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Annals of Clinical & Laboratory Science 35:240-250 (2005)
© 2005 Association of Clinical Scientists

Review

Association of Soluble Markers with Various Stages and Major Events of Atherosclerosis

James T. Wu and Lily L. Wu
ARUP Laboratories and University of Utah School of Medicine, Salt Lake City, Utah, USA, and Department of Pathology, Chang Gung Memorial Hospital, Taipei, Taiwan

Address correspondence to James T. Wu, Ph.D., ARUP Laboratories, 500 Chipeta Way, Salt Lake City, Utah 84108, USA; tel 801 583 2787; fax 801 584 5207; e-mail: wuj{at}aruplab.com.


   Abstract
Top
 Abstract
Introduction
 Various Stages of...
 Major Events Associated with...
 Importance of Measuring Multiple...
 References
 
The purpose of this review is to identify soluble markers from the recent literature that facilitate the early prevention and early detection of atherosclerosis, and that may serve as therapeutic targets. Soluble markers associated with various stages and major events of atherosclerosis were identified. We divided the process of atherosclerosis into several stages, including stages for the early risk, plaque expansion, and stable and unstable angina–though excluding the end stage of myocardial infarction. For major events taking place prior to and during the progression of atherosclerosis we included events such as endothelial dysfunction in the artery, expression of adhesion molecules at the injured endothelium, continued inflammatory responses, oxidative stress, and ischemia. We found that reactions such as cell injury, adhesion, inflammation, and oxidative stress occur not only at the early stage of risk but persist throughout the process of atherosclerosis. Most markers associated with these major events are clustered together at any time of the disease. Few markers are characteristic of individual stages. We noted that reactions such as inflammation are continuously intensified with the progression of the disease. Finally, we underscore the importance of measuring a panel consisting of minimal numbers of multiple markers with the maximal sensitivity for early risk assessment, diagnosis, and prognosis. We envision that patterns characteristic of various stages of atherosclerosis may be identifiable with the use of the multiple markers described in this review.

(received 4 April 2005; accepted 29 April 2005)

Keywords: atherosclerosis, angina, ischemia, inflammation, oxidative stress, necrosis

Abbreviations: CAD, coronary artery disease; CHD, coronary heart disease; CVD, cardiovascular disease; CRP, C-reactive protein; GP, glutathione peroxidase; IMA, ischemia modified albumin; hsCRP, high sensitive CRP; ICAM, intracellular adhesion molecule; IL-8, interleukin 8; IL-6, interleukin 6; IGF-1, insulin-like growth factor-1; MCP-1, monocyte chemoattractant protein-1; M-CSF-1, macrophage-colony-stimulating factor-1; MDA-modified LDL, malondialdehyde-modified low-density lipoprotein; Lp-PLA2, lipoprotein derived phosphate lipase A2; MI, myocardial infarction; MMP, matrix metalloprotease; MPO, myeloperoxidase; NO, nitric oxide; OxLDL, oxidized low-density lipoprotein; 8-OHdG, 8-hydroxydeoxyguanosine; PVD, peripheral vascular disease; ROS, reactive oxygen species; SAA, serum amyloid protein A; tHcy, free and bound homocysteine; TGF-ß, transforming growth factor-beta; TNF-{alpha}, tumor necrosis factor-alpha; VCAM, vascular cell-adhesion molecule; VEGF, vascular endothelial growth factor; VWF, von Willebrand factor


   Introduction
Top
 Abstract
 Introduction
Various Stages of...
 Major Events Associated with...
 Importance of Measuring Multiple...
 References
 
Atherosclerosis is the most common cause of myocardial infarction (MI), and acute MI is one of the leading causes of death in the western world. We now realize that coronary artery disease (CAD), peripheral vascular disease (PVD), stroke, and congestive heart failure can all be derived from atherosclerosis. In the past, accurate diagnosis of MI and subsequent reperfusion was the major focus of clinical practice. Since MI is often fatal and is expensive to treat, the emphasis has been shifting gradually to the early detection and early risk assessment of cardiovascular diseases (CVD).

For early risk assessment and detection of atherosclerosis at the early stage of the disease, it would be helpful if soluble markers relating to various stages of atherosclerosis were identified. The measurement of soluble markers associated with various stages and major events of atherosclerosis should facilitate early prevention, early detection, and the identification of therapeutic targets. These markers should also be useful for monitoring treatment. To facilitate the identification of markers associated with various stages, we divided the process of atherosclerosis into various stages: the early period of risk, the early development of atherosclerosis, plaque expansion, stable angina, and unstable angina. These stages take place prior to myocardial infarction (Fig. 1Go).



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  		Fig. 1. Diagram of the stages and major events of atherogenesis.

 
An effort was also made to identify circulating markers associated with major events occurring throughout the process of atherosclerosis such as injury of arterial endothelial cells, expression of adhesion molecules at the injured endothelium, inflammation, and oxidative stress [1]. We tried to include only the more important markers, based on our experience, for various stages; however, more detailed descriptions are given for individual events in order to provide a source of complete information. We believe that the measurement of these soluble markers and their circulating levels, especially in a panel of multiple markers, would facilitate early risk assessment and treatment, which may prevent the progression of the disease to MI. We envision that the panel will be modified as new information becomes available.


   Various Stages of Atherosclerosis
Top
 Abstract
 Introduction
 Various Stages of...
Major Events Associated with...
 Importance of Measuring Multiple...
 References
 
Early risks.  Atherosclerosis can be divided into major stages prior to MI (Fig. 1Go). The first stage of early risk has drawn increasing attention in recent years. Although vasoconstriction of the artery may begin with the occurrence of endothelial dysfunction, the narrowing of the blood vessel won’t start until there is the formation of foam cells and plaque. Atherogenesis at the stage of early risk is largely preventable and reversible by dietary modifications and life style changes. Many elevated soluble markers are detectable at this stage, signaling an early risk of atherogenesis (Table 1Go).


