OXIDATIVE STRESS AND HYPERTENSION

Abstract

A link between oxidative stress and hypertension has been firmly established in multiple animal models of hypertension, but remains elusive in humans. While initial studies focused on inactivation of nitric oxide by superoxide, our understanding of relevant reactive oxygen species (ROS: superoxide, hydrogen peroxide, peroxynitrite) and how they modify complex signaling pathways to promote hypertension has expanded significantly. In this review, we summarize recent advances in delineating the primary and secondary sources of ROS (NADPH oxidases (Nox), uncoupled endothelial nitric oxide synthase, endoplasmic reticulum and mitochondria), the post-translational oxidative modifications they induce on protein targets important for redox signaling, their interplay with endogenous antioxidant systems, and the role of inflammasome activation and endoplasmic reticular stress in the development of hypertension. We highlight how oxidative stress in different organ systems contributes to hypertension, describe new animal models that have clarified the importance of specific proteins and discuss clinical studies that shed light on how these processes and pathways are altered in human hypertension. Finally, we focus on the promise of redox proteomics and systems biology to help us fully understand the relationship between ROS and hypertension and their potential for designing and evaluating novel antihypertensive therapies.

INTRODUCTION

Multiple regulatory systems involving the heart, vessels, kidneys, brain and immune cells, underpin the pathophysiology of hypertension, and oxidative stress has been considered as a unifying factor linking these elements (1). The oxidative stress theory of disease is based on the premise that increased bioavailability of reactive oxygen species (ROS), comprising free radicals and non-free radicals, adversely affects cellular macromolecules, such as RNA, DNA, proteins, lipids and carbohydrates causing cell injury and death (2). As a protective mechanism, cellular antioxidant defense systems (superoxide dismutase (SOD), peroxidases, antioxidant vitamins) have evolved, which maintain oxidation-reduction (redox) status by preventing ROS accumulation. Oxidative stress was originally defined as ‘a disturbance in the prooxidant:antioxidant balance in favor of the former’, with the notion that free radicals are injurious species (3). However, following discoveries in the 1980s that i) ROS are critically involved in host:defense responses (4), ii) endothelial-derived nitric oxide (NO) is an endothelium-derived relaxing factor (EDRF) that controls vasorelaxation (5), iii) ROS generation is controlled by ‘professional oxidases’ [nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NADPH oxidases, or Noxes)] (6), and the more recent findings that levels of ROS are influenced by hormones and other factors (7–9), it became clear that cytosolic ROS are highly regulated active participants in diverse cellular processes contributing to normal signaling, cellular homeostasis and biological functions of living organisms (10,11). This led to a redefinition of ‘oxidative stress’ to incorporate the importance of redox signaling, which is now described as ‘an imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage’ (12). This is the definition used in the present review.

The significance of oxidative stress in the context of hypertension relates to the fundamental role of ROS and redox signaling in molecular, cellular and systems processes that cause endothelial damage, vascular dysfunction, cardiovascular remodeling, renal dysfunction, sympathetic nervous system excitation, immune cell activation and systemic inflammation, important in the pathophysiology of hypertension (13–16). Redox signaling is the specific oxidation-reduction post-translational modification of downstream signaling molecules induced by free radicals, such as superoxide anion (O₂⁻) and NO, and non-free radicals, such as hydrogen peroxide (H₂O₂) (17,18). Oxidative posttranslational modification (Ox-PTM) of proteins is a key molecular mechanism that controls proteins, which ultimately influence cellular biological responses (19). Normally, proteins are targets of reversible Ox-PTM, while in pathological conditions associated with oxidative stress, such as in hypertension, proteins undergo irreversible Ox-PTM leading to loss of protein function and consequent cell damage, tissue injury and target organ failure (20,21). Advances in oxidative proteomics have provided new insights on the role of redox biology in cardiovascular pathophysiology with recent studies focusing on cysteine (Cys) oxidation of proteins involved in cell proliferation, inflammation, contraction/relaxation, cytoskeletal organization and apoptosis (22,23).

Primary enzymatic sources of cardiovascular ROS are non-phagocytic Noxes, which are variably upregulated in hypertension (24–26). Emerging evidence indicates that in addition to Nox hyperactivation, nitric oxide synthase (NOS) uncoupling, endoplasmic reticulum (ER) stress and mitochondrial oxidative stress contribute to redox changes in hypertension (27–29). These processes are themselves redox-sensitive and may be considered as secondary mechanisms involved in oxidative stress in hypertension. Activation of eNOS typically generates NO, however in an oxidized milieu, NOS becomes uncoupled to generate vasoinjurious O₂⁻ instead of vasoprotective NO (30).

ER stress and the unfolded protein response (UPR) arise as a consequence of accumulation of misfolded proteins. These processes adversely affect cellular homeostasis, intracellular signaling and cell morphology leading to tissue injury (31). ER stress has been described as a novel regulator of cardiovascular disease since it plays a role in vascular cell phenotypic switching, dedifferentiation, calcification and apoptosis and contributes to endothelial dysfunction and vascular remodeling in hypertension and atherosclerosis (32,33). ROS-regulated ER stress may be an important process in hypertension-associated cardiovascular dysfunction and target organ damage (30,34). This is supported by studies showing that inhibition of ER stress protects against hypertension-induced vascular dysfunction (35), blunts development of hypertension in spontaneously hypertensive rats (SHR) (36,37), prevents cardiac impairment and hypertrophy in experimental models of hypertension and pulmonary arterial hypertension (38,39) and underlies increased angiotensin II (Ang II) signaling and sympathetic outflow (40,41).

Mitochondrial oxidative stress, which is associated with mitochondrial dysfunction and redox inactivation of mitochondrial deacetylase, Silent mating type information regulation 2 homolog 3 [Sirtuin 3 (SIRT3)], and the key mitochondrial antioxidant SOD2, may also contribute to the development of hypertension (42). These processes, which themselves cause oxidative stress, promote vascular dysfunction, cardiac fibrosis and blood pressure elevation in experimental models (43). Clinical studies support the potential role of mitochondrial oxidative stress because Sirt3 is downregulated in patients with essential hypertension (44). These findings have sparked interest in developing mitochondrial targeted interventions as vasoprotective and antihypertensive strategies (45). In particular scavenging of mitochondrial lipid peroxidation products (isolevuglandins) attenuates development of hypertension in Ang II-infused mice (46) and mitoQ, a mitochondrial-targeted SOD mimetic, ameliorates endothelial dysfunction, inhibits ROS generation and decreases blood pressure in models of hypertension (47). Similar favorable vascular responses have been demonstrated in humans treated with MitoQ, a mitochondrial antioxidant (48).

Extensive pre-clinical studies in experimental models demonstrate unambiguously that redox signaling is critically involved in cardiovascular pathophysiology and that oxidative stress is causally linked to the development of hypertension (49–51). However, this has not translated to the clinic where it has been difficult to demonstrate an etiological basis for ROS in blood pressure elevation. This may relate, in part, to a paucity of information on ROS biology in human tissue, sub-optimal methods to measure ROS in the clinical setting and inappropriately designed clinical trials to evaluate the antihypertensive effects of antioxidant therapies. Nonetheless, many clinical studies have demonstrated positive associations between systemic biomarkers of oxidative stress and blood pressure and antioxidant capacity has been shown to be reduced in hypertension (52–54).

The present review provides an up-to-date appraisal of the biology of ROS and oxidative stress in the pathogenesis of hypertension with a particular focus on emerging new concepts. Specifically we discuss i) primary (Noxes) and secondary mechanisms (NOS uncoupling, ER stress, mitochondrial stress) of ROS generation, ii) redox signaling, the ROS-regulated inflammasome and oxidative proteomics in hypertension, and iii) systems basis of oxidative stress in hypertension. Finally we provide some perspectives of the role of oxidative stress in human hypertension.

REACTIVE OXYGEN SPECIES OF RELEVANCE IN HYPERTENSION

ROS are produced as metabolic by-products of respiration, but also intentionally by enzymatic reactions, such as that catalyzed by Noxes. Over the last 30 years, their role as signaling molecules in regulating physiological functions has been well established. However, excess production of ROS or failure of antioxidant defenses in specific subcellular compartments or whole tissue results in pathological signaling as well as protein, lipid and DNA damage (24–26,55). Here we concentrate on those ROS that are intimately involved in the pathogenesis of hypertension: O₂⁻, NO, peroxynitrite (OONO⁻) and H₂O₂ (56,57). Superoxide has a very short half-life (milliseconds at neutral pH) and can react rapidly with NO to form OONO⁻ or is quickly converted to H₂O₂. While NO is vasoprotective and is an important vasodilator, OONO⁻ contributes to tissue damage and H₂O₂ reacts with various cellular proteins to modify signaling pathways, including transcription factors, protein phosphatases, kinases, ion channels and small molecular weight G-proteins.

PRIMARY MECHANISMS OF ROS GENERATION IN HYPERTENSION

One of the most important enzymatic sources of O₂⁻ and H₂O₂ in the cardiovascular system is Nox, a family of transmembrane proteins that transfer electrons across membranes (24–26,58,59) (Figure 1). Noxes catalyze the reduction of O₂ to produce O₂⁻ in an NADPH-dependent manner [NADPH + 2O₂→NADP⁺ + H⁺ + 2O₂⁻], which in turn dismutates to generate H₂O₂ (spontaneously or catalysed by SOD) (58). This cascade of reactions leads to generation of secondary ROS including the reaction of O₂⁻ with NO to form ONOO−, iron-catalysed Fenton reaction to produce hydroxyl radical (OH−), and peroxidase-catalysed generation of hypochlorous acid (HOCl). During activation, the electron donor NADPH provides two electrons that are transported across membranes to reduce O₂ to O₂⁻, with associated production of two protons that influence intracellular pH (60). Accordingly Noxes may have two functions, with the primary role the relay of electrons to generate ROS and secondly the production and transport of protons across membranes (58–60).

