Oxidative stress, Noxs, and hypertension: Experimental evidence and clinical controversies

Abstract

Reactive oxygen species (ROS) are signaling molecules that influence many physiological processes. Increased ROS bioavailability and altered redox signaling (oxidative stress) have been implicated in chronic diseases including hypertension. Although oxidative stress may not be the sole cause of hypertension, it amplifies blood pressure elevation in the presence of other prohypertensive factors (salt, renin-angiotensin system, sympathetic hyperactivity). A major source for cardiovascular ROS is a family of non-phagocytic NADPH oxidases (Nox1, Nox2, Nox4, Nox5). Other sources of ROS involve mitochondrial electron transport enzymes, xanthine oxidase, and uncoupled nitric oxide synthase. Although evidence from experimental and animal studies supports a role for oxidative stress in the pathogenesis of hypertension, there is still no convincing proof that oxidative stress is a cause of human hypertension. However, what is clear is that oxidative stress is important in the molecular mechanisms associated with cardiovascular and renal injury in hypertension and that hypertension itself can contribute to oxidative stress. The present review addresses the putative function of ROS in the pathogenesis of hypertension and focuses on the role of Noxs in ROS generation in vessels and the kidney. Implications of oxidative stress in human hypertension are discussed, and clinical uncertainties are highlighted.

Key Words::

• Antioxidant
• blood pressure
• hydrogen peroxide
• inflammation
• NADPH oxidase
• Nox
• superoxide
• vascular remodeling

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Erratum

Key messages

• NADPH oxidases are a family of enzymes that generate reactive oxygen species (ROS), which contribute to the physiological regulation of vascular function.

• Oxidative stress, through redox signaling, plays a role in cardiovascular and renal vascular inflammation and injury.

• Extensive experimental evidence indicates that oxidative stress is involved in the pathogenesis of hypertension.

• Clinical data have not yet demonstrated a causal association between oxidative stress and hypertension.

• Development of isoform-specific NADPH oxidase inhibitors may have promise as therapeutic modalities in the management of patients with NADPH oxidase-related diseases. The therapeutic potential in hypertension is unclear.

Introduction

Hypertension is a major modifiable risk factor for renal failure, cardiovascular disease, and stroke (1,2). Hypertension affects 30% of adults in the Western world and is the leading cause of morbidity and mortality worldwide (1). Although the exact etiology still remains largely unknown, it is clear that hypertension is a multifactorial, complex polygenic disorder with many interacting mechanisms contributing to its pathophysiology and involving many organ systems, including the heart, kidney, brain, vessels, and possibly the immune system (2,3). Factors implicated in the pathophysiology of hypertension include activation of the sympathetic nervous system, up-regulation of the renin-angiotensin-aldosterone system, altered G protein-coupled receptor signaling, and inflammation (4,5). Common to these processes is oxidative stress (increased bioavailability of reactive oxygen species (ROS)) due, primarily, to excess ROS generation, decreased nitric oxide (NO) levels, and reduced antioxidant capacity in the cardiovascular and renal systems (6,7).

Reactive oxygen species (ROS), including superoxide (•O₂ ⁻ ) and hydrogen peroxide (H₂O₂), were originally considered to be injurious metabolic cellular by-products. However, ROS are now recognized to have important physiological actions, including the induction of host defense genes, stimulation of transcription factors, and activation of ion transporters (8,9). In the vascular system ROS play a physiological role in controlling endothelial function and vascular tone and a pathophysiological role in processes underlying endothelial dysfunction, hyperreactivity, and vascular remodeling in cardiovascular diseases, including hypertension. Superoxide anion and H₂O₂ influence signaling pathways that regulate vascular hypertrophy, inflammation, and contraction, including mitogen-activated protein kinases (MAPK), tyrosine kinases, Rho kinase, transcription factors (NFκB, AP-1, and HIF-1), and protein tyrosine phosphatases (PTP) (10–12). http://hyper.ahajournals.org/cgi/content/ full/44/3/248 - R21 - 032003#R21 - 032003ROS also increase intracellular free Ca^{2 +} concentration ([Ca^{2 +} ]_i) and up-regulate proto-oncogene and proinflammatory gene expression and activity (13,14).

The relationship between free radicals and hypertension was first suggested in the 1960s (15), but it was some 40 years later that this association was investigated in detail when it was shown that Ang II-mediated hypertension in rats increases vascular ROS production via non-phagocytic NAD(P)H oxidase activation (16). Almost all experimental models of hypertension have evidence of oxidative excess including genetic forms (SHR, SHR-SP), surgically induced (2K1C, aortic banding), hormone-induced (Ang II, aldosterone, DOCA), and diet-induced hypertension (salt, fat) (17–20). Mice deficient in ROS-generating enzymes have lower blood pressure versus wild-type counterparts, and Ang II infusion fails to induce hypertension in these mice (21,22).

