Angiotensin-Generated Reactive Oxygen Species in Brain and Pathogenesis of Cardiovascular Diseases

Abstract

Significance: Overproduction of angiotensin II (Ang II) in brain contributes to the pathogenesis of cardiovascular diseases. One of the most promising theses that emerged during the last decade is that production of reactive oxygen species (ROS) and activation of redox-dependent signaling cascades underlie those Ang II actions. This review summarizes our status of understanding on the roles of ROS and redox-sensitive signaling in brain Ang II-dependent cardiovascular diseases, using hypertension and heart failure as illustrative examples. Recent Advances: ROS generated by NADPH oxidase, mitochondrial electron transport chain, and proinflammatory cytokines activates mitogen-activated protein kinases and transcription factors, which in turn modulate ion channel functions and ultimately increase neuronal activity and sympathetic outflow in brain Ang II-dependent cardiovascular diseases. Antioxidants targeting ROS have been demonstrated to be beneficial to Ang II-induced hypertension and heart failure via protection from oxidative stress in brain regions that subserve cardiovascular regulation. Critical Issues: Intra-neuronal signaling and the downstream redox-sensitive proteins involved in controlling the neuronal discharge rate, the sympathetic outflow, and the pathogenesis of cardiovascular diseases need to be identified. The cross talk between Ang II-induced oxidative stress and neuroinflammation in neural mechanisms of cardiovascular diseases also warrants further elucidation. Future Directions: Future studies are needed to identify new redox-based therapeutics that work not only in animal models, but also in patients suffering from the prevalent diseases. Upregulation of endogenous antioxidants in the regulation of ROS homeostasis is a potential therapeutic target, as are small molecule- or nanoformulated conjugate-based antioxidant therapy. Antioxid. Redox Signal. 19, 1074–1084.

Introduction

A ngiotensin II (Ang II), a biologically active component of the renin-angiotensin system (RAS), possesses potent effects in brain regions that are involved in the neural control of cardiovascular functions and electrolyte and fluid homeostasis (16, 19, 66). Pathologically, overproduction of Ang II or excessive activation of brain RAS contributes to the genesis of cardiovascular diseases, including hypertension and heart failure (61, 104, 108). The cellular mechanisms and signaling pathways via which brain Ang II induces cardiovascular malfunctions are currently a subject of intensive investigation. One of the most promising theses that emerged during the last decade is generation of reactive oxygen species (ROS) and activation of redox-dependent signaling cascades by Ang II. Oxidative stress, a cellular condition characterized by an imbalance between ROS production and the ability of biological systems to detoxify the reactive intermediates, plays a pivotal role in neural mechanism of hypertension and heart failure. In this review, we will discuss recent progress in our understanding of the cellular and molecular mechanisms that underlie the generation of ROS by Ang II in brain and brain redox-associated signaling pathways in Ang II-dependent cardiovascular diseases, in particular with respect to hypertension and heart failure. We will also discuss antioxidant defense systems in the protection against brain Ang II-associated hypertension and heart failure.

Sources of ROS for Brain Ang II

ROS are highly reactive oxygen moieties that are generated in cells mainly from incomplete reduction of molecular oxygen through enzymatic reactions with intracellular oxidases such as the NADPH oxidase (Nox) enzymes and xanthine oxidase, by the leakage of electrons from the mitochondrial electron transport chain (ETC), from uncoupling of nitric oxide synthases, and through cyclo-oxygenases, lipoxygenases, or cytochrome P450 reductases (100). Within the central nervous system, Nox and mitochondrial ETC are the two major sources of ROS for Ang II, particularly superoxide (O₂ ^•−) and hydrogen peroxide (H₂O₂). In addition, activated proinflammatory cytokines mediate Ang II-stimulated ROS production in brain.

NADPH oxidase

The Nox family enzymes comprise seven members, each containing a unique homolog of the catalytic subunit of the oxidase, namely, Nox1-5 and Duox1/2 (45). All Nox enzymes utilize NADPH as an electron donor and catalyze the transfer of electrons to molecular oxygen to generate ROS. Each Nox homolog exhibits a distinct cell and tissue distribution (34, 54). Nox2 and Nox4 are the most abundantly expressed Nox homologs within Ang II-sensitive sites in brain, in particular subfornical organ (SFO) of the circumventricular organ (34, 65) and hypothalamic paraventricular nucleus (PVN) (33). Both members of the Nox family enzymes are expressed in neurons and are involved in O₂ ^•− production in brain after activation of Ang II signaling (6, 22, 40, 105). Nox1 and Nox2 are expressed in the cerebral blood vessels and are essential for O₂ ^•− production in cerebral microcirculation in response to Ang II (26, 40).