Figure 1. Sources of ROS in the cardiovascular system.

The major soure of ROS in the cardioavscular system is the Nox family. Nox1–4 are p22phox-dependent oxidases, whereas Nox5, a Ca²⁺-sensitive Nox, does not require p22phox for its activation. In the cardiovascular system, Noxes are regulated by pro-hypertensive and inflammatory factors including Ang II, ET-1, aldosterone, salt, growth factors (VEGF, EGF) and TNF. ROS are also generated by eNOS uncoupling, and mitochondrial and ER mechanisms, which are influenced by Nox/ROS. MR, mineralocorticoid receptor; AT1R, Ang II type 1 receptor; ET_AR, ET-1 type A receptor; GFR, growth factor receptor; BH4, tetrahydrobiopterin.

The notion that Noxes are key players in hypertension was first suggested over 40 years ago in clinical studies when it was shown that antihypertensive drugs mediate effects through coenzyme Q10-NADPH oxidase (61) and that f-Met-Leu-Phe-stimulated NADPH-dependent O₂⁻ production in neutrophils of patients with essential hypertension is almost four-fold higher compared to that in neutrophils of normotensive individuals (62). Using a reverse translational approach, these clinical observations were tested in experimental models and in the 1990s a pivotal role for NADH/NADPH oxidase-generated ROS was demonstrated in Ang II-dependent hypertension (63,64). Since then it has been unambiguously demonstrated that Nox-derived ROS are involved in blood pressure elevation in all commonly studied models of hypertension (24–26,63–67). On the other hand, it has been more challenging to show a causal role for Nox/ROS in human hypertension (68,69)

The Nox family

The Nox family comprises seven isoforms: Nox1–5, duox1/duox2, with each isoform possessing a catalytic core (electron-transporting element), a transmembrane domain and NADPH oxidase subunits (p22phox, p47phox/Nox-activator 1 (NOXA1), p67phox/Nox-organizer 1 (NOXO1), p40phox). Nox1–4 require p22phox and different subunits for their activation, while Nox5 activation is independent of p22phox and NADPH oxidase subunits (70). All Noxes have six conserved predicted transmembrane alpha-helices with heme binding regions and a flavoprotein homology domain on the intracellular C-terminus containing binding sites for FAD and NADPH (70,71). Noxes are regulated by vasoactive agents, growth factors and physical stimuli as well as signaling molecules and localize in subcellular compartments (71–76). Nox1, Nox2, Nox4 and Nox5, which are expressed in cells of the heart, vessels, kidney and brain, play an important role in cardiovascular and cerebrovascular pathophysiology and have been linked to oxidative stress in hypertension (74–76). Many comprehensive reviews have described the role of Nox1, Nox2 and Nox4 in hypertension (77,78) and accordingly here we provide only a brief overview of these. On the other hand Nox5, the most recently characterized Nox, will be discussed in detail as it is emerging as an important mediator of vascular oxidative stress in human hypertension.

p22phox-dependent Noxes (Nox1–4) in the cardiovascular system

Nox1, the first Nox homologue to be discovered, was originally called Mox1 for ‘mitogenic oxidase’ because of its role in cell proliferation and mitogenesis (79). In addition to p22phox, it requires NOXO1 (p67 isoform), NOXA1 (p47phox isoform) and Rac1 for its full activation. It is an important driver of cardiovascular inflammation and fibrosis and inhibitors of Nox1 and Nox1/4 (GKT136901, GKT137831) prevent cardiovascular remodeling in hypertension (80,81).

Nox2 is typically expressed in neutrophils and macrophages, but also in cells of the heart, vessels, kidneys and brain (82). Activated Nox2 associates with membrane-bound p22phox, cytosolic subunits p47phox, p67phox and p40phox, and the small G proteins Rac1/2 (83). In phagocytic cells, Nox2 produces high levels of O₂⁻ in bursts, crucial in host defense responses, while in vascular and non-phagocytic cells O₂⁻ is produced at low levels in a sustained and controlled manner (84).

Nox3 is expressed almost exclusively in the inner ear (85), with little convincing evidence that it is functionally present in the cardiovascular system. However a few studies demonstrated that Nox3 is expressed in endothelial and cardiac cells (86,87).

Nox4 exists as a heterodimer linked to p22phox and does not require cytosolic subunits for its activation. Unlike other Noxes, Nox4 produces O₂⁻ and H₂O₂ and is constitutively active (88). Nox4 activity is directly linked to its abundance, cell localization, intracellular compartmentalization and association with proteins including regulatory protein polymerase delta–interacting protein 2 (poldip2) and tyrosine kinase substrate 5 (Tks5) (89,90). Nox4 is abundant in the kidney and tumour cells and has been considered to be an oncoprotein (91). It is also expressed in other cell types, including cardiomyocytes, endothelial and vascular smooth muscle cells (VSMC), osteoclasts, epithelial cells and hemopoietic stem cells. Nox4 associates with focal adhesions (92,93), is important in cell migration and localizes in the nucleus, mitochondria and ER. In the endothelium, Nox4-derived H₂O₂ has been described as an EDRF involved in vasodilation and in the stressed heart it is cardioprotective (94). Studies in Nox4 knockout and Nox4 over-expressing mice have shown that Nox4 is both cardiovascular protective and injurious (95,96), depending on the experimental model. Suggestions have also been made that Nox4 associates with Nox5, although mechanisms for this are elusive and the pathophysiological significance is unclear (97).

p22phox-independent Noxes in the cardiovascular system – importance of Nox5

Nox5 shares ≈ 50% homology with Nox2 and is unique in that it does not require p22phox or cytosolic NADPH oxidase subunits for its activation (98). Duox1/2 are also p22phox-independent isoforms and are involved in thyroid hormone synthesis (99). Duox1/2 do not appear to be important in cardiovascular pathophysiology although duox-mediated H₂O₂ generation has been described in zebrafish heart (100).

Nox5 is the most recently characterised Nox and currently is the only Nox to be crystalized (99,101). Nox5 has some distinguishing features: i) the rodent genome lacks Nox5 yet it is present in lower forms and mammals, ii) Nox5 generates O₂⁻ from a single gene product, iii) it has a unique N-terminal extension possesing Ca²⁺-binding helix-loop-helix structure domains, iv) Nox5 activation is regulated by changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and undergoes conformational changes, and v) Nox5 is regulated by post-translational modifications, including phosphorylation, oxidation, S-nitrosylation and SUMOylation, but unlike other Noxes is not glycosylated and has been described as a ‘bona fide nonglycoprotein’ (102–104).

Six Nox5 isoforms have been identified [Nox5-α, -β, -γ, -δ, -ε, -ζ] (61). Nox5α, Nox5β and Nox5γ are functionally active and generate ROS (99). Nox5α and Nox5β are the primary isoforms in human cells and are negatively regulated by Nox5ε, which inhibits Nox5-mediated O₂⁻ generation (105). Noxδ, Nox5ε and Noxζ do not produce appreciable amounts of ROS and their functional significance remains unclear. Epigenetic factors including, eg. histone deacetylase 2 (HDAC2), cause upregulation of the Nox5 gene promotor activity in VSMCs (105).

Activation of Nox5 involves Ca²⁺-binding to the EF hand on the N-terminal region (102–104). The [Ca²⁺]_i concentration needed for Nox5 activation is high, and thus additional systems involving regulatory proteins have evolved that increase sensitivity to Ca²⁺ thereby facilitating ROS generation at lower [Ca²⁺]_i. Accordingly, Nox5 can be activated directly by Ca²⁺ or indirectly by interacting with other proteins and kinases (106–110). Nox5 regulatory proteins include PKC, calmodulin, caveolin-1, c-Abl1, c-Src and chaperone molecules (Hsp90, Hsp70). These proteins differentially influence Nox5 and may stabilise the enzyme. For example PKC and calmodulin increase Nox5 sensitivity to Ca²⁺ promoting activation, while interaction with caveolin-1 promotes Nox5 inactivation. Pro-inflammatory transcription factors (NF-κB, AP-1, STAT1/STAT3) have also been shown to regulate Nox5 in human aortic smooth muscle cells.

While Nox1–4 are primarily associated with the cell membrane, Nox5 localizes in intracellular compartments mainly the perinuclear area and ER (99,104,109,111). This is functionally relevant because the ER is a Ca²⁺-rich depot and the site of protein synthesis and post-translational modification. Within cells, Nox5 traffics from intracellular compartments to the cell membrane bringing it into close proximity to regulatory proteins such as PKC and c-Src that influence its activation and downstream signaling (109–113).

Nox5 is ubiquitously expressed in human tissues and is a major ROS-generating enzyme in cells of the cardiovascular system (113,114). Of the vascular isoforms, it is the key Nox responsible for O₂⁻ production in human vessels. It is activated by vasoactive agents (Ang II, endothelin-1 (ET-1)), growth factors (PDGF, EGF) and pro-inflammatory mediators (TGFβ, cytokines) (113–117) and inhibits Nox5-induced signaling (118).

Nox5 is involved in many physiological processes including sperm motility, sperm-oocyte fusion, cell proliferation and cytokine production (113,119). In the vascular system, we identified a key role for Nox5 in redox-sensitive vascular contraction through a feedforward system where Ca²⁺-regulated Nox5 promotes redox-mediated increases in [Ca²⁺]_i that triggers contraction. Increased activation of endothelial cell Nox5 causes cell proliferation and formation of capillary-like structures, important in atherosclerosis and angiogenesis (120). In the heart Nox5 plays a role in the regulation of Ca²⁺-activated K⁺ channels involved in coronary artery contraction (121).

