Oxidative stress: Predictive marker for coronary artery disease

Abstract

The role of oxidative stress in cardiovascular disease processes, such as atherogenesis, ischemic-reperfusion injury and cardiac remodelling, has been increasingly recognized in the past few decades. Currently, an increasing number of studies suggest that levels of oxidative stress markers in body fluids correlate with atherosclerotic disease activity. This finding may lead to novel clinical approaches in patients with coronary artery disease. Assessment of oxidative stress markers could modify risk stratification and treatment of patients with suspected coronary artery disease or myocardial infarction.

Free radicals appear to be important modulators of atherovascular disease in all stages of development. They physiologically serve as signal transducers in cell communication and homeostasis including vascular gene expression and vascular cell interactions (1). When the finely regulated signalling pathways of these molecules become uninhibited, it may lead to the initiation and progression of atherosclerotic disease. New facts about the function of free radicals are emerging continuously.

OXYGEN FREE RADICALS

Reactive oxygen species

Some of the most important free radicals in biological systems are derivatives of oxygen (reactive oxygen species [ROS]) such as superoxide anion radical, hydroxyl radical, hydrogen peroxide, and triplet or singlet O₂(2). ROS molecules are characterized by one or more unpaired electrons, are unstable and highly reactive, and rapidly interact with surrounding molecules, altering their structure and functions.

ROS are produced in low amounts during cellular metabolism under normal conditions: by proton leakage during oxidative phosphorylation in mitochondria; by various enzymes such as NADH/NADPH oxidase in endothelial cells, vascular smooth muscle cells and neutrophils (3), or xanthine oxidase in endothelium; cytochrome P450; lipoxygenase/cyclooxygenase pathways; and the auto-oxidation of various substances, particularly catecholamines (4). ROS participate in various physiological functions. They regulate gene expression and post-translational modifications of proteins, serving as signalling molecules in homeostasis, mitosis, apoptosis and cell differentiation (5). Potentially toxic concentrations of ROS are enclosed in the phagosomes of phagocytes, where ROS are used as defense against exogenous microorganisms (6).

Normally, these processes are largely nonpathogenic to the host organism because low levels of ROS are maintained by enzymatic (glutathione peroxidase, catalase, superoxide dismutase) or nonenzymatic (glutathione, thioredoxin) functions of endogenous antioxidants. Some exogenous compounds (vitamins A, C and E) also act as antioxidants. Cellular structures damaged by ROS are being continuously repaired or replaced.

Oxidative stress

The fine balance between ROS and antioxidants is disturbed when excessive amounts of free radicals are produced or antioxidant capacity is decreased. This disturbance is known as oxidative stress and it plays an important role in cardiac pathophysiology (Figure 1).

Figure 1).

Scheme of free radicals effect on cells. LDL Low-density lipoprotein; MMPS Matrix metalloproteinases; mt Mitochondrial

Under conditions of oxidative stress, ROS attack biomolecules that are in close proximity. Mitochondrial and nuclear DNA damage, protein cross-linking and lipid peroxidation occurs, resulting in mutations, protein denaturation and loss of enzyme and membrane pump function. Damage to sarcolemmal and intracellular membranes impairs ATP-dependent Na⁺ and Ca²⁺ reuptake mechanisms. ROS decrease the activity of the sarcoplasmic reticulum membrane Ca²⁺ pump, which plays a crucial role in cardiac Ca²⁺ handling. Tumour necrosis factor-alpha (TNF-α) and interleukin (IL)-6 production triggered by ROS contribute to intracellular Ca²⁺ dysregulation and increase in its concentration. Cytosolic Ca²⁺ overload leads to myofibrillar hypercontracture, cytoskeletal damage and cell disruption via activation of Ca²⁺-dependent proteases and phospholipases. Mitochondrial Ca²⁺ overload causes inefficient ATP synthesis and utilization, leading to necrosis (7).

