Role of Thioredoxin in Age-Related Hypertension

Abstract

Purpose of Review

Although the roles of oxidant stress and redox perturbations in hypertension have been the subject of several reviews, role of thioredoxin (Trx), a major cellular redox protein in age-related hypertension remains inadequately reviewed. The purpose of this review is to bring readers up-to-date with current understanding of the role of thioredoxin in age-related hypertension.

Recent Findings

Age-related hypertension is a major underlying cause of several cardiovascular disorders, and therefore, intensive management of blood pressure is indicated in most patients with cardiovascular complications. Recent studies have shown that age-related hypertension was reversed and remained lowered for a prolonged period in mice with higher levels of human Trx (Trx-Tg). Additionally, injection of human recombinant Trx (rhTrx) decreased hypertension in aged wild-type mice that lasted for several days. Both Trx-Tg and aged wild-type mice injected with rhTrx were normotensive, showed increased NO production, decreased arterial stiffness, and increased vascular relaxation. These studies suggest that rhTrx could potentially be a therapeutic molecule to reverse age-related hypertension in humans. The reversal of age-related hypertension by restoring proteins that have undergone age-related modification is conceptually novel in the treatment of hypertension.

Summary

Trx reverses age-related hypertension via maintaining vascular redox homeostasis, regenerating critical vasoregulatory proteins oxidized due to advancing age, and restoring native function of proteins that have undergone age-related modifications with loss-of function. Recent studies demonstrate that Trx is a promising molecule that may ameliorate or reverse age-related hypertension in older adults.

Introduction

The aging process is linked to the aerobic metabolism that creates a sustained oxidative environment favoring progressive oxidation of biological macromolecules in the later part of mammalian life cycle. Although a number of antioxidant proteins have evolved to counter the oxidant formation and removal, these proteins themselves are prone to oxidative modifications resulting in loss-of-function. For example, the activity of total superoxide dismutase (Sod) and glutathione peroxidase (GPx) declined in older adults with hypertension [1, 2, 3•]. Therefore, the redox balance (oxidation-reduction) within a cell or tissue would shift to the predominantly oxidative state during the later phase of life cycle, modulating normal structure and function of a specific tissue type or organ. Accumulation of oxidative products, as well as reversible and non-reversible oxidative protein modifications are associated with inefficient and often times abnormal physiological and biochemical processes that give rise to serious pathological conditions in various organs limiting their normal function. Aging profoundly affects the circulatory system with structural and functional alterations resulting in manifestations of cardiac and vascular pathologies such as age-related hypertension [4•, 5•].

Hypertension is a major risk factor for cardiovascular disease and especially poses health problems for older adults. In fact, adults who are not hypertensive at 55 years of age have a 90% lifetime risk of eventually developing the disease [6]. For example, prevalence of hypertension increased from 7.3% to 23.6% to 66.6% among individuals aged 18–39, 40–59, and ≥ 60 years [7, 8••]. An increase of blood pressure (BP) as low as 140–145 mmHg makes us hypertensive and over 120 mmHg makes us pre-hypertensive [9]. A 4-year incidence of increased hypertension with a normal BP of 120/80 mmHg in 35–64 years age is about 6%, which triples to about 18% in the 65–94 years of age [7, 9]. High BP predisposes individuals to the development of left ventricular hypertrophy, heart failure, stroke, aneurysm, and other vascular and cardiac pathophysiology [4•, 5•, 10]. Hypertension is a multifactorial disease; however, the molecular pathophysiology of age-related hypertension is largely attributed to arterial stiffness, endothelial dysfunction, and loss of balance between vasodilators and vasoconstrictors [11, 12]. In this review, we will specifically discuss the effect of thioredoxin (Trx) on the vascular redox state and other factors in age-related hypertension that may provide novel approaches by which we may treat or reverse age-related hypertension.

