Pharmacological approaches to coronary microvascular dysfunction.

JPT-06702; No of Pages 20 Pharmacology & Therapeutics xxx (2014) xxx–xxx

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Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

Associate editor: Y.S. Chatzizisis

Pharmacological approaches to coronary microvascular dysfunction Giacinta Guarini, Alda Huqi, Doralisa Morrone, Paola Capozza, Giancarlo Todiere, Mario Marzilli ⁎ Cardiovascular Medicine Division, Cardio-Thoracic and Vascular Department, University of Pisa, Italy

a r t i c l e

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Keywords: Coronary microvascular dysfunction Diabetes Ischemic heart disease Cardiomyopathies Reactive oxygen species No-reflow

a b s t r a c t In recent decades coronary microvascular dysfunction has been increasingly identified as a relevant contributor to several cardiovascular conditions. Indeed, coronary microvascular abnormalities have been recognized in patients suffering acute myocardial infarction, chronic stable angina and cardiomyopathies, and also in patients with hypertension, obesity and diabetes. In this review, we will examine pathophysiological information needed to understand pharmacological approaches to coronary microvascular dysfunction in these different clinical contexts. Well-established drugs and new pharmacological agents, including those for which only preclinical data are available, will be covered in detail. © 2014 Published by Elsevier Inc.

Contents 1. Introduction. . . . . . . . 2. Diabetes . . . . . . . . . 3. Acute coronary syndrome . 4. Stable ischemic heart disease 5. Cardiomyopathies . . . . . Conflict of Interest . . . . . . . References . . . . . . . . . . .

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1. Introduction

2. Diabetes

Hypertension, obesity, diabetes, acute myocardial infarction, chronic stable angina and cardiomyopathies share a common pathophysiological denominator: coronary microvascular dysfunction. Impairment of myocardial perfusion appears to be a relevant feature for disease progression and prognosis in all these clinical settings. This is because the coronary microvascular bed is the site where myocardial blood flow is tightly adjusted to meet myocardial metabolic needs. In this review, pathophysiological changes occurring at coronary microvascular level associated with diabetes, acute myocardial infarction, chronic stable angina and cardiomyopathies will be explored. Subsequently, pharmacological approaches including well-established drugs and new agents not currently used in the clinical arena, specifically designed to treat coronary microvascular dysfunction in each of these clinical scenario, will be covered in detail.

2.1. Pathophysiology of diabetes related coronary microvascular dysfunction

⁎ Corresponding author at: Cardiovascular Medicine Division, Cardio-Thoracic and Vascular Department, Via Paradisa 2, 56124 Pisa (Pi). Tel.: +39 050996751. E-mail address: [email protected] (M. Marzilli).

Structural and functional coronary microvascular abnormalities at the level of the coronary microcirculation have been described in type 1 and 2 diabetes. Morphological changes include thickening of the arterial wall (Strauer et al., 1997) and of the capillary basement membrane, periodic acid-Schiff positive deposits in the vessel wall of small arteries (Ledet, 1976), microaneurysms, perivascular and interstitial fibrosis, and fibrosis in the wall of small coronary arteries (Fein et al., 1984). Moreover, studies in small arteries and arterioles of diabetic subjects have demonstrated that vasomotor dysfunction in microvessels precedes the appearance of morphological changes, affecting both smooth muscle- and endothelium-mediated regulatory mechanisms (De Vriese et al., 2000; Erdos et al., 2002). In humans, impaired coronary vasodilation was demonstrated after pharmacological [mainly acetylcholine (ACh)] or mechanical (cold test) stimuli; these vasomotor abnormalities were apparent in large vessels even in the absence of coronary stenosis, and were independent of other cardiovascular risk factors (Nitenberg et al., 1998). Bagi et al. found that both arteriolar

http://dx.doi.org/10.1016/j.pharmthera.2014.06.008 0163-7258/© 2014 Published by Elsevier Inc.

Please cite this article as: Guarini, G., et al., Pharmacological approaches to coronary microvascular dysfunction, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.06.008

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G. Guarini et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx

responses to myogenic and adenosine stimulation were unaffected by diabetes, whereas dilations in response to cumulative concentrations of ACh and a nitric oxide (NO) donor (NONOate) were significantly decreased in db/db mice, compared to control vessels (Bagi et al., 2003). Furthermore, arterioles of diabetic mice exhibited greatly reduced dilations to flow that were unaffected by an NO inhibitor. In carotid arteries of diabetic mice these investigators also demonstrated enhanced superoxide production (Bagi et al., 2003). Correspondingly, intraluminal administration of superoxide dismutase (SOD) significantly augmented flow-, ACh-, and NONOate-induced dilations of diabetic arterioles. Conversely, flow- and ACh-induced responses could be inhibited by L-NAME. Collectively, these findings suggested that NO mediation of agonist- and flow-induced dilations of coronary arterioles is reduced due to an enhanced superoxide production in type 2 diabetes. Furthermore, in the same year Miura et al. confirmed the impairment of coronary microvascular function (reduced vasodilation to hypoxia) in human coronary arterioles isolated from patients affected by type 1 and type 2 diabetes mellitus which was attributed, at least in part, to a decreased activity of ATP-sensitive potassium channels (Miura et al., 2003). Lately, Guarini et al. (2012) reported corruption of transient receptor potential vanilloid 1 channels- (TRPV1 channels) mediated coronary microvascular dilatation, in mice with type 2 diabetes. TRPV1 channels are involved in pathways that mediate the metabolic regulation of coronary blood flow acting via nitric oxide and BK channels. Moreover, impairment in coronary metabolic dilatation, due to enhanced ROS production, and mitochondrial DNA (mt-DNA) fragmentation, was demonstrated in diabetic rats (Guarini et al., 2010). Similar results were reproduced in lean rat in which mitochondrial dysfunction was selectively induced through mt-DNA damage, in the absence of coronary atherosclerosis and other risk factors (Guarini et al., 2011). In addition, abrogated ischemia-induced coronary collateral growth, i.e. reduced capacity to produce collaterals after brief repetitive episodes of ischemia, has been described in obese/diabetic rats (Hattan et al., 2007). The metabolic disturbances of diabetes, like hyperglycemia, hyperlipidemia, and hyper-insulinemia act as “triggers” eventually causing endothelial dysfunction through the influence of different “mediator” molecules. Several lines of evidence point to “oxidative stress” as key player in endothelial dysfunction. Similar alterations in endothelial function have been described in obese patients (Perticone et al., 2001) without overt diabetes. High circulating levels of free fatty acids and pro-inflammatory adipokines likely contribute to endothelial dysfunction early in the course of insulin resistance. Free fatty acids induce endothelial dysfunction, whereas several adipokines promote both inflammatory responses and insulin resistance (Steinberg et al., 1997). In this sense, a critical role has been established for tumor necrosis factor (TNF), leptin, and adiponectin in vascular inflammation and modulation; notably, obesity and diabetes modify plasma levels and expression of these cytokines (Ouchi et al., 2003; Yang et al., 2009). There are multiple sources of ROS in diabetes, including mitochondrial and non-mitochondrial origins. ROS are involved in several important molecular pathways of hyperglycemia-induced oxidative tissue damage. At present, four pathways are known, including: activation of protein kinase C, increased hexosamine pathway flux, increased advanced glycation end-product (AGE), and increased polyol pathway flux. This increased production of free radicals overwhelms the capacity of scavenger enzymes to neutralize ROS. Activated PKC has a number of effects on gene expression, such as decreased expression of endothelial nitric oxide synthase (eNOS), increased expressions of endothelin, vascular endothelial growth factor (VEGF), plasminogen activator inhibitor-1 (PAI-1), transforming growth factor-beta(TGF-β), nicotinamide adenine dinucleotide phosphate- [NAD (P) H] oxidases, and nuclear factor κB (NF-κB). All these in turn activate many pro-inflammatory genes in the vasculature. The activation of the AGE pathway can damage cells by three mechanisms: first, these compounds modify intracellular proteins, especially those involved in the regulation of gene transcription; second, these compound can diffuse to the extracellular space and modify

extracellular proteins such as laminin and fibronectin to disturb signaling between the matrix and the cells; and finally, these compounds modify blood proteins such as albumin, causing them to bind to AGE receptors on macrophages/mesangial cells and increase the production of growth factors and pro-inflammatory cytokines. In line with this, AGE/RAGE system has been shown to be directly involved in the development of endothelial dysfunction in coronary arterioles isolated from diabetic mice (Gao et al., 2008). Apart from the increased pool of mitochondrial and nonmitochondrial reactive oxygen species, patients with diabetes exhibit reduced antioxidant capability. Studies assessing the level of antioxidant defenses in diabetes have clearly shown that antioxidant capacity is compromised. Consistently with this, lower antioxidant activity – and specifically SOD1 activity – has been associated with both type 1 and 2 diabetes (Maxwell et al., 1997; Uchimura et al., 1999) and with endothelial dysfunction in children with Type 1 diabetes (Suys et al., 2007). This was further confirmed in human endothelial cells exposed to high glucose for 7 and 14 days. In this experiment, increased SOD1 and SOD2 protein levels were associated with reduced SOD activity, highlighting dysfunctional enzyme activity in conditions of high oxidative stress (Ceriello et al., 1996). As for the coronary microvessels, exposure of retinal endothelial cells to high glucose concentration has been proven to induce cells and mitochondrial DNA damage. Moreover, gene expression of mitochondrial-encoded proteins of the electron transport chain complexes is decreased. Other reports have demonstrated altered plasma/serum total antioxidant status or reduced free radical scavenging activity and increased plasma oxidability in type 2 diabetes, together with reduced levels of specific antioxidants such as ascorbic acid and vitamin E. In addition, the activities of antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase, and aldehyde dehydrogenase have been described as reduced in diabetics (Traverso et al., 2002; Lashin et al., 2006). Altogether, it appears that a pivotal role in diabetic microvascular dysfunction is played by oxidative stress, which causes unbalance between vasodilator and vasoconstrictor factors (Bagi et al., 2005), causing ultimately damage to the arterial wall. Loss of function/regulation of the endothelium (i.e. endothelial dysfunction) may be a critical and initiating factor in the development of diabetic micro- and macrovascular disease (Basha et al., 2012). Fig. 1 summarizes main mechanisms involved in diabetes related coronary microvascular dysfunction. Given the relevancy of mitochondrial ROS-induced vasculature damage, strategies to target mitochondria, ROS and manipulation of cardiac metabolism in diabetes are under investigation. 2.2. Pharmacological approaches to diabetes related coronary microvascular dysfunction 2.2.1. Mitochondria manipulation Only few preclinical data are available at the moment regarding pharmacological approaches to manipulate mitochondrial function. Although yet unsolved, mitochondrial uncoupling may provide beneficial effect by the means of reducing ROS generation. In this sense interestingly, a recent study has proposed that Silybin and dehydrosilybin (Tong et al., 2011) exert their protective role on cardiomyocytes from ROS by means of mitochondrial uncoupling properties (Gabrielova et al., 2010). This may open the way to further investigation in using mild/modified mitochondrial uncouplers to prevent endothelial and coronary microvascular dysfunction (Modriansky & Gabrielova, 2009). Recently, using a pharmacological approach to selectively repair mtDNA fragmentation, coronary metabolic dilation was partially restored in diabetic rats (Guarini et al., 2011). These results may provide strength for new research. 2.2.2. Controlling reactive oxygen species production and oxidative damage Several vitamins and chemical compounds with antioxidant properties and effect on mitochondria have been tested in attempt to control



