REVIEW ARTICLE

Redox biology in pulmonary arterial hypertension (2013 Grover Conference Series) Joshua P. Fessel,1 James D. West Division of Allergy, Pulmonary, and Critical Care Medicine, Department of Medicine, Vanderbilt University, Nashville, Tennessee, USA

Abstract: Through detailed interrogation of the molecular pathways that contribute to the development of pulmonary arterial hypertension (PAH), the separate but related processes of oxidative stress and cellular metabolic dysfunction have emerged as being critical pathogenic mechanisms that are as yet relatively untargeted therapeutically. In this review, we have attempted to summarize some of the important existing studies, to point out areas of overlap between oxidative stress and metabolic dysfunction, and to do so under the unifying heading of redox biology. We discuss the importance of precision in assessing oxidant signaling versus oxidant injury and why this distinction matters. We endeavor to advance the discussion of carbon-substrate metabolism beyond a focus on glucose and its fate in the cell to encompass other carbon substrates and some of the murkiness surrounding our understanding of how they are handled in different cell types. Finally, we try to bring these ideas together at the level of the mitochondrion and to point out some additional points of possible cognitive dissonance that warrant further experimental probing. The body of beautiful science regarding the molecular and cellular details of redox biology in PAH points to a future that includes clinically useful therapies that target these pathways. To fully realize the potential of these future interventions, we hope that some of the issues raised in this review can be addressed proactively. Keywords: bone morphogenetic protein receptor 2, Warburg, 3-nitrotyrosine, nitrosylation. Pulm Circ 2015;5(4):599-609. DOI: 10.1086/683814.

Considered in a positive light, the intense investigative efforts into the pathogenesis of pulmonary arterial hypertension (PAH) over the past several decades can be viewed as a major success. We continue to gain more-detailed insights into the molecular mechanisms that cause and sustain disease. New therapeutics are being successfully tested and approved for human use, with more in the developmental pipeline. Emerging data suggest not only that the thoughtful and aggressive use of existing therapies has improved the quality of life and functional status for patients with PAH but also that these therapies have actually had a measurable positive impact on survival.1 However, most investigators in the field remain driven by a lingering dissatisfaction. Although existing therapies are certainly beneficial in terms of improving functional status and quality of life and likely have a measurable survival benefit, many patients still will progress to the end points of death or need for transplantation. In tissues collected from these individuals, either at autopsy or at explantation of the diseased lungs, it appears that targeted therapies have not changed the course of the pulmonary vascular pathology.2 New drugs continue to be developed and approved for clinical use, but most of these target the same signaling pathways (e.g., prostacyclin, endothelin, and nitric oxide) as the first generation of PAH drugs.3-5 Despite impressive progress, then, we still

lack some fundamental insights into the processes that initiate and sustain PAH, and thus it remains an incurable, progressive, and ultimately fatal disease. At the 2013 Grover Conference, we discussed data from our investigations into the downstream pathogenic mechanisms that begin with loss-of-function mutations in bone morphogenetic protein receptor 2 (BMPR2) and end with what we recognize as PAH. We discussed data regarding oxidant injury and lipid peroxidation, mitochondrial dysfunction, metabolic reprogramming, and how we think these processes contribute to disease. In this review, we further expand on the separate but intimately related topics of oxidative stress and molecular metabolism, attempting to unite them under the broader heading of redox biology. We hope to highlight some “gray areas” that are in need of more in-depth investigation and clarification, as we think that there is real therapeutic benefit to be realized from using redox biology as one source for the next generation of possible treatments for PAH. We focus our discussion as much as possible on World Health Organization (WHO) group I disease, recognizing that many experimental PAH systems straddle the line between group I and other classifications (e.g., group III) and that at least some pathways may be common to more than one form of pulmonary hypertension.

Address correspondence to Dr. James D. West, Vanderbilt University, Nashville, TN 37232-0656, USA. E-mail: [email protected]. Submitted January 2, 2015; Accepted May 14, 2015; Electronically published September 21, 2015. © 2015 by the Pulmonary Vascular Research Institute. All rights reserved. 2045-8932/2015/0504-0001. $15.00.

