Pharmacological approaches to the treatment of ...

Review Special Focus: Cardiovascular Diseases For reprint orders, please contact [email protected]

Pharmacological approaches to the treatment of oxidative stress-induced cardiovascular dysfunctions Cardiovascular diseases are a growing major global health problem. Our understanding of the mechanisms of pathophysiology of cardiovascular diseases has been gaining significant advances and a wealth of knowledge implicates oxidative stress as a key causative agent. However, to date, most efforts to treat heart failure using conventional antioxidant therapies have been less than encouraging. With increasing incidences of cardiovascular disease in young as well as in aging populations, and the problem of long-term diminishing efficacy of conventional therapeutics, the need for new treatments has never been greater. In this review, we present a variety of therapeutic targets and compounds applied to treat cardiovascular diseases via inhibition of oxidative stress are presented.

Pathophysiology of oxidative stress ROS, such as superoxide anion (O2•─ ), hydrogen peroxide (H2O2), hydroxyl radical (HO •) and

peroxynitrite (OONO─ ), are generated in the cell under normal physiological conditions. The initial ROS generated, in vivo, is the O2•─ formed predominantly in the mitochondrial matrix as a byproduct of the respiratory reduction of oxygen [7–9] . Under normal conditions, only 1–2% of the electrons moving along the mitochondria respiratory complex contribute to O2•─ production [10,11] . Although O2•─ is a potentially harmful radical species, the low physiological levels of O2•─ production are thought to play a key regulatory signaling role in the coupling of myocardial energy production to energy demand [12] . This signaling regulates many important cellular functions including metabolism, cell proliferation and apoptosis [13] . Superoxide radical is also generated via XO [14] and the Nox family [15] . Under normal physiological conditions, the potentially damaging effects of these species are nearly completely eliminated by endogenous radical scavenging enzymes and antioxidants within the mitochondria (Figure 1) [16,17] . However, in certain disease states, normal cellular antioxidant defenses become inadequate, and the cell or organism is said to experience oxidative stress. In particular, there is an unregulated generation of O2•─ and its radical derivatives during ischemia and reperfusion leading to an increase in the number of cellular sites of damage [18] . In addition to the oxidation of important biomolecules including lipids, DNA and proteins, ROS can modulate signal transduction processes and transcription factors [19,20] . Mitochondrial proteins containing Fe–S clusters involved in oxidative phosphorylation are particularly sensitive

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As vaccines and other triumphs of modern medicine have prolonged human life expectancy through the suppression of endemic disease; cardio­vascular failure has risen to the position of leading cause of death in western countries [1,2] . Compounding the severity of the epidemic are the rapid rise of populations affected by risk-associated disease, such as Type 2 diabetes mellitus [3] . A myriad of mechanisms contribute to the physiological changes in the heart that constitute cardiovascular diseases which lead to heart failure. Conventional treatments for heart disease often target only a particular mechanism such as the sympathetic nervous system and the renin–angeotensin–aldosterone system. However, no ‘magic bullet’ therapeutic exists. As our understanding of the disease continues to evolve, it has become clear that a critical imbalance may lie at the root of many, if not all of the mechanisms and pathways leading to heart failure. This imbalance – commonly called oxidative stress – is characterized by an excess of harmful reactive oxygen (ROS) and reactive nitrogen species relative to the endogenous cellular antioxidant response. Many of the gross patho­physiological changes found in failing mammalian hearts – such as aortic calcification and stenosis [4] , myocardial hypertrophy [5] , and heart valve weakening and dysfunction [6] – are the direct or indirect consequences of oxidative stress.

Pedro L Zamora & Frederick A Villamena* Department of Pharmacology, & Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University, Columbus, OH 43210, USA *Author for correspondence: Tel.: +1 614 292 8215 E-mail: [email protected]

ISSN 1756-8919

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Review | Zamora & Villamena O2 [XO] [Nox4]

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Figure 1. Important reactions of O2•─. Pro-oxidative pathways are marked

with red arrows (dashed), antioxidant pathways are marked with green arrows (dashed and dotted). Superoxide is generated primarily by METC dysfunction, via XO, and the NADPH oxidase family, including Nox4. Also, during eNOS uncoupling, O2•─ production increases hence its consumption of nitric oxide (•NO) to yield OONO─, a reactive nitrogen species, leads to a reactive oxygen species and a reactive nitrogen species generation (•OH, •NO2, CO3•─) further exacerbating radical production via uncoupling of NOS. The phase II antioxidant system that is normally downregulated during oxidative stress conditions includes SOD, which catalyzes the dismutation of O2•─ to H2O2. H2O2 is then reduced to water as catalyzed by GPx (or H2O and O2 by catalase), via a 2-electron reduction process from the conversion of GSH to its oxidized form, GSSG. eNOS: Endothelial nitric oxide synthase; METC: Mitochondrial electron transport chain; O2•– : Superoxide.

to ROS [21] . Cardiac physiology is sensitive to the effects of oxidative post-translational modification by ROS, affecting the function and localization of cellular enzymes [21] .