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Fig. 1. Proposed mechanisms of microvascular dysfunction in diabetes mellitus. Microvascular dysfunction in diabetes can be induced by a combination of (1) metabolic changes, (2) inflammation, and (3) insulin resistance. Prolonged exposure to hyperglycemia is now recognized as a major factor in the pathogenesis of diabetic complications, mechanistically involving enhanced enzymatic and nonenzymatic protein/lipid glycosylation, protein kinase C activation, inflammation, and ROS production. Other factors including dyslipidemia, elevated free-fatty acids, inflammation, and insulin resistance, can cause endothelial dysfunction. “+”, activation; ET, endothelin; eNOS, endothelial nitric oxide synthase; PKC, protein kinase C; AGE, advanced glycation end products; VGEF, vascular endothelial growth factor; PAI-1, plasminogen activator inhibitor-1; TGF-β, transforming growth factor-β; NAD(P)H oxidases, nicotinamide adenine dinucleotide phosphate-oxidase; NF-KB, nuclear factor κB.

or reduce oxidative stress and prevent vascular complications associated with diabetes. The antioxidant armamentarium can count on antioxidant enzymes and co-factors (substrates, biogenic elements, combined drugs, synthetic-antioxidants), non-enzymatic antioxidants, and agents with adjunctive antioxidant activity. The enzymatic antioxidant systems, such as copper, zinc, manganese superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase may remove the ROS directly or sequentially, preventing their excessive accumulation and consequent adverse effects. Non-enzymatic antioxidant systems consist of scavenging molecules that are endogenously produced such as glutathione, ubiquinol, and uric acid or derivatives of the diet such as vitamins C and E, carotenoids, lipoic acid, selenium, etc. Other drugs used or being tested may mimic endogenous antioxidant enzymes such as the superoxide dismutase-mimetic manganase (III) tetrakis (4-benzoic acid) porphyrin [MnTRAP] and ubiquinone, or may induce scavenging enzymes like the heme-oxygenase. 2.2.3. Superoxide dismutase 2.2.3.1. Mechanism of action. The superoxide dismutase (SOD) is a family of enzymes involved in the neutralization of superoxide through its dismutation to oxygen and hydrogen peroxide, being the first line of cellular defense against elevated levels of reactive oxygen species (De Haan et al., 2003). Notably, the SOD enzymes are a key determinant of NO bioavailability, attenuating the formation of peroxynitrite. In doing so, the SOD enzymes indirectly improve NO bioavailability. 2.2.3.2. Preclinical data. Inhibition of superoxide radicals either by administration of Manganase superoxide dismutase (MnSOD) mimic such as MnTBAP (Manganese (III) tetrakis (4-benzoic acid)porphyrin

chloride) or by over-expression of MnSOD itself, prevented mt-DNA damage and protected mitochondrial-encoded genes in hyperglycemic condition (Madsen-Bouterse et al., 2010). In another study, administration of Tempol, a cell-permeable SOD mimetic, has shown improvements in diabetes associated microvascular complications, such as nephropathy and retinopathy (Schnackenberg & Wilcox, 2001; DeRubertis et al., 2007; Rosales et al., 2010). MN40403, another highly specific nonpeptide SOD mimetic, was able to reverse endothelial dysfunction ex vivo by targeting Nox-mediated superoxide production in aortae of ApoE-deficient mice (Weber & Griendling, 2003). Although, SOD and SOD mimetics have displayed some efficacy in experimental models of diabetes, their role in improving coronary microvascular function remains still unproven and far from clinical usage. Indeed, few experiments have been performed exploring their efficacy on the coronary vascular bed. General controversies exist also, related to the fact that in pathological conditions, dismutation of superoxide by SOD is linked to excessive production of hydrogen peroxide. It is possible that concurrent treatment with multiple antioxidants, acting at different level or on different ROS, may be a more effective strategy. 2.2.4. Nox inhibitors 2.2.4.1. Mechanism of action. Since the NADPH oxidase (Nox) family of enzymes is involved in the release of ROS in the vasculature, increasing interest has focused on the ways to manipulate these enzymes without compromising their physiologic activity (Bedard & Krause, 2007). Compounds that suppress/alter Nox enzymes may therefore offer therapeutic benefits to ameliorate endothelial dysfunction and atherosclerosis, especially in diabetes where Nox involvement is increasingly being appreciated (Sukumar et al., 2013; Lambeth, 2007; Dikalova et al., 2010).


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2.2.4.2. Preclinical data. Different inhibitors of the Nox enzymes have been developed and shown to have promise in experimental studies (Lassegue et al., 2001; ten Freyhaus et al., 2006; Lambeth et al., 2008). Treatment with the Nox inhibitor, apocynin, reversed the upregulation of Nox isoforms, increased eNOS function, and attenuated endothelial dysfunction in different experimental models of type 2 diabetes (Unger & Patil, 2009; Gupte et al., 2010; Olukman et al., 2010). Apocynin was also effective in preventing diabetes related cardiac dysfunction (Roe et al., 2010). Even though studies using Nox inhibitors have shown some benefits, issues of specificity and mechanisms of action slow down its applicability (Aldieri et al., 2008). For example, recent evidence has suggested that apocynin may act through Rho-kinase inhibition instead of targeting NADPH oxidase (Schluter et al., 2008). Other investigators have proposed that it may act as an antioxidant rather than a specific Nox inhibitor, as demonstrated in vascular endothelial and smooth muscle cells (Heumuller et al., 2008). Thus, a more preferable approach would be to develop Nox-inhibitors with isoform and target specificity. 2.2.5. Glutathione peroxidases 2.2.5.1. Mechanism of action. Glutathione peroxidases (GPxs) are a family of selenocysteine-containing enzymes that are involved in the neutralization of hydrogen peroxide to water utilizing glutathione (GSH) as its substrate. GPx1 is the predominant isoform expressed in the cytosol and mitochondria, where it eliminates hydrogen peroxide and reduce peroxynitrite formation (Sies et al., 1997). Indeed, recent preclinical and clinical data have shown that GPx1 plays a crucial role in protecting the cardiovascular system against oxidative stress, as that observed in diabetes and following ischemia and reperfusion (Forgione et al., 2002a,b; Torzewski et al., 2007; Lim et al., 2009; Thu et al., 2010). Moreover, it appears that GPx1 deficiency is involved in atherosclerosis (Torzewski et al., 2007), and cardiac hypertrophy and dysfunction related to hypertension (Ardanaz et al., 2010). 2.2.5.2. Preclinical data. Ebselen is a lipid-soluble seleno-organic compound, which mimics the activity of GPx1. Ebselen has been extensively studied for its therapeutic potentials in various experimental models of diabetes. In a model of type 2 diabetes, ebselen restored endotheliumdependent vasodilation, NO production and angiogenic capacity (Brodsky et al., 2004). In diabetic apolipoprotein E/GPx1 mice, ebselen diminished the expression of VEGF, Connective Tissue Growth Factor (CTGF), Vascular Cell Adhesion Molecule 1 (VCAM-1), Monocytes Chemoattranctant Protein 1 (MCP-1), and Nox2 pathways protecting from atherosclerosis progression and diabetes-related nephropathy (Chew et al., 2010). Currently, newer Ebselen analogues with a higher catalytic activity characterized by structural modifications, including a diselenide moiety, are being generated. Indeed, hypercholesterolemic LDL receptor knockout mice treated daily with diphenyl diselenide for 30 days, exhibit reduced atherosclerotic lesions, accompanied by improved vascular function, down regulation of proatherogenic genes, reduced infiltration of inflammatory cells, and lowered oxidative stress levels (Hort et al., 2011). However, there are no clinical data on the use of Ebseln in this patient population. 2.2.6. Non-enzymatic antioxidants (vitamins, alpha-lipoic acid, and polyphenols) 2.2.6.1. Mechanism of action. Vitamins C, E, A, alpha lipoic acid, and carotenoids are well-established nonenzymatic antioxidants derived from the diet, which have been studied intensively. Alpha-lipoic acid is a critical co-factor for mitochondrial dehydrogenase reactions, a compound with free radical-scavenging activity. 2.2.6.2. Preclinical data. In an animal model of diabetes, alfa-tocopherol supplementation was associated with improved endothelial functions

(Minamiyama et al., 2008). Similarly, administration of ascorbic acid early and late in the course of diabetes was associated with improved endothelial function, assessed by Ach-mediated relaxation (Sridulyakul et al., 2012). Alpha lipoic acid has provided to ameliorate cardiac fibrosis and dysfunction in an experimental model of diabetic cardiomyopathy (Li et al., 2012). 2.2.6.3. Clinical data. Population, retrospective and prospective observational studies have suggested that antioxidants may exert beneficial effects in patients with coronary artery disease and in experimental model of coronary atherosclerosis (Kinlay et al., 1999). However, welldesigned placebo-controlled randomized trials (ATBC, CARET, Physician's Health, HOPE, GISSI, PPP, HPS and HATS trials) failed to provide conclusive evidence related to their clinical effectiveness. As for patients with stable CAD, studies of the effect of ascorbic acid and tocopherol on coronary microvascular dysfunction in type 2 diabetes have yielded mixed results (Hamilton et al., 2007). For example, Vitamin E supplementation had no effect on any enzymatic activity and mRNA in both normal and hyperglycemic conditions in young adolescents with type 1 diabetes, with early signs of microangiopathy, and defective intracellular antioxidant systems (evaluated as CuZn superoxide dismutase, MnSOD, catalase, and glutathione-peroxidase activity and mRNA expression) (Chiarelli et al., 2004). Similarly, in The Heart Outcomes Prevention Evaluation Study, daily administration of vitamin E for a mean of 4.5 years did not reduce cardiovascular events in a cohort of more than 9000 patients (Yusuf et al., 2000; Lonn et al., 2005). Given these inconsistent results, antioxidant vitamin supplementation is not recommended to prevent coronary microvascular dysfunction in diabetes. Supplementation with antioxidants and/or factors essential to nitric oxide production may potentially improve endothelial function in diabetes by re-coupling eNOS and mitochondrial function, as well as decreasing vascular NAD(P)H oxidase activity (Hamilton et al., 2007). However, it is anticipated that antioxidant therapy in diabetes would prove to be beneficial only if associated with blood pressure control, management of dyslipidemia, and optimal glucose control (Jakus, 2000). 2.2.6.4. Mechanism of action. In addition to “classic” antioxidants, over the past decade, evidence has been accumulated about phenols, phenolic acids, and flavonoids (collectively known as polyphenols), an important class of antioxidants, widespread virtually in all plant foods, often at high concentrations. Among polyphenols, resveratrol has received great attention. 2.2.6.5. Preclinical data. In animal models of diabetes this molecule has been shown to restore endothelial function of cerebellar arterioles (Arrick et al., 2012), while in the cardiovascular field resveratrol has been proven to reduce coronary endothelial cells oxidative stress produced at mitochondrial level (Ungvari et al., 2009), to induce mitochondrial biogenesis (Csiszar et al., 2009), to improve neovascularization from bone marrow derived stem cells (Gan et al., 2009), to reduce infarct size and finally to improve cardiomyocytes survival after ischemia (Dekkers et al., 2008). Despite exciting results in animals, data in humans are still lacking. The overall conclusion is that published evidences are not sufficiently strong to justify a recommendation for the administration of resveratrol to humans, beyond the dose that can be obtained from dietary sources. 2.2.7. Drugs with antioxidant activity (primary or in addition to other relevant features) Because of its relevant role in the mitochondrial electron transport chain, coenzyme Q has long been studied in diabetes and obesity related disease. Coenzyme Q or ubiquinone decreases oxidative stress by quenching reactive oxidative species and by ‘recoupling’ mitochondrial oxidative phosphorylation. The net effect is to reduce superoxide production. Indeed, a possible therapeutic approach would be to synthetize