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B R I E F R E V I E W OF R E A C TI V E O X Y G EN SP E C I E S IN PAH To begin, a review of what is meant by “reactive oxygen species” (ROS) as pertains to PAH is in order. In general, this term refers to a group of small molecules formed from reactions that require the participation of at least one oxygen atom (typically from molecular oxygen or from water) and involve a configuration of electrons around the oxygen atom that renders it chemically unstable on its own. The relative reactivity of any individual ROS depends on the specific configuration of electrons around the oxygen atom, the possible alternative/resonance structures within the molecule containing the reactive oxygen atom, and local conditions outside of the molecule containing the ROS (e.g., solvent conditions). The reactivity of any given ROS is what determines how injurious it will be within a cell or tissue and also determines its ability to function in a signaling capacity. In the pathogenesis of PAH, several ROSs have been particularly well studied. Perhaps the most relevant ROS in PAH is nitric oxide (NO). NO is formed by nitric oxide synthase from the combination of the guanidino-group nitrogen in arginine with molecular oxygen to yield NO and water, converting arginine to citrulline in the process with electrons provided by NADPH and transferred by the heme iron. By virtue of its electronic configuration (with an unpaired electron in an antibonding π orbital), NO is relatively unreactive toward most biomolecules, reacting primarily with other ROSs—most notably, reacting at near the diffusion limit with superoxide to form peroxynitrite, itself an ROS that results in cellular damage by both oxidation and nitration of molecular targets. Superoxide anion, the reaction partner of NO that forms peroxynitrite, is itself an ROS formed from the 1-electron reduction of molecular oxygen. Superoxide can be produced by the specific action of NADPH oxidase as part of the oxidative burst of neutrophils. In this way, inflammation can contribute directly to oxidative stress. More commonly, superoxide is produced by an incomplete reaction cycle of enzymatic systems that typically reduce molecular oxygen completely to water. In PAH, such enzymatic systems include uncoupled nitric oxide synthase, electron-transfer enzyme complexes in mitochondria (see below), cell-free hemoglobin, and any number of other proteins containing redox-active metals. Under normal conditions, superoxide is fairly rapidly converted to hydrogen peroxide (H2O2) through the action of superoxide dismutase. H2O2 itself is considered by many to be an ROS, although its reac-

tivity is substantially less than that of most other biologically relevant ROSs (see below). These ROSs are summarized in Table 1. O X I D A T I V E ST R E S S , O XI D A N T SI G N A L I N G , A N D O XIDA N T IN JURY Mounting evidence for some time has implicated oxidative stress as an important pathogenic mechanism in PAH, both in patients with disease and in virtually every experimental model of PAH.6-10 Evidence for increased production of ROSs has been demonstrated in cells, tissues, blood, urine, and even breath condensate with a variety of analytical techniques. The often-unstated underlying assumption is that increased production of ROSs is uniformly detrimental, presumably through direct, deleterious chemical modification of proteins, lipids, and DNA. The terminology as it is most often used makes two generalizations. First, the term “oxidative stress” assumes that any observed increase in an assay for “ROSs” above the level observed in the control or normal condition is indicative of a disease-associated process. The strict definition of oxidative stress is the production of oxidizing species of all types that, in total, exceed the capacity of enzymatic and chemical antioxidant defenses and that cause a deviation from homeostasis. The second generalization is that all ROSs are more or less functionally equivalent.

The problems with “oxidative stress” The problems with these generalizations are fairly readily apparent. An increase in ROS production that is measurable but does not exceed the cell’s antioxidant capacity and/or does not cause a significant deviation from homeostasis is not creating a true stress. Also, it is clear that not all ROSs are equivalent.11 For example, H2O2 is often thought of as an ROS, as many assays used to quantify ROS measure H2O2. However, it is not an especially reactive substance by itself (a dilute, 3% solution is sold by the bottle by most pharmacies in the United States). Often, the Fenton reaction (Fig. 1) is invoked to explain how H2O2 causes oxidative stress. In the presence of free transition metals (e.g., iron or copper), H2O2 can undergo a single-electron reduction to either a hydroxyl radical or a hydroperoxyl radical, both of which are extremely reactive and will oxidatively modify nearby macromolecules with a speed at or near the diffusion limit. However, the availability of free transition metals in most biological systems is extremely low, and there

Table 1. Summary of major reactive oxygen species in pulmonary arterial hypertension Reactive oxygen species Nitric oxide (NO•) Superoxide (O2•−) Peroxynitrite (ONOO−) Hydrogen peroxide (H2O2)

Common sources

Common reaction targets

Nitric oxide synthase NADPH oxidase, mitochondrial electron transport chain, uncoupled NOS, heme-containing proteins Reaction of NO and superoxide Superoxide dismutase

Heme iron, free sulfhydryl groups Nucleotides (especially guanine), lipids (especially polyunsaturated fatty acids), proteins Tyrosine residues in proteins Metal atoms