Multiple Nox isoforms are involved in hypertension

The role of Nox-derived ROS in the development of hypertension has been demonstrated primarily in transgenic mouse models where Nox isoforms or NADPH oxidase subunits have been deleted or overexpressed (122) (Table). A direct causal role for Noxes in the development of Ang II-induced hypertension has been demonstrated in mice lacking Nox1, Nox2, Nox4, p47phox or p22phox (122–124). Nox1-deficient mice have reduced vascular O₂⁻ production and blunted pressor responses, whereas in Nox1 overexpressing mice, Ang II-mediated vascular hypertrophy and blood pressure elevation are exaggerated (123,124). However, in a model of chronic Ang II-dependent hypertension (human renin-expressing mice) development of hypertension was independent of Nox1 and Nox2 (125,126). In Ang II-infused mice treated with siRNA targeted to renal p22phox, renal Nox activity was blunted, ROS formation was reduced, and blood pressure elevation was prevented (127). Recent studies have shown that fibroblast Nox2 contributes to vascular remodeling and hypertension through a paracrine effect (128). It should be highlighted that in these Nox knockout or transgenic studies, baseline cardiovascular phenotypes of mice are unexpectedly normal and it is only when challenged, eg. Ang II or salt, that mice exhibit vascular and blood pressure changes.

Table.

Effects of increased activation/expression of Nox isoforms and mitochondria in the pathophysiology of hypertension

Source	Consequence
Nox1	↓endothelial relaxation
	↑vascular remodeling
	↑fibrosis
	↑blood pressure
Nox2	↑inflammation
	↑blood pressure
	↑neuronal excitability
	↑T cell proliferation
Nox4	↑endothelial relaxation
	↓vascular remodeling
	↓or no △ blood pressure oncoprotein renal dysfunction
Nox5 (SMC)	↑vasoconstriction
	↓endothelial relaxation no change in blood pressure in young mice hypertension with aging stroke accelerated vascular remodeling vascular calcification
Nox5 (EC)	↓endothelial relaxation
	↑systolic hypertension hypertensive retinopathy
Mitochondria	↓endothelial relaxation
	↑blood pressure

There is a paucity of information on Nox3 in hypertension, likley because this isoform is not typically expressed in the cardiovascular system. However a few studies suggest a role for Nox3 in hypertension (129–132). In two models of experimental hypertension, brain oxidative stress has been linked to Nox3 upregulation. In the cerebrum of stroke-prone SHR (SHRSP), mRNA and protein expression of Nox3 and ROS production were increased compared with Wistar Kyoto (WKY) rats (129). In the phenol renal injury model of hypertension, oxidative stress-induced activation of the sympathetic nervous system is associated with increased Nox3 expression and oxidative stress in the brain (130). In humans, data from large gene expression studies identified Nox3 as a predictor of future hypertension (131,132). In a case-control study in a Chinese population, pulmonary hypertension was associated with rs6557421 variant in Nox3 (131). In West Africans, a genome-wide search for regions linked to renal dysfunction identified Nox3 (6q25.1-q26) as a potential candidate gene implicated in hypertensive and diabetic nephropathy (132).

Nox4 seems to be especially important in salt-sensitive hypertension. It contributes to renal oxidative stress, kidney injury, hypercontractile vascular responses and blood pressure elevation in Dahl-salt-sensitive rats, DOCA-salt rats, aldosterone-salt rats and salt-fed mice with reduced renal mass (133–136). In these models Nox4-derived H₂O₂ is a major upstream regulator of mammalian target of rapamycin complex 1 (mTORC1), which contributes to salt-induced renal injury and hypertension (133). Underlying mechanisms may involve Nox4-dependent maladaptive upregulation of ENaC-regulated sodium reabsorption in the distal nephron. The importance of Nox4 in salt sensitive hypertension is supported by studies in Nox4-knockout Dahl salt-sensitive rats where renal injury and blood pressure elevation were blunted through processes that normalize renal mitochondrial bioenergetics and decreased mitochondrial ROS production (134).

Treatment of hypertensive mice with agents that inhibit Nox1, Nox2 and Nox4, such as apocynin, diphenylene iodinium, gp91ds-tat, and GKT137831, improved vascular function, normalized blood pressure and improved hypertensive cardiac remodelling, supporting the link between Nox activation, oxidative stress and hypertension (136).

Because rodents lack Nox5 it has been challenging to study Nox5 in traditional mouse or rat models of hypertension. To address this, Nox5 knock-in mice have been generated. Mice expressing human Nox5 in a podocyte-specific manner exhibited podocyte dysfunction, albuminuria and hypertension, processes that were exacerbated when mice were made diabetic with streptozotocin (137). Nox5-induced renal inflammation involves induction of cytokine expression and upregulation of toll-like receptors (TLRs), which causes a feed-forward system where TLR activation enhances Nox5-induced generation of ROS and consequent oxidative stress and renal injury (138). Expression of human Nox5 in mice in a VSMC-specific manner causes renal oxidative stress, glomerulosclerosis, mesangial expansion, renal inflammation and fibrosis, processes that accelerate progression of renal disease in diabetes (139). Vascular Nox5 has also been associated with hypertensive diabetic retinopathy (140). In oxygen-induced retinopathy in mice expressing human Nox5 in endothelial cells, retinal vascular permeability and neovascularization, and expression of angiogenic and inflammatory factors, were increased compared with wild-type littermates (140). The relationship between vascular Nox5 and blood pressure seems to be an age-dependent phenomenon, because blood pressure was not elevated in 16–20 week old mice expressing human Nox5 in VSMCs, while it was significanly elevated in 30–35 week old mice (141, unpublished data). This age-related phenomenon has also been described in mice expressing human Nox5 in an endothelial cell-specific manner (142). Aged, but not young, endothelial Nox5 knock-in mice developed systolic hypertension with endothelial dysfunction due to uncoupled NOS uncoupling (142). Increased blood pressure in endothelial Nox5 mice is associated with target organ damage and stroke (142) and vascular Nox5 expression is increased in CADASIL, a small vessel disease associated with premature vascular dementia (143).

Cardiac hypertrophy, a consequence of hypertension and a cause of heart failure, was also associated with Nox5 upregulation (144). In cardiac tissue from patients undergoing heart transplant for cardiomyopathy and heart failure Nox5 expression was increased (144). In cardiac-specific Nox5 transgenic mice, left ventricular hypertrophy, interstitial fibrosis, and contractile dysfunction induced by pressure overload or Ang II, were exaggerated compared with wildtype counterparts (144). These effects were associated with oxidative stress and activation of redox-dependent MAPK (mitogen-activated protein kinase) and procontractile Ca²⁺ signaling pathways. Nox5 has also been shown to be important in VSMC phenotypic switching relevant to vascular calcification and aneurysm formation (145,146). With the emerging evidence that Nox5 may be an attractive therapeutic target in human cardiovascular disease, interest in developing selective Nox5 inhibitors is growing and a number of promising agents have been identified including melittin, gedunin and ML090 (147–149).

SECONDARY SOURCES AND MECHANISMS OF ROS GENERATION IN HYPERTENSION

It is well established that Noxes are the primary source for ROS in the cardiovascular system and a key driver for oxidative stress in hypertension. However, growing evidence indicates that secondary sources are also important, including NOS uncoupling, ER stress and mitochondrial dysfunction (Figure 1), processes that are influenced by Nox-derived ROS and that themselves contribute to Nox regulation and redox signaling in a compartmentalized fashion.

Nitric oxide synthase uncoupling

NO, the key EDRF, mediates vascular effects through soluble guanyl cyclase (sGC)-induced activation of cyclic guanosine monophosphate (cGMP) and phosphorylation of protein kinase A (150). NO also signals through cGMP-independent pathways by altering protein structure and function through S-nitrosylation (151). Production of NO is regulated by activation of NOS, of which there are three isoforms: endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) (150). In the cardiovascular system, eNOS and nNOS are constitutively active in a Ca²⁺/calmodulin-dependent manner, whereas iNOS is activated by stress independently of Ca²⁺. In physiological conditions, in the presence of various cofactors including NADPH and tetrahydrobiopterin (BH4), NOS catalyzes L-arginine to produce L-citrulline plus NO (150). However in oxidative stress conditions, NOS removes an electron from NADPH donating it to O₂ to generate O₂⁻ rather than NO (30). This process, termed NOS uncoupling, involves S-glutathionylation and phosphorylation of eNOS and BH4 oxidation (150,151).

An important trigger for NOS uncoupling is BH4 depletion through ROS-mediated processes. Superoxide directly oxidizes BH4 to BH2, destabilizing NOS leading to its uncoupling (150). All NOS isoforms are regulated by BH4 and hence all can undergo ‘uncoupling’ in stressed conditions. A key factor determining eNOS-derived NO versus O₂⁻ involves tryptophan 447 within the BH4-binding domain of eNOS (152). When this is mutated, BH4:eNOS interaction is impaired, leading to preferential O₂⁻ generation. Mechanistic studies in transgenic mice with endothelial-targeted overexpression of GTP cyclohydrolase (rate-limiting enzyme in BH4 synthesis) crossed with mice overexpressing eNOS, demonstrated that uncoupling is an independent and direct effect of a stoichiometric discordance between the enzyme and BH4 (152). Altering BH4 levels not only perturbs NOS-derived NO production, but it influences posttranslational protein modification by S-nitrosation, especially impacting proteins of the ubiquitin-proteasome system (151,152). Hence BH4 and eNOS act as a signaling node influencing many downstream pathways.