compounds linking agents such as redox forms of quinone (ubiquinol and ubiquinone) to form alkylated triphenylposphorium compounds. Two of these lipophilic cations linking ubiquinone or vitamin E, termed MitoQ and MitoVitE respectively, have been tested so far. 2.2.7.1. Mechanism of action. These agents accumulate several hundredfold in mitochondria, because their positive charges. Once inside the mitochondria, MitoQ goes through the mitochondrial respiratory chain and is converted into its active form ubiquinol. Ubiquinol may function as an antioxidant by donating a hydrogen atom from its hydroxyl group to a lipid peroxyl radical. This active form of MitoQ has more potent and effective antioxidant properties that lead to inhibition of lipid peroxidation and reduction of oxidative stress in mitochondria, hence conferring protection against mitochondrial structural and functional damage. MitoVitE results from the conjugation of triphenylphosphonium cation to the tocopherol moiety of vitamin E. 2.2.7.2. Preclinical data. These compounds have been tested predominantly in neurodegenerative disorders, while few data are available in the setting of heart disease. In the concentration range of 100– 200 nM, MitoQ has in vitro protective effects against many cellular steps implicated in the pathogenesis of several cardiovascular diseases (Milagros Rocha & Victor, 2007). Indeed, it decreased ROS production, prevented apoptosis and improved cellular response to hypoxia and hyper-oxygenation (ischemia and reperfusion). Moreover, in vivo Mitoquininone administration has been proven to restore coronary collateral growth in a rat model of type 2 diabetes (Pung et al., 2012). MitoVitE was shown to prevent oxidative damage in vascular tissue. Mitovitamin E treatment decreased ROS production, restored mitochondrial membrane potential, inhibited cytochrome c release, caspase-3 activation and mitochondrial iron uptake and consequently reduced apoptosis in bovine aortic endothelial cells exposed to hydrogen peroxide and glucose oxidase (Dhanasekaran et al., 2004). Unfortunately, these agents have not been tested so far in the clinical arena. 2.2.8. Other drugs Large clinical trials have failed to show a clinical benefit of antioxidant treatment. However, they had the merit to drive a change in the understanding of the molecular nature of oxidative stress in diabetes and vascular disease. Consistently, oxidative stress is no longer perceived as a simple imbalance between the production and scavenging of ROS, but as a dysfunction of enzymes involved in ROS generation. NADPH oxidases are at the center of these events, underlying the dysfunction of other oxidases including eNOS uncoupling, and xanthine oxidase and mitochondrial dysfunction. Thus NADPH oxidases are important therapeutic targets. Importantly, HMG-CoA reductase inhibitors (statins) as well as drugs interfering with the renin–angiotensin– aldosterone system are able to inhibit NADPH oxidase activation and expression. Most of them, such as Angiotensin-converting enzyme (ACE) inhibitors, AT1 receptor antagonists (sartans), spironolactone or eplerenone are largely used in patients with several cardiovascular conditions and overlapping coronary microvascular dysfunction. This additive mechanism of action, often overlooked, may be in part responsible for the improved outcome. In the near future, new NADPH oxidase inhibitors such as VAS2870, VAS3947, GK-136901, S17834 or plumbagin are expected to come to the scene. This appears to be the most reasonable approach, potentially much more efficient than non-selective ROS scavenging by general antioxidant administration (Schramm et al., 2012). 2.2.9. Thiazolidinediones 2.2.9.1. Mechanism of action. The thiazolidinediones (TZDs) or ‘glitazones’ are a class of oral antidiabetic drugs that improve metabolic control in patients with type 2 diabetes through the improvement of insulin sensitivity. TZD-induced activation of Peroxisome proliferator-activated

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receptor gamma (PPAR-γ) alters the transcription of several genes involved in glucose and lipid metabolism and energy balance, reducing insulin resistance in adipose tissue, muscle and the liver. PPAR-γ is expressed most prominently in adipocytes, hepatocytes, skeletal muscle, cardiac muscle, colonic epithelium, vascular endothelial cells, renal collecting duct epithelium, and macrophages. It is possible that the effect of TZDs on insulin resistance in muscle and liver is promoted via endocrine signaling from adipocytes. Potential signaling factors include free fatty acids (FFAs) (well-known mediators of insulin resistance linked to obesity) or adipocyte-derived tumor necrosis factor-alpha (TNF-alpha), which is overexpressed in obesity and insulin resistance. 2.2.9.2. Preclinical data. In experimental models of endothelial dysfunction and atherosclerosis, troglitazone treatment was associated to inhibited expression of adhesion molecules (VCAM-1 and ICAM-1) in activated endothelial cells; moreover, in vivo PPAR-γ activation significantly reduced monocyte/macrophage homing to atherosclerotic plaques in ApoE-deficient mice (Pasceri et al., 2000). One report demonstrated that ciglitazone and a PPAR-γ synthetic ligand can stimulate the release of NO from endothelial cells (Calnek et al., 2003). However, other investigators reported conflicting results with rosiglitazone by showing that rosiglitazone reduces intracellular levels of Tetrahydrobiopterin (BH4) by inhibiting the rate-limiting enzyme of BH4 biosynthetic pathway (Linscheid et al., 2003). TZD's vasoprotective properties also include inhibition of human endothelial cell apoptosis (Artwohl et al., 2005) and improved stability of eNOS mRNA by inducing eNOS phosphorylation at Ser1177 and promoting heat shock protein 60 and eNOS interaction (Polikandriotis et al., 2005). Moreover, in a murine model of hindlimb ischemia, pioglitazone restored blood flow recovery and capillary density through increased expression of VEGF, mediated by Protein Kinase B (Akt) activation. This process was independent of its effect on glucose metabolism (Biscetti et al., 2009). In obese, spontaneously hypertensive rats with impaired relaxation in mesenteric resistance arteries and aorta to cumulative concentrations of phenylephrine, ACh, and sodium nitroprusside, glitazones elicited beneficial effects on both macrovascular and microvascular function (Mendizábal et al., 2011). 2.2.9.3. Clinical data. Few studies have specifically addressed the effect of thiazolidinediones on vascular function, and less have directly investigated their influence on the coronary circulation. In a small placebo controlled trial, 12-week troglitazone treatment improved endothelial function (evaluated with flow mediated dilatation in the brachial artery) only in the macrocirculation of patients with recently diagnosed type 2 diabetes and no clinical evidence of macrovascular disease. This improvement was strongly associated with the improvement of fasting plasma insulin concentrations (Caballero et al., 2003). In another study pioglitazone favorably affected peripheral microvascular function assessed as response to heat and Ach-mediated relaxation (Forst et al., 2005). A group of investigators has tried to evaluate the effect of Rosiglitazone on Myocardial Blood Flow Regulation in Type 2 Diabetes (ClinicalTrials.gov Identifier: NCT00549874); in this study rosiglitazone significantly reduced plasma nitrotyrosine, high-sensitivity C-reactive protein, and von Willebrand antigen, and significantly increased plasma adiponectin. No significant changes in these parameters were observed with glyburide. Treatment with glyburide, resulted in a significant deterioration in both resting and stress myocardial blood flow (MBF) that remained unchanged after rosiglitazone (Pop-Busui et al., 2009). Similar results were obtained in another report including subjects with Type 2 diabetes mellitus in which positron emission tomography (PET) failed to identify an effect of pioglitazone on resting or adenosine-stimulated MBF after 3 months of therapy (McMahon et al., 2005). Finally, a single study using adenosine triphosphate stress thallium 201 scintigraphy reported a beneficial effect of troglitazone on MBF (Sekiya et al., 2001). Other investigators have reported improved coronary microvascular


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function after long term administration of thiazolidinediones (pioglitazone or troglitazone), independent from diabetes clinical status (Murakami & Ohsato, 2007; Murakami & Ohsato, 2009; Murakami & Ohsato, 2012). Proposed mechanisms for these effects include improved endothelium-dependent vasodilation via restoration of insulin-dependent endothelial nitric oxide release, and improved vasomotor tone via increased expression of VEGF and reduced expression of endothelin-1. Despite these positive results, it is important to note that in some countries the use of thiazolidinediones has been restricted due to the observed increased risk of congestive heart failure and myocardial infarction. 2.2.10. Statins 2.2.10.1. Mechanism of action. Statins have been shown to lower lowdensity lipoprotein cholesterol by their ability to inhibit 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase, but also to improve vascular function by cholesterol-independent mechanisms (otherwise called pleiotropic effects).

BH4 is responsible for reduced NO availability. Without this critical cofactor, eNOS becomes “uncoupled”, thus resulting in the generation of superoxide radicals (O 2−) rather than NO from oxygen and NADPH. As a consequence, impaired endothelium-dependent vasorelaxation and endothelial dysfunction are evident. 2.2.11.2. Clinical data. In patients with type 2 diabetes and coronary artery disease, acute administration of intravenous L-arginine and BH4 prevented ischemia–reperfusion-induced endothelial dysfunction in the forearm, as assessed by venous occlusion plethysmography (Settergren et al., 2009). However, opposite results were lately obtained by another group of researchers. In patients with CAD, oral administration of BH4 significantly augmented BH4 levels in plasma and in saphenous vein (not internal mammary artery), but also increased levels of the BH2, which lacks endothelial nitric oxide synthase cofactor activity. There was no effect of BH4 treatment on vascular function or superoxide production. They concluded that oral BH4 treatment has no net effect on vascular redox state or endothelial function, owing to systemic and vascular oxidation of BH4 to BH2 (Cunnington et al., 2012). 2.3. Summary

2.2.10.2. Preclinical data. HMG-CoA reductase inhibitors improve endothelial function in many ways. NO bioavailability is increased not only by statin-induced reduction in ROS production but also by direct effects on eNOS enzyme. It has been suggested that statins up regulate eNOS expression and activity (Wolfrum et al., 2003; Tousoulis et al., 2008) and prolong eNOS mRNA half-life (Wolfrum et al., 2003) by a posttranscriptional mechanism involving inhibition of geranylgeranylation of Rho GTPase, and stabilization of eNOS mRNA (Laufs & Liao, 1998; Laufs et al., 1998). Statin-induced activation of phosphatidylinositol 3-kinase e Akt protein pathway also increases NO production and inhibits endothelial cell apoptosis (Wolfrum et al., 2003). In addition, several statins inhibit endothelial superoxide formation by reducing the activity of NADPH oxidase. This is partly due to the prevention of the isoprenylation of p21phox, which is critical for NADPH oxidase assembly (Wagner et al., 2000). Recently, a direct effect of atorvastatin on tetrahydrobiopterin-mediated endothelial nitric oxide synthase coupling has been elucidated. In ex vivo experiments, atorvastatin rapidly improved vascular BH4 bioavailability by upregulating GTPcyclohydrolase I gene expression and activity, resulting in improved endothelial NO synthase coupling and reduced vascular O2−. These effects were reversed by mevalonate, indicating a direct effect of vascular hydroxymethylglutaryl-coenzyme A reductase inhibition (Antoniades et al., 2011). 2.2.10.3. Clinical data. Specifically, the association of statin treatment with vascular NO bioavailability and arterial superoxide production was assessed in patients undergoing coronary artery bypass graft surgery (CABG). Statin-naive patients were randomized to atorvastatin 40 mg/d or placebo for 3 days before surgery to examine the impact of atorvastatin on endothelial function and O2− generation in internal mammary arteries. Statin treatment was associated with improved vascular NO bioavailability and reduced O2− generation in internal mammary arteries. Oral atorvastatin increased vascular tetrahydrobiopterin bioavailability and reduced basal and N-nitro-L-arginine methyl esterinhibitable O2− in internal mammary arteries independently of lowdensity lipoprotein lowering (Antoniades et al., 2011). 2.2.11. Endothelial nitric oxide synthase co-factors 2.2.11.1. Mechanism of action. Production of NO from the endothelium by endothelial Nitric Oxide synthase (eNOS) is essential for vascular homeostasis. This enzyme requires as co-factor tetrahydrobiopterin; when BH4 is present it “couples” the enzyme. BH4 bioavailability is dependent on both biosynthesis and oxidative degradation to dihydrobiopterin (BH2) and finally biopterin (B). Reduced synthesis or oxidative inactivation of