The rhythmic ejection of blood from the ventricle to the aorta produces a pulse pressure of blood flow in the artery that is optimal for microcirculatory tissue perfusion [8••, 13•]. The pressure of blood flow in the artery due to a constant ejection volume is dependent on the rigidity of arterial wall and flow-mediated dilatation (FMD) [14••]. Therefore, increased arterial stiffness and loss of factors that mediate FMD will result in a higher pressure of blood flow that will exert a radial wall pressure and longitudinal shear stress, very similar to flow of fluid in a tube with a progressively narrowing diameter when the fluid is pumped with a constant force. The major contributing factors that impact BP are structural components of vessel impacting stiffness during aging, modulation of secretion of vasorelaxing factors that dilate the blood vessels, the efficiency and coordination of pumping action by the heart, and peripheral resistance that modulates the timing and velocity of the reflected wave [13•, 14••]. All of these factors are highly impacted by the aging process due to oxidation of several proteins that are the building blocks of the vascular system and are required for maintaining the normal functional performance of vascular flow [15].

Arterial Stiffness

Arterial stiffness increases with advancing age and is a major risk factor for age-related morbidity and mortality [14••]. Stiffening of aortic wall is associated with elevated pulse wave velocity and premature wave reflection [14••]. Thus, arterial stiffness is a major determinant of pulse pressure due to combined influence on the elastic effects of the arterial wall to absorb pulsatile energy and the wave propagation effects that influence peripheral wave reflection [13•, 16•]. Stiffness of aorta leading to increased pulse pressure steadily increases during advancing age resulting in decreased distensibility of the vascular wall and consequent decrease in buffering capacity of arteries to pulsatile cardiac ejection [13•]. Cross-sectional studies have shown a strong association of aortic stiffness with hypertension, obesity, and glucose intolerance [14••]. Elevated BP may cause vascular damage and accelerate conduit artery stiffening [14••]. Conversely, aortic stiffening increases pressure pulsatility and systolic BP [16•]. Further, an increase in pulsatile hemodynamic load increases cardiac afterload, reduces diastolic coronary flow, and damages the microcirculation, specifically in high flow organs such as the kidney and brain [13•, 14••].

Molecular mechanisms that alter the stiffness of the extra-cellular matrix (ECM) of the arterial wall are directly related to the aging process, involving changes in the structural proteins such as elastin and collagen [5, 17]. Progressive loss of wall elasticity occurs due to degeneration of elastin fibers, deposition of collagen, loss of orderly arrangement of elastic fibers, and thinning, splitting, fraying, and fragmentations of elastin fibers, all of which contribute to the stiffening process [8••, 13•, 17–19]. Organization of elastic fibers is also important for elastic behavior of the vessel wall [20]. ECM remodeling is modulated by the expression of matrix metalloproteinases (MMPs) due to hemodynamic effects, oxidative stress, and inflammation [14••, 17]. At the molecular level, MMPs modulate matrix protein turnover and elasticity of vessels [20]. Additionally, BP itself plays a significant role in modulating vessel wall structure, and remodeling of wall structure occurs to accommodate wall stress [16•]. For example, when angiotensin II (Ang II) is given to mice, MMP9 activity is induced and enhanced collagen degradation occurs [16•, 21]. This improves the intrinsic distensibility of elastic arteries and therefore, blunts the rise in BP [5•]. Impairment of this compensatory mechanism may contribute to increased stiffness [16•, 21]. In humans, aortic calcification has been positively correlated with hypertension and aortic stiffness [22]. Arterial stiffness and function may therefore be important potential targets for interventions aimed at preventing incident hypertension. There are several excellent reviews on arterial stiffness that provide an understanding of role of arterial stiffness in age-related hypertension [13•, 23•].

Vascular Endothelial Dysfunction

Vascular endothelium is a key producer of vasoactive factors that impact BP during advancing age. Recent studies have shown that large artery endothelial dysfunction and microvascular function jointly antedate and potentially contribute to the development of clinical hypertension [14••]. A strong negative correlation between FMD and systolic BP in cross-sectional studies has also been reported together with speculation that impaired endothelial function may be a precursor of hypertension [14••]. Additionally, a recent report has shown that initial endothelial and microvascular function is associated with incident hypertension even after considering the potential confounding effects of initial BP as well as aortic stiffness and excessive pressure pulsatility [14••]. Higher FMD is a favorable indicator of endothelial function and hence protective association was observed [14••]. Studies have shown that endothelium-independent relaxations are unaffected by aging, suggesting that endothelial dysfunction is primarily involved in age-related hypertension [11, 12, 24]. The major relaxing factor nitric oxide (NO) is produced by endothelial NO synthase (eNOS) that maintains vascular relaxation and thus decreases wall pressure and consequently maintains normal BP. Decreased availability of NO is a major reason for impairment of vascular relaxation [11, 12]. A recent study has shown that increased eNOS glutathionylation and adverse phosphorylation of eNOS occur in aged GPx knockout mice [25], demonstrating an important role of oxidative stress in eNOS dysfunction during aging. There are several recent reviews on age-dependent endothelial dysfunction that readers may find informative [15, 26••, 27, 28•]