Key Terms Oxidative Stress: Biological imbalance between the pro-oxidant generation and antioxidant repair mechanisms in favor of the former.

Reactive oxygen species/ reactive nitrogen species: Examples of radical-ROS/RNS are O2•– /HO2•, HO •, RO2•, RO •, CO3•–, CO2•–, •NO, •NO2; nonradicals: H2O2 , HOCl, O3, O2 , ROOH, and ONOO –.

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Mitochondrial dysfunction, oxidative stress & heart failure Under conditions of prolonged hypoxia, such as during stroke or cardiac ischemia, the absence of oxygen leads to electron transport chain uncoupling and partial reduction of endogenous oxygen, and other species, within the mitochondria, become prevalent. It may seem paradoxical that production of oxygen-centered radicals increases under hypoxic conditions; however, this increase is likely more dependent on the large oxidation potentials of the highlyreduced mitochondrial electron transport chain (METC) proteins than on the concentration of oxygen [22] . The O2•─ generated through this mechanism gives rise to more potent secondary radicals that induce oxidative damage (F igure  1) . Not surprisingly, neural cells and Future Med. Chem. (2013) 5(4)

cardiomyocytes have a high oxygen demand, and hence, high concentrations of mitochondria, and are, therefore, the most susceptible to damage during ischemia and reperfusion. Under hypoxic conditions, whether moderate deprivation through a chronic condition, such as obstructive vascular disease, or via an acute event of complete blood restriction, such as during cardiac arrest or a stroke, highly aerobic cells undergo oxidative damage during reperfusion – as characterized by the return of oxygen and glucose-rich blood to the affected tissues. Without oxygen available as the final electron acceptor in the METC, oxidative phosphorylation is effectively halted and the mitochondrial membrane potential (∆Ψm) depolarizes. Thus, the cell has to rely solely on glycolysis and lactic acid fermentation to produce ATP, leading to a shortage of ATP, a decrease in cytosolic pH and an influx of Ca 2+ ions (Figure 2) . The increase in cytosolic Ca 2+ levels leads to an overload in mitochondrial matrix Ca 2+ levels when ∆Ψm and respiratory protein activity are rapidly re-established during reperfusion. The ROS generated during ischemia damage, the mtDNA and the endogenous antioxidant response is lower during reoxygenation, priming the cardio­myocytes for cell death [23] . ROS produced during ischemia contribute to peroxidation of the polyunsaturated lipids within the mitochondrial membrane, yielding reactive aldehydes [24] . The most toxic of these aldehydes is 4-hydroxynonenal, the final product of oxidation of linoleic and arachidonic acids, the major constituent fatty acids of the mitochondrial membrane [25] . 4-hydroxynonenal deleteriously modifies a variety of molecules within the mitochondria, including complex IV of the METC, resulting in impaired METC function [26] . The ROS generated during ischemia and the increase in matrix Ca 2+ concentration are key contributors to the opening of the mitochondrial permeability transition pore (MPTP) during reoxygenation, an event which directly precedes cardiac cell death [27] . The nonselective MPTP spans both mitochondrial membranes, bridging the mitochondrial matrix and the cell cytoplasm. Opening of the MPTP abolishes ∆Ψm, as ions and small molecules can freely pass through the pore. Without an electrochemical gradient across the mitochondrial inner membrane, oxidative phosphorylation is effectively halted [28] . Pore opening releases mitochondrial calcium into the cytoplasm, and allows water to enter the mitochondria, causing swelling future science group

Pharmacological approaches to the treatment of oxidative stress-induced cardiovascular dysfunctions and rupture of the outer membrane and the release of cytochrome c, resulting in apoptosis [29] . MPTP opening has also been observed to cause the release of the intramitochondrial signaling molecules, Smac/DIABLO and apoptosis-inducing factor, which participate in apoptotic initiation [30] . MPTP opening triggers a ROS-positive feedback loop, as ROS are also produced as a result of pore opening. Pore opening leads to the loss of respiratory protein components, such as cytochrome c, inhibiting oxidative phosphorylation and resulting in a

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second wave of O2•─ production [31] . Research suggests that the fate of a cell after ischemic insult depends largely on the extent of MPTP opening [32] . This may explain why in clinical trials, when using antioxidants, no clear benefit was demonstrated when treatments were administered only after the onset of reperfusion (and, hence, after the pore has already opened) [33] . In a sense, MPTP opening is like a binary ‘on’ switch for cell death. Cells that undergo only limited MPTP opening during reperfusion and do not apoptose, may recover.