Microvascular dysfunction in diabetes recognizes several structural and functional changes occurring at the coronary level, during the course of diabetes. Mainly endothelial dysfunction and increased oxidative stress are possible therapeutic targets to prevent coronary dysfunction in this condition. At the moment there are convincing data about the pathogenic role of ROS, and different pharmacological approaches are under investigation (see Fig. 2 for a comprehensive overview of pharmacological approaches to diabetes related coronary microvascular dysfunction). For some of these the clinical arena is already available, while for others more studies are needed. Cardiologists are expected in the near future to have more drugs and more targets available to improve coronary microvascular function in this clinical setting. 3. Acute coronary syndrome 3.1. Pathophysiology of acute coronary syndrome related coronary microvascular dysfunction Reperfusion therapy in acute coronary syndromes (ACSs) aims to restore myocardial perfusion in order to salvage myocardium at risk. However, several studies have conclusively demonstrated that the duration of ischemia and the effectiveness of tissue perfusion achieved (coronary microcirculation preservation) are the most important prognostic elements after acute myocardial infarction. Microvascular dysfunction/obstruction (clinically manifested as no-reflow phenomenon) predicts acute and late complications after ACSs (Hombach et al., 2005; Ito, 2006). Reperfusion initiates a cascade of events within the first minutes after restoration of flow which causes myocytes and vascular injury in a relatively short time, finally leading to irreversible cells damage and death. Ischemia–reperfusion injury (IRI) can account for up to 50% of the final infarct size (Yellon & Hausenloy, 2007). Importantly, this phenomenon is not limited to ACS patients, but has been observed in elective percutaneous coronary intervention (PCI), coronary artery bypass grafting or other cardio-thoracic surgery, that is whenever ischemia–reperfusion sequences occur. IRI is the final event of a multifactorial process, which includes the “endothelial trigger” and “the inflammatory amplification” steps (Tsao et al., 1990). The final event of IRI includes the formation of pathological, non-specific mitochondrial permeability transition pores (mPTP) in the inner mitochondrial membrane. Oxidative stress accompanied by calcium overload and ATP depletion induces also the opening of the mPTP with a high conductance resulting in matrix swelling which ultimately induce rupture of the mitochondrial outer membrane and release of pro-apoptotic proteins into the cytoplasm.



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Fig. 2. Comprehensive overview of pharmacological agents available to prevent/treat diabetes related coronary microvascular dysfunction. On the right side of the picture are listed according to their main mechanism of action the classes of agent currently tested or already available in the clinical area. See text for more information.

All these mechanisms have been the target of therapeutic approaches to prevent microvascular dysfunction, improve the success of reperfusion, reduce infarct size and enhance event-free survival. The concept of cardioprotection refers to a variety of pharmacological agents or non-pharmacological strategies that when applied before (preconditioning) or after prolonged ischemia (postconditioning) have been shown to reduce the negative impact of ischemia and attenuate the effect of reperfusion injury on the ischemic myocardium. Over the past 2 decades, several interventions have been studied as potential cardioprotective agents in IRI (Fig. 3). 3.2. Pharmacological approaches of coronary microvascular dysfunction in acute coronary syndromes 3.2.1. Adenosine 3.2.1.1. Mechanism of action. The best-characterized mediator of cardioprotection is adenosine. Indeed, adenosine has been clearly implicated in the regulation of coronary blood flow during metabolic stress such as hypoxia and ischemia (Deussen et al., 1986; Deussen et al., 1988), but mostly has been implicated in conferring cardiac protection (Liu et al., 1991; Hein & Kuo, 1999; Erga et al., 2000). Opening of the ATP-sensitive potassium channels (KATP) inside mitochondria can explain at least in part the mechanism through which adenosine elicits cardioprotection. Activation of the KATP channels prevents mitochondria Ca2+ overload and cytochrome c release, thus avoiding mPTP opening, a process which precludes cell apoptosis (Garlid et al., 1997; Korge et al., 2002). Three different types of adenosine receptors located in many tissues have been identified. A1-receptors are located on cardiomyocytes and vascular smooth muscle cells, A2-receptors on endothelial and vascular smooth muscle cells, and A3-receptors on ventricular myocytes. In addition to participating in the mechanisms involved in cardiac conditioning, adenosine replenishes high-energy phosphate stores in endothelial cells and myocytes, inhibits cytokine release from mononuclear cells, and halts oxygen free radical formation, neutrophil activation

and accumulation. It reduces cardiomyocytes apoptosis, improves microvascular function and causes preconditioning responses (Stone, 2008). 3.2.1.2. Clinical data. In a proof of concept study, Marzilli et al. demonstrated that in patients with acute myocardial infarction undergoing primary PCI within 3 h from symptoms onset, intracoronary adenosine was associated with reduced mortality and left ventricle function improvement (Marzilli et al., 2000). However, three larger randomized trials have reported not definitive results, with limited benefits in similar setting of patients. In The Acute Myocardial Infarction Study of Adenosine (AMISTAD), patients receiving thrombolytic therapy plus continuous intravenous infusion of adenosine had a smaller infarct size assessed by SPECT and a better functional recovery than those in the control group (Mahaffey et al., 1999). In The Attenuation by Adenosine of Cardiac Complications (ATTACC) study, at the six-month follow-up of 292 patients with anterior infarcts, adenosine was associated to a trend for less all-cause mortality and cardiovascular mortality. In a post hoc analysis of a subgroup with anterior infarcts and severely depressed left ventricle function, the six-month death rate was significantly lower in the adenosine group (Quintana et al., 2003). However, the Acute Myocardial Infarction Study of Adenosine II (AMISTAD-II) specifically designed to investigate the role of adenosine in 2118 patients with anterior STEMI undergoing thrombolysis or primary PCI found no difference in the primary end point of new congestive heart failure (CHF), re-hospitalization for CHF, or death from any cause within 6 months (Ross et al., 2005). Despite the lack of difference between the two groups in terms of major clinical endpoints, infarct size tended to be smaller, in a dose-dependent manner, with a marked reduction in the high-dose group. In a post hoc analysis, in patients receiving reperfusion therapy within three hours of symptoms, adenosine reduced onemonth and six-month mortality rates significantly (Kloner et al., 2006). In another study, intracoronary administration of adenosine distal to the occlusion site immediately before initial balloon inflation was not associated to increased myocardial salvage but decreased coronary microvascular obstruction (Desmet et al., 2011). Recently, adenosine


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Fig. 3. Proposed mechanisms and effectors involved in cardioprotection. Adenosine, bradykinin, opioid peptides, and other autacoids natriuretic peptides (ANP and BNP), peptide growth factors (IGF-1 and FGF-2), and TNF-alpha could play a role in induce conditioning (pre and/or postconditioning). After binding to cell surface receptors, these autacoids promote the activation of specific signaling pathways including several kinases. Evidence from some models implicates the activation of PI3K/Akt and p42/p44 ERKs. This pathway, known as the RISK pathway, is proposed to result in inhibition of mPTP opening at reperfusion, via distal components of the cascade, which include NO, and inhibition of GSK3β. Furthermore, it has been proposed that the activation of an intra-mitochondrial pool of PKC1 might cause opening of the mitochondrial KATP channel (mitoKATP), resulting in a slight increase in reactive oxygen species (ROS) formation, which eventually causes mPTP inhibition. An alternative pathway, the so-called SAFE pathway, has been proposed to play a role in ischemic postconditioning. The major components of the SAFE pathway are TNF-a, the kinase JAK, which phosphorylates the transcription factor STAT3. It is proposed that after translocation to the nucleus, STAT3 controls the transcription of factors that confer cardioprotection. Also a mitochondrial localization of STAT3 has been suggested; however, both actions of STAT3 need to be finally proven. eNOS endothelial nitric oxide synthase; GPCR G-protein coupled receptor; GSK3β glycogen synthase kinase-3β; mPTP mitochondrial permeability transition pore; ERK, p42/p44 extracellular regulated kinase; NPR natriuretic peptide receptor; pGC particulate guanylyl cyclase; PKG cGMP-dependent protein kinase; RTK receptor tyrosine kinase; SR sarcloplasmic reticulum; TNF-R TNF receptor. A question mark (?) indicates that the link between those two pathways is not completely investigated.

given intracoronarily at the time of primary PCI was associated to better ST segment resolution, and tissue perfusion at coronary angiography documented as TIMI flow grade 3 and Myocardial blush grade 3. Most importantly, the composite end-point of death, recurrent myocardial infarction, heart failure and clinically driven target vessel revascularization at 1-year follow up was significantly reduced in the adenosine arm (Grygier et al., 2013). Some of the inconsistency between studies may be related to different dosage and route of administration, but most of them should be attributed to different timing of usage. Any cardioprotective strategies cannot be beneficial if applied when there are no longer viable cells that can be salvaged (in humans N 2 h of sustained myocardial ischemia). Therefore, the most striking difference is that pharmacological protection in animals is applied within few minutes after reperfusion begins and that, most importantly, reperfusion is initiated after 30 min to 1 h of ischemia, when a large amount of myocardial cells is still viable. 3.2.2. Targeting mitochondrial oxidative stress Given the relevance of mitochondria in IRI, agents that interfere with ROS production and release from mitochondria at the time of ischemia

or during the reperfusion period have been extensively investigated. Among these, a novel class of cell-permeable peptides, the SzetoSchiller, has been developed to selectively target mitochondrial ROS. 3.2.2.1. Mechanism of action. The Szeto–Schiller (SS) peptides are relatively small water-soluble molecules that contain a similar structural motif of alternating basic and aromatic residues, which allows them to freely cross cell membranes (despite a 3+ net charge at physiological pH) (Zhao et al., 2003). Studies with fluorescent and radiolabeled SS peptides indicate that they localize into the mitochondria and concentrate at inner mitochondrial membranes (Zhao et al., 2004). One particular peptide, Bendavia (an analogue of SS-02 and SS-31; Stealth Peptides), has been shown to reduce ROS levels in isolated mitochondria and to protect cultured cells against cell death induced by a variety of chemical stressors (Szeto, 2008). 3.2.2.2. Preclinical data. Using large animal models of IRI, a group of investigators has sought to determine whether Bevandia could attenuate myocyte cell death and no-reflow. These investigators found that Bendavia treatment reduced cellular ROS generation and helped sustain



mitochondrial membrane potential (ΔΨm) in cardiac myocytes exposed to hypoxia/re-oxygenation. In particular, Bendavia reduced oxidantdependent cell death during the re-oxygenation period, having no effect on myocyte survival during hypoxia (Kloner et al., 2012). At the present no clinical data are available. 3.2.3. Hyperbaric oxygenation An alternative approach would be to selectively provide the ischemic/ hypoxic myocardium with a large amount of oxygen at the time of reperfusion, i.e. hyperbaric oxygenation (HBO).