Oxidative Stress and Antioxidant Defense

It is well established that increased levels of reactive oxygen (ROS) and nitrogen species (RNS) are generated in aged heart and arteries [29–31]. The primary ROS is the superoxide anion (O₂^·−) that is produced in excess in the mitochondria [32], by NADPH oxidase [29, 30] and due to the uncoupling of eNOS that produces O₂^·− in contrast to NO [26••, 33, 34]. When both O₂^·− and NO are produced in proximity, the highly reactive molecule peroxinitrite (ONOO⁻) is produced [26••, 33, 34]. O₂^·− dismutates to hydrogen peroxide (H₂O₂) by spontaneous reaction or with the help of SODs. H₂O₂ reacts with Fe²⁺ producing deleterious hydroxyl radicals (^·OH) that are damaging to almost all macromolecules [31, 35]. These reactive molecules attack various proteins, lipids, and carbohydrates producing secondary radicals that may inactivate a protein or produce a chain reaction of radicals such as lipid radicals [36•]. Increased ROS also depletes redox buffers such as glutathione (GSH) and creates a cellular oxidative environment [36•] that favors oxidative modification of proteins such as protein S-thiolation [37].

Several layers of the antioxidant defense system have evolved to counter the deleterious ROS generated in the aerobic mode of respiration to maximize the energy production. The primary antioxidants such as superoxide dismutases (CuZnSOD, MnSOD, and ecSOD) are first responders that neutralize the O₂^·− generated by the vascular endothelium. However, many of these antioxidant enzymes such as Sod1 and Gpx are decreased in elderly hypertensive humans [3•]. These antioxidants convert O₂^·− to H₂O₂ and molecular oxygen. The level of H₂O₂ is controlled in a manner such that only low levels of H₂O₂ are physiologically employed as signal transduction molecules [38, 39]. For example, production of low levels of H₂O₂ by Nox4 has been shown to lower BP due to its role as an endothelial derived hyperpolarizing factor (EDHF) [40]. When the production of H₂O₂ increases above threshold, the secondary antioxidants such as catalase, glutathione peroxidase, peroxiredoxins, and other peroxide scavenging enzymes and small molecule antioxidants come into play to remove high levels of H₂O₂ using electrons from various redox molecules such as NADPH via reduced GSH or Trx [36•]. Excellent recent reviews provide in-depth understanding of ROS and antioxidant defense system in the vasculature [32, 36•, 41]. Collectively, in the aged endothelium, the level of ROS is increased that can alter vascular redox homeostasis resulting in abnormal response of blood vessels to hemodynamic load.

Vascular Redox Homeostasis in the Aged Endothelium

ROS-dependent vascular damage during aging is intricately related to the antioxidant defense mechanisms of the vasculature that determines the outcome of vascular redox balance. Several layers of the antioxidant defense system in the vasculature are coupled to vascular redox systems. The major redox systems of vasculature are the glutathione system and the thioredoxin systems along with other proteins that are associated with these systems [36•]. We will only focus on the thioredoxin system in this review as the glutathione system has been the subject of several recent reviews [42, 43•]

