Blood Oxidant Ties: The Evolving Concepts in Myocardial Injury and Cardiovascular Disease

By Bashir Matata, Maqsood Elahi and Priscilla Day-Walsh

Blood Oxidant Ties: The Evolving Concepts in Myocardial Injury and Cardiovascular Disease

is an update on the recent advances in the development of antioxidant-based therapies. It starts with an overview of the mechanisms underlying the genesis of oxidative stress, summarizing the link between oxidative stress and a number of cardiovascular conditions. This is followed by an explanation of how oxidative stress interacts with lipid metabolism and the placental environment. Three chapters on the role of antioxidant-based therapy for cardiovascular diseases round up the book.

Key Features

- Outlines several cell-signaling pathways that are modulated by the interplay between reducing and oxidizing agents (redox status) and gene expression in the cardiovascular disease process

- Brings information about maternal programming environment in the placenta

- Covers development of novel nanotechnology-based antioxidant delivery systems for effective drug delivery

- Includes references for further reading

The book is aimed at a broad readership of scientific and medical professionals involved in research on cardiovascular diseases, pathophysiology, pharmacy, pharmaceutical science and life sciences. It also serves as a reference for scholars who want to understand the complex biochemical mechanisms of antioxidant agents.

• Language: English
• Publisher: Bentham Science Publishers
• Release date: Jul 18, 2023
• ISBN: 9789815165012

Book preview

Redox Signaling, Oxidative Stress in Cardiovascular Disease –basic Science and Clinical Aspects

Bashir Matata¹, *, Maqsood Elahi²

¹ Central Liverpool Primary Care Hub, 81 London Road, Liverpool, L3 8AJ, United Kingdom

² Heart & Lung Research Institute, Cardiac Eye International Foundation, Lahore, Pakistan

Abstract

The generation of certain species of biomolecules described as reactive oxidant species (ROS e.g., superoxide, O2-; hydrogen peroxide, H2O2; hydroxyl radicals (OH.)) and reactive nitrogen species (RNS e.g., peroxynitrite, OONO-; nitric oxide, •NO) is a critical step in health and disease . These species play critical roles in cell defences in both animals, and plants. They also perform an important function in the regulation of key cellular signalling pathways such as cell differentiation, proliferation, migration, and apoptosis (commonly described as redox signalling pathways). The imbalance between the levels of ROS and RNS generated to that of antioxidant species may lead to oxidative stress and biomolecular damage, especially in situations where the latter are depleted. Redox biology and oxidative stress are particularly important in ischaemia-reperfusion associated diseases in particular the pathogenesis of cardiovascular disease (CVD). CVD is a major cause of mortality on a global scale, although the exact mechanisms underlying the pathological process are not fully understood. It is believed that ROS play a pivotal role in the progression of CVD. In particular, recent evidence suggests that the development of atherosclerosis is modulated by ROS and influenced by other factors such as inflammatory responses, disturbed blood flow, and arterial wall remodelling. This chapter provides an overview of the pathways of oxidative stress and redox-regulated signalling underlying the genesis and progression of cardiovascular disease.

Keywords: Cardiovascular disease, Oxidative stress, Redox signalling pathways, Reactive nitrogen species, Reactive oxygen species.

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* Corresponding author Bashir Matata: Central Liverpool Primary Care Hub, 81 London Road, Liverpool, L3 8AJ, United Kingdom; E-mail: [e-mail]

INTRODUCTION

Oxidative stress is a biomolecular characteristic associated with the disruption of the balance between the process of production of reactive oxidant species (ROS)/reactive nitrogen species (RNS) and the effectiveness of the antioxidant

defence systems in favour of the former [1-3]. There is evidence to suggest that many drug-induced complications and diseases are associated with an adverse increase in the levels of ROS and RNS, at the same time, antioxidant defences are ineffective [4, 5].

