Date Published: September 28, 2016
Publisher: Hindawi Publishing Corporation
Author(s): Nisha Panth, Keshav Raj Paudel, Kalpana Parajuli.
Cardiovascular diseases (CVDs) have been the prime cause of mortality worldwide for decades. However, the underlying mechanism of their pathogenesis is not fully clear yet. It has been already established that reactive oxygen species (ROS) play a vital role in the progression of CVDs. ROS are chemically unstable reactive free radicals containing oxygen, normally produced by xanthine oxidase, nicotinamide adenine dinucleotide phosphate oxidase, lipoxygenases, or mitochondria or due to the uncoupling of nitric oxide synthase in vascular cells. When the equilibrium between production of free radicals and antioxidant capacity of human physiology gets altered due to several pathophysiological conditions, oxidative stress is induced, which in turn leads to tissue injury. This review focuses on pathways behind the production of ROS, its involvement in various intracellular signaling cascades leading to several cardiovascular disorders (endothelial dysfunction, ischemia-reperfusion, and atherosclerosis), methods for its detection, and therapeutic strategies for treatment of CVDs targeting the sources of ROS. The information generated by this review aims to provide updated insights into the understanding of the mechanisms behind cardiovascular complications mediated by ROS.
Researchers have been continuously studying the potential role of oxidative damage in cardiovascular diseases (CVDs) for a few decades. In a simple term, the common risk factors for CVDs like diabetes mellitus, smoking, aging, hypercholesterolemia, and nitrate intolerance can further increase the possibility of the generation of ROS. Furthermore, these risk factors can trigger several pathways such as apoptosis of endothelial cells (EC), expression of adhesion molecules, activation of metalloproteinases, induction of proliferation and migration of smooth muscle cells, lipid peroxidation, and change in vasomotor functions, collectively leading to CVDs [1, 2]. ROS are chemically reactive molecules containing oxygen. Several ROS with unpaired electrons, for instance, superoxide anion (O2∙−), hydroxyl radical (OH∙−), and lipid radicals, are considered as free radicals. ROS, such as hydrogen peroxide (H2O2), peroxynitrite (ONOO−), and hypochlorous acid (HOCl), are not free radicals but possess an oxidizing effect resulting in oxidant stress. A chain reaction leads to the production of many reactive oxygen species from one ROS (Figure 1). For example, the reactions of radicals and fatty acids (polyunsaturated fatty acids, PUFAs) within the cytoplasmic membrane result in a fatty acid peroxyl radical which can attack the adjacent side chain of the fatty acid and commence production of other lipid radicals. Lipid radicals generated in this chain reaction get collected in the plasma membrane and may have an innumerable effect on cell function, including alteration in cell membrane permeability and dysfunction of membrane-bound receptors [1, 3].
In a physiological system, the imbalance between antioxidant defense mechanism and ROS production leads to oxidative stress and subsequent pathological conditions . Most prominent ROS causing toxic insult to the human body are H2O2, O2∙−, ∙OH, and ONOO− . In the blood vessel wall, each layer can produce ROS in pathological conditions . Wattanapitayakul and Bauer reported that, within mitochondria, oxygen is usually utilized for energy production (in the form of ATP) and oxidative phosphorylation. During the mitochondrial electron transport (MET), harmful ROS are formed but they are balanced by antioxidant defense. However, in case of ischemia or hypoxia, MET is imbalanced, leading to ATP depletion, acidosis, mitochondrial depolarization, collection of noxious metabolites, intracellular Ca2+ overload, and cell death . For example, approximately 1–3% of molecular oxygen is converted to unstable/reactive O2∙− in mitochondrial complexes I and III through a pathway involving oxidative phosphorylation . In general, cardiac myocytes consume a high level of oxygen due to considerable higher number of mitochondria than other cells . For this reason, cardiac myocytes also release ROS and cause oxidative stress to other cells . But ROS do not have only a negative side, since production of ROS at physiological levels promotes cellular activities, controls the hormone level, maintains chemical balance, strengthens synaptic plasticity, and induces