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Figure 2. Mitochondrial dysfunction during ischemia/reperfusion in a cardiomyocyte. Blue arrows indicate normal

cellular functions while red arrows indicate events during oxidative stress. The initiating event is loss of molecular oxygen at the level of oxidative phosphorylation. Electron ‘leakage’ from the METC proteins results in O2•– generation and loss of ATP. Without oxidative phosphorylation, pyruvate generated in glycolysis is converted to lactic acid to generate ATP. A consequence of this is the acidification of the cytoplasm. Protons are shunted out of the cell via the Na + /H + exchange protein. Excess sodium is pumped out of the cell in exchange for calcium via the Na + /Ca2+ exchange protein, leading to an increase in calcium concentration. Excess calcium that would normally be pumped out of the cell via the Ca2+ ATPase is trapped due to low ATP. The calcium instead enters the mitochondrial matrix via the MCAU where it initiates the opening of the MPTP, leading to further ∆Ψm depolarization, reactive oxygen species generation, and apoptosis. ∆Ψm: Mitochondrial membrane potential; ER: Endoplasmic reticulum; MAM: Mitochondria-associated membrane of the endoplasmic reticulum; MCAU: Mitochondrial calcium uniporter; METC: Mitochondrial electron transport chain; MPTP: Mitochondrial permeability transition pore; O2•– : Superoxide.

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Review | Zamora & Villamena Thus, treatments that block or partially inhibit the MPTP opening or expression may prevent more O2•─ production and cell death, by keeping respiratory proteins localized to the mitochondrial matrix and by maintaining ∆Ψm. MPTP effectors If the opening of the MPTP is responsible for a myriad of adverse effects on cell function and structure, as mentioned above, compounds designed to block the pore or inhibit pore opening may serve as a valuable therapeutic strategy against cardiac failure. Minocycline (Figure 3) is a synthetic tetracycline derivative with a safe clinical track record, mainly used as an acne treatment [34] . Like many drugs originally developed as broad-spectrum antibiotics, it demonstrates biological effects which are unrelated to its antimicrobial actions. An increasing number of studies report the potential use of minocycline as a cytoprotectant in neurological disorders, such as acute cerebral ischemia [35,36] . It has been proposed that the observed protection conferred by minocycline is derived from its antioxidant properties, including blockade of inducible nitric oxide synthase [37] and free radical-scavenging ability [38] . However, the precise mechanisms of its cytoprotection remains a matter of debate with a recent study implicating direct involvement of minocycline in mitochondrial permeability transition [39] . MPTP opening was induced in neurons via an oxidative damage cascade just as would occur in a cerebral or cardiac ischemic event. Using the environmental toxin, rotenone, a complex ketone and inhibitor

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Figure 3. Mitochondrial-targeted drugs. (A) Mitochondrial permeability transition pore effectors minocycline, and (B) a Szeto–Schiller peptide. (C) The mitochondrial calcium uniporter blocker Ru360.

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of complex I of the METC, matrix calcium levels were elevated and MPTP opening was induced. Treatment with minocycline was more effective in protecting cells and restoring ∆Ψm than cyclosporin A, a known MPTP blocker. Minocycline had previously been shown to inhibit MPTP opening in mitochondria of different cell types, including brain [40] and liver [41] cells, but direct inhibition of pore opening had not been shown. The study provided direct evidence for inhibition of the MPTP by minocycline, even at high matrix calcium concentrations [39] . As cyclosporin A has previously been demonstrated to reduce cardiac ischemia/reperfusion injury [42] presumably via its MPTP inhibitory actions, it is logical then that minocyline should be further investigated as a cardiac therapeutic. Another promising compound is from a novel class of cell-permeable peptides that selectively target the mitochondria. The Szeto–Schiller peptides (Figure  3) are small, relatively water soluble and contain a structural motif of alternating basic and aromatic residues that allow them to freely cross cell membranes [43] . A recent study investigated a Szeto–Schiller peptide analog, termed Bendavia™, and concluded that it could protect the myocardium from reperfusion injury [44] . Radiolabeling studies had previously shown that Bendavia localizes to, and accumulates within the mitochondria [45,46] . Furthermore, uptake of Bendavia appears to be independent of ∆Ψm [44] . This is considered an advantage of Bendavia over treatments that target the mitochondria via an intact ∆Ψm, such as the mitochondria-targeted triphenyphosphonium cation conjugates of SkQ1, MitoQ, MitoE and MitoSOD. The experiments conducted also produced the conclusions that Bendavia reduces cellular ROS generation, helps sustain ∆Ψm, and reduces oxidant-dependant cardiomyocyte death during reperfusion. The authors hypothesized that Bendavia maintained ∆Ψm by inhibiting the opening of the MPTP and inner membrane ion channels, however, it does not directly block the pore (as cyclosporine A does, for example) but rather prevents pore opening by keeping ROS level low [44] . Besides its sensitivity to ROS generated during ischemia, the MPTP is also strongly regulated by calcium, and the calcium influx during ischemia/reperfusion is the primary contributor to pore opening [47] . Experiments have demonstrated calcium transport between endoplasmic reticulum (ER) and mitochondria via a stable mitochondria–reticulum zone of interaction future science group