Reactive Oxygen Species and Reactive Nitrogen Species

There are different types of ROS that at low-level production, maintain redox regulation of physiological signalling broadly divided into oxygen radicals (e.g., superoxide, O2-.), hydroxyl radical (.OH), and peroxynitrite (ONOO-) or non-radicals (e.g., hydrogen peroxide (H2O2)). Commonly, superoxide is formed by the one-electron donation to molecular oxygen (Equation 1) in a reaction catalysed by Nicotinamide Adenine Dinucleotide Phosphate Hydrogen (NADPH) oxidase (Equation 2), with electrons supplied by NADPH [6]:

Superoxide is a short-lived molecule that acts locally and at low pH, it spontaneously dismutases to hydrogen peroxide (equation 3):

A number of endogenous free radical scavengers keep levels of hydrogen peroxide in check. Hydrogen peroxide is more stable than superoxide and can diffuse widely and accounts for the majority of distinct effects on redox regulation and underlying established specificity of ROS signalling [6]. However, higher levels of hydrogen peroxide generate hydroxyl radicals in the presence of metal ions via the Fenton or Haber-Weiss reactions [6]. Hydroxyl radicals are extremely reactive [6] and would react with the first molecule they contact with and also have a very short half-life.

Nitric Oxide Synthases and the Generation of Reactive Nitrogen Species

Reactive nitrogen species (RNS) also play an important role in redox biology and pathophysiology of diseases with nitric oxide (NO), laying a central role [7, 8]. NO is a vasodilator and inhibitor of platelet aggregation, leukocyte adhesion, and smooth muscle cell proliferation [7, 8]. Endothelial NO modulates vascular tone and blood pressure by cyclic guanosine monophosphate (cGMP)–stimulated smooth muscle relaxation, inhibition of platelet aggregation and adhesion to the endothelium, and prevention of smooth muscle proliferation (prevents vascular wall thickening) [9].

Three distinct mammalian isoforms of NO synthase (NOS) enzymes responsible for NO synthesis have been identified i.e., neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) [10]. The isoforms are products of different genes and have different localization and regulation properties, with distinct differences in the rate of NO production by these enzymes and the inhibition of NO production by different inhibitors of these enzymes [10, 11].

All three NOS enzymes can catalyse the 5-electron oxidation of L-arginine to L-citrulline [11, 12] in a process that involves the oxidation of Nicotinamide adenine dinucleotide phosphate (NADPH) to the reduced form NADP+. Molecular oxygen acts as a co-substrate for the reaction and tetrahydrobiopterin (BH4), flavin adenine dinucleotide, Flavin mononucleotide and haem are the cofactors involved in the catalytic process [11, 12].

The production of NO occurs twice as fast for nNOS as it does through eNOS although the output by eNOS is significantly higher compared with nNOS [13, 14]. Both eNOS and nNOS are constitutive enzymes (cNOS) with NO production by eNOS and nNOS being calcium-dependent where a calcium/calmodulin complex is needed for NOS activation [13]. iNOS, which is calcium-independent, is a very high output but a slow rate of enzyme activity. NO is produced by cNOS in a pulsatile manner, whereas the production of NO by iNOS is continuous [15]. Unlike nNOS and iNOS, eNOS adjusts in response to a change in the environment or status and is often targeted to the plasmalemma terminal caveolae [15]. The interaction of eNOS with some domains of caveolin-I causes the eNOS to become inactive [13]. However, interaction with the calcium/calmodulin complex with eNOS permits electron transfer through the enzyme and the oxidation of L-arginine [13].

Under some pathological conditions, the vascular endothelium becomes dysfunctional and generates a much greater amount of O2- than the normal endothelium. These conditions favour the production of high concentrations of superoxide that reacts with NO to form peroxynitrite, which is directly cytotoxic and in turn reduces NO bioavailability [6]:

Peroxynitrite promotes oxidative modification of proteins through nitration of protein targets such as tyrosine residues, producing a distinct molecular signature for nitric oxide-derived oxidants called nitrotyrosine [12-14].

Nitrating oxidants promote oxidative damage, cell injury, and conversion of low-density lipoprotein (LDL) into an atherogenic form [7-11].

Potential Sources of Reactive Oxygen Species (ROS)

There is a multitude of potential sources of ROS [14, 15] ranging from (nicotinamide adenine dinucleotide phosphate hydrogen) NADPH oxidases (NOX1-5, DUOX1-2) and the mitochondrial electron chain, to xanthine oxidase, monoamino oxidase(s), cyclooxygenase(s), lipoxygenase(s), lysyl oxidase(s), cytochrome P450, or Molecules Interacting with CasL (MICAL) family members as prominent examples [15].