enzymes. Moreover, ROS also helps to fight against invading pathogens and induce an immune response against the pathogenic influence . To a certain extent, ROS are neutralized by intracellular antioxidant enzymes such as glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase and consumption of other nonenzyme antioxidants like β-carotene, ascorbic acid, and tocopherols as a supplement . In spite of being necessary to carry out cell signaling pathways, overproduction of ROS leads to injury of the cell membrane integrity causing altered permeability, change in proteins expression, and DNA damage . For the majority of CVDs, the enzymatic sources of ROS include NAD(P)H oxidase, lipooxygenase, cyclooxygenase (COX), xanthine oxidase (XO), uncoupled nitric oxide synthases (NOS), cytochrome P450, and mitochondrial respiration [12–14] (Figure 2). The process of increased O2∙− generation, facilitated by XO enzyme, can be antagonized by a therapeutic approach with XO inhibitor, like allopurinol, to ameliorate cardiac conditions . NADPH oxidase (Nox), commonly found on the cellular membrane, is stimulated during phagocytosis leading to increased ROS release . In particular, the overexpression of Nox2 and Nox4 is linked to the remarkable oxidative stress observed during CVDs. A study done by Kuroda et al. showed that Nox4 knockout mice showed a low level of cardiac O2− revealing that Nox4 is a potential source of superoxide in cardiac myocytes. Nox4 overexpression worsened the cardiac function and induced apoptosis and fibrosis in a mouse with response to pressure overload. Thus, Nox4 is a key contributor of oxidative stress in the mitochondrial redox systems leading to cardiac impairment during pressure overload. Therefore, the physiological role of Nox, translocating electrons throughout the membrane, can be deregulated in CVDs leading to cardiac dysfunction . However, some pathways associated with ROS mediated CVDs are yet to be clarified. However, researchers are trying to reveal good, bad, and ugly roles of ROS in the physiological system. In contrast to the good face of ROS on signaling and immune response, at high concentrations, ROS can exhibit the deleterious effect on redox homeostasis leading to intracellular components damage as seen in neurodegenerative diseases, CVDs, and pulmonary disorders .
Endothelial cells are lining the interior surface of blood and lymphatic vessels cells. Endothelial cells play an important role in homeostasis and immune and inflammatory reactions. EC regulates vascular tone by releasing various vasodilator factors such as nitric oxide (NO) endothelium derived hyperpolarizing factor, prostacyclin or vasoconstrictive factors such as thromboxane (TXA2), and endothelin-1 (ET-1). Endothelial dysfunction (ED) is a pathological state of the endothelium, which is a predictor of various CVDs, and is caused by imbalance between vasodilating and vasoconstricting substances . ROS are considered as signaling molecules that contribute to ED in experimental and clinical atherosclerosis [3, 18]. NO is a potent vasodilator produced by the endothelium. Besides vasorelaxation, nitric oxide exerts various functions like antiplatelet, antithrombotic, and anti-inflammatory properties and permeability decreasing properties . It is reported that ONOO− which is formed by the reaction of superoxide and free radical NO can oxidize tetrahydrobiopterin. If formed in a small amount, ONOO− exerts a similar physiological activity like NO. However, at a high concentration, it shows injurious activity by converting to harmful peroxynitrous acid and causing alteration of protein structure . ED is associated with polymorphisms of various genes which include cytochrome P450, methylene tetrahydrofolate reductase, p22phox, angiotensin convertase enzyme, and glutathione-S-transferase . Excess amount of ROS damages the endothelium, especially the terminal arteries leading to alteration of the intracellular reduction-oxidation homeostasis . In a patient with diabetes mellitus, small vessel disease linked with mitochondrial disorders might also be due to oxidative stress. The result of diabetes mellitus in atherosclerosis is stimulated mainly by oxidative stress . ROS may also activate mitogen activated protein kinase, which regulates the expression of monocyte chemoattractant protein 1 (MCP-1) and favors the chemotaxis of circulating monocyte to the site of atherosclerotic lesion. This demonstrates a potential link between arterial wall strain and atherosclerosis .