It is a general consensus that generation of Nox-derived ROS by brain Ang II is mediated via activation of angiotensin type 1 receptor (AT₁R) (6, 70, 105). Whereas Nox1-derived O₂ ^•− is important for AT₁R signaling (2, 35), it is Nox2-derived O₂ ^•− that is generated in response to activation of AT₁R normally expressed on the cell surface (2). At the cellular level, Nox subunit p47^phox (27) and gp91^phox (87) are present in somatodendric and axonal profiles containing AT₁R in neurons of nucleus tractus solitarii (NTS), an Ang II-sensitive brain region that plays an important role in cardiovascular regulation. Intriguingly, chronic Ang II administration results in a redistribution of p47^phox subunits away from intracellular organelles in the distal dendritic compartment toward nonvesicular targets in less distal, intermediate areas of NTS neurons. This suggests that it is likely that production of ROS in NTS neurons by Nox is subject to subcellular plasticity in response to Ang II (27). In addition, Ang II increases generation of O₂ ^•− in microglial cells from mesencephalic cultures via activation of NADPH oxidase (70). Both mRNA and protein of major Nox subunits, including gp91^phox, p67^phox, p47^phox, and p40^phox in brain are upregulated by Ang II (6, 22, 60, 64, 108) via protein kinase c (PKC) (6, 22, 86) and tyrosine kinase c-Src (30) pathways. Ang II-induced upregulation of Nox subunits is accompanied by an increase in enzymatic activity of the oxidase through activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) (105), a small GTPase required for assembly and activation of Nox (28), and phosphorylation of p47^phox (6), leading to generation of ROS in SFO, PVN, NTS, and rostral ventrolateral medulla (RVLM) (6, 8, 12, 22, 23, 30, 40, 64, 65, 80, 86, 105).

Mitochondrial ETC

Because of the indispensible dependence of neurons on ATP supply via aerobic metabolism, mitochondrial respiration is the major source of O₂ ^•− in the central nervous system (13). During aerobic metabolism, the oxidoreduction energy of mitochondrial electron transport is converted into the high-energy phosphate bond of ATP via a multicomponent NADH dehydrogenase complex in the inner membrane (47). Molecular oxygen is the final electron acceptor for Complex IV and is ultimately reduced to water. However, a small quantity of oxygen may be incompletely reduced, and the leakage of single electron from the ETC causes the reduction of oxygen to O₂ ^•− in the mitochondria. A majority of the O₂ ^•− is generated in Complexes I and III of the ETC (84). Electrons derived from the substrate of Complex II can also undergo reverse transport into Complex I and generate O₂ ^•−(44).

In contrast to the vast amount of reports on vascular smooth muscle cells and endothelial cells, evidence on generation of mitochondrion-derived ROS in brain by Ang II has emerged only recently. In RVLM, a brain stem site that maintains sympathetic vasomotor activity, Ang II reduces enzyme activities of Complexes I, II, and III in the mitochondrial ETC, and suppresses the electron transport capacity between Complexes I and III and Complexes II and III, resulting in an increase in the mitochondrial H₂O₂ level (11). Generation of mitochondrial ROS by Ang II is further confirmed in differentiated PC-12 cells and catecholaminergic CATH.a neurons (58, 96). In cultured dopaminergic neurons, Ang II decreases mitochondrial inner membrane potential and increases mitochondrial ROS production (69). A reversal of Ang II-induced ROS production by pretreatment with a mitochondrial ATP-sensitive potassium channel blocker suggests that activation of these channels may contribute to mitochondrial ROS production by Ang II (69).