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Figure 1. Reactive oxygen species (ROSs) and products of redox reactions exist along a spectrum from oxidant injury to oxidant signaling. The ROSs that are most commonly studied under the umbrella of “oxidative stress” can be separated on the basis of how they behave as injurious stimuli versus how they behave as signaling species/second messengers. A partial listing is shown in this figure. For example, the hydroxyl radical covalently reacts essentially at the diffusion limit with any nearby macromolecule (lipid, protein, or nucleic acid), kinetics that are too rapid for regulated signaling and that functionally can only drive oxidant injury. By contrast, at the other end of the spectrum is nitric oxide, a free radical that is quite chemically stable and has a very well established role as a bona fide second messenger. Similarly, the products of free-radical reactions with macromolecules can be ordered along a similar spectrum. Products such as F2-isoprostanes and protein carbonyls have predominantly been shown to be markers of oxidant injury (although F2-isoprostanes can signal, there does not appear to be tightly regulated production and degradation that would qualify them as an oxidant signal). However, production of 8-oxoguanosine has been shown to exhibit regulated production to control gene expression. GSSG: glutathione disulfide; HNE: hydroxynonenal.

is some debate as to whether Fenton chemistry happens to any appreciable degree in vivo.12 The Haber-Weiss cycle (Fig. 1) has been proposed as an alternative mechanism for how H2O2 and superoxide anion together can result in hydroxyl radical production. This may have more relevance to in vivo settings, although in the absence of free transition metals to catalyze this series of reactions, the Haber-Weiss cycle is fairly slow and would require relatively high concentrations of H2O2 and superoxide to proceed with any efficiency.13 Further, a strong case can be made for H2O2 as a bona fide signaling molecule, possessing characteristics of regulated synthesis and breakdown, specific molecular targets, localized cellular production, and measurable biologic effects that exist along a spectrum of intensity that coincides with that of a signal.14-16 Superoxide anion, while more reactive than H2O2, is still a relatively stable ROS; the reactivity of the hydroxyl radical is between 5 and 10 orders of magnitude greater. NO, which is itself a free radical (and one could debate whether it is an ROS or a reactive nitrogen species), is so stable as to serve an almost exclusively signaling role and becomes damaging only after reacting with superoxide to form the peroxynitrite anion. Many colorimetric and fluorimetric assays cannot distinguish between the different ROSs, and the functional differences are substantial.17,18 These are not merely semantic distinctions, as evidence exists in the literature to suggest that more-specific determinations about

the oxidants produced in PAH and their role in disease pathogenesis could have a significant impact on novel therapeutic development. As suggested above, the differences between oxidant injury and oxidant signaling are separable, and these processes should thought of as related to but distinct from each other (Fig. 2). Oxidant injury more clearly refers to the end result of oxidative macromolecule modification in an essentially unregulated fashion. Rather than relying on techniques that provide some readout of the presence and production of ROSs, assessing oxidant injury involves quantification of the stable end products of macromolecular damage—lipid peroxidation products, oxidatively modified nucleotides, protein oxidation products, and carbohydrate oxidation products. By contrast, oxidant signaling involves the coordinated, regulated production of ROSs to achieve a specific, measurable biological end point. Oxidant signaling possesses other hallmarks of signal transduction cascades, including signal amplification, signal termination, and a relatively narrow spectrum of downstream responses. Both oxidant signaling and oxidant injury have been demonstrated in PAH.

Methods for assessing “oxidative stress” Before discussing the particulars of oxidant injury and oxidant signaling, we present a brief review of the methods for measuring ROSs and the results of injurious chemical reactions driven by ROSs. Several methods exist for quantifying ROSs themselves.

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Oxidant signaling in PAH

Figure 2. Summary of the Fenton reaction and the Haber-Weiss cycle. The Fenton reaction and the Haber-Weiss cycle are often invoked as important sources of damaging reactive oxygen species (particularly the hydroxyl radical) in vivo. There is some debate as to whether either of these processes proceeds with any appreciable efficiency in living cells and tissues.

Electron paramagnetic resonance (EPR)/electron spin resonance (ESR) spectroscopy can be used to quantify specific radical species, either through spectroscopic examination of the radicals themselves or through the use of spin traps that form more-stable radical species to permit study. EPR/ESR is quantitative and specific and can be used with biological samples, but the technique requires a great deal of technical expertise and specialized equipment. As in the use of spin traps, there are a variety of dyes that become fluorescent upon reaction with particular ROSs. These are notable for being easy and relatively inexpensive to use, but as discussed above, specificity and true quantitation are significant drawbacks to many of these dyes. Aside from quantifying ROSs themselves, many methods are available for quantifying the myriad products of ROS interactions with biological macromolecules. Lipid peroxidation products (e.g., isoprostanes, isofurans, hydroxynonenal, malondialdehyde, and total lipid hydroperoxides) can be quantified by mass spectrometry, the TBARS (thiobarbituric acid reactive substances) assay, enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography, and various colorimetric assays. Similarly, nucleotide oxidation products (e.g., 8-oxoguanosine) can be quantified by many of these same methods. Protein oxidation can be quantified by measurement of “protein carbonyls,” a general measure of protein oxidation, or by more specific assays, such as quantification of 3-nitrotyrosine by ELISA, Western blot, mass spectrometry, or other methods. All of these methods vary in their performance characteristics (e.g., limits of detection, interfering substances and tolerance of sample quality, specificity of the species quantified), and while a complete review is well beyond the scope of this work (and the topic has been amply reviewed elsewhere19-24), the specific technique employed in any given experiment should be tailored to the question being asked.