Thioredoxin Redox System

Trx is a multifunctional protein that maintains cellular redox state. It was originally discovered as an electron donor for ribonucleotide reductase (RNR) [44]. The Trx system, which includes thioredoxin reductase (TrxR) in addition to Trx, utilizes NADPH as the source of reducing equivalents to perform protein-disulfide reduction [45•, 46]. The Trp-Cys-Gly-Pro-Cys active site of Trx is highly conserved across species. The mammalian Trx has five cysteine residues at positions 32, 35, 62, 69, and 73 [46]. Whereas Cys32 and Cys35 perform the direct transfer of electrons to a disulfide, Cys62, 69, and 73 perform regulatory functions [46]. For example, nitrosylation of Cys73 results in increased activity of hTrx [47–49], and oxidation of C62–C69 decreases the disulfide reductase activity of hTrx [50]. Although Trx does not scavenge superoxide anion (O₂^·−), it scavenges hydroxyl radicals and quenches singlet oxygen [51]. However, the mutant protein (C32S and C35S) can also scavenge hydroxyl radicals and quench singlet oxygen similar to the native protein. Hence, these functions are redox-independent [51]. Further, Trx induces MnSOD in endothelial cells [52, 53••]. Additionally, Trx induces the expression of peroxiredoxin (Prx) and also donates electrons to Prx for its peroxidase activity [54, 55]. Thus, Trx is a powerful antioxidant that can neutralize major ROS (directly scavenging ^·OH and ¹O₂, and indirectly O₂^·− via MnSOD induction, and H₂O₂ via Prx) in the vascular cells. Trx also controls signal transduction via a disulfide-thiol exchange reaction [53••, 56]. Recent studies show that it can reduce specific residues in Map kinase kinase 4 (MKK4) that plays a significant role in the transcription of MnSOD in endothelial cells via activation of NFκB [52, 53••]. Although Trx is loosely considered as an antioxidant enzyme, in fact, it is not an enzyme, but a protein containing redox-active sites Cys32 and Cys35 that transfer electrons directly to disulfides for reduction. The reduction process is non-enzymatic but stoichiometry determines the rate of reduction [45•, 46, 57, 58]. Taken together, thiol-disulfide exchange by Trx transduces signals similar to protein phosphorylation and dephosphorylation.

In addition to cytosolic Trx there also is a mitochondrial form of thioredoxin (Trx2), which is synthesized in cytosol and translocates to mitochondria [45•, 58]. Structurally, the Trx2 protein lacks the regulatory cysteines found in the cytosolic Trx, although the catalytic cysteines that take part in reduction are conserved [45•, 58]. Loss of Trx2 induces massive apoptosis of heart suggesting that it may be important to protect against mitochondrial-triggered apoptotic signaling [59]. Overexpression of Trx2 in young mice protects against hypertension caused by Ang II infusion, and this effect involves attenuation of NADPH oxidase-mediated oxidative stress [60]. This study also determined that Trx2 had no effect on eNOS levels or NO production [60]. In contrast, Zhang et al. [61] observed increased NO bioavailability in their Trx2-overexpressing transgenic mice. Both of these studies examined young adult mice, so the protective effects of high levels of Trx2 expression they described are not likely to be related to the effects of Trx in aging mice, since the mechanisms associated with age-related hypertension are different than in angiotensin-dependent experimental hypertension.

Effect of Trx on Factors that Contribute to Age-Related Hypertension

Trx is a multifunctional protein involved in redox control of gene expression, antioxidant defense, thiol-disulfide exchange reaction in a variety of proteins, and regeneration of oxidatively inactivated proteins. The diversity of its impact on structure and function of proteins and cellular redox control makes it a unique redox molecule. In the following paragraphs, we will discuss the effect of Trx on several factors in the development of age-related hypertension.