Proinflammatory cytokines

It is well recognized that Ang II upregulates expression of adhesion molecules, cytokines, or chemokines and exerts proinflammatory effects on leucocytes, endothelial cells, and vascular smooth muscle cells (4, 36, 76, 93). Several recent studies unveiled a strong relationship between brain inflammation and generation of ROS by Ang II. Chronic Ang II infusion increases the number of activated microglial cells, and upregulates the expression of proinflammatory cytokines, including interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in PVN (73) and RVLM (Fig. 1). These responses are accompanied by an increase in the tissue level of O₂ ^•− (38), which is alleviated by blockade of proinflammatory cytokines (38, 39). The origin of those proinflammatory cytokines at least includes endothelial cells and perivascular macrophages in the cerebral microvasculature (72, 97, 98). On the other hand, other recent studies (37, 102) reported that ROS triggered by Ang II induces inflammation in brain. Ang II infusion leads to greater immune–endothelial interaction and higher blood–brain barrier permeability in cerebral microvasculature, which are attenuated by treatment with the O₂ ^•− scavenger, tempol (102). Together, a new integrative concept has emerged that depicts an interplay between inflammation and oxidative stress that converges on excessive ROS production in brain by Ang II (Fig. 2).

FIG. 1.

Increase in proinflammatory cytokine expression in rostral ventrolateral medulla (RVLM) by peripheral Ang II. The expression of proinflammatory cytokines, including interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α), in the RVLM is significantly increased in rats subjected to intraperitoneal infusion of Ang II. These induced increase in proinflammatory cytokines can be prevented when the cyclo-oxygenase inhibitor, NS398, is infused into the forth ventricle. *p<0.05 versus saline group, #p<0.05 versus Ang II group.

FIG. 2.

Suggested vicious cycle of interplay between oxidative stress and neuroinflammation to produce excessive ROS in response to Ang II. Activation of Nox and inhibition of mitochondrial ETC by Ang II result in oxidative stress and redox-dependent neuroinflammation. Ang II, at the same time, may induce microvascular inflammation, activation of microglia and production of cytokines to elicit neuroinflammation, which in turn induces oxidative stress. The resultant vicious cycle accounts for the excessive production of ROS in brain by Ang II. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

Interplay between Nox and mitochondria in ROS generation

A feed forward mechanism that engages interplay between Nox and mitochondria to produce excessive ROS in brain by Ang II has been recently put forward (9, 11, 58). According to this mechanism, Ang II increases intracellular Ca²⁺ concentration via activation of Nox, and an increase in mitochondrial Ca²⁺ uptake leads to mitochondrial ROS production (Fig. 3). Given as evidence is the observation that gene knockdown of p22^phox subunit of Nox attenuates Ang II-induced O₂ ^•− and H₂O₂ production in the mitochondrial fractions from RVLM via a redox-dependent impairment of mitochondrial ETC complexes (11). Similarly, gene transfer with dominant negative Rac1 reduces the mitochondrial ROS level (58). Mitochondrial accumulation of Ca²⁺ produces ROS in several cell systems, including neurons (32). Ang II increases intracellular Ca²⁺ concentration via Nox-derived ROS (107) by a mechanism that involves reversible thiol oxidation of the cysteine residues present on ion channels and transporters. Using p-trifluoromethoxycarbonylcyanide phenylhydrazone to prevent Ca²⁺ uptake into the mitochondria, Nozoe et al. (58) demonstrated that Ca²⁺ may function as an interposing mediator for Ang II-elicited mitochondrial ROS production. In addition, an excessive production of cellular ROS may damage mitochondrial DNA, and the resultant impairment in the synthesis of some components of the mitochondrial ETC leads to further ROS production (68). Whether mitochondrial ROS activates cytoplasmic Nox to constitute a vicious cycle in maintaining chronic oxidative stress in brain following Ang II stimulation, however, awaits further delineation. Once identified, it would establish a signaling cascade involving ROS generators in separate compartments that amplify the initiating signal by Ang II across subcellular domains. This vicious cycle of interplay between the Nox and mitochondria in ROS production was recently proposed for the manifestation of chronic oxidative stress induced by Ang II in vascular endothelial cells (18).

FIG. 3.