Since inhibition of ROS-generating enzymes, antioxidants, and ROS-scavengers lowers blood pressure whereas pro-oxidants increase blood pressure, it has been suggested that ROS are causally associated with hypertension, at least in animal models. However, in clinical hypertension the evidence is not so convincing. Most human studies examining ROS are based on associations between plasma markers of oxidative stress and blood pressure. Biomarkers of systemic oxidative stress, including levels of plasma thiobarbituric acid-reactive substances (TBARS) and 8-epi-isoprostanes, are elevated in patients with hypertension (23,24). Factors implicated in oxidative stress in human hypertension include decreased antioxidant activity, reduced levels of ROS scavengers, and a http://hyper.ahajournals.org/cgi/content/full/44/3/248 - R36 - 032003#R36 -  032003ctivation of ROS-generating enzymes (25–28). A causal link between ROS and high blood pressure has not yet been definitively established in humans. Only a few small clinical studies showed a blood pressure-lowering effect of antioxidants (25,29), whereas many large antioxidant clinical trials failed to demonstrate any cardiovascular benefit and blood pressure reduction (30,31).

Production of ROS in the cardiovascular system

Reactive oxygen species are formed as intermediates in reduction–oxidation (redox) reactions leading from O₂ to H₂O. Of the ROS produced in the vascular system, •O₂ ⁻ and H₂O₂ are particularly important. In biological systems, •O₂ ⁻ is short-lived and unstable owing to its rapid reduction to H₂O₂ by superoxide dismutase (SOD) (32). The charge on the superoxide anion makes it unable to cross cellular membranes except possibly through ion channels. H₂O₂ has a longer life-span than •O₂ ⁻ , is relatively stable, and is easily diffusible within and between cells. The distinct chemical characteristics between •O₂ ⁻ and H₂O₂ and their specific sites of localization mean that different species of ROS activate diverse signaling pathways, leading to divergent, and potentially opposing, cellular responses.

ROS are products of normal cellular metabolism and derive from many sources in different cellular compartments. Enzymatic sources of ROS in cardiovascular disease and hypertension include: xanthine oxidoreductase, uncoupled NO synthase (NOS), mitochondrial respiratory enzymes, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (33–36), found in many cell types in vessels, the heart, kidney, and central nervous system. Unlike most ROS-generating enzymes, which produce •O₂ ⁻ and/or H₂O₂ either as by-products of their catalytic activity or as a result of abnormal functioning in pathological conditions, NADPH oxidase has as its sole function the generation of ROS, hence termed a ‘professional’ ROS-producer (37). NADPH oxidase appears to be the major source of ROS in the cardiovascular and renal systems and is the focus of the present review.

Nox family NAD(P)H oxidases

NADPH oxidase was originally considered to be expressed only in phagocytic cells involved in host defense and innate immunity. It is now evident that there is a family of NAD(P)H oxidases, based on homologs of the catalytic subunit gp91phox, that are functionally active in non-phagocytic cells. The new homologs, along with gp91phox, are designated the Nox family of NAD(P)H oxidases (38) and are important in vascular ROS production. The prototypical gp91phox-containing phagocytic NAD(P)H oxidase (now termed Nox2) comprises five subunits: p47phox (‘phox’ stands for phagocyte oxidase), p67phox, p40phox, p22phox, and the catalytic subunit gp91phox (39). In basal conditions p47phox, p67phox, and p40phox exist in the cytosol, whereas p22phox and gp91phox are in the membrane, where they occur as a heterodimeric flavoprotein (cytochrome b558). Upon stimulation p47phox and p67phox form a complex that translocates to the membrane, where it associates with cytochrome b558 to assemble the active oxidase, which transfers electrons from the substrate to O₂, forming •O₂ ⁻ (40). Activation also requires Rac2 (or Rac1) and Rap1A.

The mammalian Nox family comprises seven members: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1, and Duox2 (38–42). All are transmembrane proteins that have a core catalytic subunit (Nox) and numerous regulatory subunits. Nox1, Nox2, Nox4, and Nox5 have been identified in cardiovascular and renal tissue. Hyperactivation of Noxs leads to excessive ROS generation that disrupts redox networks, normally regulated by thiol-dependent antioxidant systems. This results in oxidative stress, triggering molecular processes, which, in the vasculature, contributes to vascular injury. The Noxs have been extensively reviewed (43–46), and only an overview of recent developments is discussed here.

Nox1

Nox1, originally identified in colon tumor cells (47), is expressed in vascular and cardiac cells. It localizes to the cell membrane, caveolae/lipid rafts, and endosomes. Nox1 is similar to Nox2 in that it requires p22phox, p47phox (or its homolog NoxO1 (Nox organizer 1)), p67phox (or its homolog NoxA1 (Nox activator 1)), and Rac1 for its activity. New Nox1 regulators have been identified, Tks4 and Tks5, which resemble p47phox and NoxO1, and which interact with NoxA1 (48). Nox1 localizes with p22phox and is expressed at low levels in physiological conditions. Nox1-derived •O₂ ⁻ is increased in a stimulus-dependent manner, involving complex interactions between regulatory subunits and the redox chaperone protein disulfide isomerase (PDI) (49,50). Nox1 has been implicated in vascular smooth muscle cell migration, proliferation, and extracellular matrix production, effects mediated by cofilin (51).