The overriding factor promoting NOS uncoupling is oxidative stress leading to decreased NO production, increased O₂⁻ generation and formation of unstable and reactive ONOO, which induces BH4 oxidation and nitration of prostacyclin synthase. This feedforward system where Nox-mediated oxidative stress begets oxidative stress through NOS uncoupling has been demonstrated in SHR, SHRSP, Ang II-induced hypertension and DOCA-salt rats and in patients with hypertension (153,154). However, not all hypertension models involve eNOS uncoupling, because sepiapterin, a BH4 precursor, had no blood pressure lowering effect in glucocorticoid-mediated hypertension (155).

Reversing eNOS uncoupling is vasoprotective

Normalizing NOS activity by increasing BH4 levels and eNOS transcription enhancing agents (4-fluoro-N-indan-2-yl-benzamide (AVE9488) and 2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid indan-2-ylamide (AVE3085)) has been suggested as a potential therapeutic strategy in cardiovascular disease (156). BH4 treatment normalized endothelial function in rodent hypertension and diabetes and improved endothelial function in patients with hypertension and atherosclerosis (156). High dose oral BH4 improved endothelial NO bioavailability and had a significant antihypertensive effect in patients with resistant hypertension (156). BH4 may have protective effects beyond preventing NOS uncoupling. In experimental hypertrophic cardiac disease, BH4 suppressed inflammatory responses and myocardial macrophage infiltration without impacting NOS activity (157). In patients with chronic kidney disease, BH4 ameliorated the exaggerated blood pressure response to exercise by decreasing sympathetic activity (156). Not all studies have demonstrated protective effects of BH4. In patients with established coronary artery disease and endothelial dysfunction, oral BH4 had no effect on vascular redox state or endothelial function and failed to decrease blood pressure in some patients with hypertension (158).

Nox:NOS interplay

While eNOS uncoupling may be an important source of O₂⁻ in cardiovascular disease, this is likely a secondary mechanism because the process is driven a priori by oxidative stress, which in hypertension is due mainly to Nox activation. The interplay between these systems may involve cross-talk between Noxes and NOS. For example p47phox and NOXO1-dependent activation of Nox1, but not other Noxes, mediates uncoupling of vascular eNOS in diabetes, effects that are ameliorated by Nox1 downregulation (159). A similar relationship between Nox1 and uncoupled eNOS was demonstrated in mice overexpressing Nox1 in VSMCs, where impaired endothelium-dependent relaxation was attributed to oxidative stress and eNOS uncoupling (160). In aortic aneursyms, Nox4 upregulation is linked to NOS uncoupling (161). Further adding to the complexity, there is evidence that mitochondria play a role in Nox-regulated eNOS uncoupling highlighting tight associations between ROS-generating systems, NOS and redox regulatory pathways in cardiovascular disease (162).

Endoplasmic reticulum, ROS generation and oxidative stress

The ER is increasingly being recognized as an important player in redox pathophysiology in the cardiovascular system (163–165). This is supported by: i) ER enzymes such as ER oxidoreductin (Ero1) and its thiol redox partner protein disulfide isomerase (PDI) are involved in ROS generation, ii) the ER is the site of p22phox and Nox synthesis, maturation and post-translational modification (glycosylation, phosphorylation, oxidation), iii) Nox interacts with the ER chaperone PDI, promoting Nox activity, iv) some Noxes, especially Nox4, are active in the ER, v) the ER communicates with mitochondria through mitochondria-associated ER membranes enabling Ca²⁺ and ROS exchange between compartments, especially important for Nox5, which is redox-and Ca²⁺-sensitive, and vi) the ER is critically involved in redox protein folding and stress responses (163–167). Moreover the ER provides a functional structure comprising molecular machinery for compartmentalized ROS production and redox signaling.

Protein folding in the ER is a tightly regulated redox-sensitive process. In stressed conditions the influx of unfolded proteins into the ER exceeds its capacity to fold them (ER stress) resulting in activation of ER stress signaling pathways, called the unfolded protein response (UPR) (168). The UPR comprises three major axes including, inositol-requiring protein 1 (IRE1), activating transcription factor-6 (ATF6) and PRKR-Like ER Kinase (PERK), which upon sensing ER stress, signal to downstream molecules. Activation of the UPR causes protein accumulation and misfolding leading to apoptosis, phenotypic switching, de-differentiation and trans-differentiation, processes that underlie cardiovascular remodeling and injury in hypertension (169,170). Noxes, ROS and oxidative stress are integrally associated with ER stress/UPR (171,172). Oxidative stress can directly trigger UPR, through oxidation of ER stress signaling molecules. In addition, ROS are generated during the UPR primarily through Nox4 activation and possibly Nox2. This likely involves ER chaperones, eg. PDI, which connects Noxes to ER-mediated functions.

Interplay between ER stress and ROS in hypertension

Interactions between ER stress, UPR and oxidative stress are implicated in the pathophysiology of hypertension and associated target organ damage (Figure 2). In Ang II-induced hypertension, the UPR proteins, binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP), are upregulated in the aorta, mesenteric arteries and heart, as well as in the circumventricular subfornical organ of the brain (173–175). Treatment with ER stress inhibitors 4-PBA and TUDCA reduced blood pressure, decreased cardiovascular UPR activity and restored endothelial function in Ang II-induced hypertension and in SHR (176). In DOCA-salt hypertension, ER stress inhibition ameliorated hypertension-induced cardiac dysfunction by improving ER stress-associated calcium mishandling, apoptosis, inflammation and fibrosis (177). Pro-hypertensive effects of ER stress involve inactivation of redox-regulated SERCA2 and impaired sodium homeostasis, as demonstrated in transgenic mice expressing irreversibly oxidized SERCA (SERCA2 C674S) (178). ER stress has been suggested as a common factor in the synergistic effects of hypertension and diabetes in cardiovascular injury and target organ damage (179). In human hypertension, markers of ER stress and gene expression of ATF6, IRE1 and PERK are increased (180).

Figure 2. Interplay between oxidative stress and ER stress in hypertension.

Increased Nox-induced ROS generation promotes activation of ER stress signaling pathways and the unfolded protein response that influence vascular function in hypertension.

Mitochondrial dysfunction and oxidative stress

Mitochondria are multifunctional organelles involved primarily in ATP synthesis via the TCA cycle and the electron transport chain (ETC), which comprises five multienzyme complexes (I, II, III, IV, V). The transfer of electrons across the ETC is coupled with the transport of protons, establishing the electrochemical gradient that generates ATP, which regulates K⁺ channels, that coordinate organelle performance and cellular energy requirements (181). Normally this is an efficient process with minimal electron leak. However in pathological conditions mitochondrial function is impaired with increased electron leak leading to O₂⁻ production (182,183). Within the mitochondria, SOD dismutates O₂⁻ to H₂O₂, which crosses the mitochondrial membrane to the cytosol by diffusion or via mitochondrial pores and voltage-dependent anion channels (182,183).

Mitochondrial oxidative stress and hypertension

The contribution of mitochondrial O₂⁻/H₂O₂ to cellular ROS status is minor in physiological conditions but in pathological conditions, including hypertension, mitochondrial impairment contributes to cellular oxidative stress, a process itself that promotes mitochondrial dysfunction (184,185). In hypertension, cytosolic Nox-derived ROS traverses the mitochondrial membrane promoting electron leak and mitochondrial oxidative stress (185,186), processes that also involve NO because L-NAME prevents Ang II-stimulated mitochondrial O₂⁻ formation. These findings indicate important cross-talk between Noxes and mitochondrial oxidases through ROS/NO and suggest that the mitochondria may function as a central amplification system further contributing to mitochondrial and global oxidative stress in hypertension (187). Of the Noxes Nox2 is especially important in this (188).

Mitochondrial-targeted antioxidants

Antioxidants targeted to the mitochondria ameliorate mitochondrial dysfunction and reduce blood pressure in experimental hypertension. Inhibitors of cyclophilin D, which opens the mPTP, prevent development of hypertension in Ang II-infused mice (189). MitoQ, the mitochondrial-targeted antioxidant, attenuates hypertension-associated target organ damage and reduces blood pressure in SHRSP, salt-sensitive hypertension, pre-eclampsia and pulmonary hypertension (47,48). Clinical studies demonstrated that 6-week treatment with MitoQ improved endothelial function (brachial artery flow-mediated dilation) and reduced plasma biomarkers of oxidative stress in elderly individuals (48).

Mitochondrial dysfunction, SIRT3 and hypertension

Another mitochondrial system that is gaining attention is SIRT3, which is a key mitochondrial deacytelase that activates cyclophilin D and the mitochondrial anti-oxidant SOD2 (42). The antioxidative function of SIRT3 is attributed mainly to its deacytelation and activation of SOD2. In hypertension, vascular SIRT3 levels are reduced and SOD2 is hyperacetylated, leading to mitochondrial O₂⁻ accumulation and redox-sensitive vascular dysfunction (40–44). In SIRT3 knockout mice, Ang II- and DOCA-salt-induced hypertension, vascular oxidative stress, endothelial dysfunction and blood pressure elevation were exaggerated, responses that were normalized in hypertensive mice overexpressing SIRT3. The relationship between SIRT3 and blood pressure seems to be especially important in females, since SIRT3 knockout only in females was associated with cardiac dysfunction and hypertension, suggesting a sex-specific role for endothelial SIRT3 (190). In cell-based systems, Ang II-induced epithelial-mesenchymal transition was associated with decreased SIRT3 expression and oxidative- and mitochondrial stress (191), effects attenuated by SIRT3 overexpression (191). Decreased expression and activity of other SIRT isoforms, SIRT1 and SIRT6, which localize in the nucleus, have also been implicated in cardiovascular disease and hypertension (192,193).