Proposed scheme of interplay between NADPH oxidase (Nox) and the mitochondrial electronic transport chain (ETC) in the generation of reactive oxygen species (ROS) in brain by Ang II. Angiotensin II (Ang II) acts on angiotensin type 1 receptors (AT₁R) to activate Nox via phosphorylation of protein kinase c (PKC) and c-Src tyrosine kinase (c-Src), resulting in an increase in ROS production and redox-sensitive increase in intracellular Ca²⁺ concentration. Uptake of Ca²⁺ by the mitochondria and inhibition of ETC enzyme complexes by ROS lead to mitochondrial ROS production. Rac-1, Ras-related C3 botulinum toxin substrate 1. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

ROS in Brain and Ang II-Associated Cardiovascular Diseases

An engagement of brain ROS in the pathogenesis of hypertension and heart failure triggered by Ang II was supported by studies (6, 20, 38, 42, 60, 81) showing that prevention of Ang II-induced cardiovascular responses after central application of superoxide dismutase (SOD) mimetics or O₂ ^·− scavengers. A detailed study that deciphers ROS derived from different cellular compartments in brain Ang II-induced cardiovascular effects was first reported by Zimmerman et al. (106), who demonstrated that overexpression of mitochondrial manganese SOD (MnSOD or SOD2) and cytosolic copper–zinc SOD (CuZnSOD or SOD1), virtually abolishes central Ang II-induced pressor response, but not the effects of another central pressor agent, carbachol. Although SOD1 may also be present in liver mitochondria (59), the role of mitochondrial and extramitochondrial ROS in brain on cardiovascular responses to Ang II is largely supported by the study. Following this pioneer work, tremendous progress has been made during the last 9 years to further characterize the role of ROS derived from Nox and mitochondria in the pathogenesis of brain Ang II-dependent hypertension and deleterious sympathoexcitation associated with heart failure.

Nox-derived ROS

A role for Nox-derived ROS in brain in Ang II-induced hypertension and heart failure is derived mainly from three lines of evidence using pharmacological tools, gene manipulation, and RNA silencing to dissect the system. The three most often used pharmacological agents to inhibit enzymatic activity of the Nox are enzyme inhibitor diphenyleneiodonium chloride, Nox assembly inhibitor apocynin, and Nox peptide inhibitor gp91ds-tat. These inhibitors, when applied to the lateral ventricle (5, 22, 38 –40, 52, 60, 62, 105) or microinjected directly into SFO (94), PVN (60, 103), NTS (86), or RVLM (6, 51, 60), attenuate ROS production, hypertension, sympathoexcitation associated with chronic heart failure, and baroreflex impairment, all of which are attributable to the elevated central Ang II. A causal role for ROS derived from Nox in the pathogenesis of hypertension and heart failure is further consolidated by compelling data derived from studies that used genetically modified mice that lack components of the Nox (24, 46, 50), or by gene knockdown with antisense (6, 9, 12), or dominant negative mutant of the Nox subunits (105). Recently, adenoviral vectors expressing small-interfering RNA to silence Nox2 (AdsiNox2) or Nox4 (AdsiNox4) expression in brain have been developed (33, 65). Using this technique, mice treated with AdsiNox4 in PVN showed significant diminishment in sympathetic outflow and marked improvement in cardiac function in myocardial infarction-induced heart failure (33). In addition, both members of Nox enzymes in SFO are required for the full vasopressor effects of brain Ang II, but only Nox2 is coupled to Ang II-induced water intake (65). These results revealed for the first time differential involvements of Nox subunits in different central effects of Ang II.

Mitochondrion-derived ROS

Brain is uniquely vulnerable to oxidative stress-induced damage due to a large quantity of mitochondria. The involvement of mitochondrion-produced ROS in modulating central Ang II-induced cardiovascular responses was first reported in a study showing that overexpression of mitochondrial SOD2 in SFO of mice markedly attenuates acute, central Ang II-induced pressor responses (106). Similarly, SOD2 overexpression in RVLM inhibits cardiovascular responses to microinjection of Ang II to this nucleus (7, 58) or reduces sympathoexcitation and hypertension in spontaneously hypertensive rats (SHR) caused by elevated Ang II (7). Furthermore, preservation of the mitochondrial electron transport capacity in RVLM with a highly mobile electron carrier, coenzyme Q₁₀, reduces arterial pressure in SHR and attenuates the pressor response of normotensive Wistar-Kyoto rats to Ang II infusion (11). Long-term treatment with AT₁R blocker losartan improves mitochondrial ETC function and coenzyme Q content in brain mitochondria and prevents brain dysfunction in hypertension (77). The Ang II-induced hypertension is also attenuated in transgenic mice overexpressing mitochondrial SOD2 (18). In addition, decreased mitochondrial SOD2 levels in RVLM are observed in rabbits with chronic heart failure (21). Together, these data indicate that mitochondrion-produced O₂ ^·− is involved in the pathogenesis of hypertension and heart failure associated with increased brain angiotensinergic signaling.