In cultured endothelial and vascular smooth muscle cells Nox1 is up-regulated by mechanical factors (shear stress), vasoactive agents (Ang II, aldosterone), and growth factors (EGF, PDGF) (52,53). Ang II-induced induction of Nox1 may involve mitochondria, suggesting an interaction between Nox1 and mitochondria, possibly through a Ca²⁺ -dependent mechanism (54). Nox1 expression/activity is increased in the vasculature in models of cardiovascular disease including hypertension, atherosclerosis, diabetes, and hypercholesterolemia (55). Studies from Nox1 knock-out and transgenic mice suggest a possible role for Nox1 in acute, but not chronic, forms of Ang II-dependent hypertension (56,57), in atherosclerosis, restenosis post injury, endothelial dysfunction, and stroke. Mice genetically deficient in Nox1 also display decreased expression of aortic AT₁R (58), which may contribute to blunted hypertensive effects of Ang II infusion in these mice. Although there are extensive experimental data implicating Nox1 in cardiovascular disease, there is little information in humans, although expression of Nox1 and NoxA1 is increased in human atherosclerotic vessels (59).

Nox2

Nox2 is the catalytic subunit of the respiratory burst oxidase in phagocytes but is also expressed in cardiovascular cells (60). Nox2 is unstable without p22phox and requires p47phox, p67phox, and Rac1/2 for its full activation. In neutrophils Nox2 localizes to intracellular and plasma membranes, and in vascular cells it also localizes with the cytoskeleton, lipid rafts/caveolae, and in the perinuclear compartment. The Nox2 gene is inducible and is highly regulated by Ang II and stretch. Vascular Nox2, derived from resident macrophages or vascular cells, is up-regulated in experimental hypertension (61,62), atherosclerosis, ischemia-reperfusion injury, and neointimal formation. Although Nox2 has been shown to be important in models of Ang II-infused hypertension, it does not seem to play a role in blood pressure elevation or cardiac hypertrophy in a model of chronic Ang II-dependent hypertension (63). Nox2 is also implicated in stroke in experimental models. Nox2-deficient mice exhibit significant reduction in cerebral infarct size compared with wild-type controls (64). In humans, NADPH oxidase has been shown to play a role in endothelial function, since patients with chronic granulomatous disease (CGD) who have an X-linked Nox2 mutation exhibit a significant increase in forearm-mediated vasodilation with increased NO bioavailability (65), suggesting that Nox2-based NADPH oxidase influences endothelial function and NO biology in humans. In patients with CGD with mutations in Nox2 or p47phox, endothelial ischemia-reperfusion injury was blunted, indicating a role for NADH oxidase-derived ROS in human ischemia-reperfusion injury (66).

Nox4

Nox4, of which 4 splice variants have been identified (NOX4B, NOX4C, NOX4D and NOX4E), is found in vascular cells, fibroblasts and osteoclasts and is abundantly expressed in the kidney (66,67). In vascular smooth muscle cells, Nox4 co-localizes with p22phox and vinculin in focal adhesions and has been implicated in cell migration, proliferation, tube formation, angiogenesis, and cell differentiation (69). Nox4 has been identified in the endoplasmic reticulum, mitochondria, and nucleus of vascular cells. Nox 4 does not seem to require p47phox, p67phox, p40phox, or Rac for its activation, although polymerase (DNA-directed) delta interacting protein 2 (Poldip2), a Nox4-binding protein, has recently been shown to be important (70). Poldip2 is a Nox4/p22phox-interacting protein and is a potent positive regulator of Nox4 activity in VSMCs. The Nox4/p22phox–Poldip2 complex regulates Rho-dependent cytoskeletal reorganization and cellular migration (70). Another regulator of Nox4, potentially important in vascular fibrosis and differentiation, is transforming growth factor-β (TGF-β), which up-regulates expression and activity of Nox4 in many cell types, including vascular smooth muscle cells (71).

Unlike Nox1 and Nox2, Nox4 is constitutively active, producing primarily H₂O₂ rather than •O₂ ⁻ (72). The difference in the species generated may underlie Nox-specific actions in cell signaling. Nox4 contributes to basal ROS production through its constitutive activity and to increased ROS generation when stimulated by Ang II, glucose, TNFα and growth factors. The pathological role of Nox4 is unclear, although it has been implicated in hypertension, atherosclerosis, and cardiovascular and renal complications of diabetes and in remodeling of pulmonary arteries in pulmonary hypertension (73). Nox4-derived ROS has also been suggested in cellular senescence and aging (74) and in insulin-mediated differentiation of adipocytes (75). Recent studies demonstrated that Nox4 may have protective effects. In mice with a genetic deletion of Nox4 or a cardiomyocyte-targeted overexpression of Nox4, basal cardiac function was normal in both models, but Nox4-null animals developed exaggerated contractile dysfunction, hypertrophy, and cardiac dilatation during exposure to chronic overload, whereas Nox4-transgenic mice were protected (76). Nox4-derived H₂O₂ may act as a vasodilator in some vascular beds, which could explain why mice with targeted endothelial Nox4 overexpression in the endothelium exhibit lower blood pressure and improved endothelium-dependent vasodilation compared with wild-type controls (77). The exact (patho)physiological role of Nox4 in the cardiovascular system remains unclear, because many of the in-vivo studies interrogating Nox4 were performed in transgenic mice where Nox4 was up- or down-regulated.