Enzymatic Generation of NADPH Oxidase as a Source of ROS

There are a number of known enzymes able to produce ROS [15], most notably the NADPH oxidase that spans the cell plasma membrane. NADPH oxidase is associated with the production of ROS, mostly through superoxide and hydroxide in addition to certain non-radicals, such as hydrochlorous acid and ozone [15].

The NADPH oxidase enzyme also known as NOX comprises six different subunits that interact to form an active enzyme complex capable of producing O2-. [16]. Other NOX subunits, such as gp91phox (also known as the β subunit) and p22phox (also known as α subunit), are integral membrane proteins that together form the large heterodimeric subunit called flavocytochrome b558 (cyt b558). Regulatory protein subunits called p40phox, p47phox and p67phox exist within the cytosol which exists as a complex in the unstimulated state [17]. Upon stimulation, the subunit p47phox undergoes phosphorylation, and the entire complex translocates to the membrane and where it binds to cyt b558 to form the active oxidase enzyme [17].

The activated complex promotes the transfer of electrons from the substrate to oxygen through a prosthetic group composed of a Flavin adenine dinucleotide (FAD) and a haem group(s), which act as electron carriers [18]. The activation of the complex is initiated by two low-molecular-weight guanine nucleotide-binding proteins, Rac2 and Rap1A [19]. The molecule Rac2 is present in the cytosol in the form of a dimeric complex together with Rho-GDI (guanine nucleotide dissociation inhibitor), while Rap1A is a membrane protein [20, 21]. Once activated, Rac2 binds guanosine triphosphate (GTP) and translocates to the membrane along with p40phox, p47phox, and p67phox [22-24].

The NOX family exists as a group of gp91phox, which shares a common origin (homologs) [24]. This includes NOX1, NOX2, NOX3, NOX4, NOX5, and Dual oxidase (Duox) proteins (Duox1 and Duox2) [24]. The magnitude of expression of NOX family proteins among various tissues varies, as illustrated by the review by Panday et al. [24]. For example, NOX1 is common in tissues such as the colon, vascular smooth muscles, prostate, uterus, and placenta, specifically in certain cell types including endothelial cells, osteoclasts, retinal pericytes, neurons, astrocytes, and microglia. The review by Panday et al. [24] also cite authors that demonstrated the expression of the NOX2 (gp91phox) in cell types including stimulated phagocytes or granulocytes, and human umbilical vein endothelial cells. Panday et al. [24] also cite the evidence of high NOX3 expression in fetal kidneys, liver, lung, and spleen with low levels also shown in the adult colon and kidney, and inner ear. The expression of NOX4 is present in the tissues such as the kidneys, the liver, ovary, and eyes [24], while the NOX5 is present in tissues such as the spleen, testis, mammary glands, cerebrum, fetal brain, heart, kidneys, liver, lungs, skeletal muscle, thymus, prostate, lymphatic tissue, and endothelial cells [24]. The review by Panday et al. [24], cite evidence that Duo1 and Duo2 show their expression in the thyroid, cerebellum, lungs, ileum, cecum, floating colon, respiratory tract epithelium, islets and prostrate, stomach, duodenum, jejunum, and the sigmoidal colon.

The Mechanisms of Assembly and Activation of NOX Enzymes

The activation of the NOX complex begins with the phosphorylation of NOX1 and NOX2 by cytosolic proteins NOXO1 and p47phox, respectively [25]. The activated NOX forms a complex with other cytosolic proteins p40phox, p67phox, and Rac. The activated complex subsequently translocates from the cytosol to the membrane [26-28] which in turn results in the transfer of electrons to oxygen molecules [25-28].

The mechanism of activation of NOX3 is similar to that of NOX1 and NOX2 [25-29]. The main difference is that the activation of NOX3 is catalysed through a p22phox-dependent pathway, which is not dependent on binding to Rac.

NOX4 activation, on the other hand, involves binding to p22phox in a process catalysed by a protein known as polymerase delta interacting protein-2 (Poldip2) [25-29]. Poldip-2 acts as a positive regulator of the NOX4 activity [27-29]. Recent in vitro studies have shown that Poldip2 first associates with p22phox, which then binds and activates NOX4 [30].