Arterial Stiffness and Trx

Vascular matrix remodeling during aging is an important biological process responsible for age-associated arterial stiffness and the development of hypertension. Vascular matrix remodeling includes processes of vascular smooth muscle cell proliferation (VSMC), fibrosis, and calcification [62•, 63, 64]. As age advances, the increase in vascular inflammation and oxidative stress triggers activation of MMPs and inhibition of tissue inhibitors of matrix metalloproteinases (TIMPs) [65–68]. Activated MMP degrades collagen and elastin and releases growth factors from the basement membrane to create a proinflammatory environment [69]. MMP inhibitors were shown to suppress age-associated inflammatory signaling, collagen deposition, decreases in elastin level and reduce BP [70]. Activation of MMP2 is associated with arterial elastolysis [70], arterial fibrosis [71], and calcification [72], which collectively contribute to the stiffness of arteries. In fact, Wang et al. [73] detected activated MMP2 in aged arteries. Trx was shown to inhibit MMP2 activity [74] and therefore, can directly reduce the stiffness of arteries. Trx also was shown to inhibit ultraviolet radiation (UVA)-induced MMP-1 and collagen 1α expression in dermal fibroblasts [75]. Further, Trx was included as an important component of an anti-aging therapeutic cocktail [75]. In another study, Trx was shown to interact with a disintegrin and metalloprotease 17 (ADAM 17) to reduce its activity [76]. Additionally, it has been shown that nuclear overexpression of Trx stabilizes Nrf2-ARE activity that mitigates the exaggerated TGF-β1 expression and fibrosis induced by chronic alcohol ingestion following bleomycin-induced lung injury [77]. Anti-inflammatory role of Trx is further evidenced by a study that showed the atheroprotective effect of Trx by promoting macrophage differentiation into the M2 anti-inflammatory phenotype [78]. Proinflammatory signals and MMP activation remodel the vascular ECM that transforms VSMCs from the contractile to synthetic phenotype. These secretory and migratory VSMCs cause intimal-medial thickening and synthesis of more collagen via activation of TGF-β/SMAD signaling [62•]. Aged VSMCs expressed higher levels of platelet-derived growth factor receptor-BB (PDGF-BB) and proliferate more in response to PDGF-BB [79]. PDGF-BB signaling inactivates protein tyrosine phosphatase B1 (PTPB1) by cysteine oxidation and Trx was shown to reactivate oxidized cysteines of PTPB1 in vitro and in cell-based systems [80–82]. Collectively, Trx might prevent aged VSMC proliferation via PTPB1 reactivation. It has been shown that aged SOD2^+/− mice develop arterial stiffening due to increased expression and activity of MMP-2, collagen, and decreased elastin expression [83]. Further aging diminished SOD2 expression in arteries and TEMPOL administration ameliorates arterial collagen deposition and stiffening [84]. We have shown that Trx upregulates SOD2 expression in human endothelial cells; thus, the presence of active Trx may prevent arterial aging via SOD2 expression [53••].

In our recent study, we provided direct evidence that high levels of Trx in aged Trx-Tg resulted in significant reduction of arterial stiffness [31]. Using high-resolution ultrasound imaging, we found that peak systolic blood velocity (PSV) and end diastolic blood velocities (EDV) were higher in young and aged Trx-Tg compared to age-matched NT and dnTrx-Tg mice, where redox-active cysteines C32, C35 are mutated to serine [31, 85]. The resistance index, calculated as the difference between PSV and EDV divided by PSV, was similar between young NT, Trx-Tg, and dnTrx-Tg mice, but increased with aging in NT, but not in Trx-Tg mice [31]. The inner diameter of the superior mesenteric artery (SMA) was increased in aged Trx-Tg mice, indicative of outward structural remodeling [31]. Further, wall thickness was significantly increased in SMA from young dnTrx-Tg. In addition, we observed a thicker wall, characteristic of hypertrophic remodeling with aging, in SMA from NT and dnTrx-Tg mice, whereas SMA from Trx-Tg mice showed no change in wall thickness. We also found a greater stiffness of the SMA in aged NT and dnTrx-Tg compared to aged Trx-Tg mice [31].

At the molecular level, studies have shown S-nitrosylation post-translational modification mediated by NO via cycle GMP-dependent pathways, where the protein cysteine thiol undergoes covalent modification by a NO group and generates S-nitrosothiol [86]. The S-nitrosylation process of the tissue transglutaminase (TG2) protein has been shown to be involved in the calcium-dependent, TG2-mediated modification of the vascular ECM through formation of collagen cross-linking affecting stiffness [87]. Since Trx is also known to cause denitrosylation of proteins [88•], it can contribute to decreased stiffness by denitosylation of TG2. The reduced S-nitrosylation of TG2 that takes place with reduced production and bioavailability of NO causes secretion of the protein to the extracellular space. Increased activity of matrix TG2 has been shown to be associated with increased aortic PWV in TG2 knockout mouse models [89]. Studies in aging rats and TG2 and eNOS knockout mice models have shown that a reduction in the bioavailability of NO as occurred with aging, inflammation, and endothelial dysfunction in general is associated with cellular mechanisms contributing to arterial stiffness [90]. Therefore, high levels of Trx could potentially alter this pathway resulting in decreased arterial stiffness during aging.