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phenomenon; secondary end points were continuous ST elevation, Q-wave myocardial infarction (MI), and non-Q-wave MI. Incidence of no-reflow/slow-flow phenomenon was significantly lower in the nicorandil group, as well as incidence of persistent ST-segment elevation and non-Q-wave MI. The authors concluded that use of intracoronary Nicorandil attenuates ischemia–reperfusion and ameliorates microcirculatory function. However, in another study intracoronary infusion of Nicorandil failed to show improved outcome at 30 days follow up as well as to reduce cardiac events during hospitalization, despite better TIMI flow grade in the treatment arm (Lee et al., 2008).

3.2.3.1. Mechanism of action. Aqueous oxygen (AO) is a relatively newly developed solution containing extremely high oxygen concentrations (1–3 ml O (2)/mL saline). The AO system mixes AO solution with a patient's blood from an arterial puncture and delivers the hyperoxemic blood to targeted ischemic myocardium via an infusion catheter for regional correction of hypoxemia and production of hyperoxemia. The system precisely controls the level of pO (2) without clinically significant microbubble formation.

3.2.5. Statins

3.2.3.2. Preclinical data. In a rat model of IRI, a cardioprotective effect was observed by hyperoxia + hyperbaria reperfusion and this mechanism was mediated by NOS (Cabigas et al., 2006) and catalase induction (Kim et al., 2001). Similar results were observed in other experimental models of acute myocardial ischemia (Sterling et al., 1993; Spears et al., 2002; Johnson et al., 2004).

3.2.5.2. Preclinical data. The immediate use of statins either before the onset of ischemia (Iliodromitis et al., 2010) or at the time of reperfusion (Sanada et al., 2004) has been proven to reduce infarct size regardless of hyperlipidemia.

3.2.3.3. Clinical data. A small proof of concept study has provided safety and efficacy data about HBO treatment (Bartorelli, 2003). The hyperoxemic reperfusion with AO was further investigated in the AMIOTH (Acute Myocardial Infarction in the Hyperoxemic Therapy) trial which included 269 patients undergoing primary or rescue PCI within 24 h of symptom onset (O'Neill et al., 2007). Patients were randomized after successful PCI to receive AO (TherOx Inc., Irvine, California) delivered through a system that mixes oxygen with blood, achieving a pO2 of 760 to 1000 mm Hg, or normoxemic blood autoreperfusion, delivered into the proximal portion of the infarctrelated artery. No improvement in regional wall motion, ST-segment resolution, or final infarct size was observed, though, in a post-hoc analysis, improved regional function in patients with anterior myocardial infarction treated within 6 h of symptom onset was reported. As for studies administering adenosine, inconclusive results may be related to the time at which the strategies are applied. 3.2.4. Nicorandil 3.2.4.1. Mechanism of action. Nicorandil is a nicotinamide ester with a dual mechanism of action. Its primary pharmacological effect is to open KATP channels. Activation of the KATP channels induce stabilization of the inner mitochondrial membrane and prevents membrane uncoupling, similar to the inhibition of mPTP opening due to preconditioning (Costa & Garlid, 2008) and by inhibiting Glycogen synthase kinase 3 (GSK-3β) (Gross et al., 2004). Nicorandil also acts as a nitrate-like factor, dilating systemic veins and epicardial coronary arteries. 3.2.4.2. Clinical data. The effect of nicorandil administration in the acute phase of an ischemic event has been tested in several clinical trials with inconsistent results (Ishii et al., 2005; Kobatake et al., 2011). In a subset of the J-WIND study, Nicorandil failed to show any infarct limitation in the acute phase, although oral administration during follow-up increased LV ejection fraction (Kitakaze et al., 2007). In another study the effectiveness of Nicorandil over Verapamil was tested regarding the ability to prevent no reflow/slow flow (surrogate indices of coronary microvascular dysfunction) in patients undergoing primary PCI and rotational coronary atherectomy (Matsuo et al., 2007). In this study the primary end point was incidence of no-reflow/slow-flow

3.2.5.1. Mechanism of action. Recent studies investigating the effects of statins have clearly reported that statins exert some of their effects by inducing cardioprotection. Evidence strongly suggests that cardioprotection induced by statins goes beyond lipid lowering and involves the signaling cascades of preconditioning and postconditioning, such as Akt activation and oxidative stress reduction (Merla et al., 2007b).

3.2.5.3. Clinical data. The same cardioprotective effects were also reported in humans (Merla et al., 2007a). Importantly, although in experimental models different statins have been attributed distinct action mechanisms (Meijer et al., 2009; Shen et al., 2010), benefits appear to be reproducible in clinical trials with most of statins used. In addition, as shown in the MIRACL trial, administration of statins reduced adverse outcomes when used as late as 24 h after reperfusion. Similar results have been obtained in patients with stable angina undergoing elective PCI (Briguori et al., 2004, 2009). 3.2.6. Mitochondrial permeability transition pore opening inhibitors 3.2.6.1. Mechanism of action. Cyclosporine A inhibits the opening of mPTP by binding to cyclophilin D at the inner mitochondrial membrane. Other potential mechanisms involve ROS generation, iNOS and heat shock protein 70 (Hsp70). (Chen et al., 2002) 3.2.6.2. Preclinical data. In isolated cardiomyocytes under condition of simulated ischemia Cyclosporin A permitted the maintenance of electromechanical function and, improved the post-ischemic functional recovery. Cyclosporin A reduced the ischemia-induced lactate dehydrogenase and troponin I release, and lowered subsequent rise in heat shock protein mRNA observed after ischemia and reoxygenation. Moreover, cyclosporin A improved the resumption of the mitochondrial function (Bes et al., 2005). Pharmacologic inhibition of mPTP opening by intravenous administration of cyclosporine or with sanglifehrin A, immediately before reperfusion, has been shown to reduce myocardial infarct size in animal studies by up to 50% (Clarke et al., 2002; Gomez et al., 2008) 3.2.6.3. Clinical data. In humans, cyclosporine A has demonstrated to reduce infarct size of approximately 40%, measured in terms of CK release, and confirmed by magnetic resonance imaging (MRI) (Piot et al., 2008). The findings of protection by cyclosporine are now extended to include endothelial cells after ischemia–reperfusion in humans (Okorie et al., 2011), but seem still preliminary and need further confirmation in large-scale clinical trials 3.2.7. Glycoprotein IIb/IIIa Inhibitors Glycoprotein IIb/IIIa inhibitors (Gp IIb/IIIa) are an attractive strategy to preserve coronary microvascular function, targeting platelets aggregation


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and vascular clotting, mechanisms implicated in microvascular damage due to IRI. 3.2.7.1. Mechanism of action. Three Gp IIb/IIIa antagonists are currently used in clinical practice: integrilin, abciximab and tirofiban. Integrilin is a cyclic heptapeptide; abciximab is a Fab fragment of a mouse/ human chimeric antibody against Gp IIb/IIIa; Tirofiban is a synthetic, non-peptide inhibitor of the Gp IIb/IIIa receptor (Phillips et al., 2005). 3.2.7.2. Preclinical data. In animal models, Gp IIb/IIIa receptor antagonists have been beneficial in the setting of IRI. In a canine model of IRI, tirofiban administered 45 min before reperfusion improved microvascular flow and reduced infarcted area (Kunichika et al., 2004). Tirofiban was more effective than aspirin and clopidogrel in attenuating noreflow in a swine infarction/reperfusion model (Yang et al., 2006). 3.2.7.3. Clinical data. Despite positive results in animal models of IRI, in humans these agents have shown less evident beneficial effects. Clinical reports suggest that abciximab improves epicardial flow, microvascular perfusion and left ventricular recovery in patients who were referred for rescue angioplasty after failed thrombolysis (Kaul et al., 2001). In a randomized study, Abciximab therapy was associated with better myocardial perfusion as determined by thrombolysis in myocardial infarction (TIMI) frame count (Petronio et al., 2005). In a small retrospective study, in patients who developed the no-reflow phenomenon, post-procedural abciximab was associated to a better 6-month followup outcome as compared to controls. The authors concluded that ‘rescue’ administration of abciximab may be an effective option for the treatment of the no-reflow phenomenon determining significant prognostic improvements (Picchi et al., 2008). A meta-analysis of 12 trials, including over 20,000 patients undergoing PCI reported reduced 30-day mortality in patients receiving glycoprotein IIb/IIIa antagonist, with 2–8 lives saved per 1000 patients (Kong et al., 2003). 3.3. Summary Since the identification of vulnerable atherosclerotic plaques as the substrate of acute coronary syndromes, great efforts have been made in order to design and test strategies to limit infarct size and improve clinical outcomes. Approximately over 95% of occluded vessels can be easily reopened in the setting of STEMI. Unfortunately, the clinical impact of these interventions falls short of expectations and a large fraction of patients surviving the acute phase develop left ventricular remodeling and heart failure. Several factors contribute to these disappointing results; the most important include incomplete understanding of the time course of myocardial cell death and underestimation of the contribution of reperfusion injury to final infarct size. All strategies used to protect myocardial infarction are time-sensitive and can be beneficial only if applied when ischemic cells are still viable and can be salvaged by reperfusion. In fact the most important difference among animal and humans studies is that pharmacological conditioning in animals is applied very early after reperfusion and, most importantly, that reperfusion is initiated after 30 min to 1 h of ischemia. These important concepts are well demonstrated with the results of the study of Marzilli et al. (2000) that administered adenosine downstream the coronary blockage, before initiating reperfusion, and including only patients with less than 3 h of ischemic time. 4. Stable ischemic heart disease 4.1. Pathophysiology of ischemic heart disease associated coronary microvascular dysfunction

a precarious balance between oxygen supply and metabolic requirements to the myocardium, due to significant coronary obstruction (CAD). However, approximately 30% of these patients present with angiographically normal epicardial coronary arteries (Cannon, 2009). A number of pathogenic mechanisms, including microvascular dysfunction, endothelial dysfunction, altered vascular tone, coagulation abnormalities and altered metabolic switch in response to increased myocardial energy demand have been proposed (Cannon & Epstein, 1988; Kaski et al., 1995). Among others, endothelial dysfunction probably represents the most widely studied condition and has been associated with variable clinical presentations, ranging from reduced vasodilator reserve to microvascular coronary abnormalities and, ultimately, myocardial ischemia (Panza et al., 1990; Hurst et al., 2006). Interestingly, endothelial dysfunction, as evidenced by the loss of acetylcholine-induced coronary vasodilatation, has been documented in patients with atherosclerosis, hyperlipidemia (Flavahan, 1992), hypertension (Linder et al., 1990; Panza et al., 1990), diabetes mellitus and smoking, all factors known to predispose to microvascular angina (Cai & Harrison, 2000; Kaski et al., 2004). Despite these evidences, many patients receive no therapy when coronary angiography reveals no coronary obstructions (Panza, 2002; Phan et al., 2009). However, new data suggest that this approach may no longer be appropriate. Indeed, the prognosis of such patients is not as benign as previously thought, in this way underscoring the relevance of proper identification and treatment (Johnson et al., 2006). Although studies indicate structural/functional microvascular changes as the pathophysiological basis, the first line treatment agents in microvascular angina are represented by conventional drugs, including nitrates, calcium channel blockers and beta blockers (Kaski & Valenzuela Garcia, 2001). These are the so called ‘hemodynamic agents’ which act by lowering oxygen requirements by producing decreased rate-pressure product and/or systemic venodilation, thereby lowering left ventricular end-diastolic pressure and volume and myocardial wall tension. However, the development of drugs (e.g. trimetazidine, ranolazine, ivabradine, nicorandil, etc.) with alternative action mechanisms (i.e. metabolic agents and agents that induce endothelial vasodilation) has gained particular interest in the group of patents with angina and no obstructive CAD. 4.2. Pharmacological approaches of coronary microvascular dysfunction in IHD 4.2.1. Beta-blockers 4.2.1.1. Mechanism of action. Beta-blockers (BBs) inhibit the action of endogenous catecholamines (epinephrine and norepinephrine in particular) on adrenergic receptors. Their anti-angina effects are mediated through a reduction in ventricular inotropy, heart rate and a decrease in the maximal velocity of myocardial fiber shortening, therefore keeping myocardial oxygen demand below the threshold at which angina occurs. Available BBs differ in their effects on the 3 adrenergic receptors (β1, β2, and α) and in their duration of effect. Beta-blockers can be considered as the first line treatment option for patients with chronic angina, in particular those with exercise induced symptoms and/or those with increased sympathetic activity (Romeo et al., 1988; Fragasso et al., 1997). 4.2.1.2. Clinical data. Among other BBs, Nebivolol, a highly selective β1-adrenergic receptor-blocker of third generation, has been found to significantly improve coronary flow reserve by stimulating nitric oxide release from endothelial of angina patients (Togni et al., 2007). 4.2.2. Nitrates