Although many of the cardiovascular effects of Ang II appear to require Nox- and mitochondria-derived O₂ ^·−, some are specific for H₂O₂. In this regard, administration of the H₂O₂-scavenging enzyme catalase into RVLM reduces the elevated ROS level in stroke-prone SHR (42). In addition, catalase expression in RVLM of SHR is decreased, and transfection of adenoviral vectors expressing SOD1, SOD2, or catalase results in similar degrees of prolonged antihypertension (7), implicating the involvement of H₂O₂ in the response. By contrast, CuZnSOD-produced H₂O₂, at least in PVN, is not involved in the manifestation of sympathoexcitation associated with myocardial infarction-induced heart failure (33). As pointed out by Zimmerman (104), since catalase is localized primarily to peroxisomes, scavenging H₂O₂ by this antioxidant may not affect the hydroxyl radicals produced in other subcellular compartments. Additional studies using H₂O₂-scavenging enzymes, for example, glutathione peroxidases and peroxiredoxins present in other subcellular locations, are therefore required to decipher the role of H₂O₂ in central Ang II-mediated cardiovascular responses.

Brain Redox-Sensitive Signaling Pathways Underlie Ang II-Induced Cardiovascular Responses

Ang II acts in brain to contribute to hypertension and heart failure via activation of sympathetic activity. Although it is generally well accepted that the increased generation of cellular ROS is critical in Ang II-induced sympathoexcitation, the precise redox-sensitive signaling cascades mediating ROS-induced sympathetic overexcitation in brain are still unsettled. Growing evidence suggests that Ang II-induced sympathoexcitation is mediated via cellular repertories that involve redox-dependent activation of ion channels, protein kinases, or transcription factors.

Ion channels

Ca²⁺ and K⁺ channels are two ion channels that have been implicated in a redox-sensitive increase in neuronal excitability after Ang II stimulation. Ang II in brain enhances neuronal activation by increasing voltage-gated Ca²⁺ current (87, 107) and inhibiting K⁺ current, in particular the delayed rectifier K⁺ current (78, 79), resulting in an increase in neuronal discharge rate in SFO, PVN, or RVLM, and overexcitation of sympathetic outflow commonly associated with hypertension and heart failure. Microinjection of a voltage-gated K⁺ channel blocker, 4-aminopyridine, into RVLM evokes sympathoexcitation and hypertension in rats with chronic heart failure (20). Evidence from both in vivo and in vitro (primary neurons isolated from rat hypothalamus and brain stem or a catecholaminergic neuronal cell line, CATH.a neurons) studies further suggests that Ang II-mediated modulation of Ca²⁺ and K⁺ currents involves ROS derived from Nox (20, 78) and mitochondria (96), as well as activation of redox-sensitive protein kinase Ca²⁺/calmodulin kinase II (CaMKII) (79, 96). Recently, Ang II-induced reduction of an inward rectifier current in the small conductance Ca²⁺-activated K⁺ (SK) channels was reported to play a significant role in increasing neuronal excitability of a group of sympathetic activity-related and RVLM-projecting neurons in PVN (14). Whether modulation of SK channel activity by Ang II depends on brain ROS signaling, however, awaits further delineation. The modes of action via which ROS modulates channel activity, be it a direct oxidation of amino acid residues in channel proteins or indirect modulation of protein kinases that control gating property of the channels, also remain to be determined.

Mitogen-activated protein kinase

The mitogen-activated protein kinase (MAPK) family represents a major class of redox-regulated signaling molecules in the cardiovascular system. The significance of redox-sensitive MAPKs signaling in the pathogenesis of hypertension and heart failure associated with heightened Ang II has been extensively reviewed (41, 57, 101). This review will focus on the role of redox activation of MAPKs in two Ang II-sensitive brain regions, namely, RVLM and PVN, in neural mechanisms of hypertension and heart failure.