Role of Trx in Endothelial Dysfunction in Age-Related Hypertension

One of the underlying mechanisms associated with age-related vascular endothelial dysfunction is the chronic oxidation of vascular proteins that occurs over the life span of mammals resulting in the loss of arterial relaxation and consequent increase in BP. Accumulating evidence suggests that bioavailability of NO, a critical endothelium-derived relaxing factor produced by eNOS, becomes impaired during aging resulting in increased vascular resistance and high BP [27, 91–95]. A dysfunctional eNOS produces O₂^·− by transferring electrons to molecular oxygen (O₂) instead of L-arginine, resulting in the uncoupling of eNOS-L-arginine pathway of production of NO [96]. Further, O₂^·− produced by dysfunctional eNOS is also known to react with NO, resulting in the production of vascular peroxynitrite (ONOO⁻), which is a powerful oxidizer, and could cause further oxidative damage to aging vessels. Therefore, bioavailability of NO is decreased with advancing age [26••]. Chen et al. [97] showed that eNOS glutathionylation at Cys689/908 resulted in uncoupling of eNOS and formation of O₂^·−. We found that eNOS glutathionylation was elevated in aged arteries [31] and Trx directly deglutathionylates eNOS in a redox-dependent manner and restores NO formation by eNOS [98••]. Soluble guanylate cyclase (sGC) is the major and most sensitive receptor of eNOS generated NO [99]. Kloss et al. showed that aging desensitized sGC in the aged rat associated with hypertension [100]. Even though the mechanism of aging associated sGC desensitization is not known, it has been shown that cysteine nitrosylation in sGC caused its inactivation and resulting in hypertension [101]. Recently, Huang et al. [102] demonstrated that Trx is associated with dysfunctional (nitrosylated) sGC and restored its active state via denitrosylation [102]. A recent study has also identified nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase 3 (Cyb5R3) in the VSMC is a critical regulator of sGC heme iron reductase that controls sGC sensitivity to NO and cGMP levels [103]. Although the role of Cyb5R3 depletion has been shown to modulate GSH/GSSG ratio in VSMC [103], its effect on the Trx redox system has not been delineated. Trx could be an important regulator of the NO-sGC-cGMP signal transduction pathway of vasorelaxation in the VSMC due to its redox control of sGC [103] and its role in denitrosylation.

Increased oxidative load with decreased antioxidant defense promotes protein modification in the aged vasculature that significantly impacts structural and functional attributes of vessels. For example, arteries from aged wild-type mice, but not Trx-Tg mice, show high levels of eNOS glutathionylation [31], suggesting a protective role of Trx either in the formation of S-glutathionylated eNOS or deglutathionylation of SG-eNOS by Trx. Indeed, our recent publication has shown that Trx is an efficient deglutathionylating agent that deglutathionylates eNOS in response to ischemia-reperfusion injury in the heart [98••]. Functionally, this is a novel property of Trx that constitutes an effective mechanism in protection against dysfunctional PrS-SG proteins including eNOS-SG. This function is especially important in aging heart as Grx1, the major enzyme known to deglutathionylate PrS-S-G proteins, remains inactivated in the mitochondria due to advancing age (Fig. 1). In contrast to Grx1, the activity of Trx in the heart mitochondria does not decrease during aging [104], suggesting an important role of Trx in age-related Pr-S-SG deglutathionylation compared to Grx1.

Fig. 1.

In younger vessels, redox homeostasis favors reducing conditions that maintain eNOS and Grx1 in the active state; thereby, NO production is maintained in a sustained manner resulting in decreased arterial stiffness and normal vascular relaxation. Aging oxidizes GSH to GSSG and also decreases the activity of Grx1 that oxidizes critical cofactor BH4 of eNOS. Also, high levels of GSSG induce eNOS glutathionylation that produces high levels of O₂^·− with decreased vasorelaxation and increased arterial stiffness. High levels of Trx deglutathionylate eNOS and induce NO generation resulting in vascular relaxation. In this process, reduced Trx is oxidized and is further regenerated by thioredoxin reductase (TrxR) utilizing NADPH