Nox5

Nox5 is the most recently identified of the Nox enzymes and has unique features compared with other family members. Nox5 is a Ca^{2 +} -sensitive homolog found in testes, spleen, and lymphoid tissue, but also in kidney and vascular cells (78). While all Noxs are present in mice, rats, and man, the rodent genome does not contain the nox5 gene (79). Unlike other vascular Noxs, Nox5 possesses an amino-terminal calmodulin-like domain with four binding sites for Ca^{2 +} (EF hands), and unique to Nox5 is its lack of requirement for p22phox or other subunits for its activation. Nox5 is directly regulated by intracellular Ca^{2 +} ([Ca^{2 +} ]_i), the binding of which induces a conformational change leading to enhanced ROS formation (78). The biological significance of vascular Nox5 is unknown, although it has been implicated in cell proliferation, angiogenesis, and migration and in oxidative damage in atherosclerosis (79). Vascular Nox5 is activated by thrombin, PDGF, Ang II, and ET-1 (80), and its expression is regulated by the Ca^{2 +} -sensitive transcription factor CREB. Increased Nox5 expression has been demonstrated in coronary arteries from patients with coronary artery disease (81).

Networking between Noxs and mitochondria

Mitochondrial enzymes and NADPH oxidase are concomitantly activated in pathological processes. Nox4 has been localized within mitochondria, and it has been suggested that cross-talk between mitochondria and Noxs may be involved in dysregulated ROS formation (82–85). Such cross-talk may be important in oxidative stress associated with nitroglycerin-triggered vascular dysfunction, myocardial infarction, cardiac failure, vascular remodeling, and endothelial dysfunction (83). Mitochondrial-Nox interaction may also regulate vascular oxygen-sensing mechanisms (84). Mechanisms linking these ROS-generating systems include ERK1/2, PKC, the mitochondrial permeability transition pore, and ATP-sensitive potassium channels (83–86).

Antioxidant defense systems

In biological systems, enzymatic and non-enzymatic systems protect against injurious oxidative stress. Major enzymatic antioxidants are SOD, catalase, glutathione peroxidases, thioredoxin, and peroxiredoxin (87–89). Non-enzymatic antioxidants include ascorbate, tocopherols, glutathione, bilirubin, and uric acid. SOD catalyzes the dismutation of •O₂ ⁻ into H₂O₂ and O₂. Of the three SOD isoforms eSOD is the main vascular SOD.

Low antioxidant bioavailability promotes cellular oxidative stress and has been implicated in cardiovascular and renal oxidative damage associated with hypertension (90). Activity of SOD, catalase, and GSH peroxidase is lower, and the GSSG/GSH is higher in plasma and circulating cells from hypertensive patients than normotensive subjects (91). In mice deficient in EC-SOD and in rats in which GSH synthesis is inhibited, blood pressure is significantly elevated, demonstrating that reduced antioxidant capacity is associated with elevated blood pressure (87). In angiotensinogen-overexpressing mice, which exhibit hypertension and renal dysfunction, catalase overexpression prevented blood pressure elevation and protected against kidney damage (92). Failure to up-regulate antioxidant genes and reduced antioxidant capacity are associated with age-accelerated atherosclerosis (93).

Production of ROS in the vascular and renal systems in hypertension

Mechanisms whereby ROS influence the development of hypertension involve oxidative damage of multiple systems including the heart, kidneys, central nervous system, and vasculature (Figure 1). In pathological conditions ROS are involved in inflammation, endothelial dysfunction, cell proliferation, migration, and activation, extracellular matrix deposition, and fibrosis. These effects are mediated through redox-sensitive signaling pathways including MAPK, PTP, tyrosine kinases, proinflammatory genes, ion channels, and Ca^{2 +} (94–96).

Figure 1. ROS and vascular remodeling in hypertension. In healthy vessels, endothelial cells produce NO and ROS, where they play a role in cellular signaling related to the control of endothelial function and maintenance of vascular integrity. In hypertension, increased ROS production, due to increased activity of pro-oxidant enzymes and decreased antioxidant defense systems, and decreased NO bioavailability, are associated with endothelial dysfunction and vascular remodeling. Exposure of endothelium to high concentrations of ROS induces hypertrophy and expression of adhesion molecules with infiltration of inflammatory cells. In VSMCs, ROS activate profibrotic, proliferative, and apoptotic pathways leading to increased extracellular matrix (ECM) accumulation (fibrosis), hypertrophy, and fibrosis. The adventitia is also responsive to high levels of ROS and, through adventitial fibroblasts and adipocytes, plays a role in the regulation of cellular responses in vascular remodeling.

Changes in vascular function and structure probably relate to oxidative stress-induced endothelial dysfunction, reduced vasodilation, increased contraction, vascular inflammation, and structural remodeling, causing increased peripheral resistance and elevated blood pressure (Figure 2) (96). Centrally produced ROS by NADPH oxidase in the hypothalamic and circumventricular organs are implicated in central control of hypertension, in part through sympathetic outflow (97,98).