Pharmacological approaches to the treatment of oxidative stress-induced cardiovascular dysfunctions whereby calcium that floods the cell during an ischemic event are channeled quickly and accumulate to the mitochondria [48] . Thus, interventions that reduce mitochondrial calcium overload could inhibit MPTP opening and, hence, prevent ∆Ψm and the other associated oxidative injuries. Ruthenium red is a well-known inhibitor of the mitochondrial calcium uniporter (MCAU), and had been shown to exhibit cytoprotective effects in reperfused rat hearts [49,50] . However, it has also been demonstrated that ruthenium red interacts with many proteins involved in the cardiac excitation–contraction cycle, altering the contractile response in normal hearts and affecting other excitable tissues [51,52] . Promisingly, recent studies have demonstrated that Ru360, a ruthenium red analog (Figure 3) , exerts specific inhibition of the mitochondrial calcium uniporter, preventing MPTP opening when perfused into isolated rat heart [53] . NADH/NADPH oxidase family The Nox family is a major source of ROS generated outside of the mitochondria and have been found to be implicated in cardiac failure, hypertrophy, and cell death [54] . There is evidence that approximately 60% of the O2•─ in diseased human coronary arteries is derived from Nox’s [55] . The Nox complex consists of two membrane-bound catalytic subunits (a p22phox and one of five Nox protein homologs, termed Nox’s 1–5) and several cytosolic regulatory subunits (p47phox, p67phox, p40phox and the GTPase Rac1). This complex catalyzes the transfer of high energy electrons from NADPH, across a biological membrane, to oxygen, producing O2•─ and H2O2. The active site of Nox complexes contains two heme groups in the N-terminal transmembrane region and an NADPH-binding and FAD-binding sites in the C-terminal cytoplasmic region, forming a complete pathway to transfer electrons from NADPH to O2, via FAD and the two heme groups [56] . Of particular importance is Nox4, which is the predominant Nox isoform in endothelial cells [57,58] , and is also present in significant concentrations in vascular smooth muscle cells [59] and cardiac tissues in general [60] . Research suggests that Nox4 localized in the mitochondria in cardiac myocytes is a major source of ROS production during cardiac failure, leading to mitochondrial dysfunction, apoptosis of cardiac myocytes, and left ventricular remodeling and dysfunction [61] . Nox4 is also unique among the Nox family of proteins in that it generates predominantly O2•─ and, later, future science group

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H2O2 via O2•─ dismutation [62] . This may explain why O2•─ specific radical scavengers have little therapeutic benefit in treating reperfused tissue compared with broad-spectrum radical scavengers. This also suggests Nox as a major source of ROS production during ischemia/reperfusion, other than electron leakage from METC, and thus presents another viable target for a cardiac antioxidant-therapeutic. Nox4 & 2 effectors Three Nox isoforms predominate in endothelial cells, Nox2, 4, and 1, but only Nox2 and 4 have been found to majorly contribute to ROS production [63] . Nox 2 localizes to the plasma membrane and ER membrane, and Nox 4 localizes to the intracellular membranes, including the ER membrane and mitochondrial membrane [64] . Unlike other Nox enzymes which exhibit stimulusdependant dormant and active isoforms, Nox4 seems to be constitutively active. Studies have shed light onto the structural differences between Nox4 and other Nox enzymes. For example, substitution of the complete Nox4 C-terminal region with the analogous region in Nox2 converts Nox4 to a stimulus-dependant enzyme [65,66] . Compounds inhibiting Nox family proteins, such as ampocynin and diphenylene iodonium, have been available for years, however these inhibitors are not entirely selective for Nox proteins. Diphenylene iodonium, as a general flavoprotein inhibitor, also inhibits XO, another source of O2•─ during ischemia, but in addition inhibits eNOS, which casts doubt as to its therapeutic potential for treating cardiac failure [67–69] . Statins, commonly used as cardio­protective agents for their cholesterollowering activity, have also been observed to inhibit Nox activity and lower cardiomyocyte O2•─ production and cardiac hypertrophy in animals, via modulation of the Rac1-activating mevalonate-IPP synthesis pathway [70] . This pathway has multiple metabolic branches leading to formation of various products, including cholesterol. Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme of the mevalonate pathway [71] . Intermediates of the mevalonate pathway are necessary for the function of GTPases such as the Rho family of proteins [72] . The Rho Rac1 is responsible for the translocation of the p40Phox, p47Phox, p67Phox complex in the cytosol to the p22Phox–Nox membrane complex [73] . Only through this interaction is electron transfer from NADPH to O2 possible [74] . This suggests the cardioprotective effects observed with statin use is, in some part, www.future-science.com