In RVLM, the activities of MAPKs, in particular p38 MAPK and extracellular signal-regulated kinase (ERK), are potentiated by Ang II via processes that involve AT₁R-depedent activation of PKC-Nox-ROS signaling cascade (6, 8). Functionally, redox activation of p38MAPK and ERK in RVLM differentially participates in Ang II-induced hypertension (Fig. 4). Whereas ROS-p38 MAPK signaling mediates the short-term pressor response of Ang II by enhancing glutamatergic neurotransmission in RVLM (6), ROS-dependent ERK phosphorylation leads to long-term pressor response to Ang II via transcriptional upregulation of AT₁R mRNA expression (8). These findings, at the same time, suggest that Ang II upregulates mRNA expression of its own receptor in the RVLM by a redox-sensitive ERK signaling. The AT₁R-Ras-ROS-p38 MAPK/ERK pathway in RVLM is related to manifestation of hypertension phenotype in stroke-prone SHR (43). In addition, the Ang II-induced downregulation of voltage-gated K⁺ channel expression in CATH.a neurons is mediated by AT₁R-ROS-p38 MAPK signaling (20). In PVN, Ang II triggers phosphorylation of p38 MAPK, ERK, and c-Jun N-terminal kinase (JNK) (88, 90). Both AT₁R-ROS-ERK and AT₁R-ROS-JNK, but not p38 MAPK, signaling contribute to upregulation of AT₁-R (88, 89) and sympathoexcitation (89) in rats with heart failure. Intriguingly, ROS-MAPK signaling participates in Ang II-induced upregulation of AT₁-R mRNA expression in RVLM and PVN, and mediates sympathoexcitation associated with Ang II-dependent hypertension and heart failure. This redox-sensitive MAPK signaling cascade may likely to be a common denominator in the Ang II-sensitive brain regions for the pathogenesis of cardiovascular diseases, including hypertension and heart failure.

FIG. 4.

Potential signaling pathways in RVLM mediating short-term and long-term pressor response to Ang II. Ang II binds to the AT₁R to induce superoxide (O₂ ^•−) production via activation of Nox or inhibition of mitochondrial electron transport enzyme complexes in RVLM. Ang II-induced O₂ ^•− mediates short-term pressor response by phosphorylating p38 mitogen-activated protein kinase (p38 MAPK) to increase glutamatergic neurotransmission. O₂ ^•− may mediate long-term pressor effect of Ang II via phosphorylation of extracellular signal-regulated kinase (ERK), which in turn upregulates AT₁R mRNA expression via activation of the transcription factor, c-Fos. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

In addition to MAPK members, the activity of serine/threonine protein kinases Akt and Rho has been reported to be redox sensitive and to play an active role in the risk of cardiovascular disorders (29, 56, 82). There is little evidence on Akt and Rho in the brain on hypertension (31, 85) and sympathoexcitation associated with heart failure (21). It will thus be of interest to investigate the role of brain ROS in Akt and Rho signaling for a better understanding of redox-sensitive cellular signaling in Ang II-stimulated neurocardiovascular disorders. We (Chan, unpublished data) recently found that in SHR, transfection into RVLM of adenoviral vectors expressing SOD1 or SOD2 suppressed heightened phosphorylated status of Akt, indicating that both mitochondrial and cytosolic ROS may modulate Akt activity under hypertensive condition (Fig. 5).

FIG. 5.

Redox-dependent phosphorylation of Akt in RVLM by Ang II. Representative gels demonstrating phosphorylation of serine/threonine protein kinases (Akt) at both the serine 473 and threonine 308 residues 30 min after microinjection of Ang II bilaterally into RVLM. Ang II-induced Akt phosphorylation was inhibited by microinjection into the bilateral RVLM of adenoviral vectors encoding superoxide dismutase 1 (AdSOD1) and SOD2 (AdSOD2).