Figure 2. Potential effects of reactive oxygen species on the development of hypertension—an integrated system. The damaging effects of oxidative stress involve multiple organ systems. Exposure of the central nervous system to increased ROS induces production and release of neurotransmitters responsible for the regulation of vascular resistance, cardiac function, and blood pressure control. Effects of redox-sensitive neurotransmitters may be direct or indirect through activation of the renin-angiotensin-aldosterone system, which influences peripheral resistance by influencing vasoconstriction, volume status (increased sodium reabsorption), target tissue inflammation, and fibrosis. Systemic ROS, lipid peroxidation, and high levels of Ang II/aldosterone lead to increased cardiac contractility, remodeling, inflammation, and fibrosis, reflecting increased cardiac output. ROS also influence the vasculature, where peripheral resistance is increased due to endothelial dysfunction and vasoconstriction as well as to induction of vascular remodeling (as in Figure 1). All of these processes could contribute to blood pressure elevation.

The kidney, and particularly the renal medullary circulation, plays a fundamental role in modulating long-term blood pressure control and fluid balance (99,100). ROS are important regulators of medullary blood flow (100). Elevation of •O₂⁻ or reduction of NO in the renal medulla decreases medullary blood flow and Na⁺ excretion, resulting in sustained hypertension. Oxidative stress within the renal medulla makes the kidney functionally more vulnerable to effects of Ang II and salt and promotes renal dysfunction (101). Superoxide and H₂O₂ augment afferent arterial tone and reactivity and enhance renal vascular resistance. Renal ROS influence glomerular filtration rate, tubuloglomerular feedback response, and Na ⁺ transport. NO inhibits absorption of NaCl in the thick ascending limb, whereas •O₂⁻ enhances NaCl reabsorption. Moreover, Ang II and oxidative stress promote mesangial cell proliferation, mesangial matrix accumulation, and podocyte injury, which are hallmarks of glomerulonephritis and diabetic nephropathy (102).

Increased renal ROS production induces up-regulation of proinflammatory genes, such as hypoxia-inducible factor and AP-1, implicated in inflammation, fibrosis, and sclerosis in hypertension-associated kidney damage. Renal ROS generation is especially important in severe and salt-dependent forms of hypertension such as Dahl salt-sensitive rats, DOCA-salt rats, and stroke-prone SHR (SHR-SP) (103). In these models blood pressure-lowering effects of the SOD mimetic tempol were associated with reduced renal excretion of 8-isoprostane PGF2α, decreased vascular resistance, increased GFR, and enhanced diuresis and natriuresis (104).

Renal oxidative stress is also important in kidney disease associated with Ang II-dependent forms of hypertension. In TG(mRen2)27 (Ren2) transgenic rats, which overexpress the mouse renin gene, AT₁R blockade reduced blood pressure and tissue oxidative stress, improved glomerular filtration barrier integrity, and prevented albuminuria, thereby slowing progression of kidney disease (105).

A major source of renal •O₂⁻ is NAD(P)H oxidase. All of the major Noxs and NAD(P)H oxidase subunits are present in the renal cortex, medulla, and renal vessels (106,107). These proteins are expressed primarily in renal arterioles, glomeruli, and the distal nephron and are abundantly expressed at the luminal border of macula densa cells and in podocytes. In SHR, Ang II-induced hypertension and in salt-sensitive hypertension, renal expression of NAD(P)H oxidase subunits are increased and activity of Nox is enhanced.

Oxidative stress and hypertension—from the bench to the bedside

The link between oxidative stress and increased blood pressure was first suggested in the early 1990s both in patients with essential hypertension (108) and in SHR (109). Oxidative stress precedes development of hypertension in SHR and is implicated in fetal programming and development of hypertension later in life (110). Markers of oxidative stress, such as TBARS and F_2α-isoprostanes, tissue concentrations of •O₂⁻ and H₂O₂, and activation of NADPH oxidase and xanthine oxidase are increased, whereas levels of NO and antioxidant enzymes are reduced in experimental hypertension (111–113).

Ang II-dependent hypertension is particularly sensitive to ROS derived from NAD(P)H oxidase (114,115). In Ang II-infused rats and mice, expression of Noxs (Nox1, Nox2, Nox4), oxidase activity, and ROS generation are increased. In p47phox knock-out mice and in gp91phox (Nox2) knock-out mice Ang II infusion failed to induce hypertension, and these animals do not show the same increases in •O₂² production, vascular hypertrophy, and endothelial dysfunction observed in Ang II-infused wild-type mice (116). In Ang II-infused mice treated with siRNA targeted to renal p22phox, renal NADPH oxidase activity was blunted, ROS formation was reduced, and blood pressure elevation was attenuated (117). On the other hand, overexpression of vascular p22phox was associated with increased oxidative stress and vascular dysfunction, but no significant increase in blood pressure (118). Treatment with apocynin or diphenylene iodinium, non-specific pharmacological inhibitors of NAD(P)H oxidase, or gp91dstat, a novel specific inhibitor of NADPH oxidase, reduced vascular •O₂² production, prevented cardiovascular remodeling, and attenuated development of hypertension in Ang II-treated mice (119,120). Nox1-deficient mice have reduced vascular •O₂^- production, and blood pressure elevation in response to Ang II is blunted (121), whereas in transgenic mice in which Nox1 is overexpressed in the vascular wall, Ang II-mediated vascular hypertrophy and blood pressure elevation are enhanced (122). In these models, Ang II was infused for a short time period (1–3 weeks), inducing an acute hypertensive response. In a model of chronic Ang II-dependent hypertension, where we crossed transgenic mice expressing human renin (which exhibit an Ang II-sensitive hypertensive phenotype) with Nox2−^/− or Nox1−^/− mice, development of hypertension was not prevented even though oxidative stress was reduced, suggesting that Noxs may be more important in acute than in chronic hypertension (123,124). It should be stressed that in these Nox knock-out or transgenic studies, baseline cardiovascular phenotypes of mice are surprisingly normal, and it is only in the context of a challenge, such as with Ang II or salt, that mice exhibit vascular and blood pressure alterations (125–128).