Together these studies suggest that upregulating SIRTs may be an attractive strategy to prevent tissue fibrosis, hypertension and target organ damage. A machine-learning strategy supports this notion. Agents that activate SIRTs such as polyphenols (eg, resveratrol, quercetin) and modulators of acetylation (eg, curcumin, honokiol, oroxilyn A, quercetin, epigallocatechin-3-gallate, bakuchiol, tyrosol, and berberine) have been shown to reduce blood pressure and cardiovascular injury in experimental hypertension (194). However, not all studies have shown a protective effect of SIRT3 in hypertension. Sunitinib-induced hypertension and cardiotoxicity in mice were associated with increased SIRT3 levels in cardiovascular tissue (195). These cardiovascular toxicities were attenuated in SIRT3-knockout.

ROLE OF ANTIOXIDANT ENZYMES IN PERTURBED REDOX BALANCE IN HYPERTENSION

Redox homeostasis in cardiovascular pathophysiology is tightly regulated by antioxidant enzymes including SOD [cytoplasmic Cu/Zn SOD (SOD1), mitochondrial SOD (SOD2), extracellular SOD (SOD3)], glutathione peroxidase (GPX), catalase, and thioredoxin-peroxiredoxin (TRX-Prdx), and non-enzymatic antioxidants (α-tocopherol, β-carotene, bilirubin and uric acid) (196,197). SOD dismutates O₂⁻ into H₂O₂ while catalase catalyzes H₂O₂ into O₂ and H₂O. The peroxidases catalyze oxidation/reduction processes and promote reduction of oxidized proteins. A major transcription factor controlling cellular antioxidants is Nuclear factor erythroid 2-related factor 2 (Nrf2), the master regulator of antioxidant genes and hence of antioxidant status.

Altered antioxidant systems, including decreased expression and activity of antioxidant enzymes and reduced plasma levels of vitamins A/C/E, have been consistently demonstrated in experimental hypertension (198–200). Administration of exogenous SOD supports the notion that ROS play a pathophysiological role in hypertension. Membrane permeable SOD and SOD mimetics improve hypertension, endothelial dysfunction, cardiovascular remodeling, renal injury and oxidative stress in experimental models of hypertension (200–202). In transgenic mice deficient in antioxidant enzymes, eg. SOD1 and GSH synthase, blood pressure is elevated (203). Epigenetic silencing of SOD2 has been demonstrated in pulmonary hypertension (204). On the other hand, overexpression of antioxidants, catalase, SOD1 and SOD3, prevented blood pressure elevation and protected against kidney damage in Ang II-dependent hypertension and models of pulmonary hypertension (205). SOD knockout or overexpression do not influence blood pressure in normotensive animals, indicating that beneficial antioxidant effects occur in the context of oxidative stress and hypertension.

A common factor that may be responsible for dysfunctional antioxidant enzymes in hypertension is Nrf-2 (206,207). In two kidney-one clip (2K1C) hypertensive rats, nuclear accumulation of Nrf-2 and expression of Nrf2-regulated genes are reduced (206). Treatment with nitrites decreased oxidative stress, improved vascular function and reduced blood pressure by upregulating Nrf-2 and increasing antioxidant enzymatic activity. It has also been suggested that activation of the protective axis of the renin angiotensin system mediates effects by upregulating Nrf2, since ACE2 overexpression in mice was associated with increased Nrf-2-sensitive antioxidant genes (207).

Clinical studies have also demonstrated abnormal antioxidant status in patients with hypertension. Plasma levels of vitamin C are inversely related to blood pressure and antioxidant capacity in circulating cells is decreased in hypertension (208). In the Alpha-Tocopherol, Beta Carotene Cancer Prevention study, a 30-year prospective cohort analysis, higher baseline serum alpha tocopherol levels were associated with reduced cardiovascular disease risk and overall mortality (209). Moreover, an evaluation of dietary carotenoids and hypertension in the National Health and Nutrition Examination Survey 2007–2014 showed that higher intake of alpha-carotene, beta-carotene, beta-cryptoxanthin, lycopene and lutein was associated with lower risk of hypertension (210). Antioxidant vasoprotective effects seem to be amplified with aging as evidenced by the negative correlation between SOD and age in patients with hypertension (211).

Antioxidants in hypertension

Studies in experimental models of hypertension and pre-eclampsia consistently demonstrated amelioration of oxidative stress and blood pressure-lowering by antioxidant vitamins, SOD mimetics and resveratrol (212,213) Antioxidants also improved endothelial function, normalized vascular remodeling and reduced arterial stiffness, in part by decreasing Ox-PTM of proteins and restoring normal redox signaling in these models (212,213). A meta-analysis on the SOD-mimetic tempol clearly showed beneficial effects in experimental hypertension (214). However, these favourable responses have not translated to the clinic. Except for a few small studies, large antioxidant trials, such as the GISSI and HOPE trials, have been mostly negative (215,216). Reasons for this are multifactorial. To address some of these concerns, it has been suggested that compartment-specific agents including MitoQ or antioxidants that directly influence protein oxidation may have beneficial effects in patients (217).

Indirect antioxidants and Nrf2 agonists – new approaches to reduce oxidative stress

Most antioxidant therapies investigated clinically have direct effects by donating one or two electrons. A new concept suggests that agents may have indirect actions where agents augment endogenous antioxidant responses without being antioxidants themselves. Indirect antioxidants include the polyphenol resveratrol and Nrf-2 agonists (218,219). Resveratrol increases expression of antioxidant enzyes SOD, catalse and GPX, enhances Nrf-2-heme oxygenase-1 pathway, restores activity of eNOS and reduces expression of ROS-generating Nox4 (218,219). Hypertensive patients treated acutely with resveratrol showed improved vascular function, but without blood pressure-lowering effect (220). When added to standard anti-hypertensive therapy, resveratrol further reduced blood pressure in patients with hypertension (221). Based on these promising findings, randomized, crossover, double-blinded, placebo-controlled trials are underway to assess efficacy of resveratrol in controlling hypertension (222). Nrf2 activators, such as bardoxolone methyl and sulforaphane, are also indirect antioxidants and induce effects by enhancing expression of antioxidant and cytoprotective genes. Clinical efficacy of bardoxolone methyl and sulforaphane have been tested in many conditions including diabetes, kidney disease, cancer and pulmonary hypertension, with some favorable outcomes (222,223). Whether this is also evident in essential hypertension has yet to be demonstrated.

THE MOLECULAR BASIS OF OXIDATIVE STRESS IN HYPERTENSION

Molecular mechanisms whereby ROS contribute to hypertension are rooted in their effects on protein structure and function and associated redox signaling. ROS activate and inactivate transcription factors, membrane channels and metabolic enzymes, stabilize cytoskeletal proteins, and regulate Ca₂⁺-sensitive and phosphorylation signaling pathways (224–226). Proteins are highly sensitive to the action of O₂⁻ and H₂O₂ and constitute ~70% of ROS targets. Specific ROS effects are mediated by covalent modification of redox-sensitive amino acids including histidine, tyrosine and cysteine. Oxidation and reduction of Cys-containing thiol proteins are amongst the key processes whereby ROS integrate into intracellular signaling pathways influencing cell function. Under normal conditions proteins are targets of reversible oxidative modifications, involved in signal transduction and cell homeostasis. In pathological conditions, as a result of oxidative stress, proteins undergo irreversible oxidation, which triggers protein degradation pathways and cell death (227). Dysregulated Ox-PTM of proteins is emerging as an important molecular process in cardiovascular disease (228).

ROS are highly reactive. Production of O₂⁻ in the vicinity of NO, or as a result of eNOS uncoupling, results in inactivation of NO and impairs endothelium-dependent relaxation (30). Peroxinitrite reacts with tyrosine to form 3-nitrotyrosine. Whether, and how, 3-nitrotyrosine affects protein function remains unclear. H₂O₂, on the other hand, reacts with deprotonated Cys groups to form sulfenic (R-SOH), sulfinic (R-SO₂H) or sulfonic (R-SO₃H) acids, as well as disulfides (RSSR) (229). While formation of sulfinic and sulfonic acids are reversible, sulfonic modifications are not (226,227). Sulphenic acids can also react with glutathione (GSH), resulting in S-glutathiolation. Both disulfides and s-glutathiolated residues can be reduced back to the thiol by thioredoxin/thioreductase or GSH/glutathione reductase. Alternatively, reactive Cys can interact with NO to generate S-nitrosothiols. By virtue of the fact that these reactions are fast, reversible and localized, all of these modifications can be classified as redox-sensitive intracellular signals that influence cell function (Figure 3).

Figure 3. Post translational oxidative modification of proteins.

Main reactions leading to reversible and irreversible oxidation of proteins in cardiovascular cells.

Redox signaling in hypertension

Oxidative modifications affect the structure and function of proteins, with multiple molecular and cellular consequences; for example, oxidation of protein tyrosine phosphatases reduces activity and oxidation of protein kinases such as Src or ASK-1 activates them (228–230) (Figure 4). Relevant to control of vessel tone, oxidation of protein kinase G activates it independently of cyclic GMP leading to vasorelaxation (229). Moreover, oxidant-based activation of MAP kinases, including ERK, JNK and p38MAPK, contributes to vascular and cardiac inflammation, proliferation, hypertrophy and migration (230) all of which are involved in cardiovascular remodeling and hypertension-induced end organ damage. Along with redox-sensitive activation of Src and Rho kinase, the MAP kinases can regulate contraction (231). Similarly, other tyrosine kinases, including PI3K/Akt and receptor tyrosine kinases (VEGFR, EGFR, PDGFR) can be modulated by ROS (232,233).

Figure 4. Schematic of redox-sensitive signaling pathways involved in hypertension.