Two recent studies from the pulmonary hypertension literature provide fairly clear, illustrative examples of oxidant signaling in PAH. Al-Mehdi et al.25 published an elegant series of studies in 2012 showing that chemical oxidation of nucleotides (specifically, guanosine bases) in the promoter region of the vascular endothelial growth factor (VEGF) gene is required for efficient hypoxiainducible factor (HIF)-mediated transcription of the gene under hypoxic conditions. The oxidation of the promoter region was a carefully orchestrated event that involved translocation of mitochondria to a perinuclear region, followed by a nuclear-specific increase in ROSs. Prevention of mitochondrial clustering prevented the oxidation of the VEGF promoter, decreased the binding efficiency of HIF-1α, and decreased production of VEGF messenger RNA without changing the amount of HIF-1α in the nucleus. In 2010, Archer and colleagues26 demonstrated that in the fawn-hooded rat model of PAH, superoxide dismutase 2 (SOD2) is epigenetically silenced, leading to a hyperproliferative, apoptosis-resistant phenotype in pulmonary artery smooth muscle cells (PASMCs). Interestingly, in the absence of normal SOD2 expression, PASMCs showed less ROS production (with data support ing H2O2 as the relevant ROS). Restoration of SOD2 expression or replacement with a pharmacologic SOD2 mimetic (MnTBAP) increased ROS production, restored normal apoptosis, normalized PASMC proliferation, and ameliorated the PAH phenotype in vivo. The fact that increasing ROS production led to restoration of programmed cell death—a tightly regulated process required for normal cellular homeostasis— strongly supports an argument for oxidant signaling.

Oxidant injury in PAH Oxidant injury, as defined above, has also been demonstrated to associate with and to contribute to PAH pathogenesis. Cracowski and colleagues10,27 found that levels of F2-isoprostanes—a robust biomarker for lipid peroxidation and oxidant injury in vivo—measured in the urine of PAH patients at the time of diagnostic right heart catheterization independently predicted increased mortality in both unadjusted and adjusted risk prediction models. Previously, Robbins et al.28 had published that the major urinary metabolite of F2-isoprostanes decreased in PAH patients after the initiation of epoprostenol therapy. Oxidized guanosine and 3-nitrotyrosine levels have also been demonstrated to be elevated in experimental models of PAH and in lung tissue from patients.29-31 We and others have shown that disruption of extracellular superoxide dismutase (EC-SOD), either globally or in specific cell populations, results in exacerbation of experimental PAH, with accompanying increases in markers of oxidant injury, and that enhancing EC-SOD activity ameliorates disease.32-34 To help solidify a causative link between oxidant injury and PAH, we have shown that driving mitochondrial oxidant injury (quantified by measurement of lipid peroxidation products) using brief daily exposure to hyperoxia worsens PAH in BMPR2-mutant mice and also drives dysregulation of glucose homeostasis, suggesting a link between oxidant injury and cellular metabolism.35

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Ambiguity in distinguishing oxidant injury from signaling It can sometimes be difficult to distinguish between oxidant injury and oxidant signaling, and there is certainly a gray area in the middle. For example, 3-nitrotyrosine is formed from the nonenzymatic nitration of tyrosine residues by peroxynitrite, an aggressive pro-oxidant produced by the reaction of superoxide anion with NO. Thus, 3-nitrotyrosine is appropriately considered a product of oxidant injury. However, nitration of tyrosine residues does not result in widespread, nonspecific protein dysfunction. Indeed, in pulmonary microvascular endothelial cells from BMPR2-mutant mice (Fig. 3, left) and in skin fibroblasts from heritable PAH patients with BMPR2 mutations (Fig. 3, right), a Western blot for 3-nitrotyrosine shows multiple specific bands of higher-intensity staining than is seen in wild-type cells. Similarly, a group of investigators at the University of Illinois in Chicago36-38 has very nicely demonstrated that knockout of caveolin-1 results in both increased superoxide and increased NO production, causing nitration of specific protein targets (e.g., protein kinase G, p190RhoGAP-A), and leads to the development of experimental PAH. In keeping with these findings, mutations in caveolin-1 in humans have been shown