Unlike cell-based studies where Ang II alone is sufficient to induce oxidative stress and activation of redox-sensitive pathways that regulate vascular function, growing in-vivo evidence indicates that, in Ang II-dependent models of hypertension, Ang II alone is probably insufficient to induce oxidative stress and that additional stressors are needed. Such co-inducers include, amongst others, salt, activation of the sympathetic and/or central nervous system, humoral factors, and involvement of peripheral T cells and the immune system. As an example are studies demonstrating that redox signaling in the subfornical organ (SFO) in the forebrain is critical in Ang II-mediated regulation of blood pressure and hypertension, because SFO-targeted ablation of SOD3 causes a significant increase in basal blood pressure and enhances the sensitivity to systemic Ang II at a concentration that does not normally affect blood pressure in mice (126,127). Moreover, SOD3 ablation in the SFO is sufficient to increase vascular ROS, an effect associated with increased sympathetic output and increased circulating CD69 ⁺ T lymphocytes. Taken together these data suggest that increased •O₂⁻ in the forebrain through SOD3 deletion leads to peripheral T cell activation and vascular oxidative stress and inflammation, promoting an increase in blood pressure (128). This paradigm links the central nervous system, immune system, vasculature, and oxidative stress in the pathophysiology of hypertension, at least in experimental models of Ang II-induced hypertension.

Additional sources of NADPH oxidase-derived ROS in Ang II-induced hypertension are cytokines from inflammatory cells, such as T lymphocytes. In RAG1^-/- mice, which lack lymphocytes, pressor responses to Ang II are reduced, a response that is restored by adoptive transfer of T lymphocytes, but not of B lymphocytes (129). T cells influence blood pressure elevation by interacting with B7 ligands (CD80 and CD86) and the T cell co-receptor CD28, and through dysregulation of T regulatory and T effector cells (129). Importance of the immune/inflammatory system is further demonstrated by studies showing that T regulatory lymphocytes (Tregs), which suppress T effector lymphocytes, attenuate vascular oxidative stress, inflammation, and blood pressure in Ang II-induced hypertension (130).

There is also evidence for ROS involvement in the pathogenesis of hypertension independent of direct Ang II actions. In SHR, vascular, renal, and cardiac •O₂⁻ production is enhanced compared with normotensive controls. In stroke-prone SHR, aortic expression of Nox1 and Nox4 is significantly increased compared with WKY (131). In DOCA salt-induced mineralocorticoid hypertension, vascular •O₂₋ production involving elevated NAD(P)H oxidase activity, uncoupling of endothelial NOS, and mitochondrial sources, in part through the endothelin-1 (ET-1)/ETA receptor pathway, is increased (132). Infusion of ET-1 increases NAD(P)H oxidase-dependent •O₂₋ production; however, preventing this increase in ROS generation does not inhibit development of hypertension in these animals. Overexpression of human ET-1 in mice also induces vascular remodeling and impairs endothelial function, via activation of NAD(P)H oxidase. To support further a role for oxidative stress in experimental hypertension, treatment with antioxidant vitamins, SOD mimetics (tempol (4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl)), free radical scavengers, or tetrahydrobiopterin (BH4) has been shown to attenuate or prevent development of hypertension and associated target organ damage (133–135).

Unlike experimental studies, where there is strong evidence that oxidative stress plays a role in the pathogenesis of hypertension, clinical data are less convincing, and most studies have been based on indirect evidence demonstrating increased plasma markers of oxidative stress in patients with hypertension (Table I) (136,137). http://hyper.ahajournals.org/cgi/content/full/44/3/248 - R34 - 032003#R34 - 032003 http://hyper.ahajournals.org/cgi/content/full/44/3/ 248 - R35 - 032003#R35 - 032003. It should be stressed that biomarkers may simply reflect associations, and not cause, between ROS and blood pressure.

Table I. Indirect evidence supporting a role for oxidative stress in human hypertension.