Several researches support the fact that ROS are involved in ischemic occlusion leading to cardiac damage [39, 40]. Apart from carrying out the function of cellular O2 storage and supply by oxymyoglobin (Mb), it also acts as a potent preventive source against I/R injury . During the outset of I/R injury, O2∙− release has been observed in an isolated rat heart. Zhu and Zuo speculate that generation of O2∙− is linked with Mb because of the lower myocardium oxygen tension. Results revealed that the rise of fluorescence in the ischemic heart was terminated by a SOD mimic, carbon monoxide (CO), or by Mb gene knockout. Likewise, O2∙− was not formed in intracellular EC but rather from the myocytes, which are considered a potential source of Mb. This suggests that Mb is an important factor responsible for production of O2∙− during ischemia . An enzyme responsive to stress, named sirtuin-6 (SIRT6), displays cardiac protection from I/R injury as revealed in partial SIRT6 knockout mice as well as in vitro cultured cardiomyocyte. SIRT6 is a deacetylase and mono-ADP ribosyltransferase enzyme responsive to oxidative stress and can protect the cell against oxidative stress. This protective activity was achieved by initiating the expression of catalase and manganese SOD antioxidant-encoding gene resulting in reduced cellular oxidative stress . Restoration of the coronary artery blood flow reverse to the ischemic myocardium can have a detrimental effect on the microvascular function, causing arrhythmias . In the endothelium, the rise of ROS release and the opening of mitochondrial permeability transition (MPT) pore play an important role in the protection from I/R damage . However, Kim and Lemasters showed that mitochondrial ROS, accompanied by normalization of pH, stimulate initiation of MPT pore followed by death of myocytes after reperfusion. However, Ca2+ overloading does not promote onset of MPT pore . Research demonstrated that the reason behind the protective effect was the involvement of ROS and potent vasodilator NO in regulating downstream pathways by stimulating adenosine triphosphate sensitive potassium channel in mitochondria . After open cardiac surgery, the I/R injury can influence postsurgical consequences because of the lipid peroxidation mediated by ROS . In comparison to the heart obtained from juvenile rat, the cardiac dysfunction due to I/R was dramatically concealed in the heart obtained from congenital heart disease (CHD) model rats. Moreover, the ratio of n-3/n-6 PUFA was remarkably raised in I/R phase in CHD rats, whereas it was not observed in juvenile rats suggesting that the rise in n-3/n-6 ratio could result in the upregulation of cell defense system against oxidation via n-3 PUFA oxidation product 4-hydroxy-2-hexenal causing higher tolerance to I/R damage .
Excess production of ROS plays an important role in inflammation, disturbed blood flow/abnormal shear stress, and arterial wall remodeling. ROS causes remodeling through proliferation of smooth muscle cell and increased inflammation . Repeated continuous exposure to nonstreamline shear stress of arterial regions generates O2 induced by endothelial Nox resulting in adhesion of monocytes . The upregulation of adhesion molecules including P-selectin, VCAM-1, and E-selectin causes further inflammation by adhesion of white blood cells. Development of inflammatory response increases ROS production by phagocytosis, which is important in the early stage of atherosclerosis [48, 49]. The Nox family of superoxide producing proteins is an important source of ROS in signal transduction. Nox are found to be expressed in phagocytic cells, EC, smooth muscle cells, and fibroblasts. Experiments conducted on arteries from human volunteers with coronary artery disease and animal experimental model with hypertension, diabetes, or atherosclerosis demonstrated that Nox1, Nox2, and Nox5 stimulate endothelial dysfunction, inflammation, and programmed cell death; however, isoform Nox4 protects the vascular system by increasing bioavailability of nitric oxide and stoppage of cell death pathways . Some research presents the controversial role of Nox4 displaying either protective or a deleterious role of Nox4.
Mitochondria play an important role in cellular signaling pathways, particularly in the modulation of calcium stores within the cell, generation of ROS, respiration, and biogenesis. So, changes in mitochondrial function lead to development of human diseases [55, 56]. Mitochondrial DNA (mtDNA) damage is linked to the atherosclerotic lesions in apolipoprotein E (apoE) knockout mice and also introduces atherogenesis in young apoE knockout mice . Raised levels of mtDNA damage have been seen in the vascular tissue of CVD patients . Mitochondrial dysfunction is due to decreased manganese SOD, increased damage of mtDNA, and increased atherosclerosis in apoE knockout mice . There is excessive mitochondrial damage in atherosclerosis model. Oxidized-LDL stimulates mitochondrial complex I activity which depends on the induction of oxidative stress [60, 61]. Composed of 46 subunits, human mitochondrial complex I is the key enzyme responsible for oxidative phosphorylation. Dysfunction of the mitochondrial oxidative phosphorylation in a physiological system is responsible for occurrence of CVDs in humans, and mitochondrial diseases are linked to mitochondrial respiratory-chain pathologies and mutations of mitochondrial DNA. Studies reported that various stress induced in the cells causes structural and functional disturbance of mitochondria [62, 63]. Dysfunction of mitochondria provokes a signaling pathway for cell death resulting in organ failure and diseases. Mitochondria based pathological conditions including obesity, cancer, stroke, diabetes, neurodegenerative diseases, heart failure, and aging, however, are caused by intrusion of mitochondrial Ca2+, ATP, or ROS metabolism [61, 63]. Myocardial ischemia-reperfusion injury leads to mitochondrial Ca2+ overload and consequent generation of ROS and opening of the mitochondrial permeability transition pore [60–62], resulting in apoptosis. Compounds which can reduce mitochondrial Ca2+ overload, decrease mitochondrial ROS collection, and prevent mitochondrial energy generation are all potential sources of therapies for preventing disease. Mitochondria produce oxidative stress which plays an important role in mediating programmed cell death (apoptosis) and damage to mtDNA and leads to human aging, cancer, and CVDs. Oxidative damage of the mitochondrial membrane results in depolarization of membrane and uncoupled oxidative phosphorylation and altered cellular respiration. Altered mitochondrial respiratory chain can hinder the pivotal role of providing the energy to the cell as ATP, leading to various disease progression [60–64].