Transcription factors

Transcription factors that are involved in Ang II-induced cardiovascular phenotypes and whose expression is redox sensitive may at least include NF-κB, activator protein-1 (AP-1), and c-Fos. In PVN, the AT₁R-dependent increase in O₂ ^·− production contributes to NF-κB activation and neurohumoral excitation in Ang II-induced hypertension (38). In catecholaminergic CATH.a neurons, activation and nuclear translocation of NF-κB mediate AT₁R upregulation in response to Ang II stimulation (55). In RVLM, redox-sensitive upregulation of AP-1, c-jun, or c-fos were observed in animals with hypertension (8) or chronic heart failure (48, 49), along with an increase in DNA binding activity of AP-1 in RVLM (108). Together with the demonstration of an Ang II-Nox signaling in the regulation of AT₁R expression (8, 23), these data suggest that a positive feedback mechanism involving redox-sensitive activation of transcription factor expression and DNA binding contributes to the pathological consequences of AT₁R upregulation in heart failure (108).

Brain ROS As Therapeutic Target Against Ang II-Induced Hypertension and Heart Failure

The growing body of evidence on a pivotal role for ROS in brain in the pathogenesis of hypertension and heart failure prompts the use of antioxidants as a therapeutic target in the treatment of cardiovascular diseases associated with aberrant Ang II signaling in brain. In principle, any component of the highly elaborated antioxidant system that modulates the level of ROS, comprising both enzymatic ROS scavengers, such as SOD, catalase, glutathione peroxidase, thioredoxin, glutaredoxin, peroxiredoxin, heme oxygenase, and paraoxonase, and non-enzymatic ROS scavengers, such as glutathione, vitamins, lipoate, urate, and ubiquinone (99), is a potential target.

SODs are by far the most studied antioxidants for the treatment of hypertension and heart failure that are associated with enhanced redox-sensitive Ang II signaling in brain. Scavenging O₂ ^·− with the SOD mimetic, tempol, administered either to the cerebral ventricle (5, 38, 49, 63, 103) or directly to Ang II-sensitive brain sites such as RVLM (6, 7, 42, 51, 52, 60), ameliorates oxidative stress and attenuates pressor responses and sympathoexcitation associated with elevated brain Ang II. This antioxidant, when intravenously injected (91) or orally administered in drinking water (74), is also effective in reducing neuronal activity of presympathetic neurons in PVN and RVLM (91), and normalizing the increased sympathetic activity in chronic heart failure (74). These results suggest that tempol could readily cross the blood–brain barrier to exert antioxidant effects in brain. Other O₂ ^·− scavengers, including tiron (52) and Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP; 63, 81), as well as the hydroxyl radical scavenger (83), edarovone, have also been reported to be beneficial for brain Ang II-associated cardiovascular dysfunctions.

A large collection of literature over the past decade has clearly validated transfection of viral vectors carrying sod genes as another effective approach to increase the expression and enzyme activity of SOD in brain. This method has been used to overexpress SOD1 (7, 33), SOD2 (6, 8, 106, 105), and extracellular SOD (EcSOD or SOD3) (105) in various Ang II-sensitive sites to protect against oxidative stress-associated hypertension and heart failure. Ang II-induced hypertension is also significantly attenuated in mice overexpressed of human thioredoxin 2, a mitochondria-specific antioxidant enzyme (92) or in rats with overexpression of human catalase (7). Although adenoviral vector-mediated gene transfection may seem to be an excellent means to increase antioxidant activity in brain, the elevated potential for inflammatory response, toxicity (17), and the inability of viral vectors to target the CNS after peripheral administration (15) hinder the therapeutic impact of viral vector-mediated SOD treatment on cardiovascular diseases associated with heightened brain Ang II signaling.

Alternatively, nanotechnology-driven delivery has received great interest in recent years because nanocarriers such as liposomes or polymers that entrap, encapsulate, or bind to therapeutic agents, including proteins, DNA, or drugs, can penetrate physiological barriers, prolong presence in circulation by avoiding renal clearance, reduce the frequency of administration by releasing drugs in a sustained manner, and minimize systemic adverse effects by delivering drugs preferentially to target tissues (25). SOD1 conjugated with poly(ethyl oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymer (95) or electrostatically bound to a synthetic poly(ethyleneimine)-poly(ethyleneglycol) polymer to form a polyion complex (SOD1 nanozyme) (71) has recently been developed. These nanoformulated SOD1 conjugates have been demonstrated to penetrate neuronal cell membrane to elevate SOD1 function (95). Moreover, an intracarotid injection of SOD1 nanozyme into rabbits significantly attenuates a central Ang II-induced pressor response (71). Nanoformulated conjugation may thus be a new delivery system for antioxidants into neurons and is therapeutically beneficial for brain Ang II-related cardiovascular diseases.