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Review | Zamora & Villamena due to modulation of products of the mevalonate pathway besides cholesterol [75] . It can be assumed that inhibitors specific to the Nox family, and to Nox2 and Nox4 in particular, would be the most beneficial in treating complications arising from an acute ischemic episode. Recent advances in the development of selective Nox inhibitors introduce promising therapeutic strategies. For example, a selective Nox1/Nox4-inhibiting class of compounds, termed pyrazolo-pyridine diones (Figure  4), have demonstrated excellent potency in reducing ROS production on human Nox1/Nox4 membranes [76] . The pyrazolo-pyradine dione GKT137831 (Genkyotek, Switzerland) is a core inhibitor of Nox1/Nox4 activity and a candidate drug for the treatment of diabetic neuropathy currently undergoing Phase I clinical trials [77] . The ROS responsible for the pathology of diabetic neuropathy and cardiomyopathy are derived from Nox4 by the same mechanisms seen in cardiac failure [78] . Thus, the pyrazolopyradine diones may logically be indicated for treatment of cardiac failure. Another method of decreasing Nox activity is via inhibition at the level of transcription. Recent studies have shown that HDACs can regulate Nox4 transcription in human endothelial cells [79] . Treatment of human endothelial cells with HDAC inhibitor scriptaid (Figure 4) [80] led to a marked decrease in Nox4 mRNA and protein, and Nox4-derived ROS [79] . Nox2 inhibitors are less common in the literature, but are still an area of active interest. A Nox2 assembly-inhibiting peptide, Nox2ds, has been demonstrated to specifically inhibit O2•─ production by Nox2 without interfering with production by Nox1 or Nox4 [81,82] . NO & eNOS Not all radical species generated in the cell are deleterious agents. Nitric oxide (NO), a reactive nitrogen species of cellular origin, is an

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important signaling molecule [83] and plays key roles in modulating cardiac cell function [84] . Together, NO and O2•─ are the two main constituents of the so-called ‘nitoso-redox’ balance in the cardiovascular system; a key indicator of cardiac health or pathogenesis. During an ischemic episode, the excess O2•─ generated can scavenge NO by forming ONOO─, causing vasoconstriction [85] . Under normal conditions, excess O2•─ is removed by SOD, creating H2O2, which acts as a membrane-permeable vasodilator at normal physiological levels [86] . However, recent investigations challenge the hypothesis that NO removal by ROS, generated during ischemia, is the primary cause of the observed vasoconstriction [87] . The ROS formed lead to the induction of electron-‘uncoupled’ eNOS. Uncoupled eNOS is itself a significant source of O2•─, which can perpetuate oxidative stress leading to the observed vasoconstriction [88] . The active site of NOS contains a heme iron coupled to the sulfur of a cysteine thiolate that serves as a redox catalyst in the synthesis of NO [88] . Tetrahydrobiopterin (BH4) acts as a cofactor at the heme site in the NO synthesis. During a crucial step, BH4 transfers an additional electron to ferrous–dioxy complex (FeII–O2 or FeIII–O2–) intermediate, forming the +•BH4 radical and the guanidine–hydroxylating species, iron−oxo species (FeIV=O) [88] . In the absence of BH4, the electron flow is uncoupled to form O2•─ [89] , thus, the +•BH4radical must be reduced back to BH4 before the NO synthesis cycle can occur [90] . BH4 availability is reduced under conditions of oxidative stress [91] , presumably because ROS either directly oxidize BH4 or scavenge its radical form. This also may explain the prolonged and reduced bioavailability of NO following ischemia/reperfusion. Nonetheless, targeting eNOS uncoupling appears as a viable therapeutic strategy. The endothelial isoform of nitric oxide synthase is heavily expressed in cardiac cells, and

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Figure 4. NOX inhibitors. Examples of pyrazolo-pyridine diones with reported NOX4 inhibition constants of (A) 72 nM; (B) 156 nM. (C) Human histone deacetylase scriptaid.

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Pharmacological approaches to the treatment of oxidative stress-induced cardiovascular dysfunctions the neuronal isoform is also present. Recent findings [92] suggest that of these two, eNOS is the principal isoform activated by angiotensin-II and H2O2, whereas b-adrenergic receptor activation is more coupled to neuronal isoform-derived NO synthesis (angiotensin-II and b-adrenergic pathways being two main targets of classical interventions in cardiac failure). eNOS effectors Perhaps because of the link eNOS has to heart failure, a variety of treatments have been in existence that appeared to provide benefits via the vascular NO system. One such established class of drugs, the lipid-lowering statins, posses a wide range of biological effects including modulation of the eNOS system. Statins have been demonstrated to increase eNOS expression by stabilizing eNOS mRNA [93] , enchance eNOS availability by decreasing caveolin levels (caveolins are scaffolding proteins of the caveolae that serve to compartmentalize and sequester integral membrane proteins such as eNOS) [94] , as well as inhibit the activity of Nox enzymes, thereby ameliorating BH4 oxidation and decreasing eNOS uncoupling [95] . Statins have also been observed to enchance mRNA levels of GTP cyclohydrolase I, the rate-limiting enzyme in the first step of BH4 synthesis, in a parallel observation with increased BH4 levels and eNOS mRNA levels in vascular endothelial cells [96] . Recent novel therapeutics modulate eNOS activity via specific targets. The recently identified naturally derived pentacyclic triterpenes, ursolic acid [97] and betulinic acid [98] (Figure 5) , upregulate eNOS expression by increasing eNOS promoter activity, however the exact molecular mechanisms underlying these effects have not been clarified. Additionally, both compounds reduce Nox protein expression in human endothelial cells via protein kinase C-independent mechanisms, thereby increasing NO levels by increasing production and decreasing removal by ROS. Endogenous antioxidant system As a general definition, antioxidants are small molecules or biomolecules that protect the cell from the damaging effects of ROS when present in a low concentration compared with the concentration of oxidizable substrate [99] . Oxidizable substrates are broadly defined as lipids, proteins, and DNA. Free radical-scavenging enzymes are included in this definition because they catalytically remove radicals from the cell. Enzyme scavengers of the future science group