Figure 3. Pulmonary arterial hypertension–causing bone morphogenetic protein receptor-2 (BMPR2) mutations drive tyrosine nitration of specific targets. In both murine pulmonary microvascular endothelial cells (PMVECs) and skin fibroblasts from patients, expression of any one of several mutant BMPR2 isoforms is sufficient to drive nitration of tyrosines in multiple protein targets, as shown in this Western blot from total protein lysates probed with an antinitrotyrosine antibody. Fold increase is indicated by the numbers and calculated as a ratio of the densitometry in the BMPR2 mutant sample to that in control/wild-type cells (average densitometry for the two, in the case of the two different BMPR2 mutations represented by the human fibroblasts). Of note, bands appear as a ladder in both wild-type and mutant cells, indicating that tyrosine nitration is not a stochastic event. Further, only certain bands (arrows) show increased intensity in the BMPR2 mutant cells, suggesting further specificity.

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to cause heritable PAH.39 Thus, only a subset of proteins within cells exhibit significant 3-nitrotyrosine formation, and certain tyrosines within those proteins are preferential targets for nitration. Moreover, the presence of 3-nitrotyrosine has specific effects on the function of individual proteins, with some exhibiting significant loss of function (e.g., SOD2, prostacyclin synthase) while others show notable gain of function (e.g., increased peroxidase activity of cytochrome c after tyrosine nitration).40 With a narrower range of possible targets and target-specific functional effects that are observed with nitration of a relatively small percentage of the total possible targets, formation of 3-nitrotyrosine has several features of a signaling pathway, much like other posttranslational modifications, such as phosphorylation. As implied in the above discussion, mitochondria are a key site of convergence for both oxidant injury and oxidant signaling. The mitochondria are one of the most important cellular sources of ROSs, which can be directed into oxidant signaling pathways or which can escape control and mediate oxidant injury. Mitochondria contain high concentrations of redox-active metals (e.g., hemecontaining cytochromes, iron-sulfur clusters) and are the primary site for single-electron redox reactions in the cell. Mitochondria contain many of the enzymes for the key pathways involved in maintaining redox balance in the cell by way of synthesizing cellular reducing equivalents, such as NADH. The convergence of energy metabolism, regulation of apoptosis, and redox homeostasis allows mitochondrial function to influence and to be influenced by oxidant fluxes and the products of oxidant signaling and injury.

CAR B O N M E TA B OL IS M A N D RE D O X B I O L O G Y IN PAH : B E YO ND G L U CO S E OX I D A T I O N The past 10–15 years have seen a renaissance of interest in intermediary metabolism, and PAH research has definitely been part of this resurgence. The role of molecular metabolic program as a primary driver of disease in the modern era is commonly traced back to the work of Otto Warburg in the early twentieth century. Warburg41,42 demonstrated that a shift to lactate-producing glycolysis as a primary means of carbon utilization, even in the presence of ample oxygen to allow complete glucose oxidation, not only was a feature of malignant cells but also was central to malignant transformation. The term “Warburg effect” is often used as shorthand for any cell or tissue that appears to undergo a change in metabolic program to a reliance on lactatogenic glycolysis under normoxic conditions. Evidence for the Warburg effect in PAH came first from experimental models, such as the monocrotaline-treated rat and the fawn-hooded rat, and was followed by studies supporting increased lactatogenic glycolysis as a primary carbon-use program in cells and tissues from PAH patients.43-46 Several underlying molecular mechanisms have been proposed, with the weight of data in support of normoxic activation of HIF and increased “oxidative stress” by a variety of mechanisms (e.g., epigenetic silencing of SOD, a direct increase in superoxide flux from mitochondria). This subject has been very thoroughly reviewed elsewhere, with the summary being that a shift from glucose oxidation to lactatogenic glycolysis

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is a key pathogenic mechanism in multiple models of experimental PAH.47-53

Glucose oxidation versus fat oxidation: which to inhibit, which to activate? In normally functioning cells, glucose oxidation exists in a reciprocal relationship with fatty acid oxidation by way of a feedback mechanism termed the glucose–fatty acid cycle, or Randle’s cycle, first described by endocrinologist Philip Randle54,55 in the 1960s. As fatty acid oxidation proceeds, rising concentrations of multiple intermediates provide feedback inhibition that limits carbon flux through the glycolytic pathway.56 Specifically, rising acetyl coenzyme A (CoA) levels and NADH levels inhibit pyruvate dehydrogenase, and rising citrate levels inhibit phosphofructokinase and, indirectly, hexokinase. Reciprocal inhibition of fatty acid oxidation is provided by the conversion of glucose-derived citrate to malonyl-CoA through the sequential action of adenosine triphosphate (ATP) citrate lyase (which converts citrate to acetyl-CoA) and acetyl-CoA carboxylase. Malonyl-CoA provides feedback inhibition of carnitine palmitoyltransferase 1, which limits the availability of acyl chains for oxidation and shifts the balance toward fatty acid esterification. As malonyl-CoA accumulates, then, glucose oxidation is required to meet the energetic needs of the cell. These reciprocal relationships are summarized schematically in Figure 4. Much of the work that has been done thus far to leverage molecular metabolism in PAH has been focused on the restoration of glucose oxidation. This has been accomplished in experimental PAH in a variety of ways. Administration of inhibitors of