ROS influence many signaling molecules that regulate cardiovascular function including kinases, phosphatases, Ca²⁺ channels, transcription factors, genes. These processes involve oxidative post-translational modifications (Ox-PTM) of downstream redox-sensitive targets. Oxidative stress causes abnormal redox signaling leading to cardiovascular dysfunction and remodeling in hypertension. Decreased antioxidants contribute to oxidative stress. PTP, protein tyrosine phosphatases; MMP, matrix metalloproteinases; UPR, unfolded protein response.

Other proteins particularly susceptible to regulation by oxidative stress are cytoskeletal proteins, including actin (234). Actin oxidation not only influences its polymerization rate, but also its interaction with actin-binding proteins such as vinculin (235). Focal adhesion regulation by FAK as well as turnover are modulated by Nox4-derived H₂O₂ (234,236). Similarly, studies have shown that high ROS concentrations have adverse effectS on cardiac myofilament function (237). Perhaps more importantly, ROS activate transcription factors, most notably NFκB and HIF-1, which in turn regulate expression of proinflammatory chemokines and cytokines, leading to activation and recruitment of macrophages and immune cells, leading to cardiovascular inflammation and fibrosis, hallmarks of hypertension-induced end-organ damage (238). Other redox-sensitive transcription factors include STAT3 and AP-1, as well as Nrf-2/ Kelch-like ECH-associated protein 1 (KEAP1) (239). Nrf-2 is normally bound to and targeted for degradation by Keap1/Cullen-3, but when Cys residues on Keap1 are oxidatively modified, Nrf-2 is stabilized and translocates to the nucleus, inducing expression of antioxidant genes. Many of these pathways are protective, such that activation of Nrf-2 reduces oxidative stress and lowers blood pressure in SHR.

An important class of redox-sensitive signaling proteins in the cardiovascular system are Ca²⁺-binding proteins and channels (240), but the functional consequences are complex. Transient receptor potential (TRP) channels, store-operated Ca²⁺ channels, inositol trisphosphate receptors (IP₃Rs) and voltage-gated Ca²⁺ channels are subject to regulation by ROS and many are directly regulated by cysteine oxidation (242). TRP channels, and in particular TRPM2 and TRPM7, are activated by ROS (241,242). In contrast, oxidation of ORAI1 channels by H₂O₂ inhibits activity, and STIM2, which senses endoplasmic reticulum calcium levels and gates ORAI channels, can also be oxidized, resulting in further inhibition of activity (243). H₂O₂ also downregulates IP₃Rs, leading to reduced Ca²⁺ efflux in VSMCs (244). The overall effect of ROS on Ca²⁺ signaling thus depends on the interplay of multiple cascading targets.

Redox signaling and compartmentalization

Another important consideration for ROS-mediated signaling is subcellular compartmentalization. Because antioxidants are prevalent throughout the cell, O₂⁻ and H₂O₂ act upon signaling molecules in their immediate vicinity. For Nox1, that means lipid rafts and internalized vesicles (245) while for Nox4, signaling occurs mainly in the ER and potentially in the focal adhesions (246). Nox2 is found in neutrophil phagosomes where it is released extracellularly, but in other cell types, including endothelial cells, it is found in lamellipodial focal complexes, membrane ruffles and endosomes (247). Mitochondrial oxidants act upon ferrochelatase in the inner mitochondrial membrane to reduce heme and indirectly inhibit soluble guanylate cyclase (248). In addition, ROS-induced ROS release can amplify and spread signal generation, as is the case for Nox2-induced mitochondrial ROS production in mice treated with Ang II (249). Many redox-sensitive signaling proteins localize in cholesterol-rich microdomains, especially caveolae and lipid rafts, which facilitates ROS production with redox-sensitive targets in close proximity (250).

Redox signaling and the inflammasome in hypertension

The inflammasome, important in immune and inflammatory cells as well as VSMCs and endothelial cells, is increasingly being recognized to play a role in vascular inflammation in hypertension (251,252). Several inflammasomes have been described, but the nucleotide-binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is best characterized (251). The NLRP3 inflammasome contains ASC (apoptosis-associated speck-like protein) adapter protein and procaspase-1. In the presence of immune activators, cell stresses and oxidative stress, the conformation of NLRP3 is altered giving rise to the NLRP3 inflammasome, which triggers cleavage of procaspase-1, generating caspase-1, which induces pyroptosis, (caspase-1-specific form of cell death leading to inflammation), and IL-1β and IL18 production and consequent induction of pro-inflammatory signaling pathways (251, 252). ROS have an important role in activation of the inflammasome by activating NFκB or by directly activating the inflammasome (253) (Figure 5). ROS act as second messengers and activate inflammasomes through Nox and redox-sensitive pathways involving ERK1/2, PI3K, thioredoxin and apolipoprotein C3 (ApoC3) (254). TRP cation channels that function as polymodal sensors and scaffolding proteins have also been implicated in inflammasome activation (255). Of significance, TRPM2, which we described as a point of cross-talk between redox and Ca²⁺ signaling in VSMCs (256), is an important link between ROS and the inflammasome (257). Because of its association with injurious signals as well as its dependency on ROS, activation of inflammasome is increasingly being considered as an important mechanism leading to chronic inflammatory processes in cardiovascular disease, including hypertension.

Figure 5. Oxidative stress and the inflammasome in hypertension.

Prohypertensive factors induce activation of NLRP3 inflammasome through ROS. ROS influences signal 1 (cytokines, pathogen-associated molecular patterns (PAMPS), danger-associated molecular patterns (DAMPS)), leading to activation of NFkB and gene expression of components of the inflammasome. ROS also influence signal 2, by PAMPS, DAMPS, oxidized LDL (oxLDL), oxidized phospholipids (oxPL), ATP, Ang II, ET-1, aldosterone (aldo) and cations (K⁺, Ca²⁺, Na⁺). These processes lead to assembly of the inflammasome complex (NLRP3, ASC and pro-caspase 1), and consequent activation of caspase 1, cleavage of pro-IL-1β and pro-IL-18, and production of active forms of IL-1β and IL-18, which increase the inflammatory response, fibrosis and vascular remodelling in hypertension. MR, mineralocorticoid receptor; ASC - Apoptosis-Associated Speck-Like Protein Containing CARD; question mark (?) indicates possible effect.

Activation of NLRP3 inflammasome has been shown in Ang II-mediated and DOCA/salt hypertension, and in preeclampsia, pulmonary hypertension, and hypertensive nephropathy (252,258,259). NLRP3 inflammasome activation contributes to Ang II-induced VSMC phenotypic transformation, cell proliferation, vascular remodeling and hypertension (260). In Ang II-infused NLRP3^−/− mice, development of hypertension, vascular remodeling and VSMC phenotypic switching (contractile to proliferative) were blunted compared with wildtype mice (261). The NLRP3 inflammasome is also involved in Ang II-induced podocyte injury, mitochondrial dysfunction, renal dysfunction and cardiac hypertrophy (262). In Ang II-infused mice, hypertension-associated cardiac remodeling, which involves the serum- and glucocorticoid-inducible kinase (SGK1), was ameliorated by EMD638683, a highly selective SGK1 inhibitor, by suppressing NLRP3 expression, caspase-1 activation and IL-1β secretion (263). Benefical effects of EMD638683 on cardiac fibrosis were abolished by supplementation with exogenous IL-1β and the NLRP3 inflammasome inhibitor MCC950, indicating that EMD638683 reduces Ang II-induced cardiac inflammation and fibrosis by inhibiting the NLRP3 inflammasome/IL-1β secretion axis. This inflammasome process may be important in aldosterone-induced redox-mediated cardiac fibrosis and hypertension because aldosterone signals through SGK1 (264).

The thiol proteome in hypertension

Advances in LC-MS and biotin-switch methodologies have enabled proteome-wide assessments of Ox-PTMs formation in cardiovascular tissue (22,265). We demonstrated increased protein sulfenylation and hyperoxidation of protein tyrosine phosphatases and peroxiredoxins in vessels from experimental models of hypertension and showed that protein hyperoxidation is associated with oxidative and ER stress through upregulation of plasmalemmal-Nox1 and ER-Nox4 (21–23). Using a redox proteomics approach, protein targets of carbonylation in genetically obese hypertensive rats have been described. These rats have increased carbonylation of proteins associated with lipid metabolism, redox regulation and protein chaperone activity and decreased carbonylation of catalase, processes that promote metabolic syndrome and increased cardiovascular risk (266). Increased carbonylation has also been demonstrated in salt-sensitive hypertension in Dahl-salt-sensitive rats (267). In SHR kidney, total free thiol content was significantly lower than that of control rats, indicating increased oxidation of sulfhydryls (268). This was attributed, in part, to reduced thioredoxin reductase activity. Similar profiles have been described in human hypertension, where plasma levels of protein carbonyls and advanced protein oxidation products were increased with associated decreased levels of total thiols (269). Plasma protein carbonyl may be a biomarker of oxidative stress, while also playing a role in the pathogenesis of hypertension (270).

One of the most abundantly oxidized vascular proteins of relevance in hypertension is actin, critically involved in cytsoskeletal organization and cell contraction (234,235). Exposure of VSMC to H₂O₂ causes cytsoskeletal rearrangement, F-actin fragmentation, impaired polymerization and phenotypic changes, processes causing vascular dysfunction and arterial remodeling in hypertension (271). In conditions associated with severe oxidative stress, such as end stage heart failure and hypertension-associated target organ damage, carbonylation (irreversible form of Ox-PTM) of actin was associated with loss of cell viability and contractile dysfunction (271).