Since ROS are highly unstable and very reactive, researchers always face the problem of precisely monitoring them in biological systems. One way to find out the possibility of ROS in CVDs subjects involves exploring experimental proof of oxidative reactions. Fluorescent probes and electron spin resonance probes tools for detection of ROS are limited in animal and human experiment due to technical problems . The following list of direct methods can show at least in part indirect evidence of ROS effect in CVDs (Table 1).
During skeletal muscle contraction, ROS are generated which can affect muscle adaptation and function. Zuo et al. studied whether ROS are generated in the process of muscle contraction in isolated single skeletal muscle fibers (using Xenopus laevis muscle), as well as whether these ROS generated by contraction have an impact on fatigue development. To detect the ROS generation, myofibers were loaded with fluorescent probe (dihydrofluorescein-diacetate) which reacts with ROS to form fluorescein. Fluorescein signal was raised remarkably in both the first (42 ± 14%) and the third periods (39 ± 10%) of maximal tetanic contraction. However, with the treatment of reference antioxidant compound, ebselen, there was no rise of fluorescein during the second contractile period suggesting that ROS generation is high during contractile activity and antioxidant treatment can halt ROS production without any effect on myofiber contractility . In spite of the various pathways of ROS generation, the study of key pathways of their production is still undergoing. In particular, ROS generation in response to exercise, hypoxia, and heat in the diaphragmatic skeletal muscle (a key muscle during respiration) is a topic of interest [85, 86]. During the state of heat stress, O2∙− is generated by skeletal muscle which can be quantified by cytochrome c reduction as it is correlated with arachidonic acid metabolism. The blockage of enzyme phospholipase A2 using manoalide remarkably reduced O2∙− release. However, neither the blockage of COX with nonselective COX inhibitor indomethacin nor the blockage of CYP P-450 contingent monooxygenase with SKF-525A reduces O2∙− generation. In contrast, lipoxygenase blockage with common inhibitors cinnamyl-3,4-dihydroxy-α-cyanocinnamate and 5,8,11,14-eicosatetraynoic acid drastically halted the signal. Moreover, O2∙− generation was notably reduced by diethylcarbamazine (5-LOX inhibitor) suggesting that metabolism of arachidonic acid involving LOX is a key mediator of generation of extracellular O2∙− in skeletal muscle . The role of ROS in myocardial I/R injury has been widely studied . Vanden Hoek et al. proposed the generation of a high amount of ROS in case of ischemia before reperfusion by an in vitro experiment in isolated cardiomyocyte during simulated I/R. The fluorescent probes 2′,7′-dichlorofluorescein and dihydroethidium (DHE) were significantly oxidized during ischemia, revealing ROS production. After an hour of ischemia, reperfusion leads to further generation of OH− and H2O2. In contrast, treatment of antioxidant compounds (1,10-phenanthroline and 2-mercaptopropionyl glycine) during ischemia injury halted oxidant production, raised the viability of cardiomyocytes, and opposed contraction following ischemia. The ROS production in response to residual O2 as in case of ischemia causes cellular injury observed in the reperfusion stage . In a similar study on cardiomyocytes model of ischemia performed by Becker et al., an inhibitor of mitochondrial site III (myxothiazol) reduced oxidation. However, the inhibitor of mitochondrial site IV (cyanide) along with NOS inhibitor (nitro-L-arginine methyl ester), XO inhibitor (allopurinol), and Nox inhibitor (apocynin) showed no effect, suggesting that excessive O2∙− production is observed in ischemia prior to reperfusion through ubisemiquinone area of the MET chain . Another study suggests that sublethal H2O2 production in isolated cardiomyocytes during the period of simulated ischemia modulates cell death later in reperfusion step, mainly due to the burst of reperfusion oxidant .
The exact mechanism of CVD is complex and is not yet fully understood. ROS plays an important role in the progression and development of CVD. There is a link between ROS and the pathophysiology of CVD. We have developed a greater understanding of production of ROS, detection of ROS, and therapeutic strategy to prevent production of ROS and cardiovascular disease. However, more works to improve the detection and treatment of the ROS mediated dysfunction are necessary in the upcoming days.