The Biological Effect of NOX-activation

Evidence suggests that the activation of NADPH oxidase leads to the release of reactive oxidant species (ROS), that play key roles in physiological and human pathological changes [31]. Indeed, ROS is known to have a significant impact on conditions such as cardiovascular disease, immunodeficiency, and pulmonary diseases [31-35]. The release of ROS also is integral to the generation of oxidative burst, an important inflammatory mechanism for killing invading microorganisms by macrophages and neutrophils [36].

In cardiovascular disease, there is evidence of an upregulation of NOX2 and NOX4, a process linked with worsening hypertrophy and elevated oxidative stress [37]. In contrast, the calcium-dependent activation of NOX5 and Duox has been associated with antioxidant properties [38]. For example, Duox is known to have a peroxidase-like domain which in a mature form that modulates the rapid transition of superoxide into molecular oxygen and hydrogen peroxide [36-38].

Defects in the NOX System and its Consequences

As summarised in Fig. (1.1), the NOX system is central to the biological functions of most organisms as part of their redox signalling pathway [38, 39]. Indeed, NOX2 performs a key function in modulating the host immune system affecting genetic disorders such as chronic granulomatous disease (CGD) [40-42]. CGD is a condition characterised by altered NOX2 production and resultant defective neutrophil killing capacity due to low phagocytic respiratory bursts [24, 40-42].

Intracellular Reactive Oxidant Species as By-products of Mitochondrial Respiration

During mitochondrial respiration, NADH or flavoprotein-linked dehydrogenases release electrons in a process termed electron transport chain [43]. The electron transport chain is responsible for the chemical transformation of molecular oxygen into water and ATP through a process called oxidative phosphorylation by mitochondrial complexes coded by both nuclear and mitochondrial DNA [43]. In certain pathological conditions, 2-5% electrons may escape the electron transport system and react with O2 leading to the production of copious amounts of intracellular reactive oxidant species (ROS) within mitochondrial complexes I and III [43-45].

Under physiological conditions, the electron transport system is highly regulated and most of the ROS produced remain inside the intact mitochondria [46]. In contrast, under pathological conditions, there are elements of the mitochondrial outer membrane such as monoamine oxidases that produce NO or H2O2 which in turn leads to the accumulation of free radicals within the mitochondria [47]. The coupling state of the mitochondria is the key process linked to the synthesis of ATP that determines the rate of mitochondrial respiration and ROS formation [47]. This process is controlled by internal and external Ca²+ levels, and the activities of some antioxidants such as that of manganese superoxide (Mn-SOD) located in the mitochondrial matrix [47].

In many physiological processes, molecules may undergo chemical modifications through a reducing and oxidizing reaction with other reactive molecules. This may happen if a molecule with an unpaired electron combines with another molecule capable of donating an electron. The processes of donation and gaining of an electron are termed as oxidation and reduction respectively commonly termed as a redox state. During reduction or oxidation, the reduced molecule may become unstable, and therefore, free to react with other molecules. Depending on the target molecule, this may cause damage to cellular and sub-cellular components such as membranes, proteins, and DNA. The redox state is a term that describes the balance between oxidants and antioxidants within the mitochondria. A redox state describes an essential component of physiological functions [24].

In pathological conditions, an initial small burst of reactive oxidants triggers the opening of the mitochondrial permeability transition pore (MPTP) [48, 49]. The opening of the MPTP leads to the depolarisation of mitochondrial membrane potential and an increased electron flux [48, 49]. The increased electron flux through the MPTP is associated with further ROS production, energy uncoupling (No associated ATP production) and oxidative stress-mediated damage to the tissue [50].

The overproduction of mitochondrial ROS/NO has also been shown to result in early atherosclerosis, and is strongly associated with patients with hypercholesterolemia [51]. In addition, the perturbation of the mitochondrial ROS is also strongly associated with hyperglycaemia-induced glycation and protein kinase C activation [52]. The mitochondria also act as a source of H2O2 which in turn regulates flow-mediated arterial dilatation and resistance, in human coronary arteries [53].

In endothelial cells, the onset of hypoxia triggers ROS generation which decreases activator protein activator protein-1 (AP-1) transcriptional activity [54]. Mitochondrial ROS modulates hypoxia-induced signalling as demonstrated by myothioxol inhibition of mitochondrial cytochrome bc1 complex (coenzyme Q - cytochrome c reductase) by blocking the electron transport chain [54].