S-nitrosothiols in the cardiovascular system regulate multiple processes that are mainly protective, including NOS activity and vasodilation, cardiomyocyte Ca²⁺ homeostasis and myocardial contraction, ATP production and mitochondrial bioenergetics and inhibition of platelet aggregation and hemostasis. In hypertension S-nitrosylation of various proteins has been implicated in vascular dysfunction (272). For example, S-nitrosylation of eNOS (Cys101) promotes eNOS uncoupling and ROS production, while S-Nitrosylation of cyclooxygenase-2 (COX-2), Ca²⁺ channels and angiotensinogen (Cys18, Cys138) influence vascular tone (273). In heart failure, Cys294 S-nitrosylation and S-glutathionylation of ATP synthase are negatively correlated with ATP hydrolysis, indicating that Ox-PTMs cause inactivation of enzymatic activity and reduced ATP production and myocardial injury (274).

The number of identified proteins that undergo Ox-PTM in cardiovascular disease and hypertension continues to grow (21–23). In addition to those above, other proteins of interest include ryanodine receptor 2 (RyR2), Keap/Nrf-2, bone morphogenic proteins (BMP), NFATc4/ERK/AKT, Trx1, histone deacytelase 4 (HDAC4) and TRPM2 (275–277). We demonstrated in human VSMCs, that under basal conditions 92% of proteins were similarly oxidized in cells from normotensive and hypertensive patients, while ~ 3% were hyperoxidized and ~ 5% showed reduced oxidation in the hypertensive group (278). Some of the potentially interesting candidate vascular proteins that were highly oxidized included β-actin, annexin A1, galectin-1, FK506 binding protein polymerase I and transcript release factor (PTRF, −1.92) and vimentin (278). Of significance, many of these proteins play an important role in cytoskeletal organization and contraction.

While there have been enormous technological developments in proteomics, the field of redox proteomics in human physiology and disease mechanisms is still immature. However, this is an emerging field in cardiovascular research and new discoveries defining the oxidative proteome in hypertension will help delineate which proteins undergo Ox-PTM, how these underpin redox mechanisms of disease and whether they are useful biomarkers to predict and track development of hypertension.

OXIDATIVE STRESS AND ORGAN SYSTEMS IN HYPERTENSION

Oxidative stress is a unifying mechanism of tissue injury in multiple systems and organs that play a role in the pathophysiology of hypertension (Figure 6). Moreover, hypertension-associated target organ damage is a consequence of oxidative stress.

Figure 6. Systems biology, oxidative stress and hypertension.

Functional effects of oxidative stress in regulatory systems and organs in the pathophysiology of hypertension.

The vascular system

Vasculopathy, consisting of impaired endothelium-dependent relaxation, increased arterial stiffness, enhanced contractility, inflammation, vascular calcification and remodeling is both a cause and consequence of hypertension (279,280). These processes that occur with aging, are accelerated in hypertension (280). Oxidative stress and redox-sensitive signaling contribute to these dysfunctions, as has been demonstrated both in vitro and in vivo (279–281). Many pro-hypertensive factors instigate and amplify ROS production in endothelial cells, VSMCs, adventitia, and perivascular adipose tissue, leading to vascular injury.

Ang II was the first vasoactive agent shown to induce the production of ROS in vascular cells in vitro and in aortas of hypertensive mice (282). Vessels from Ang II-infused mice and resistance arteries from hypertensive patients show increased vascular expression of Nox1 and Nox2 and increased ROS production, which in turn activate complex signaling pathways including MAP kinases, Src, Rho kinase and Ca²⁺ channels (282,283). These processes cause vascular hyperreactivity, endothelial dysfunction, vascular remodeling, influx of inflammatory cells into the vessel wall, increased wall stiffness and fibrosis. In hypertensive animals, genetic deletion of Noxes, pharmacological Nox inhibitors, ROS scavengers or antioxidants inhibit vascular remodeling, reduce inflammation and normalise endothelial function with associated blunting in blood pressure elevation (284).

Other ROS-dependent contributors to hypertension include aldosterone, ET-1, growth factors, immune factors and salt (285,286). Aldosterone, through mineralocorticoid receptors, signals via genomic and non-genomic pathways to increase ROS in VSMC and endothelial cells by activating Nox1 and Nox4 respectively. Inhibition of mineralcorticoid receptors attenuates Ang II-induced ROS production in the vasculature, while AT1 receptor signaling is required for aldosterone-induced oxidative stress, suggesting interplay between these two pro-hypertensive systems (285). Mice overexpressing endothelial ET-1 are hypertensive and exhibit oxidative stress, with associated increase in Nox1 and Nox2 expression (287). Ang II and ET-1, which signal through G-protein coupled receptors, transactivate growth factor receptors such as PDGF, EGF and IGF-1, which amplifies redox-dependent vascular processes including medial hypertrophy, vascular remodeling, inflammation and fibrosis (288).

Another tissue source of ROS, besides the endothelium and vascular media, that may influence vascular function in hypertension is perivascular adipose tissue. Adipose tissue possesses functionally active Nox4, which generates ROS, important in adipocyte function and adipokine production (289). Perivascular adipose tissue influences vascular tone through adipocyte-derived vasoactive factors and ROS, processes that are amplified in hypertension, obesity and diabetes mellitus.

The renal system

The kidneys play a critical role in the pathophysiology of hypertension through multiple processes that involve ROS (290,291). Increased generation of ROS in the kidney can trigger blood pressure elevation and accelerates development of hypertension in experimental models. The major source of renal ROS is Nox4, although other Noxes also play a role. Increased O₂⁻ causes vascular and tubular dusfunction, while H₂O₂ seems to be important in regulating pressure natriuresis by decreasing preglomerular vascular reactivity and increasing medullary blood flow (291). In vivo studies of single nephron function and in vitro studies with the double-perfused juxtaglomerular apparatus preparation have shown extensive interaction between O₂⁻ and NO in macula densa to regulate afferent arteriolar tone mediated by the tubuloglomerular feedback response (291). Oxidative stress in the kidney also promotes renin release, activates renal afferent nerves, causes dysfunction of glomerular cells and disrupts Na⁺ and water homeostasis, processes that contribute to the development of hypertension (290,291). Moreover production of O₂⁻ in specific nephron segments increases reactivity to Ang II. Recent evidence suggests that renal dopamine receptors (D1-D5R) regulate the redox state by decreasing ROS generation and that dopamine receptor dysfunction promotes oxidative stress and hypertension (292). The impact of renal oxidative stress on hypertension occurs early in life because oxidative stress plays a pathogenic role in developmental programming of kidney disease (293). Dampening oxidative stress in the kidney using ROS scavengers, antioxidants and Nox inhibitors lead to improved renal function and ameliorates blood pressure elevation in experimental hypertension (294). The involvement of renal oxidative stress in hypertension is complex and has been extensively reviewed (290–293).

The central nervous system

Oxidative stress in the brain plays an important role in the development of hypertension. Nox-induced ROS production in the nucleus tractus solitarii (NTS) and rostral ventrolateral medulla (RVLM) causes sympathoexcitation in hypertensive rats, through mechanisms that involve NO and pro-inflammatory processes (296). Redox signaling in the subfornical organ (SFO), an important forebrain circumventricular organ, is critical for sympathetic activation, driving the elevation in blood pressure in Ang II-infused mice (297). This was demonstrated in mice in which p22phox was deleted in the SFO, where Ang II failed to elicit a hypertensive response. Mechanisms through which the SFO mediates ROS-related blood pressure effects in the brain involve activation of the PVN causing increased plasma vasopressin, upregulation of ET-1 in cerebral small arteries and activation of ETA and AT1 receptors (297,298).

The brain has an active renin angiotensin system involved in the development of hypertension through oxidative mechanisms. Acute administration of Ang II to the paraventricular nucleus (PVN) caused an increase in blood pressure (299) and intracerebroventricular infusion of Ang II leads to an increase in Nox subunit expression and O₂⁻ production in the in the RVLM (300). Moreover, chronic Ang II infusion leads to elevated ROS in the subfornical organ, and administration of SOD selectively to this organ inhibits the associated increase in blood pressure (301). Additional work has shown that, similar to the vasculature, Nox-derived ROS induce dysfunction of mitochondrial electron transport, which in turn contributes to neurogenic hypertension.

Less is known about the redox targets in the brain that regulate the hypertensive response. In general, Ang II signaling in neurons increases Ca²⁺, activates Noxes, stimulates mitochondrial ROS production, and regulates voltage-gated K⁺ channels (302,303). In particular, O₂⁻ mediates Ang II-induced influx of extracellular Ca²⁺ and inhibition of the outward K+ current leading to increased neuronal excitability (303). Calcium/calmodulin-dependent protein kinase II (CaMKII) regulates these processes. Adenoviral-mediated overexpression of CaMKII in the subfornical organ enhanced Ang II-induced inhibition of the voltage-gated K⁺ current and increased the pressor response to intracerebroventricular injection of AngII. In addition, evidence shows that targeting c-Src in the PVN inhibits NFκB, pro-inflammatory cytokine production, and oxidative stress induced during salt-sensitive hypertension (304). Ang II administration to the RVLM of rats made hypertensive by exposure to repeated foot shocks and noise resulted in an increase in SAPK/JNK activity, which could be blocked with apocynin and contributed to elevation of blood pressure (305). Oxidative stress caused by intracerebroventricular infusion of Ang II can be attenuated by overexpression of ACE2, which in turn upregulates Nrf-2 (306).