1. Increased plasma and urine levels of markers of oxidative stress (e.g. TBARS, isoprostanes)

2. Increased vascular generation of superoxide anion

3. Decreased plasma levels of antioxidant vitamins

4. Inverse association between plasma ascorbate levels and blood pressure in epidemiological studies

5. Blood pressure-lowering effect of vitamin C in small clinical studies

6. Antihypertensive drugs reduce ROS production and decrease oxidative stress by inhibiting activation of NADPH oxidase and through intrinsic antioxidant properties

Hypertensive patients exhibit higher levels of plasma H₂O₂ than normotensive subjects (138). Normotensive subjects with a family history of hypertension have greater H₂O₂ production than blood pressure-matched normotensives without a family history of hypertension, suggesting that there may be a genetic component that leads to elevated production of hydrogen peroxide (139–141). Racial differences in oxidative stress and inflammation have been demonstrated. Human umbilical venous cells (HUVEC) from African-Americans exhibited higher levels of NO, IL-6, p47phox, Nox2, and Nox4 and lower superoxide dismutase activity than HUVECs from Caucasians (142). Moreover in African men, but not women, plasma ROS was positively associated with systolic blood pressure (142).

We showed that ROS production is increased in vascular smooth muscle cells from resistance arteries of hypertensive patients and that this is associated with up-regulation of vascular NADPH oxidase (143,144). The importance of NADPH oxidase in oxidative stress in human cardiovascular disease is supported by studies showing that polymorphisms in NADPH oxidase subunits are associated with increased atherosclerosis and hypertension. In particular, the −930(A/G) polymorphism in the p22(phox) promoter may be a novel genetic marker associated with hypertension (145). The C242T CYBA polymorphism is associated with essential hypertension, and hypertensive patients carrying the CC genotype of this polymorphism exhibit features of NADPH oxidase-mediated oxidative stress and endothelial damage and are prone to cerebrovascular disease (146). Polymorphisms −337GA and 565 + 64CT of xanthine oxidase gene have been shown to be related to blood pressure and oxidative stress in hypertension, further supporting a role for xanthine oxidase in hypertension (147).

In addition to excess ROS generation, decreased antioxidant defense mechanisms contribute to oxidative stress in patients with hypertension. Hypertensive patients have reduced activity and decreased content of antioxidant enzymes, including SOD, glutathione peroxidase, and catalase (148). Decreased levels of antioxidant vitamins A, C, and E have been demonstrated in newly diagnosed, untreated hypertensive patients compared with normotensive controls (149). Antioxidant vitamins reduced blood pressure and arterial stiffness in patients with diabetes (150), but had no effect in postmenopausal women or in healthy subjects (151). In patients with white-coat hypertension serum protein carbonyl (PCO, indicating protein oxidation) was increased, and endogenous antioxidant proteins (protein thiol, SOD, glutathione) were decreased compared with normotensive individuals, further supporting a relationship between oxidative stress and hypertension (151).

Human studies examining ROS and hypertension are primarily correlative, and there is still no definitive proof that oxidative stress is a cause of human hypertension. There is no direct evidence that Noxs play a pathophysiological role in human hypertension. However, NADPH oxidase-derived ROS have been implicated in the regulation of vascular function, because patients with chronic granulomatous disease due to mutations in genes encoding for NADPH oxidase subunits demonstrate blunted ischemia-reperfusion-induced flow-mediated dilation.

Targeting ROS as a therapeutic strategy in clinical hypertension

The potential of antioxidants in treating conditions associated with oxidative stress is supported by experimental investigations, observational findings, small clinical studies, and epidemiological data. http://hyper.ahajournals.org/cgi/content/full/44/3/248-R44 - 032003#R44 - 032003. http://hyper.ahajournals.org/cgi/content/full/44/3/248-R45 - 032003#R45 - 032003. However, findings are inconsistent, and clinical trial data are inconclusive (152). Recent large clinical trials examining effects of antioxidant vitamins (vitamins C and E) in the prevention of pre-eclampsia and gestational hypertension were also negative (153,154). Possible reasons for these disappointing outcomes relate to 1) type of antioxidants used, 2) patient cohorts included in trials, and 3) the trial design itself. With respect to antioxidants, it is possible that agents examined were ineffective and non-specific and that dosing regimens and duration of therapy were insufficient. It is also possible that orally administered antioxidants may be inaccessible to the source of free radicals, particularly if ROS are generated in intracellular compartments and organelles. Furthermore, antioxidant vitamins do not scavenge H₂O₂, which may be more important than •O₂⁻ in cardiovascular disease. Another factor of importance is that antioxidants do not inhibit ROS production. Regarding cohorts included in large trials, most subjects had significant cardiovascular disease, in which case damaging effects of oxidative stress may be irreversible. Another confounding factor is that most of the enrolled subjects were taking aspirin prophylactically. Since aspirin has intrinsic antioxidant properties, additional antioxidant therapy may be ineffective. Moreover, in patients studied in whom negative results were obtained, it was never proven that these individuals did in fact have increased oxidative stress. To date, there are no large clinical trials in which patients were recruited based on evidence of elevated ROS formation. Also, none of the large clinical trials were designed to examine effects of antioxidants specifically on blood pressure.