Apart from the direct toxic effects on surrounding molecules, excessive levels of ROS trigger a number of regulatory chain reactions. ROS are involved in apoptotic pathway control (eg, TNF-α receptor/caspase pathway) and activation of the cascade of transcriptional factors, proinflammatory cytokines (TNF-α, IL-1β, IL-6) and adhesion molecule expression (ie, intercellular adhesion molecules, vascular cell adhesion molecules, macrophage colony stimulating factor, monocyte chemoattractant protein, etc). As a result, massive recruitment of inflammatory cells, especially neutrophils, occurs (8). They penetrate the endothelium of the vessels in the affected area and highly express NADPH-oxidase, an additional important source of free radicals, which gives rise to a vicious cycle (9–11). In addition to neutrophils, smooth muscle cells, fibroblasts and T lymphocytes generate ROS. Leukocyte attraction and activation can also cause white thrombi formation in microvessels, increased platelet aggregability, microvascular cell edema and dysfunction, resulting in aggravation of ischemia and tissue stunning (12).

Laboratory assessment

To measure ROS levels in tissue or blood, various techniques can be applied (Table 1). Because of the high reactivity and very short half-life of ROS, the possibilities are limited, especially in clinical conditions. Several methods for the detection of ROS (eg, lucigenin enhanced chemiluminescence [13,14], fluorescence microtopography [15,16] or electron paramagnetic resonance spectroscopy [17]) have been described. Regarding the instability of ROS, detection using these techniques is currently only applicable under experimental conditions rather than clinical situations. The most frequently used methods detect more stable downstream products of free radicals in body fluids, such as oxidized DNA, advanced oxidation protein products (AOPPs), lipids, or assess altered defense mechanisms (superoxide dismutase levels, oxidized/reduced glutathione ratio) (18–22).

TABLE 1.

Examples of oxidative stress assessment

	Target molecule(s)	Measured oxidation product	Method
Direct measurement of free radicals			Lucigenin enhanced chemiluminescence, fluorescence microtopography, electron paramagnetic resonance spectroscopy
Indirect measurement
Downstream oxidation products	Lipids	Malonyldialdehyde, isoprostanes	ELISA, high-performance liquid chromatography
	Proteins, lipoproteins	AOPP, PB-DOPA, ox-LDL	ELISA
	DNA products	5-OHdC, 8-OHdG, DNA strand-break frequency	ELISA, high-performance liquid chromatography variations, comet assay
Decreased antioxidant capacity	Nonenzymatic	Cysteine/cystine ratio	RPLC, RPLC-MS
		GSH/GSSG ratio, thioredoxin	HPLC, ELISA
	Enzymatic	Superoxide dismutase, catalase	ELISA, RIA, spectrophotometry

5-OHdC 5-hydroxy-2’-deoxycytidine; 8-OHdG 8-hydroxy-2′-deoxyguanosine; AOPP Advanced oxidative protein products; GSH Glutathione; GSSG Oxidized GSH; MS Mass spectrometry; ox-LDL Oxidized low-density lipoprotein; PB-DOPA Protein-bound 3,4-dihydroxyphenylalanine; RIA Radioimmunoassay; RPLC Reversed-phase liquid chromatography;

OXIDATIVE STRESS AND CORONARY ARTERY DISEASE

Oxidative stress as a marker of coronary artery disease activity

The pathological processes underlying atherovascular disease remain incompletely understood; however, there is growing evidence that oxidative stress and inflammation are positively associated with the instability of atherosclerotic plaque and the incidence of acute coronary syndrome (ACS). ROS-induced initiation of inflammatory cascades and low-density lipoprotein (LDL) oxidation leads to the formation of macrophage-derived foam cells, differentiation and proliferation of vascular smooth muscle cells, activation of vascular matrix metalloprotein-ases and impairment of the extracellular matrix (ECM) of the affected site. This may culminate in atherosclerotic plaque rupture (23,24). Ehara et al (25) demonstrated increased plasma levels of oxidized LDL in cases of ACS.

Kaneda et al (26) showed that plasma levels of AOPP were significantly higher in patients with coronary artery disease than in those without. AOPP levels correlated with severity score of CAD according to the the Gensini scoring system. In contrast, Azumi et al (9) observed that even when there was no significant difference in angiographic stenosis, the generation of ROS was significantly higher in unstable angina pectoris patients compared with stable angina patients.

Another marker of oxidative stress, thioredoxin, correlates with atherosclerotic activity. Thioredoxin is a stress-inducible protein that contains a redox-active dithiol/disulfide in the active site and provides cytoprotection against oxidative stress. Plasma thioredoxin levels are significantly increased in patients with unstable angina compared with those with stable angina (18). Miwa et al (27) showed that patients with coronary spastic angina had a higher serum thioredoxin level associated with a lower serum level of antioxidant vitamin E.