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H H

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Figure 5. eNOS effectors. (A) Ursolic acid and (B) betulinic acid.

most biological relevance include SOD, GPX, GR catalase, and glutathione S-transferase. In addition to the radical-scavenging enzymes, nonenzymatic antioxidants such as GSH, vitamin E, vitamin C, caratenoids, flavanoids and polyphenols can protect the cell from oxidative damage. These endogenous antioxidant systems provide sufficient protection against oxidative damage during normal conditions, but are overwhelmed during hypoxic episodes. In fact, the endogenous antioxidant response during such events is often lower than baseline, due to vast oxidative damage to pathways responsible for facilitating the response. For example, the mitochondrial matrix is the primary site of ROS generation in ischemic attack, in which the nonenveloped mtDNA is left subject to damage. Encoded within the mitochondrial genome is the gene expressing mitochondrial superoxide dismutase, an enzyme that facilitates the dismutation of O2•─ to H2O2, as SOD does, but it is localized to the mitochondria. Mitochondria with, impaired ability to generate mitochondrial superoxide dismutase, have been linked to Alzheimer’s ‘diease-like’ states in human neurons [100] . Thus, early efforts to treat reperfusion injury focused primarily on SOD mimetics and catalase. These strategies reduced infarct size in some studies [101–103] , but others produced contradictory results [104] . Despite some positive results in animal models, these strategies do not appear to provide the same benefits in clinical trials [105] . The reasons for the failed transition to clinical medicine likely involve cell permeability problems or the fact that SOD mimetics selectively scavenge only O2•─ [44] , while a plethora of reactions within the cell are likely to rapidly transform O2•─ into the secondary bioradicals that propagate the damage. Antioxidant supplementation A logical strategy for treating oxidative heart failure is supplementation with antioxidants, however, to date, clinical trials designed to www.future-science.com

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Review | Zamora & Villamena study cardioprotection by long-term administration of vitamins E and C have failed to demonstrate clear benefits [106–108] . It may seem as if the window of efficacy of antioxidant use in the clinical setting is very narrow during the onset of ischemia, and prior to reperfusion. Thus, focus has shifted away from orally administered, long-term preventative antioxidant therapy to acute, remedial intervention immediately following myocardial infarction or ischemia by direct (intravenous) route [109] . The value of direct, nonspecific radical-scavenging compounds in mediating the initial oxidative damage following ischemia cannot be overemphasized, considering that the more target-specific therapeutics being investigated often act on the time frame of transcription plus translation and may, therefore, not provide instantaneous benefits. Furthermore, investigation of more powerful radical scavengers is needed to treat the acute oxidative overload of ischemia/ reperfusion as, for example, O2•─ reacts with NO at a rate 105-fold greater than it does with ascorbic acid (vitamin C) [110] . The nitrone spin traps are a class of radical scavenging compounds originally designed to form stable adducts with oxygen-derived free radicals in electron paramagnetic resonance (EPR) studies. Experiments have previously shown that the cyclic nitrone 5,5-dimethyl-pyrroline N-oxide (DMPO) (Figure 6) , an extensively studied nitrone spin trap, can protect animal hearts from post-ischemic ventricular fibrillation  [111] . Recent studies have provided further promise for the therapeutic use of nitrones. In rat heart models of ischemia/reperfusion injury, DMPO treatment significantly reduced O2•─ production both during ischemia and following reperfusion, reducing infarct size by 37% [112] . The same study observed that the mitochondria of DMPO treated hearts exhibited increased recovery of function of respiratory complexes I, II and IV, suggesting a robust mechanism of protection via both direct radical scavenging and restoration of normal cellular

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Figure 6. Nitrone spin traps. (A) 5,5-dimethyl-pyrroline N-oxide and (B) a-phenyl N-tert-butyl.