Based on the lack of evidence to prove the benefits from use of antioxidants to prevent cardiovascular disease, antioxidant supplementation is not recommended for the prevention or treatment of hypertension. However, most therapeutic guidelines suggest that the general population consumes a diet emphasizing antioxidant-rich fruits and vegetables and whole grains. The low-sodium DASH diet has been shown to reduce oxidative stress and improve vascular function in salt-sensitive individuals (155). Another important life-style modification that may have cardiovascular-protective and blood pressure-lowering effects by reducing oxidative stress is exercise. In experimental models of hypertension and in human patients with coronary artery disease, exercise reduced vascular NADPH oxidase activity and ROS production, ameliorated vascular injury, and reduced blood pressure (156). However, in elderly patients, combining antioxidant therapy with exercise negated beneficial blood pressure-lowering effects of exercise (157).

Recent clinical studies examining effects of xanthine oxidase inhibitors (158), tetrahydrobiopterin (sapropterin dihydrochloride (6r-bh4)) (159), and N-acetylcysteine (160) have demonstrated improved vascular function and blood pressure lowering in patients with hypertension, chronic kidney disease, and pulmonary hypertension. Some of the beneficial effects of classical antihypertensive agents such as β-adrenergic blockers, ACE inhibitors, AT₁ receptor antagonists, and Ca^{2 +} channel blockers may be mediated, in part, by decreasing vascular oxidative stress (161,162). http://hyper.ahajournals.org/cgi/content/full/44/3/248-R41 - 032003. These effects have been attributed to direct inhibition of NAD(P)H oxidase activity and to intrinsic antioxidant properties of the drugs. However, some studies failed to show changes in oxidative stress despite significant blood pressure lowering by classical antihypertensive drugs (163,164).

NADPH oxidase and Nox isoforms as therapeutic targets—clinical potential

Based on experimental evidence NADPH oxidase subunits and Nox isoforms are potential therapeutic targets for cardiovascular disease and hypertension. Because of this there has been enormous interest in the development of agents that inhibit NADPH oxidases in an isoform-specific manner (46,165). Different strategies have been employed, including small molecule inhibitors, peptide NADPH oxidase inhibitors, and siRNAs (Table II). Several compounds have been registered as NADPH oxidase inhibitors in the patent literature (165). However, none has gone through clinical trials, and some have not yet completed preclinical studies. To date two different classes of compounds have been claimed as potent and orally active bioavailable NADPH oxidase inhibitors: pyrazolopyridines (GKT136901 and GKT137831) and triazolopyrimidine derivatives (VAS2870 and VAS3947). Although the mechanisms of inhibition have not yet been clarified, GKT compounds may act as competitive substrate inhibitors, since structurally they resemble NADPH. Although much research is still needed to confirm the clinical use of NADPH oxidase inhibitors in humans, these drugs hold promise in the management of patients with Nox-associated pathologies (46,165).

Table II. Compounds that have been registered as NADPH oxidase inhibitors (165). None of these have yet been tested clinically.

Compound	Name
I. Small molecule NADPH oxidase inhibitors:
Pyraolopyridine derivatives	GKT136901, GKT137831
Pyrazoloprimidine derivatives
Triazolopyrimidine derivatives	VAS2870, VAS3947
Tetrahydoindole derivatives
Fulvene and fulvalene analogues
Synthetic polyphenol	S17834
Apocynin
Bicyclic pyridazine derivatives
AEBSF (aminoethyl benzenesulfono fluoride)	Pefabloc
Aryl sulfonates and benzamide derivatives
II. Peptide NADPH oxidase inhibitors
Gp91ds-tat
Proline-rich polypeptides
NADPH oxidase cytosolic cofactor mutant peptides
(Noxa1, p67phox mutants)
III. NADPH oxidase siRNAs
14– 30 nucleotides
siNox1
18– 40 nucleotides
siNox2
siNox4
siNox5
siDuox2
siNoxO1/2
siNoxA1/2

Conclusions

In physiological conditions, ROS play an important role in cardiovascular and renal biology through highly regulated redox-sensitive signaling pathways. Uncontrolled production/degradation of ROS results in oxidative stress, which induces cardiovascular and renal injury and activation of the sympathetic nervous system with associated increase in blood pressure. Although oxidative damage may not be the sole cause of blood pressure elevation, together with a constellation of prohypertensive factors, such as salt-loading, activation of the renin-angiotensin system, and sympathetic hyperactivity, it amplifies the development of hypertension. Convincing findings from experimental and animal studies suggest a causative role for oxidative stress in the pathogenesis of hypertension. However, in humans there is still no solid evidence that oxidative stress is fundamentally involved in the pathogenesis of hypertension. Further research in the field of oxidative stress and human hypertension is warranted. In particular, there is an urgent need for the development of sensitive, specific, and reliable biomarkers and assays to assess the redox status of patients. Also needed are clinical trials designed specifically to address the role of ROS in the development of hypertension. With a better understanding of processes regulating ROS metabolism and identification of factors that promote oxidative excess in humans, it should be possible to target therapies more effectively so that damaging actions of oxygen free radicals can be reduced. Such therapies could have potential in the management of redox-sensitive diseases associated with cardiovascular and renal damage, including hypertension (46,165).

Declaration of interest: Work from the author's laboratory was supported by grants 44018 and 57886, both from the Canadian Institutes of Health Research (CIHR). R.M.T. is supported through a Canada Research Chair/Canadian Foundation for Innovation award and A.C.M. by a fellowship from the CIHR. The authors report no conflicts of interest.