Ischemic heart disease (IHD) is a leading cause of mortality and stable angina represents the most frequent clinical presentation (Go et al., 2013). Chronic stable angina is often considered as the result of

4.2.2.1. Mechanism of action. Nitrates represent a mainstay therapy for symptom control of patients with CAD. Although the predominant



effect of nitrates is to reduce preload, with a greater activity in the venous than arterial beds, at higher doses a direct effect upon arteries becomes also evident and results in a reduction in blood pressure (BP) and afterload. These effects translate into reduced myocardial oxygen consumption and a higher threshold level before angina is triggered. 4.2.2.2. Clinical data. Their efficacy appears to be disappointing in patients with chronic stable angina and no obstructive CAD. Indeed, the majority of studies have shown only a modest effect of sublingual and/or intracoronary nitrate therapy in time to angina and peak STsegment depression of patients with microvascular angina (Bugiardini et al., 1993; Radice et al., 1994). In a recently published study, Russo et al. have assessed the effects of short-acting nitrates on exercise stress test (EST) results and the relation between EST results and coronary blood flow (CBF) response to nitrates in 29 patients with microvascular angina as compared to 24 patients with obstructive CAD. CBF response to nitroglycerin was assessed in the left anterior descending coronary artery by transthoracic Doppler-echocardiography. ST-segment depression N/=1 mm (STD) was induced in 26 (90%) and 23 (96%) microvascular and CAD patients, respectively (p = 0.42). Following sublingual nitrate administration STD was induced in 25 (86%) and 14 (56%) microvascular and CAD patients, respectively (p = 0.01). Time and rate pressure product at 1 mm STD increased following sublingual nitrate administration in CAD but not in microvascular angina patients (Russo et al., 2013) Keeping in mind that many stable angina patients may present with overlapping microvascular/macrovascular spasm (Horimoto et al., 2002), the rationale for the use of nitrate therapy in the subset of patients with stable angina and no obstructive CAD is more than justified (Ong et al., 2012). 4.2.3. Calcium channel blockers 4.2.3.1. Mechanisms of action. Calcium channel blockers (CCBs) are potent coronary and systemic arterial vasodilators that reduce BP as well as cardiac contractility. CCBs bind to and inhibit L-type calcium channels, reducing calcium influx into cells. Intracellular calcium deprivation relaxes smooth muscle cells, causing vasodilation in the peripheral and coronary beds and increased coronary blood flow. There are two classes of CCBs. They differ not only in their basic chemical structure, but also in their relative selectivity toward cardiac versus vascular L-type calcium channels. The most smooth muscle selective class of CCBs is that of dihydropyridines. Because of their high vascular selectivity, dihydropyridines are primarily used to reduce systemic vascular resistance and therefore to treat hypertension. They are not, however, generally used to treat angina because their powerful systemic vasodilator and pressure lowering effects can lead to reflex cardiac stimulation (tachycardia and increased inotropy), which can dramatically increase myocardial oxygen demand. Non-dihydropyridynic CCBs include verapamil (phenylalkylamine class) and diltiazem (benzothiazepine class) which have more selective cardio-inhibitory properties. 4.2.3.2. Clinical data. CCBs have been proven to lower the frequency of angina, reduce the need for nitrates, extend treadmill-walking time, and improve ischemic ST-segment changes on exercise testing and electrocardiographic monitoring in patients with CAD. However, their role in stable angina patients with no obstructive CAD remains controversial. In a small randomized, double-blind, placebo-controlled study, 1-month therapy with calcium channel blockers which included nifedipine or verapamil significantly improved angina and exercise tolerance in patients with chest pain and reduced coronary vasodilator reserve (Cannon et al., 1985). Similar results were obtained in other studies testing the benefit of felodipine and nisoldipine (Montorsi et al., 1990; Ozcelik et al., 1999). However, in another study Sutsch et al. investigated the effects of diltiazem on coronary flow reserve in patients with microvascular angina. Coronary blood flow was determined at rest,

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after dipyridamole (0.5 mg/kg) and following intravenous administration of diltiazem (10 mg) using coronary sinus thermodilution technique. Patients with normal coronary flow reserve (coronary flow reserve N 2.0) received either dipyridamole alone (group 1, controls, n = 6) or dipyridamole and diltiazem (group 2, n = 5), whereas patients with reduced coronary flow reserve (coronary flow reserve b 2.0) were administered dipyridamole and diltiazem (group 3, n = 5). Resting coronary flow was identical in the three groups, but after maximal vasodilation with dipyridamole, coronary flow increased significantly more in groups 1 and 2 than in group 3 (P b 0.05). Coronary flow reserve was 2.5 in group 1 and 2.3 in group 2, but was significantly reduced in group 3 (1.3; P b 0.05). Intravenous diltiazem failed to increase coronary blood flow in groups 2 and 3. The authors concluded that despite the reported clinical benefits of calcium channel blockers in this patient population, diltiazem did not affect coronary flow reserve in patients with microvascular angina, in this way suggesting that other factors (i.e. structural abnormalities in the microcirculation or functional abnormality in smooth muscle relaxation not responsive to calcium channel blockade) are probably responsible for the occurrence of myocardial ischemia in patients with microvascular angina (Sutsch et al., 1995). Indeed, while calcium channel blockers can induce regression of myocardial hypertrophy, the same agents have not been shown to reverse microvascular dysfunction (Vogt et al., 1994). 4.2.4. Trimetazidine 4.2.4.1. Mechanism of action. Trimetazidine (1-[2,3,4-trimethoxybenzyl] piperazine dihydrochloride) is metabolic modulator that partially inhibits fatty acid oxidation, thereby increasing glucose oxidation through an interdependence mechanism, called the ‘Randle cycle’ (Randle et al., 1963; Kantor et al., 2000). This effect results in an increase in phosphate dehydrogenase (PDH) activity, which compensates the reduced availability of acyl-CoA, derived from β-oxidation of fatty acids, by providing them through glucose oxidation. Stimulation of glucose oxidation increases glycolysis/glucose oxidation coupling, resulting in decreased proton production, less tissue acidosis, less intracellular calcium overload and less free radical production. As a consequence, the ischemic myocardium can more efficiently use the limited oxygen supply, which clinically translates into an amelioration of angina symptoms (Kantor et al., 2000). 4.2.4.2. Clinical data. Trimetazidine has been used in Europe for over three decades and its efficacy in alleviating chronic angina has been confirmed in several randomized clinical trials. However, only few studies have tested the efficacy of trimetazidine in patients with microvascular angina. Nalbantgil et al. tested the effects of trimetazidine in a placebocontrolled, double blind study consisting of two 4-week treatment periods. Symptom-limited exercise testing was assessed in thirtyfive patients. Trimetazidine prolonged total exercise time and time to 1 mm ST depression and reduced maximum ST depression when compared with placebo (Nalbantgil et al., 1999). Rogacka et al. sought to determine the effect of trimetazidine (20 mg three times a day) on angina symptoms and exercise tolerance in 34 patients with microvascular angina. Treatment with trimetazidine was associated with negative exercise tests at follow up, improved tolerance to exercise and increased time to ST-segment depression (Rogacka et al., 2000). Conversely, in a head-to-head comparison study with atenolol, trimetazidine did not exert any significant effect on any of the analyzed variables in 16 patients with microvascular angina (Leonardo et al., 1999). 4.2.5. Ranolazine 4.2.5.1. Mechanism of action. In 2006, ranolazine was approved for the relief of angina in patients who remained symptomatic despite BBs, CCB, or nitrates (Chaitman, 2006). It is a piperazine structurally related to trimetazidine, which was shown to have anti-ischemic properties


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through promotion of glucose oxidation at the expense of fatty acid oxidation since the early nineties (McCormack et al., 1996). However, additional properties such as reduction in intracellular calcium overload through inhibition of the late sodium channels have gained more attention recently (Wasserstrom et al., 2009). In fact, ranolazine is currently considered a first generation of a new drug category (i.e. inhibitor of late sodium currents). Nonetheless, it is important to note that therapeutic concentrations at which a reduction in calcium overload is observed are similar to those at which an increase in glucose oxidation has been documented (McCormack et al., 1996).

4.2.7. Nicorandil

4.2.5.2. Clinical data. Regardless of the main action mechanism, ranolazine has been shown to confer significant clinical benefit in angina patients, including patients with microvascular angina. Venkataraman et al. demonstrated that short term Ranolazine therapy (4 weeks) improves myocardial perfusion, evaluated by SPECT, and decreases the ischemic burden (Venkataraman et al., 2009). Villano et al. randomized 46 patients with stable microvascular angina who had symptoms inadequately controlled by standard anti-ischemic therapy to ivabradine (5 mg twice daily), ranolazine (375 mg twice daily), or placebo for 4 weeks. The Seattle Angina Questionnaire (SAQ), EuroQoL scale, and exercise stress test were assessed at baseline and after treatment. Coronary microvascular dilation (in response to adenosine and to cold pressure test) and peripheral endothelial function (measured by flow-mediated dilation) were also assessed. Both drugs improved SAQ items and EuroQoL scale compared with placebo (p b0.01 for all). Time to 1-mm ST-segment depression and EST duration were improved by ranolazine compared with placebo. No effects on coronary microvascular function and on flow-mediated dilation were observed with drugs or placebo (Villano et al., 2013; Vitale et al., 2013). In another study Mehta et al. conducted a pilot randomized, double-blind, placebocontrolled, crossover trial in 20 women with angina, no obstructive CAD, and N/=10% ischemic myocardium on adenosine stress cardiac magnetic resonance imaging. Participants were assigned to ranolazine or placebo for 4 weeks separated by a 2-week washout. Compared with placebo, patients on ranolazine had significantly better SAQ scores, including physical functioning (p = 0.046), angina stability (p = 0.008), and quality of life (p = 0.021). There was a trend toward better mid-ventricular perfusion (p = 0.074) on ranolazine. Among women with coronary reactivity testing (n = 13), those with CFR b/=3.0 had a significantly improved perfusion on ranolazine versus placebo compared to women with CFR N 3.0. This phase 2 study provided the basis to conduct a definitive large clinical trial to evaluate the role of ranolazine in microvascular coronary dysfunction (Mehta et al., 2011).