These findings suggest that coronary artery disease status may be determined by oxidative stress activity rather than the degree of coronary stenosis.

Ischemia-reperfusion injury of myocardium and predictive value of oxidative stress in recurrent cardiac events

Oxidative stress plays a key role in ischemia-reperfusion injury (2,5,28,29) and subsequent cardiac repair. Interruption of blood flow in the coronary arteries causes ischemia of adjacent tissues, leading to cell injury, which may result in cell necrosis and apoptosis. The duration of ischemia determines the extent of damage to the myocardium and related tissues (30). During ischemia, cellular defenses against oxidative injury are impaired, with lower activities of antioxidants such as superoxide dismutase and glutathione peroxidase. Furthermore, greater amounts of ROS are produced, for example, by xanthine dehydrogenase, which is converted to xanthine oxidase, a potent generator of O₂⁻ and hydrogen peroxide (31).

Timely blood supply restoration, especially in acute coronary artery occlusion, is essential for myocardial salvage. Nevertheless, abrupt blood flow restoration (thrombolysis, percutaneous coronary intervention or spontaneous) in ischemic tissue causes a precipitous increase in ROS levels, which may result in paradoxical tissue damage rather than improvement. ROS- and cytokine-initiated apoptosis after reperfusion participate in tissue damage and heart remodelling in the ensuing postinfarction period (32).

Oxidative stress contributes to the occurrence of cardiac events after reperfusion therapy in ACS. In a study by Feng et al (22), oxidative stress marker-plasma AOPP concentration correlated positively with an increased incidence of major cardiac events in patients treated with percutaneous coronary intervention for ST-segment elevation myocardial infarction during a six-month follow-up. Naruko et al (33) showed a correlation between persistently high levels of oxidized LDL in patients after myocardial infarction who underwent primary coronary stenting and stent restenosis (>50% diameter stenosis) after a six-month follow-up.

Results of the study by Nagayoshi et al (34), who studied the dynamics of urinary 8-hydroxy-2′-deoxyguanosine related to creatinine levels in patients with acute myocardial infarction, support this theory. In patients who did not experience subsequent cardiac events, 8-hydroxy-2′-deoxyguanosine/creatinine levels decreased to normal by 24 h after reperfusion therapy. However, in the cardiac event group, the levels at 24 h remained higher than those in the noncardiac event group. Hokamaki et al (18) demonstrated that after treatment (not specified) of unstable angina, recurrent angina attacks at rest occurred more frequently in patients with high thioredoxin levels than in patients with low thioredoxin levels.

Long-term predictive value of oxidative stress

There is a correlation among oxidative stress, ventricular remodelling and progressive dilation leading to end-stage heart failure. The remodelling process in postischemic myocardium is the result of multiple underlying structural and signalling changes comprising myocardial cell contractile dysfunction, necrosis, apoptosis, inflammation, microvascular dysfunction and ECM changes. Following myocardial infarction, ROS-activated matrix metalloproteinases and fibroblast proliferation lead to structural and functional rearrangement of the ECM. The migration of cytokine-attracted neutrophils and macrophages producing additional free radicals is facilitated. The neuroendocrine system, ROS and inflammatory cytokines are important regulators of the remodelling process (11,35). The extent of ischemia-reperfusion damage may be more widespread than the area primarily affected, with apoptosis, necrosis, interstitial fibrosis and remodelling occuring not only at the site of infarction, but also in remote areas of the myocardium (8). Heart failure under both acute and chronic conditions is associated with increased levels of oxidative markers (eg, malonyldialdehyde, glutathione peroxidase [36], thioredoxin [37] or superoxide dismutase [38]). Mitochondria damaged by ischemia-reperfusion injury are one of the sources of persistently elevated ROS levels (39,40). Increased renin-angiotension-aldosterone system activity stimulates NADH/NADPH oxidase, as shown by Griendling et al (41) and Rajagopalan et al (42).

PERSPECTIVE: OXIDATIVE STRESS – A NEW PROGNOSTIC MARKER?

In the past two decades, numerous studies have demonstrated the importance of oxidative stress in the development of atherosclerosis and ischemia-reperfusion injury. Elevated concentrations of a variety of oxidative stress markers were linked with a more frequent occurrence of cardiac events. These findings could help assess risk stratification, diagnosis and prevention of ACS, both in patients with and without previous cardiac history. This topic needs to be further studied.