pyruvate dehydrogenase kinase, such as dichloroacetate (DCA, most commonly used to treat inborn errors of metabolism), has long been known to activate pyruvate dehydrogenase and to facilitate entry of glucose-derived carbon into the tricarboxylic acid (TCA) cycle to permit its oxidative metabolism, effectively counteracting the Warburg effect.57 DCA has shown efficacy in several models of experimental PAH, ameliorating pathologic changes in the pulmonary vasculature as well as maladaptive right ventricle (RV) remodeling.8,45,48,58-62 A small phase I, open-label trial of DCA in PAH has been conducted by the University of Alberta and Imperial College London (DCA for the treatment of PAH; ClinicalTrials.gov identifier: NCT01083524), and the forthcoming results are eagerly anticipated. Strategies to leverage Randle’s cycle have also shown efficacy in experimental PAH.61,63,64 By inhibiting fatty acid oxidation pharmacologically with compounds such as ranolazine or trimetazidine or by increasing malonyl-CoA through genetic deletion of malonylCoA decarboxylase (the major enzyme that breaks down malonylCoA), glucose oxidation can be facilitated and at least some of the PAH pathology improved in experimental models. Currently, there are 4 registered trials examining ranolazine’s safety and initial efficacy in pulmonary hypertension and one registered trial to study trimetazidine in PAH. While it is likely that facilitating pyruvate entry into the TCA cycle by using DCA should have some beneficial effect, the actual realized benefit of inhibition of fatty acid oxidation is less clear. Indeed, the data are unclear regarding what happens to fatty acid oxidation in the normal progression of the molecular metabolic pathology that characterizes PAH. With inhibition of glucose oxidation, other things being equal, one might expect a compensa-

Figure 4. Schematic of Randle’s cycle. A reciprocal relationship exists in most cells between glucose oxidation and fatty acid oxidation. As shown here and discussed in the text, acetyl coenzyme A (CoA) and citrate feed back to inhibit glucose oxidation, mainly at pyruvate dehydrogenase (PDH) and phosphofructokinase (PFK) and indirectly at hexokinase and glucose transporter 4. Malonyl-CoA will feed back to inhibit fatty acid oxidation through inhibition of carnitine palmitoyltransferase 1 (CPT1). Note that a number of enzymes (e.g., adenosine triphosphate citrate lyase) have been left out to allow focus on the key nodes of regulation between the two pathways that constitute Randle’s cycle.

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tory increase in oxidative fat metabolism, based on Randle’s cycle. Indeed, Zhao and colleagues65 reported an upregulation of the expression of genes involved in fatty acid oxidation as well as increases in metabolic intermediates in lung tissue from PAH patients, compared to controls. In this setting, then, inhibition of oxidative fat metabolism would make sense. However, metabolic remodeling in the heart in PAH is a different story. Normal myocardium generates up to 70% of its ATP needs from fat oxidation.66 In several models of PAH, fatty acid oxidation has been shown to be decreased in the myocardium of the RV, compared to that in normal animals.62,67,68 Supportive evidence for suppression of fat oxidation has also been found in RV myocardium from humans with PAH.69 With this in mind, it is more difficult to imagine how further inhibition of fatty acid oxidation will ultimately be beneficial, particularly given that RV function is the major determinant of survival in patients with PAH.

Broader metabolic reprogramming: the TCA cycle and redox balance More importantly, if the data regarding suppression of both glucose and fat oxidation in PAH (or at least in the RV in PAH) are taken at face value, then it appears that a more fundamental breakdown in metabolic regulation underlies PAH. If Randle’s cycle were functioning as expected, suppression of glucose oxidation should result in upregulation of fat oxidation, and vice versa. The fact that this seems not to happen in PAH suggests that additional coexisting metabolic dysregulation is a key part of the molecular pathogenesis of PAH. One piece of the puzzle is likely a disruption of the normal mechanisms that regulate the inflow and outflow of carbon in the TCA cycle through pathways other than pyruvate/acetyl-CoA. The inflow reactions are collectively called anaplerosis, and the outflow reactions are collectively referred to as cataplerosis. We have published that in pulmonary endothelial cells expressing PAH-causing BMPR2 mutations, several key anaplerotic pathways are disrupted compared to wild-type endothelium.70 This has also been demonstrated in the RV with respect to a specific anaplerotic pathway, glutaminolysis.71 The amino acid L-glutamine can be converted to glutamate and then to 2-oxoglutarate (also called α-ketoglutarate), which enters directly into the TCA cycle through a route that involves neither glucose nor fatty acids. Glutaminolysis has been shown to be upregulated in the RV in PAH, and this may indeed be part of the broader metabolic dysregulation that would allow for simultaneous suppression of glucose oxidation and fatty acid oxidation. Other anaplerotic pathways (e.g., formation of oxaloacetate directly either from pyruvate via pyruvate carboxylase or from aspartate via aspartate aminotransferase) have not been fully explored in PAH, although we have shown that the metabolism of multiple amino acids is altered in the PAH endothelium.70