Endothelium-Derived Hyperpolarizing Factor and Trx

Although NO and prostacyclin are major vasorelaxing factors in the endothelium that regulate BP, a significant amount of residual vasorelaxation remains in spite of inhibition of acetylcholine or prostacyclin in the endothelium [40]. This unidentified relaxing factor is known as EDHF and is more prevalent in resistance arteries and is considered as a critical mechanism in BP control [105, 106]. Recent studies have identified H₂O₂ and cytochrome P-450 derived lipid mediators as EDHF [40, 107]. H₂O₂ functions as EDHF in several vascular beds including mouse and humans [38, 106]. H₂O₂ activates PKG1α by inducing the formation of a disulfide bond between two PKGα1 polypeptides that causes activation of this kinase independent of NO-cycling guanosine monophosphate (cGMP) pathway and induces vasorelaxation [105, 106]. A single atom substitution in mouse PKG1α (C42S), a redox-inactive version of PKG1α, inhibits the vasodilatory action of H₂O₂ in resistance vessels and causes hypertension in vivo [106]. We have previously shown that endothelium-dependent hyperpolarizing (EDH) relaxations in small mesenteric arteries are dependent on in vivo redox condition of vessels [35]. Our study demonstrated that increased Trx expression in Trx-Tg mice results in an increased EDH response in small mesenteric arteries [35]. In contrast, mice that are deficient in active Trx show severely blunted endothelium-dependent relaxations due to both a reduced NO- and EDH-mediating relaxation [35]. Functionally, a reduced NO and EDH response in dnTrx-Tg was reflected by a significantly higher systolic blood pressure; in contrast, increased NO and EDH response was correlated with decreased blood pressure in Trx-Tg mice [35], confirming the important role of endothelial K_Ca channels in modulating BP [35]. Additional in vitro studies show that the EDH relaxations could be modulated by exogenous addition of sulfhydryl modifying agents such as diamide or DTT, where diamide blunted but DTT enhanced the EDH relaxation [35]. Further, we demonstrated a pivotal role for IK1 channel activation in mediating the EDH response via redox state modulation [35]. We have also shown increased EDH-mediated relaxation responses in the presence of L-NAME and indomethacin [35] in the resistance arteries of Trx-Tg mice compared to WT mice or dnTrx-Tg mice [35]. Consistent with our observation, other studies have found no effect of catalase on inhibition of EDH-mediated relaxing responses [108]. However, the specific effect of H₂O₂ on K_ca channels remains unknown [108]. Our study demonstrated that a reduced form of Trx is able to increase the EDH response, which increases IK1 channel activity and lower systolic BP [35]. Conversely, loss of active Trx or oxidation of Trx resulted in decreased NO and EDH-mediated relaxing responses to acetylcholine [35], demonstrating a redox control of EDH response by Trx.

In Vivo Modulation of Vascular Trx Redox and Age-Related Hypertension

We have recently shown that high levels of Trx in mice protect against endothelial dysfunction and the development of age-related hypertension [31]. Aged Trx-Tg mice showed higher Trx activity in the vessels compared to WT or dnTrx-Tg mice. Additionally, the redox state of Trx in the endothelium of aged Trx-Tg mice was found to be more reduced compared with aged WT or dnTrx-Tg mice suggesting that high levels of vascular Trx outcompete the oxidation of endogenous Trx, but loss of active Trx resulted in increased oxidation of vascular Trx in vivo [31, 35]. Aged Trx-Tg mice were normotensive compared to aged WT or dnTrx-Tg mice that were hypertensive [31]. Trx-Tg mice showed decreased arterial stiffness concomitant with enhanced NO generation with significant decrease in the level of glutathionylated eNOS in the aorta [31]. Further, endothelium-dependent relaxation was preserved in superior mesenteric artery (SMA) of aged Trx-Tg mice, but severely blunted in aged dnTrx-Tg mice [31]. Additionally, high amount of Trx in the vasculature decreased the SMA flow resistance in aged Trx-Tg, but not dnTrx-Tg [31]. Electron paramagnetic resonance (EPR) spectrometry studies demonstrated eNOS to be the major O₂^·− producing protein in aged artery, but not Nox [31]. Additionally, eNOS phosphorylation and expression were increased in the mesenteric artery of aged Trx-Tg mice, but not in SMA of WT or dnTrx-Tg mice (Fig. 2).