The mitochondrial uncoupling protein (UCP) was identified recently (9) as an important endogenous natural antioxidant in the regulation of ROS homeostasis. UCP in the inner membrane of the mitochondria mitigates ROS production by reducing the transmembrane potential across the mitochondrial inner membrane (1). This antioxidant, whose transcription is activated by Ang II via cellular events that involve Nox-ROS-p38 MAPK signaling cascade, plays an active role in the feedback inhibition of ROS produced by Ang II in RVLM (Fig. 6). Microinjection into RVLM (9) or oral intake (12) of rosiglitazone, an activator of the transcription factor peroxisome proliferator-activated receptor gamma, upregulates UCP2 expression to protect RVLM from oxidative stress and Ang II-dependent hypertension. Intriguingly, this natural mitochondrial antioxidant also plays an active role in the feedback regulation of Ang II-induced ROS production via Nox-dependent transcriptional upregulation of brain-derived neurotrophic factor for protection against Ang II-induced long-term pressor response (10).

FIG. 6.

Schematic diagram illustrating intracellular signaling cascade that mediates feedback maintenance of ROS homeostasis via transcriptional upregulation of mitochondrial uncoupling protein 2 (UCP2) in RVLM during oxidative stress. Ang II binds to the AT₁R to induce O₂ ^•− production via activation of Nox. O₂ ^•− in turn induces transcriptional upregulation of mitochondrial UCP2 via phosphorylation of p38 MAPK. UCP2 then exerts feedback inhibition on oxidative stress and redox-associated pressor response induced by Ang II. APPRE, PPAR-responsive element. Reproduced by permission from Chan et al. (9). (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

Despite the exciting results from animal models on the beneficial effects against Ang II-associated cardiovascular disorders by targeting brain ROS, clinical trials designed to test the therapeutic efficacy of antioxidants, in particular vitamins C and E, in cardiovascular diseases have not been successful (3, 53, 67). Moreover, there is yet no current clinical trial designed to test the therapeutic efficacy of antioxidants against brain Ang II-related cardiovascular diseases. A few hypotheses have been put forward (3, 75, 104) to rationalize the failure of antioxidant therapy in cardiovascular diseases. First, there are limitations for the antioxidants to reach the target tissue, cells, or subcellular compartments where ROS is being primarily generated. Second, insufficient presence of antioxidants at the target sites prevent optimal therapeutic efficacy in terms of scavenging the ROS. Third, there are difficulties with the specificity of the antioxidants to scavenge the particular ROS that drives the pathophysiological condition. Fourth, there is a shortage of the therapeutic window for the antioxidants to scavenge ROS generated through multiple feedback mechanisms. Based on these concerns, the small molecule- or nanoformulated conjugate-based antioxidant therapy will thus hold promise for future clinical trials because they could posses specific ROS scavenging activities and may be designed to target ROS generation in specific tissue, cells, and even subcellular compartments (104).

Summary and Conclusion

A large body in the literature during the last decade has clearly identified that production of ROS, in particular O₂ ^•− and H₂O₂, and activation of redox-sensitive cellular events as important signaling intermediates that lead to an increase in neuronal activity and elevated sympathetic output in brain Ang II-dependent cardiovascular diseases. ROS in brain therefore presents itself as an important target for therapeutic management of these diseases. Future studies are needed to fully elucidate intra-neuronal signaling and the downstream redox-sensitive proteins involved in controlling neuronal discharge rate, sympathetic outflow, and the pathogenesis of cardiovascular diseases, including hypertension and heart failure. Moreover, the cross talk between Ang II-induced oxidative stress and neuroinflammation in neural mechanisms of cardiovascular diseases also warrants further elucidation. Perhaps more importantly, additional studies are needed to identify new redox-based therapeutics that work not only in animal models, but also in patients suffering from these prevalent diseases.

Footnotes

Acknowledgments

This work was supported in part by a research grant from the National Science Council, Taiwan (NSC97-2320-B-075B-002-MY3 to J.Y.H.C).

Abbreviations Used

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