Metabolism in service to redox balance The end result of most of the carbon metabolism pathways in the cell is the generation of reducing equivalents that participate in

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critical cellular redox reactions. The reducing equivalents produced include NADH, produced from NAD+ by glycolysis, fatty acid oxidation, and the TCA cycle; NADPH, produced from NADP+ primarily by the pentose phosphate pathway, with additional contributions from isoforms of malic enzyme and isocitrate dehydrogenase (IDH); and FADH2, primarily from flavoproteins involved in fatty acid oxidation (electron transport flavoprotein complex) and the TCA cycle (e.g., pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, and succinate dehydrogenase). Not only is the balance between the reduced and oxidized forms of these electron carriers a key determinant of the overall redox balance within the cell, but levels of these intermediates also regulate the metabolic pathways themselves. As mentioned above, NADH produced from fatty acid oxidation provides feedback inhibition to pyruvate dehydrogenase to limit the complete oxidation of glucose-derived carbon. This is one of the key molecular underpinnings of Randle’s cycle. In trying to understand the complex regulation of molecular metabolism in PAH, perhaps more focus should be placed on understanding redox balance at the level of electron carriers in specific cellular compartments. For example, one possible mechanism by which glucose oxidation and fatty acid oxidation could both be simultaneously suppressed is generation of NADH through an alternative pathway, such as upregulation of the activity of NAD+dependent IDH isoforms. Indeed, we have shown that IDH1/2 activity is increased in the plasma of PAH patients.70 As a second example, recent publications have suggested that decreased activity of one or more sirtuin isoforms may be an important contributor to PAH pathogenesis.72 The sirtuins are lysine deacetylases in the same superfamily as histone deacetylases. Several sirtuin isoforms have been shown to be localized to mitochondria (Sirt3, Sirt4, and Sirt5) and to be master regulators of metabolic program. Their metabolic regulatory function, particularly in the case of Sirt3, is thought to be directly linked to their use of NAD+ as a required cofactor, allowing their activity to be dynamically regulated by the redox state of the cell. Although decreased expression of sirtuin isoforms could certainly contribute to the development of PAH, it is at least as likely that sirtuin activity is regulated by cofactor availability. Indeed, the concept of cofactor availability as a regulatory mechanism for posttranslational modifications has been very nicely captured in other work from this same group.73 The most dynamic regulation of sirtuin activity almost requires that a significant portion of sirtuin activity be regulated by factors other than expression levels, such as cofactor availability and subcellular localization. Viewed in this way, the availability of NAD+ as a cofactor would be a critical regulator of sirtuin function in PAH, and any change to steady state that decreased NAD+ concentrations (e.g., conversion of NAD+ to NADH by increased activity of IDH1/2) would decrease sirtuin activity. Although existing data certainly do not support the above IDH-dependent mechanism as even one of the critical molecular metabolic alterations underlying PAH, the above cases are nonetheless illustrative of the fact that we are only just beginning to understand the complex interplay between redox (im)balance and metabolic dysfunction in PAH.

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M IT O C H O N D R I A I N P A H : TH E C E N T E R OF CELL UL AR RED OX BIOLO GY As discussed in the above sections, the processes of oxidant injury, oxidant signaling, redox balance, and cellular metabolism are all intimately interconnected. The main point of integration for all of these processes is the mitochondrion.74 Oxidative carbon-substrate metabolism and the resultant electron carriers converge on the mitochondrial electron transport system to generate the proton gradient that lies at the heart of chemiosmotically driven ATP synthesis. The proton gradient (or proton-motive force) is generated by the protonpumping action of several mitochondrial complexes and is powered by the free energy liberated in sequential electron transfers. The ultimate electron acceptor in this process is molecular oxygen, which is converted to water when completely reduced. (Note that the overall stoichiometry of the combination of carbon substrate with oxygen to release carbon dioxide, water, and energy is equivalent to a combustion reaction.) However, no process happens with perfect efficiency, and there are multiple points within the electron transport system where electrons can be prematurely transferred to molecular oxygen (with cytochrome c oxidase—also known as complex IV—being the site where oxygen is normally reduced to water). A 1-electron reduction of molecular oxygen results in the formation of superoxide anion, and in this way there is a direct link between metabolism and oxidant signaling and injury.