Fig. 2.

In age-related hypertension dysfunctional eNOS (eNOS-SG), activated NADPH oxidase, and decreased activity of Sod results in generation of high levels of O₂^·− in the vessel that creates an oxidative and inflammatory environment, which activates MMPs and induces arterial stiffness with decreased vascular relaxation that give rise to age-related hypertension. High levels of Trx can decrease arterial stiffness and increase vascular flow by decreasing activity of MMPs, inducing eNOS-mediated NO production, decreasing O₂^·− production by NADPH oxidase and activated mitochondria. The restoration of these proteins to their native state results in decreased age-related hypertension that is maintained for a longer time period

These findings suggest a profound protective effect of high levels of human Trx expression in age-related vascular dysfunction and hypertension in Trx-Tg mice.

Novel Therapeutic Role of Trx in the Reversal of Age-Related Hypertension

Since increased O₂^·− is produced in a chronic manner by oxidatively modified vascular proteins due to advancing age, an intervention that prevents age-related oxidation of these proteins is likely to protect the development of hypertension. Further, an agent that reverses the oxidatively modified proteins to their native state is expected to reverse age-related hypertension. In contrast, removal of O₂^·− due to increased expression of antioxidant enzymes or by injecting therapeutic levels of superoxide dismutase (SOD) mimetic is expected to remove O₂^·− produced by the dysfunctional eNOS or NADPH oxidase that may prevent further oxidation of vascular proteins, but these agents will not regenerate the dysfunctional eNOS to a functional state, or reverse other oxidized proteins or enzymes to their native state. Consistent with this reasoning, a study using Sod1^+/− mice has shown that TEMPOL, a SOD mimetic, had no effect in decreasing hypertension in aged mice; however, TEMPOL was effective in decreasing endothelial dysfunction in Sod1^+/− mice indicating that TEMPOL could compensate for 50% of Sod1 that was unavailable in Sod1^+/− mice [109]. However, this study showed that the activity of Sod1 did not change between young or aged mice, although the BP was noted to be significantly higher in aged mice [109]. In contrast, a recent study has shown that antioxidative therapy with TEMPOL could improve arterial stiffness and decrease endothelial dysfunction in mice, but it had no effect on BP in contrast to many other studies that have shown increased BP in aged mice [84, 110]. Our recent study has shown that direct injection of recombinant human Trx lowered BP in aged WT mice, decreased eNOS glutathionylation, and decreased the ROS generation in the vessels of aged WT mice, suggesting therapeutic efficacy of recombinant Trx in reversal of age-related hypertension. Since hypertension is a multifactorial disease and Trx is a multifunctional protein that regulates several determinants of age-related hypertension, Trx is a promising molecule to treat or reverse age-related hypertension. This finding provided a new approach to reverse age-related hypertension. Additionally, new therapies could also be designed based on this finding to reverse age-related hypertension (Fig 2).

Conclusion

Age-related hypertension is a multifactorial disease and is an underlying mechanism for several cardiovascular disorders in older adults. Age adversely impacts arterial stiffness, eNOS function, vascular redox balance, and generation of ROS, all of which contribute to the development of age-related hypertension. Therefore, any therapeutic intervention that targets a single factor is expected to provide only temporary amelioration of hypertension. For example, an AT₁ receptor blocker (ARB) would decrease normal contraction of vessels due to angiotension II and thereby would induce vascular relaxation, which decreases BP. However, an ARB would not be expected to provide sustained lowering of BP in the older persons. Trx is a multifunctional protein; it can regenerate eNOS that would produce NO for a considerable longer time period. Trx can also reverse arterial stiffness and promote beneficial vascular remodeling and would also be able to decrease ROS production by increasing antioxidant enzyme restoration and maintaining the redox homeostasis of the aged vessel. Recent exciting experimental findings demonstrate that indeed Trx can effectively perform all of these functions, resulting in prolonged lowering of BP in aged mice. This finding introduces a novel treatment strategy for older persons. Further, once Trx is tested in a clinical trial, it would be clear whether humans could use high levels of Trx to restore the aging blood vessel structure and function resulting to reverse age-related hypertension.