Generation of Reactive Oxidant Species by the Xanthine Oxidoreductase System

As illustrated in Fig. (1.1), there are two forms of the interchangeable xanthine oxidoreductase system, which are xanthine dehydrogenase and xanthine oxidase [55, 56]. The xanthine dehydrogenase reduces NAD+ whereas xanthine oxidase reacts with molecular oxygen, to form a superoxide anion and hydrogen peroxide [57]. On the other hand, xanthine oxido-reductase catalyses oxidative hydroxylation of hypoxanthine to xanthine, and from xanthine to uric acid [57], a strong antioxidant and a free radical scavenger (Fig. 1.1). The double role played by xanthine oxidase suggests that it has an important function as a regulator of the redox state within the cell [57].

Under pathophysiological stress conditions, xanthine oxidoreductase is an important source of reactive oxidant species [58]. Indeed, in experimental atherosclerosis models [59], it was demonstrated that oxypurinol, a xanthine oxidase inhibitor abrogated excess superoxide production. In addition, for patients with coronary heart disease or heart failure-associated contractile dysfunction is strongly related to elevated xanthine oxidase-generated ROS [60].

Fig. (1.1))

The proposed mechanism of xanthine oxido-reductase pathways (Scheme modified from Puig et al 1989) [53].

NOS Uncoupling as a Source of Reactive Oxidant Species in the Cell

Commonly, NO Synthases (NOS) are major sources of endogenous NO. In certain conditions, endothelial NOS (eNOS) may become uncoupled resulting in the generation of ROS instead of NO that cause vascular endothelial dysfunction [61-64]. The nitric oxide synthase, eNOS, is a cytochrome P450 reductase-like enzyme [65] that plays an important role in the catalysis of the flavin-mediated electron transport from the electron donor NADPH to a prosthetic haem group [63]. eNOS can produce both nitric oxide (NO) via its oxygenase function and through its reductase function, the latter being dependent on the availability of NADPH [65]. eNOS enzyme catalytic function requires tetrahydrobiopterin (BH-4) co-factor bound near the haem group to transfer electrons to guanidino nitrogen of L-arginine to form NO [63]. Uncoupling of eNOS contributes to ROS when there is a deficiency of L-arginine or BH-4 [64]. In the absence of L-arginine or BH-4, eNOS instead can produce O2·− and H2O2 [60]. The product of the reaction between NO, and O2·−can oxidize BH4 and this may lead to further eNOS uncoupling [65].

Physiological processes depend on NO as a major cell signalling molecule, mediating functions such as neurotransmission, regulation of vascular dynamics and immune system regulation [66]. The release of NO release may be the result of either vascular shear stress or by eNOS activation in response to cytokine activation [67] and plays a protective role in suppressing abnormal proliferation of vascular smooth muscle cells (VSMCs) following various pathological situations [68]. NO reacts with ROS resulting in direct inactivation [69] whereas, O2·− reacts with NO to produce peroxynitrite, which reduces the bioavailability of NO and produces more damaging secondary species. The presence of ROS also impacts NO responses by oxidizing sites on the protein that reacts with NO or modulation of allosteric NO binding. The concentration of NOS in blood vessels is therefore dependent on the balance between the production of NO on one hand and the destruction by ROS on the other [69]. NO may inhibit xanthine oxidase and NADPH oxidases, suggesting that NOS activity also regulates free radical production to maintain ROS/NO homeostasis [70]. Evidence of eNOS contribution to cellular ROS is present in the context of hypercholesterolemia [71], atherosclerosis [72], coronary artery disease [73], aging and diabetes [74]. An imbalance between the endothelial NO and ROS production is one of the major contributors to endothelial dysfunction which plays an important part in atherosclerosis and cardiac disease [75].

Redox Signalling Pathways

ROS can act as biochemical messengers that regulate various intracellular components of biological functions (illustrated in Fig. (1.2) below) including development, cellular processes, pathophysiological changes, and disease [38]. For example, ROS have been implicated in the regulation of calcium (Ca²+) induced signalling in the vasculature. This, in turn, activates calcium-dependent protein kinases activity such as PKC and calcineurin [76]. It is also known that intracellular ROS affect the activity of protein kinase pathways by influencing the redox state of the cell [76]. The presence of certain types of ROS leads to alterations in the redox state of protein