The immune system

Inflammation has long been associated with hypertension, ranging from early studies reporting that immunosuppression lowers blood pressure following renal infarction to current work focused on the role of the innate and adaptive immune system in hypertension and searching for initiating signals. In 2007, Harrison’s group showed that mice lacking both T and B cells due to deletion of the recombination-activating gene 1 (RAG) had a reduced hypertensive response to angiotensin II or deoxycorticosterone acetate (DOCA) plus salt. Adoptive transfer of T cells restored the blood pressure response (307). Similar findings were made in RAG1-deficient Dahl salt-sensitive rats and in SCID mice, which are deficient in lymphocytes (308,309). Patients with hypertension tend to have an increased number of circulating “immunosenescent” proinflammatory CD8+ T cells, which generate excessive IFN-γ and TNF-α compared to CD8+ T cells from non-hypertensive individuals (310). CD4+ T cells also have a role in hypertension, particularly with regard to increased production of IL-17, which can raise blood pressure and impair endothelium-dependent dilatation (311). In addition, depletion of B cells using a CD20 antibody or using mice deficient in B-cell-activating factor receptor (which prevents B cell maturation) partially inhibits the blood pressure increase caused by angiotensin II infusion (312). Monocyte and macrophage depletion can also normalize the hypertensive response to angiotensin II (313).

Most relevant to the role of oxidative stress in hypertension are the findings that dendritic cells derived from monocytes of hypertensive mice generate excess ROS, which leads to formation of isolevuglandin adducts that act as neoantigens for T cells (314). Importantly, sodium uptake via an amiloride-sensitive channel in dendritic cells stimulates ROS by activating Nox2, resulting in activation of T cell proliferation and production of IL-17, both of which exacerbate hypertension (315). In addition, mice with CD4-targeted Nox2 deficiency had a reduced blood pressure increase following angiotensin II infusion that was accompanied by an increase in tissue-resident regulatory Tregs and a reduction in infiltrating effector T cells (316). In support of this finding, it has been shown that T-cell Nox2 is critical to the development of hypertension in Dahl salt-sensitive rats (317). It should also be noted that as T-cells become activated in hypertension, they secrete cytokines that act upon surrounding tissue and can increase ROS production by resident cells (317). In the case of the vasculature, cytokine-stimulated ROS production leads to inactivation of NO and impaired endothelium-dependent relaxation.

CLINICAL PERSPECTIVES

Despite the overwhelming pre-clinical findings that oxidative stress is causally linked to the development of hypertension, this has been more difficult to prove in humans. Nonetheless there is growing evidence that Nox activity, ROS generation and redox signaling are amplified in cardiovascular and renal tissue and VSMCs from resistance arteries from patients with hypertension, recapitulating some findings in experimental models (278,280,284,318,319). To further support a role for Nox/ROS in vascular regulation in humans, studies in patients with genetic Nox deficiency (chronic granulomatous disease) demonstrated higher flow-mediated dilation, lower intima-media thickness, reduced urinary isoprostanes and serum Nox2 activity, increased NO bioavailability, and higher serum nitrite/nitrate compared with controls, suggesting reduced vascular damage (320).

Genetic factors have been implicated in oxidative stress-related hypertension in humans. Normotensive individuals with a positive family history of hypertension have greater ROS production than blood pressure-matched subjects without a family history of hypertension (321,322). Polymorphisms in Nox subunits (CYBA (p22phox)) and Nox isoforms (Nox3) are associated with hypertension in different populations (131,132,323,324). More recently, findings from a large GWAS of almost half a million people identified Nox4 and Nox5 as new blood pressure-associated genes (325).

Beyond genetics, epigenetic factors influence development of hypertension, especially during critical windows in the early stages of development. Oxidative stress is an important epigenetic modulator since ROS regulate histone modifications (methylation, acetylation), DNA modifications expression of non-coding RNAs and ATP-dependent chromatin modelling (326). Numerous vascular targets for epigenetic programming have been identified in essential hypertension such as redox-sesnitive genes encoding for vasorelaxation/vasoconstriction, vasoactive peptides, inflammatory mediators and VSMC phenotypes. In addition redox-regulated DNA modification of genes involved in endocrine hypertension have been described including hydroxysteroid (11-beta) dehydrogenase 2 (HSD11B2), somatic angiotensin-converting enzyme (sACE), Na⁺/K⁺/2Cl⁻ cotransporter 1 (NKCC1), angiotensinogen (AGT), and α-adducin (ADD1) gene (326,327). Also a number of miRNAs implicated in hypertension pathophysiology have been linked to oxidative stress, such as miR-200 family members that play a role in redox-dependent endothelial dysfunction, and miR-210 a key player in mitochondrial metabolism (326,328). Furthermore histone acetylation plays an important role in endothelial gene expression of eNOS through processes involving redox-regulated HDAC1 (326). Histone methylation and acetylation are reversible processes, accordingly interventions that reduce oxidative stress may be an attractive strategy to normalize epigentic changes involved in early programming of essential hypertension.

Most studies examining ROS in humans are based on associations between circulating or urine biomarkers of oxidative stress and blood pressure. Hypertensive patients exhibit higher levels of plasma H₂O₂, increased plasma and urine markers of oxidative stress such as thiobarbituric acid-reactive substances (TBARS), oxidized low-density lipoprotein (oxLDL), and 8 iso-prostane compared with normotensive individuals (329,330). Biomarkers of oxidative stress have also been demonstrated in patients with secondary hypertension (hyperaldosteronism) and preeclampsia (331,332). Because of the potential predictive value of biomarkers, there is growing interest in identifying more specific and direct indices of oxidative stress including oxidized proteins in serum, plasma, and urine. Patient studies have shown that plasma levels of reactive carbonyl species correlate with blood pressure and predict future hypertension, while serum levels of nitrosylated proteins are increased in patients with hypertension (333,334).

Markers of decreased antioxidant defense systems have also been demonstrated in human hypertension. Patients with essential hypertension exhibit reduced activity and decreased plasma levels of antioxidant enzymes, including SOD, glutathione peroxidase, and catalase (335–337). Ferric reducing antioxidant power (FRAP), an index of antioxidant status, and plasma levels of antioxidant vitamins A/C/E are lower in newly diagnosed untreated hypertensive patients compared with normotensive controls (336,337). In patients with white coat hypertension, serum protein carbonyl (indicating protein oxidation) was increased and endogenous antioxidant proteins (protein thiol, SOD, glutathione) were decreased compared with normotensive individuals, further supporting a relationship between low antioxidant capacity, increased oxidative stress, and hypertension (338). Population studies have demonstrated an inverse association between plasma vitamin C levels and blood pressure and a meta-analysis reported that vitamin C supplementation reduced systolic and diastolic blood pressure (339).

Considering the potential importance of oxidative stress in the pathophysiology of hypertension and the convincing experimental data, it is reasonable to imagine that reducing ROS bioavailability through antioxidants, ROS scavengers, and Nox inhibitors would have protective and blood pressure-lowering effects. Interest in developing therapies to target Nox/ROS is growing as highlighted in recent reviews (136,147–149). However, it should be emphasised that currently there are no clinically approved Nox/ROS inhibitors or antioxidants for use in the treatment of hypertension. Although further clinical research and drug development in the field are still needed, clinical utility of agents that reduce oxidative stress to manage hypertension and target organ damage is promising.

CONCLUSIONS

Evidence from animal studies and mechanistic studies in cultured cells have established a strong causative link between ROS, redox signaling, and hypertension. An important pathophysiological role for Noxes, uncoupled eNOS, mitochondrial ROS and ER stress in experiemnatl hypertension is clear. Common to these elements is oxidative stress. While antioxidant trials in humans have been largely negative, much remains to be learned about the timing, targeting, delivery and efficacy of novel agents that target ROS. Drugs that prevent production of ROS in specific subcellular compartments or drugs that activate endogenous antioxidant defenses show promise. However, to fully evaluate their potential as anti-hypertensives better biomarkers are needed to ensure that the expected targets are in fact inhibited and to assess the ability of these new therapies to interfere with hypertensive signaling while maintaining redox homeostasis. Redox proteomics offers a promising new approach to not only identify biomarkers to test the efficacy of antioxidant therapies, but also to provide mechanistic insights into the actual molecular targets of elevated oxidative stress. Additional delineation of the intersection between inflammasomes, ER stress, ROS, and mitochondrial function can be derived from systems biology and experimental medicine approaches in clinical samples or tissues from hypertensive animal models. These new technologies offer hope to finally resolve the paradox between the clearly established role of ROS in hypertension in animals and the current equivocal evidence in human hypertension.

ABBREVIATIONS

Ang II

Angiotensin II

ATF6

Activating transcription factor 6

BiP

Binding-Immunoglobulin Protein (Endoplasmic Reticulum Chaperone)

cGMP

Cyclic guanosine-3′,5′-monophosphate

CHOP

CCAAT-enhancer-binding protein homologous protein

EDRF

Endothelium-derived relaxing factor

ER

Endoplasmic reticulum

ET-1

Endothelin 1

H₂O₂

Hydrogen Peroxide

HDAC

Histone Deacetylase

IRE-1

Inositol-requiring enzyme 1

MAPK

mitogen-activated protein kinase

mTORC1

mammalian target of rapamycin complex 1

NO

Nitric Oxide

NOS

Nitric oxide synthase

NOX

NADPH oxidase

O2

Anion superoxide

Ox-PTM

Oxidative post-translational modification

4-PBA

4-phenylbutyric acid

PERK

Protein kinase RNA-like endoplasmic reticulum kinase

PKA

Protein kinase A

PKG

Protein kinase G

ROS

Reactive oxygen species

SERCA2

Sarcoplasmic/endoplasmic reticulum calcium ATPase 2

sGC

Soluble guanylyl cyclase

SHR

Spontaneously Hypertensive Rats

SHRSP

Spontaneously Hypertensive Stroke Prone Rats

SIRT

Sirtuin

SOD

Superoxide dismutase

TUDCA

Taurine-conjugated ursodeoxycholic acid

UPR

Unfolded protein response