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redox functions. Both DMPO and the linear nitrone a-phenyl N-tert-butyl (PBN) demonstrated inhibition of H2O2-mediated cytotoxicity and apoptosis in endothelial cells [113] . Treatment with either DMPO or PBN resulted in the induction and restoration of phase II antioxidant enzymes (including GSH, GR, GSR and GPX) via nuclear translocation of Nrf-2 in oxidatively challenged cells. The nitrones were also found to inhibit mitochondrial depolaritzation, and to down-regulate pro-apoptotic proteins p53 and Bax, and upregulate anti-apoptotic proteins Bcl-2 and p-Bad. Clinical trials initially reported significantly improved outcomes in patients suffering from acute ischemic stroke treated with the PBN-derived nitrone NXY-059 [114] . Based on the promising results, a larger second trial was conducted, but failed to find a significant difference in outcomes between NXY-059 treated stoke patients and controls [115] . Controversy ensued, because for such circumstances to occur either the results of one the studies would have to be due to chance, or there was some overlooked variation in conditions between the two trials. However, these findings should not dissuade further investigations into the therapeutic use of nitrones for cerebral stroke, but more so for other conditions such as cardiac failure. Another promising class of cardioprotective radical scavengers is the water-soluble fullerene derivatives (Figure 7) . Fullerenes have been long known for their ability to act as ‘radical sponges’, with radicals adding across their aromatic pi bonds leading to a high radicals removed/mol ratio [116] . Recently, fullerene derivatives have been a hot biomedical research topic as they have been observed to be nontoxic [117,118] , neuro­protective [119] , anti-inflammatory [120] , antiapoptotic [121] and cardioprotective [122,123] . A recent study demonstrated that water-soluble fullerenes have excellent radicalscavenging ability for all physio­logically relevant radicals, including, O2•─ and HO • [124] . The study investigated the radical scavenging abilities of three water-soluble fullerenes: the hydroxylated C60 (OH)22, the malonic acid-substituted C 60 (C(COOH) 2 ) 2 and the transition metal gadolinium-encaging metallofullerenol Gd@C82 (OH) 22 and the compound’s efficacy was measured using EPR. The weakest EPR signals (and, hence, strongest scavenging ability) came from Gd@C82 (OH) 22 (88% reduction in signal intensity for HO • adduct), followed by C60 (OH)22 (67% reduction) and then future science group

Pharmacological approaches to the treatment of oxidative stress-induced cardiovascular dysfunctions CH2OH O

CH2OH O O OH O OH

OHO HO

HO O HO

HOH2C

CH2OH O HO O HO

HOH2C OH O OH O

| Review

CH2OH OH OOH O

HO O HO O OH HO O

O

(OH)n

CH2OH

HOH2C

O

O

Gd

OH OH

HO HO O

O (OH)22

Figure 7. Examples of water-soluble fullerenes. (A) Two g-cyclodextrins enclose a C60 molecule; (B) a C60 fullerenol; (C) a malonic acid substituted C60 fullerene; and (D) a gadolinium metal enclosing C82 metallofullerenol.

C60 (C(COOH)2)2 (55% reduction). This same order of efficacy was observed for O2•─ scavenging, and in cytoprotection and lipid peroxidation-inhibition experiments where all three fullerenes were found to stabilize ∆Ψm, reduce intracellular ROS production, and inhibit lipid peroxidation under H 2O2-induced oxidative stress in endothelial cells. The Gd@C82 (OH)22 was hypothesized to be such a powerful radical scavenger due to its higher electron affinity. Another study in rats examined the effects of the fullerenol C60 (OH)24 on cardiotoxicity caused by the anthracycline antibiotic doxorubicin (DOX). The adverse cardiotoxic effects of DOX have been linked to an increase in ROS production and oxidative damage in heart cells [125] . The study found that fullerenol significantly ameliorated DOX-induced cardiodilation, eliminated DOX-induced lipid peroxidation, and maintained normal levels of antioxidant enzymes SOD, GPX, and GR. Other water-soluble fullerenes with demonstrated anti­oxidant potential include g-cyclodextrin-bicapped C60 [126] . Future perspective Cardiometabolic disease (CMD) is a combination of various risk factors, such as high blood pressure, abnormally high glucose and elevated triglycerides, which in combination, lead to an increase risk of developing Type II diabetes milletus (T2DM) and cardiovascular diseases. Both atherosclerosis and T2DM are proto­t ypical future science group

chronic inflammatory diseases that involve heightened redox stress involving multiple pathways. CMD is the leading cause of mortality in the USA and worldwide. Current therapeutic approaches using antioxidants that target globally are not very effective in treating CMD. Development of innovative therapeutic strategies for the prevention of CMD is, therefore, critical. Results in experimental models of diabetes have demonstrated an important effect of a-lipoic acid in improving mitochondrial function and biogenesis [127,128] , benefits with respect to glycemic control, improved insulin sensitivity, oxidative stress, and neuropathy have been demonstrated to be promising [129] . a-lipoic acid also reduced NF-kB activation in human monocytic cells and reduced inflammation [130] , induce phase II enzymes via NRf-2 nuclear translocation [131] . However, targeting of Golgi and ER by antioxidants has not been explored; ER [132] and Golgi stresses [133] play critical roles in the development of CMD. By subcellularly targeting these antioxidants to the Golgi and ER, the authors expect to optimize clinical efficacy and attenuate endothelial dysfunction in vitro and in vivo. Since T2DM and heart failure are prime risk factors for cardiovascular diseases, therapeutic interventions that may have synergistic effects to improve insulin sensitivity and vascular functions are particularly attractive. Therefore, targeting post-translational regulation by antioxidants of NOS activity and/or suppression of signal transduction and gene induction www.future-science.com

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Review | Zamora & Villamena processes to subcellular locations of NOS (i.e., plasma membrane, sarcoplasmic [endoplasmic] reticulum, or Golgi), by antioxidants,

could salvage or reverse eNOS uncoupling where

it is most susceptible – potentially serving as a powerful pharmacological strategy. As such,

Executive summary Pathophysiology of oxidative stress „„

Reactive oxygen species (ROS) are generated in the cell under normal physiological conditions but are regulated by endogenous antioxidant enzymes and molecules.