4.2.7.2. Clinical data. In patients with CAD and stable angina pectoris, nicorandil has been shown to prolong time to the onset of angina and ischemic ECG changes, extend exercise duration (Chen et al., 1997; Markham et al., 2000), and reverse ischemia-related impairment in regional wall motion. Less data are available in patients with microvascular angina. In a study involving 11 patients with hypertension, diabetes mellitus and nearly normal coronary angiograms, Yamabe et al. evaluated the antianginal effects of intravenous nicorandil. Nicorandil was found to improve both extent and severity of exercise induced thallium myocardial perfusion defects and to reduce both exercise-induced angina and ST segment changes (Yamabe et al., 1995). In a randomized placebo controlled trial 2-weeks of oral treatment with nicorandil improved both time to 1 mm of ST segment depression and peak exercise capacity. However, the agent failed to significantly improve the magnitude and the incidence of exercise induced ST segment changes (Chen et al., 1997).

4.2.6. Ivabradine 4.2.6.1. Mechanism of action. In 2005, Ivabradine was approved as an anti-angina drug in Europe. This novel drug selectively inhibits the hyperpolarization activated, mixed sodium/potassium inward If current, a primary senatorial node (SAN) pacemaker current, thereby decreasing rest and exercise heart rate responsiveness (Borer et al., 2003). Importantly, Ivabradine does not affect contractility, AV nodal conduction, nor alter hemodynamic. 4.2.6.2. Clinical data. The only study in which the effects of Ivabradine were tested in patients with microvascular angina is the previously mentioned study by Villano et al. in which Ivabradine was shown to improve clinical parameters of chronic angina, without affecting measured parameters of coronary microvascular function (Villano et al., 2013). However, in a small study including patients with stable CAD, Ivabradine treatment improved hyperemic coronary flow velocity and CFR. These effects were evident even after heart rate correction, suggesting improved microvascular function (Skalidis et al., 2011).

4.2.7.1. Mechanism of action. Nicorandil is structurally a nicotinamide derivative with a nitrate moiety and a dual mechanism of action. First, it increases potassium ion conductance by opening adenosine triphosphate (ATP)-sensitive potassium channels, in turn activating the enzyme guanylate cyclase. Second, nicorandil shares the smooth muscle-relaxing property of nitrates to vasodilate, lowering preload through venodilation. The drug also reduces afterload and promotes expression of endothelial NO synthase and microvascular dilation (Hongo et al., 1995; Jahangir et al., 2001).

4.2.8. Aminophylline 4.2.8.1. Mechanism of action. Different pathophysiological mechanisms have been attributed to adenosine in patients with microvascular angina, including mediation of nociception, arteriolar dilatation with microvascular “steal” phenomenon and modulation of antiplatelet aggregation activity (Sylven & Lagerquist, 1990; Aurigemma et al., 2009). Given such evidences aminophylline, an adenosine receptor blocker, has represented an intense research area. 4.2.8.2. Clinical data. Short-term intravenous administration of aminophylline was shown to improve angina, exercise capacity and exercise induced ST segment changes (Yoshio et al., 1995; Yesildag et al., 1999). Similarly, oral aminophylline significantly improved exercise tolerance, particularly time to angina, but failed to show a relevant effect on other variables such as chest pain and ST segment depression (Elliott et al., 1997). In a randomized cross-over trial, when compared to nitrates, acute administration of aminophylline improved the time to exercise induced angina, time to 1 mm of ST segment depression and the magnitude of ST segment depression in 20 patients with microvascular dysfunction (Lanza et al., 1997). The beneficial effects of adenosine antagonists have been postulated to be in relation to prevention of steal mechanisms occurring in microvascular dysfunction by attenuating excessive hyperemia in relatively well-perfused areas. This therapeutic strategy can be particularly useful in patients with persistent, microvascular dysfunction who also have chronic obstructive airways disease, bronchial asthma and unexplained shortness of breath as a major component of the syndrome. 4.2.9. Rho-kinase inhibitors 4.2.9.1. Mechanisms of action. Rho kinase (ROCK) is an important intracellular enzyme, which by phosphorylating several proteins affects a number of cellular functions. Among other actions, ROCK can phosphorylate myosin resulting in smooth muscle cell contraction and therefore vasoconstriction. Fasudil, a ROCK inhibitor has been investigated both in the bench and later on in the bedside.



4.2.9.2. Preclinical data. In animal models of IHD, inhibition of myosin phosphorylation by fasudil resulted in a decrease of vascular smooth muscle hypercontraction and reduction of ischemic ST segment depression. It has also been shown to afford cardioprotection through postconditioning (Jiang et al., 2013).

4.2.9.3. Clinical data. These effects were reproduced in a phase II, doubleblind, placebo controlled, randomized clinical trial of 84 patients with chronic angina (Vicari et al., 2005). Fasudil has also been shown to induce a further dilation at the site of coronary spasm in patients with vasospastic angina, who had already been treated with nitroglycerin (Otsuka et al., 2008). Mohri et al. sought to determine the effects of fasudil in patients with microvascular angina attributable to coronary microvascular spasm. While ACh reproducibly induced myocardial ischemia in the saline group, 11 of the 13 patients pre-treated with fasudil had no evidence of myocardial ischemia during the second infusion of ACh (p b 0.01). This finding was associated with improved lactate extraction ratio (Mohri et al., 2003). Fukumoto and colleagues more recently confirmed the beneficial effects of fasudil in chronic microvascular dysfunction in another study. Fasudil significantly increased oxygen saturation in coronary sinus vein and ameliorated pacinginduced myocardial ischemia in patients with effort angina, without exerting significant hemodynamic changes (Fukumoto et al., 2007). However, despite the proven clinical efficacy and safety, the drug is not yet commercially available for clinical use.

4.2.10. Alpha-1 blockers Increased vasoconstrictor tone, involving α-1 receptor, has been supposed as one of the pathogenic mechanisms of microvascular angina and reduced vasodilator capacity of the coronary microvessels (Camici et al., 1994). Both doxazosin (Botker et al., 1998) and clonidine (Cannon et al., 1994) – α1 and α2 agonists – were tested in small studies but failed to improve daily-life chest pain or exercise test variables. Thus, despite their theoretical benefits, these agents do not seem to have a practical role in the management of patients with coronary microvascular angina.

4.2.11. Angiotensin-converting enzyme inhibitors

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4.2.12. L-Arginine 4.2.12.1. Mechanism of action. Abnormal vasomotion is a well-recognized contributor to myocardial ischemia in patients with microvascular angina (Maseri et al., 1991). Endothelium-derived nitric oxide is a potent vasodilator that plays a critical role in maintaining vascular homeostasis through its anti-atherogenic and antiproliferative effects on the vascular wall. NO is synthesized from its precursor, L-arginine, by endothelial NO synthase (Deanfield et al., 2007). Supplementation with L-arginine facilitates production of nitric oxide and improves endothelium-dependent vasodilation and clinical status (Creager et al., 1992; Piatti et al., 2003). 4.2.12.2. Clinical data. In a small placebo-control study, intracoronary infusion of L-arginine (50 mg/mm) antagonized acetylcholine-induced coronary vasoconstriction of patients with microvascular angina (Egashira et al., 1996). However, intracoronary infusion of L-arginine did not alter attenuated acetylcholine-induced endothelium-dependent coronary vasodilation in patients with CAD and hypertension, suggesting that the mechanisms of endothelial dysfunction may differ depending on the cause and severity of vascular disease (Hirooka et al., 1994). 4.2.13. Vitamins and co-factors supplementation It has been suggested that plasma concentrations of some group B vitamins (vitamin B6, vitamin B12 and folic acid) inversely correlate with cardiovascular risk. Several experimental and clinical studies, mostly retrospective and case–control studies indicate a defect of such vitamins as capable of promoting the progression of atherosclerosis. Since all these vitamins are implicated in homocysteine metabolism, and since homocysteine has a recognized relationship with cardiovascular risk, the simplest hypothesis to explain the relationship of vitamin B6, vitamin B12 and folic acid on the one hand, and cardiovascular risk on the other is that this relationship is mediated by plasma levels of homocysteine. Endothelial dysfunction may be one underlying cause leading to pro-atherogenic effects associated with hyperhomocysteinaemia. However, the mechanisms, which lead to impaired endothelial function in hyperhomocysteinaemia, are not fully understood. Recent evidence suggests that homocysteine may interact with physiological mediators of the endothelial matrix. Oxidative mechanisms and decreased biological activity of endothelium-derived nitric oxide may also contribute to homocysteine-associated endothelial dysfunction.

4.2.11.1. Mechanisms of action. Angiotensin-converting enzyme (ACE) and local angiotensin II (AT-II) levels have been shown to modulate coronary vascular resistance (Banes-Berceli et al., 2007). In particular, angiotensin II stimulates ROS production, which increases nitric oxide catabolism, favoring endothelial dysfunction. Angiotensin II also mediates vascular remodeling by increasing cell growth and decreasing apoptosis. All these events mediate vascular disease (Griendling & Alexander, 1997). Based on these principles, it has been postulated that ACE inhibition may efficiently restore the appropriate homeostatic balance between these vasoactive systems (Mancini et al., 1996).

4.2.13.1. Mechanisms of action. B vitamins are essential cofactors in the metabolism of homocysteine to methionine via the remethylationpathway (vitamin B12, folic acid) and to cystathionine via the transsulfuration-pathway (vitamin B6).

4.2.11.2. Clinical data. Indeed, blockade of the renin–angiotensin system (RAS) has been shown to positively affect microvascular function, particularly in the subset of patients with diabetes and/or hypertension (Kaski et al., 1994). Moreover, ACE inhibition has been reported to attenuate sympathetic coronary vasoconstriction in patients with coronary artery disease (Perondi et al., 1992). In a sub-study of the Women's Ischemia Syndrome Evaluation (WISE trial), 61 women with angina, microvascular dysfunction and no obstructive CAD were randomly assigned to either quinapril or a placebo. Treatment with ACE-inhibitor significantly reduced angina symptoms and improved CFR (Pauly et al., 2011). Given the pharmacological properties together with the well tolerability, it is appropriate to suggest that microvascular angina patients may benefit from ACE inhibitor treatment.

4.2.13.3. Clinical data. In 2008 a group of research investigated the effect of homocisteine-lowering therapy on mortality and cardiovascular events in patients undergoing coronary angiography (stable CAD or ACS). This trial was terminated early because of concern due to preliminary results from a contemporaneous Norwegian trial suggesting adverse effects from vitamins B and acid folic supplementation. No significant benefit was associated with the study regimen (Ebbing et al., 2008). Recently the effects of B-vitamin therapy and acid folic supplementation on coronary flow and vascular function in patients with established coronary artery disease (CAD) have been examined (Bleie et al., 2011). In this trial, forty patients with stable CAD were randomly assigned to daily oral treatment with 0.8 mg of folic acid and 0.4 mg of vitamin B12 or placebo, and 40 mg of vitamin B6 or

4.2.13.2. Preclinical data. The most convincing data for a relationship with cardiovascular risk are for vitamin B6 and folic acid. These vitamins, however, have also a series of in vitro effects indicating a direct anti-atherogenic action, and the results of several clinical studies, especially for vitamin B6, indicate an inverse relationship with cardiovascular risk at least in part independent of homocysteinemia.


