygen species in endothelial cells causes activation of KCa channels on endothelial and VSMC resulting in hyperpolarization. Endothelial hyperpolarization may also spread to underlying VSMCs through gap junctions. Endothelial [Ca2+]i elevation induced by •O2 − increases eNOS activity leading to nitric oxide (NO) production, which in the presence of •O2 −, forms peroxynitrite (ONOO−). NO, and possibly ONOO−, relax VSMCs through cyclic GMP (cGMP)-dependent mechanisms. Depending on the species, concentration, cellular location absence or presence of endothelium and agonist, O2-derived radicals appear to induce differential vasoactive responses.Hence, it is evident that reactive oxygen species are capable of inducing actions that could promote both vasodilation and vasoconstriction. Major factors underlying the differential vascular responses to activated oxygen metabolites could relate to the blood vessel studied, the presence or absence of the endothelium, the concentration and species of free radical studied and the compartment in which •O2– or H2O2 predominate. Vascular •O2–, which is generally cell membrane-impermeable, is located primarily intracellulary in adventitial fibroblasts and vascular smooth muscle cells, whereas H2O2 is easily diffusible and migrates to the extracellular milieu or traverses the vascular wall to reach the endothelium. Accordingly, increased •O2– in vascular smooth muscle cells may indeed lead to elevated [Ca2+]i and consequent vasoconstriction, whereas predominantly increased endothelial or extracellular H2O2 would promote vasodilation (Fig. 1). These processes may explain, at least in part, why •O2– did not seem to be a vasoconstrictor in the present study. NADP, the NAD(P)H oxidase substrate, was administered exogenously to activate the endogenous enzyme to generate •O2–. However, NADP is cell membrane-impermeable and therefore may not have reached the intracellular site of activated NAD(P)H oxidase. Thus, vascular smooth muscle cell •O2– was probably not generated in high enough concentrations to increase Ca2+ influx and induce its vasoconstrictor action. Although the study was designed to evaluate the role of •O2– in angiotensin II-stimulated vasoconstriction, the data are more supportive of a vasodilatory effect of H2O2. This is highlighted by the fact that NADP induced vasodilation independently of diphenylene iodinium, a flavoprotein inhibitor that inhibits NAD(P)H oxidase, suggesting that the NADP-mediated effect is not through the oxidase. Another factor pointing to an important role of H2O2 relates to the inhibitory actions of catalase. Exogenously added catalase, which catalyses H2O2 to H2O and O2, resulted in decreased bioavailabilty of H2O2 with consequent decreased vasodilation and potentiated vasoconstrictor effects of angiotensin II. Thus, compartmentalization of different species of activated oxygen molecules may be crucial in the overall vasoactive effects of angiotensin II. There are some limitations that warrant consideration when interpreting the findings of the study under consideration [15]. First, numerous pharmacological agents were used in whole vessel preparations. It should be emphasized that in such preparations where cell type is heterogeneous and where different species of free radicals reside in different vascular and subcellular compartments, the specificity and sensitivity of the pharmacological inhibitors/mimetics used are questionable. Second, there was no evidence that angiotensin II did in fact increase vascular •O2– concentration. Thus, the premise that angiotensin II-induced generation of •O2– causes vasoconstriction is in doubt from the beginning. Third, vessels were studied in organ baths and not as pressurized vessels. Normally, vessels are exposed to both mechanical forces (pressure, stretch, shear stress) and humoral stimulation (such as angiotensin II, norepinephrine, endothelin-1). Because pressure itself is an important activator of NAD(P)H oxidase-derived reactive oxygen species [32], in the absence of such a stimulus, angiotensin II possibly failed to significantly increase •O2– formation by the oxidase in the vessels studied. Nevertheless, the study performed by Schuijt et al. [15] certainly has strengths that need to be underlined. First, unlike previous studies where exogenous O2– and/or H2O2 was added at pharmacological concentrations, this is one of the few studies to investigate the vasoactive effects of endogenously produced reactive oxygen species by angiotensin II [15]. Second, the vessels under investigation were from large animals and humans, making the findings physiologically and clinically more relevant than studies performed in vessels from rats or mice. Third, intact endothelium-containing vessels were examined. Finally, the findings from this study shed light on the possible mechanisms whereby angiotensin II, through endothelial AT1 receptors, may induce vasodilation. In conclusion, the results of the study by Schuijt et al. [15], taken together with those obtaioned in previous studies, highlight the diverse functions and complexities relating to the vasoactive role of reactive oxygen species. At present, it is still unclear exactly what the functions of •O2– and H2O2 are with respect to vascular contraction/dilation in physiological conditions, and it is even more confusing and perplexing when trying to unravel the role of these complex processes in pathophysiological conditions. Hence, additional studies such as the one by Schuijt et al. [15] are important because they could lead to an understanding of whether reactive oxygen species are indeed important regulators of vascular tone and, if so, what the underlying mechanisms might be. Furthermore, such studies could elucidate the significance of endogenous free radicals in the regulation of vascular function in both healthy individuals and those with cardiovascular disease.