„„

The primary ROS normally generated in the cell is O2•─, produced by electron leakage from the mitochondrial electron transport chain.

„„

ROS are also produced by enzymes such as the Nox’s, XO and uncoupled eNOS.

„„

When ROS production exceeds endogenous antioxidant capacity and oxidative damage occurs on a greater-than-normal level, the cell or organism affected is said to experience oxidative stress.

During ischemia, O2•─ production in the mitochondria increases, leading to secondary radical generation and oxidative damage of proteins, lipids, DNA, and oxidative modification of crucial signaling molecules, such as the mitochondrial permeability transition pore (MPTP). Mitochondrial dysfunction, calcium & heart failure „„

„„

Loss of mitochondrial respiration and ATP production during ischemia leads to low cellular pH, and high mitochondrial matrix Ca2+, causing additional ROS generation.

„„

ROS and high [Ca2+] in the mitochondrial matrix causes opening of the MPTP, leading to complete loss of mitochondrial membrane potential (∆Ψm) and the release of pro-apoptotic signaling molecules into the cytoplasm.

„„

The ruthenium red analog Ru360 is a selective mitochondrial calcium uniporter blocker that prevents excess calcium from entering the mitochondria, ameliorating oxidative stress and preventing MPTP opening and apoptosis in cardiac cells.

„„

The tetracycline antibiotic, minocycline, and the Szeto–Schiller peptide, Bendavia™, inhibit the mitochondrial permeability transition pore opening, thereby maintaining the ∆Ψm and preventing oxidative damage and apoptosis.

NADPH oxidases & heart failure „„

The Nox enzymes are a major source of cellular ROS outside of the mitochondrial electron transport chain.

„„

Nox enzymes catalyze the transfer of electrons from NADPH to O2, forming O2•─ and H2O2.

„„

Nox4 is the primary Nox isoform in endothelial cells, and is thus a target for cardioprotective drugs.

„„

The pyrazolo–pyridine diones are a class of compounds that have shown excellent selective inhibition of Nox4/Nox1 and the ability to greatly reduce ROS produced by these enzymes.

The HDAC scriptaid has shown the ability to inhibit Nox4 transcription in human endothelial cells, thereby reducing the total Nox4 protein and reducing ROS production. eNOS, NO & cardiac function „„

„„

NO has long been recognized as a key signaling molecule in cardiovascular function. NO removal by ROS such as O2•─ leads to vasoconstriction and loss of vascular tone.

„„

eNOS can become electron uncoupled by ROS, leading to production of O2•─ instead of NO.

The pentacyclic triterpenes, ursolic acid and betulinic acid have demonstrated the ability to upregulate eNOS expression and reduce Nox expression, providing dual cardioprotective actions. Endogenous & supplemented antioxidants „„

„„

Past approaches to treating heart failure have used mimetics of endogenous antioxidant systems, mostly SOD. However, SOD only removes O2•─ and not other harmful ROS, thus, the lack of encouraging clinical data regarding SOD mimetics.

„„

Heart failure treatments with conventional non-enzymatic antioxidants such and vitamins E and C have also not translated well into clinical studies.

„„

Use of powerful, broad-spectrum radical scavengers prior to reperfusion may be beneficial.

„„

The nitrone spin traps 5,5-dimethyl-pyrroline N-oxide and PBN have shown excellent radical scavenging abilities and cardioprotective properties, including maintenance of ∆Ψm, and upregulation of endogenous antioxidant enzymes.

„„

Clinical trials with nitrones for cerebral ischemia have produced mixed results, however, nitrone treatments in cardiac disease are worth further investigation.

„„

The fullerenes act as ‘radical sponges’ by reacting with a high number of radicals per mole of fullerene. Water-soluble fullerenes have demonstrated low toxicity, and excellent radical-scavenging abilities in cells.

„„

Fullerene compounds should be investigated further for the treatment of ischemic oxidative damage, as they may scavenge enough radicals quickly enough to prevent reperfusion damage.

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Future Med. Chem. (2013) 5(4)

future science group

Pharmacological approaches to the treatment of oxidative stress-induced cardiovascular dysfunctions antioxidant therapies not only directly scavenge ROS and increase NO bioavailability, but can also indirectly regulate ROS production via induction of antioxidant enzyme activity [113] , NOX inhibition, improve Ca 2+ homeostasis [134] , reverse eNOS uncoupling, suppress maladaptive signal transduction processes [113] , and prevent eNOS dysfunction via (de)-phosphorylation [135] and may represent promising drug leads for the prevention of complications due to CMD. References 1

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Hydroxy-methylglutaryl-coenzyme A

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