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Fig. 4. Comprehensive overview of available pharmacological agents to treat microvascular angina. A: Drugs that act at the cardiac and macrovascular level, mainly composed of “conventional anti-anginal agents”; B: Drugs that act at microvascular level, mainly composed of “non conventional anti-anginal agents”; C Drugs acting at the level of myocardial intracellular system, mainly composed of “non conventional anti-anginal agents”. Acronyms: ACEI: Angiotension-converting enzyme inhibitors; BBs = beta-blockers; CCBs = calcium channel blockers; FAO = fatty acid oxidation; HR = heart rate; L-A = L-arginine; ROCK = Rho kinase; s.m.c. = smooth muscle cell; TMZ = trimetazine.

placebo, using a 2 × 2 factorial design. Coronary blood flow was assessed by coronary angiography and Doppler flow-wire measurements during intracoronary infusion of saline (basal), incremental doses of acetylcholine, adenosine, and nitroglycerin. The authors found a significant increase in basal (P b 0.02) and adenosine-induced (P b 0.05) coronary blood flow in patients who received folic acid/vitamin B12 for 24 months, compared with placebo or vitamin B6 alone. Folic acid/vitamin B12 or vitamin B6 treatment did not change endothelial-dependent response to acetylcholine infusion or flow-dependent proximal dilatation in response to adenosine-induced maximal hyperemia. Long-term treatment with a combination of folic acid and vitamin B12 increased basal and adenosine-induced maximal coronary blood flow; the authors suggested that this effect might reflect improved microvascular function in patients with stable CAD. 4.3. Summary Although microvascular angina affects a significant number of patients, currently there are no standardized and evidence-based treatment recommendations. Quite paradoxically, most pharmacological agents utilized in microvascular angina exert their effects mainly at non-microvascular level (Fig. 4). Even though their utility has been questioned, conventional anti-anginal drugs (beta blockers, calcium channel blockers and nitrates) are recommended as the first line therapy. On the other hand, nonconventional agents principally affecting endothelial function and intracellular systems appear to be promising candidates, but conclusive evidence is still missing. 5. Cardiomyopathies 5.1. Pathophysiology of cardiomyopathies related coronary microvascular dysfunction Microvascular dysfunction is the leading cause of disease progression in two of the most frequent cardiomyopathies: idiopathic dilated cardiomyopathy (IDCM) and Hypertrophic cardiomyopathy (HCM). Virtually all cardiomyopathies seem to share some degree of coronary

microvascular dysfunction, which can be detected even at early stages and is related with disease progression and long-term outcome (Paul et al., 2012; Patel et al., 2013). 5.2. Idiopathic dilated cardiomyopathy In explanted hearts from patients with end-stage heart failure due to idiopathic dilated cardiomyopathy (IDCM), myocardial blood flow is severely reduced at rest independent from myocardial fibrosis which does not involve more than 20% of the myocardial mass (Parodi et al., 1993). In addition, up to 82% of these patients show reduced myocardial blood flow, at rest and during metabolic and pharmacological vasodilation, due to coronary microcirculatory dysfunction. Moreover, the severity of flow abnormalities, and hence of microvascular dysfunction (since CAD is ruled out), is able to predict the evolution of the disease towards progressive ventricular dysfunction and heart failure (Neglia et al., 2002a). Associated to global and regional coronary vasodilation capacity impairment, cardiac metabolism abnormalities have been described, particularly glucose metabolism (Vercesi et al., 1978), a similar pattern of flow-metabolism “mismatch” described in patients with coronary artery disease and hibernated myocardium (van den Heuvel et al., 2000; Neglia et al., 2002b). 5.3. Hypertrophic cardiomyopathy Similar data support a role for microvascular dysfunction in hypertrophic cardiomyopathy (HCM). Using cardiac SPECT wall motion abnormalities and regional perfusion defects were evidenced, while PET demonstrated an inadequate increase in myocardial blood flow after intravenous administration of dipyridamole, although resting myocardial blood flow was unchanged (Camici et al., 1991; Lorenzoni et al., 1998; Choudhury et al., 1999). In addition, the degree of microvascular dysfunction was a strong, independent predictor of clinical deterioration and death at follow up (Cecchi et al., 2003). Indeed cardiac magnetic resonance (CMR) studies have revealed areas of myocardial fibrosis close to or in regions of reduced MBF (Petersen et al., 2007). Structural abnormalities of intramural coronary arterioles, characterized



by thickening of the intima and/or media layers of the vessel wall have been recognized in this patient population (Basso et al., 2000). These abnormalities may mediate microvascular dysfunction in this clinical context. Unfortunately, despite the modern recognition of cardiomyopathies, and in spite of considerable advances in its phenotyping and molecular genetics, current pharmacological treatment has remained largely empiric and based on heart failure studies. 5.4. Pharmacological approaches to coronary microvascular dysfunction in cardiomyopathies 5.4.1. Beta-blockers In patients with ICDM, carvedilol treatment was associated to an increase in left ventricle ejection fraction, in a proportion similar or even more consistent than in patients with heart failure due to ischemic heart disease; this effect seems to be independent of the level of cardiac sympathetic nervous system impairment (Gerson et al., 2002). Neglia et al. (2007) also demonstrated that coronary flow reserve significantly increased following carvedilol treatment, while stress-induced regional perfusion defects decreased after carvedilol treatment. 5.4.2. Angiotensin-converting enzyme inhibitors and angiotensin-converting enzyme receptor blockers 5.4.2.1. Mechanism of action. More recently, experimental models have investigated the beneficial effects of ACE receptor blockers on IDCM. Angiotensin converting enzyme-2 (ACE-2) is a monocarboxypeptidase that metabolizes angiotensin (ANG)-II into angiotensin 1-7 (ANG 1-7), thereby functioning as a negative regulator of the renin–angiotensin system. 5.4.2.2. Preclinical data. The cardioprotective effect of telmisartan was evaluated in rats with dilated cardiomyopathy induced by experimental autoimmune myocarditis. In this study, myocardial protein and mRNA expressions of inflammatory markers [CD68, iNOS, NF-kB, interleukin-1β, interferon-γ, monocyte chemotactic protein-1], expression of markers of myocardial fibrosis and hypertrophy [OPN, CTGF, TGF-β1 and collagens I and III and atrial natriuretic peptide and GATA-4, respectively], protein expressions of NADPH oxidase subunits [p47phox, p67phox], superoxide production, and level of mitogen-activated protein kinase (MAPK) signaling molecules were significantly decreased after telmisartant treatment. Telmisartan treatment also significantly improved LV systolic and diastolic function (Sukumaran et al., 2012). Similar results were obtained in another strain of rats with olmesartan medoxomil, an ANG-II type 1 receptor blocker. These effects were due in part to suppression of oxidative stress, endoplasmic reticulum stress and inflammatory cytokines (Sukumaran et al., 2011), and partially due to ACE-2/ANG 1-7 receptor modulation (Sukumaran et al., 2012).

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5.4.3.2. Clinical data. The effects of trimetazidine on cardiac performance and left ventricular function have been estimated in patients suffering from non-ischemic dilated cardiomyopathy. However, in idiopathic dilated cardiomyopathy, trimetazidine decreases cardiac free fatty acid oxidation modestly, by only 10%; therefore it raises the possibility of additional mechanisms, such as whole-body metabolic effects with increased insulin sensitivity, suggesting decreased whole body FFA oxidation, as found by Fragasso et al. in diabetic ischemic patients (Fragasso et al., 2013). Tuunanen et al. demonstrated that EF was increased by 15% during the treatment in the trimetazidine population study, and this effect is enhanced in patients under beta-blocking agents, probably due to an additive metabolic impact. In fact both drugs inhibit different enzymes in the FFA path and improve insulin resistance, taking synergic positive effects on systolic left ventricle function. The last effect could be due to antioxidative and protective effect, as increased HDL cholesterol levels were reported in the study (Tuunanen et al., 2008). 5.4.4. Perhexiline 5.4.4.1. Mechanism of action. Perhexiline is an antianginal agent thought to act by shifting myocardial substrate use from fatty acids to carbohydrates, through inhibition of Carnitine Palmitoyl Transferase (CPT-1) and, to a lesser extent, CPT-2, resulting in increased glucose and lactate utilization (Kennedy et al., 2000). 5.4.4.2. Clinical data. Abozguia et al. showed that perhexiline significantly improved myocardial ratios of phosphocreatine to adenosine triphosphate by 31P CMR spectroscopy, and normalized the abnormal prolongation of heart rate normalized time to peak filling between rest and exercise in non-obstructive HCM. These changes were accompanied by an improvement in primary end point (peak VO2, p = 0.003) and New York Heart Association functional class (NYHA) (p b 0.001) (Abozguia et al., 2010). Unfortunately, its beneficial anti-ischemic effects are dramatically resized during chronic intake because of the induced hepatotoxicity (Roberts et al., 1981) and peripheral neuropathy (Sack et al., 2000). 5.5. Summary Recent studies have clearly demonstrated that the presence and the extent of coronary microcirculatory dysfunction are independent and relevant predictors of worse prognosis in patients with hypertrophic and dilated cardiomyopathy. Actually no specific drugs are used with the aim to improve microvascular function in these conditions; however it is anticipated to be a relevant target in the near future. Agents with metabolic activity (such as trimetazidine, ranolazine and perhexiline) may play a pivotal role in this sense, by improving endothelial, myocytes and smooth muscle cell function. Conflict of Interest

5.4.3. Trimetazidine “The Authors declare that there are no conflicts of interest.” 5.4.3.1. Mechanism of action. Trimetazidine has been reported to have a potential benefit in patients with HF (Lionetti et al., 2011; Horowitz et al., 2010). Trimetazidine may affect myocardial substrate use by inhibiting oxidative phosphorylation and shifting energy production from free fatty acids (FFAs) to glucose oxidation. It may also contribute to the preservation of intracellular levels of phosphocreatine and ATP, reduce calcium overload and free radical-induced injury, inhibit cell apoptosis and improve endothelial function (Lionetti et al., 2011). More recently, trimetazidine has been suggested to inhibit cardiac fibrosis through an NADPH oxidase-reactive oxygen species connective tissue growth factor pathway (Liu et al., 2010). Furthermore in experimental models trimetazidine inhibits cardiomyocytes apoptosis in ischemic–reperfusion damage.

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Coronary Microvascular Dysfunction, Microvascular Angina, and Management.

Coronary microvascular dysfunction: an update.

Coronary microvascular dysfunction: mechanisms and functional assessment.

Obesity Related Coronary Microvascular Dysfunction: From Basic to Clinical Practice.

Myocardial perfusion echocardiography and coronary microvascular dysfunction.

Stable microvascular angina: instrumental evaluation of coronary microvascular dysfunction with coronary angiography and myocardial scintigraphy.

Acceleration time of systolic coronary flow velocity to diagnose coronary stenosis in patients with microvascular dysfunction.

Coronary microvascular dysfunction: sex-specific risk, diagnosis, and therapy.

Seeing is believing: new updates on coronary microvascular dysfunction.

[Coronary microvascular dysfunction and aortic stenosis: an update].

Coronary microvascular dysfunction in chronic inflammatory rheumatoid diseases.

Impact of left ventricular hypertrophy on impaired coronary microvascular dysfunction.

Pharmacological approaches to cocaine dependence.

Pharmacological approaches to appetite suppression.

Role of mitochondrial dysfunction in hyperglycaemia-induced coronary microvascular dysfunction: Protective role of resveratrol.

Mineralocorticoid receptors: an appealing target to treat coronary microvascular dysfunction in diabetes.

Migraine and coronary microvascular dysfunction: what about the insulting factor for both cerebral and coronary endothelia?

Thermodilutional Confirmation of Coronary Microvascular Dysfunction in Patients With Recurrent Angina After Successful Percutaneous Coronary Intervention.

Novel non-pharmacological approaches to heart failure.

New pharmacological approaches to therapy of NIDDM.

Non-pharmacological Approaches to Cognitive Enhancement.

Headache management: pharmacological approaches.

Takotsubo cardiomyopathy and microvascular dysfunction.

Using Modified Sample Entropy to Characterize Aging-Associated Microvascular Dysfunction.