A number of conclusions can be derived from the relationships depicted in Figure 5. For example, one would predict that different fuel sources would yield different efficiencies when expressed as the ratio of electrons transferred to superoxide produced. For each turn of the TCA cycle, electrons are introduced via NADH oxidation by complex I and by conversion of succinate to fumarate by complex II. However, only complex I serves as a major site of superoxide generation, and so free-radical leak is minimized. For the same number of electrons transferred through the electrontransfer flavoprotein complex (responsible for fatty acid oxidation), one would predict a much higher rate of superoxide formation, as the route of entry for electrons into the electron transport system is the same as the route of inappropriate egress that results in superoxide formation. Indeed, increased fatty acid oxidation has been shown to lead directly to increased generation of ROSs.75 Hypoxia and redox biology in PAH. The case of hypoxia deserves special mention with regard to redox biology in PAH. Chronic hypoxia was one of the earliest models of experimental pulmonary hypertension, and although the presence of hypoxia in WHO group I PAH is secondary to vessel dropout and falling cardiac output (as opposed to being primarily causative in WHO group III pulmonary hypertension), many publications on experimental PAH use hypoxia as part of the model system. The effects of hypoxia to activate HIF and cause metabolic remodeling, char-

Figure 5. Major sites for superoxide generation in the mitochondrial electron transport system. Shown are the major sites for free-radical leak, the overall direction of electron flow, and the overall direction of proton flow in the mitochondrion. ADP: adenosine diphosphate; ATP: adenosine triphosphate; CoQ: coenzyme Q/ubiquinol/ubiquinone; Cx: complex; cyt c: cytochrome c; ETF: electron transport flavoprotein system; FAO: fatty acid oxidation; Pi: phosphate ion.

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acterized by increased glycolysis, decreased carbon flow through pyruvate dehydrogenase, increased reliance on glutamine metabolism to support fatty acid synthesis, and isoform switching of cytochrome c oxidase, are all fairly well known.76-80 However, there is a great deal of literature regarding increased oxidative stress under conditions of hypoxia. On the face of it, this is somewhat difficult to understand. With relatively less molecular oxygen available, it would seem that there should be an absolute reduction in superoxide production, at least from the mitochondria. The reverse has been demonstrated, in which increased molecular oxygen concentrations (hyperoxia) increase superoxide production from mitochondria, increase mitochondrial oxidant injury, and increase PAH.35,81-84 For the case of hypoxia, Hoffman and Brookes,85 in 2009, published a beautiful set of experiments in isolated mitochondria that quantitatively answers some of these questions. The summary is that, in hypoxic conditions with ample substrate, the proportion of molecular oxygen converted to superoxide can and does increase, but the absolute number of molecules of oxygen available for 1-electron reduction is far lower. The net result is that absolute superoxide generation actually decreases in hypoxic conditions. Moreover, these and other published studies suggest that the site(s) of maximum superoxide generation are a complex function of oxygen availability, relative substrate concentrations, relative expression of different components of mitochondrial metabolism, and perhaps even regulatory signaling outside the mitochondria.75,86,87 Finally, and perhaps not surprisingly, there is mounting evidence that pulmonary hypertension driven by hypoxia, while sharing some mechanisms and even some of the features of the redox pathobiology associated with heritable and idiopathic PAH, is a fundamentally different disease. Indeed, some of the disrupted metabolic regulatory mechanisms that play a role in PAH pathogenesis have been shown not to affect pulmonary hypertension driven by chronic hypoxia.88 Of course, these mechanisms are central to the pathogenesis of WHO group III pulmonary hypertension.

S U MM A R Y AN D F U T U R E D I R E C T IO N S Investigations examining the facets of redox biology, including oxidant signaling and stress and molecular metabolic reprogramming, have taught us much about the pathogenesis of PAH. There are many promising new therapeutic targets that come from these mechanisms, some of which are already being explored in PAH while others are currently under development in cancer. To continue to advance our understanding and to maximize the likelihood of truly disease-modifying therapies being developed for PAH, we must redouble our efforts to elucidate fine mechanistic details in relevant model systems and then verify those mechanisms in humans. The devil, as ever, is in the details. We must be as specific as possible when studying “oxidative stress” and its effects in any given model system. We should seek to examine all of the relevant interrelationships between metabolic pathways and identify regulatory nodes that, when disrupted or altered, explain more of the variability in molecular metabolism in PAH than our current models. We must broaden our thinking about redox biology in PAH to move beyond old concepts and interventions.

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