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  		Table 1. Major soluble markers associated with various stages of atherosclerosis.
 
As shown in Fig. 1Go, events such as endothelial dysfunction, expression of adhesion molecules, and recruitment of leukocytes all take place prior to the augmentation of atherosclerosis [1,2]. Markers associated with these events (Table 1Go) have been employed to assess the early risk for atherogenesis. Increasing evidence indicates that damage of arterial endothelial cells invariably leads to inflammation and oxidative stress [3,4]. If not prevented, inflammation and oxidative stress will cause foam cell formation, formation of fibrous plaque, plaque rupture, thrombosis, and finally, myocardial infarction.

When the arterial endothelium is injured by any of the risk factors, adhesion molecules expressed at the injured site recruit leukocytes to the site of the lesion and augment the inflammatory reaction [5,6]. Among several adhesion molecules detectable in the circulation, the vascular cell adhesion molecule-1 (VCAM-1) appears to respond most specifically to endothelial cell injury in the artery [7] even though several other adhesion molecules are detectable at the same time. Detection of elevated VCAM-1 indicates that the arterial endothelium has been injured and leukocyte derived inflammation will follow. Several markers of systemic inflammation appear in the circulation following endothelial dysfunction. These markers, which are powerful predictors of cardiovascular events and have prognostic implications, include fibrinogen, C-reactive protein (CRP), serum amyloid protein A (SAA), and proinflammatory cytokines.

High sensitive CRP (hsCRP) is selected because a highly sensitive assay is required for early risk assessment. CRP has been extensively studied as a marker of early risk [8,9], although a recent large prospective study indicates that the contribution of CRP to cardiovascular disease is less impressive than previously believed [10]. Inflammation-associated chemokines, such as monocyte chemoattractant protein 1 (MCP-1) and interleukin 8 (IL-8), are also detectable at the early risk stage.

Plasma myeloperoxidase (MPO), a heme enzyme secreted by activated leukocytes at sites of inflammation, has been reported to promote oxidative stress and lipid peroxidation and to predict the risk for atherogenesis [11]. Plasma MPO level can be quantified by ELISA and has good correlation with an oxidative marker, F2-isoprostane. Urine F2-isoprostanes are products of the peroxidation of arachidonic acid, catalyzed by free radicals and myeloperoxidase. Quantification of urine F2-isoprostane is technically more difficult than MPO measurement. Therefore, MPO is recommended to replace F2-isoprostane at this stage as an index of lipid peroxidation in vivo. It was reported recently that a single measurement of plasma MPO independently predicts the early risk of myocardial infarction [12].

Recent reports emphasize the importance of monitoring urine microalbumin as an index of the risk of atherogenesis [13]. Urine microalbumin is not simply a marker of diabetic nephropathy. An elevated urine microalbumin level is now considered a signal of systemic vascular leakage, which is closely associated with endothelial dysfunction and inflammation. Urine microalbumin is simple and inexpensive to measure. Urine microalbumin seems to be the most valuable marker for early risk assessment of diabetic nephropathy, CVD, metabolic syndrome, and cancer [14].

Two independent markers, plasma homocysteine (tHcy) and plasma uric acid, have also been found useful for predicting risk of atherogenesis. The risk of atherogenesis is related to the oxidative stress and endothelial dysfunction caused by an elevated plasma level of homocysteine [15,16]. Voutlainen et al [17] found that in the presence of hyperhomocysteinemia there was increased lipid peroxidation and increased plasma F2-isoprostane concentration. Okumura et al [18] reported that plasma tHcy concentrations are significantly elevated in diabetic patients with clinical macroangiopathy, including coronary artery disease (CAD), stroke, and peripheral vascular disease (PVD).

Recent evidence suggests that serum uric acid is a sensitive marker for predicting the mortality of patients with heart disease [19,20]. It appears that serum uric acid is not a risk factor for CVD. Hyperuricemia does not lead to the development of CVD. Serum uric acid is rather a sensitive marker reflecting the presence of various risk factors for atherogenesis. In addition to gout, hyperuricemia is associated with metabolic syndrome. Higher quartiles of uric acid levels have been shown to be associated with increased death rates from ischemic heart disease and with higher blood pressure, higher serum cholesterol level, increased body mass indices, raised serum creatinine level, increased alcohol consumption, and diabetes. Conceivably, serum uric acid may be considered as a sensitive marker for predicting the risk of atherogenesis.

We hope that the markers listed in Table 1Go will be sufficient for detecting early risk of atherosclerosis. If not, studies may be needed to determine what is the least number of markers required for maximal sensitivity, since there are more markers detectable at this stage. We are uncertain whether or not insulin-like growth factor-1 (IGF-1) and its receptor should be included in Table 1Go, since serum IGF-I and IGF-binding protein-1 have been implicated in the development of CVD. IGF-I has been shown to stimulate nitric oxide production from both the endothelium and vascular smooth muscle cells (VSCM), to increase forearm blood flow, and to stimulate proliferation of coronary VSMC [21].

Early stage of atherosclerosis.  It is not easy to separate the stage of early risk from the early stage of atherosclerosis based on the measurement of circulating markers. With few exceptions, most markers detectable during the stage of early risk can also be found at the beginning and during the early development of atherosclerosis. It should be interesting to find out whether there are differences in the levels of various markers between these two stages and whether there are characteristic patterns of markers associated with individual stages.

It is difficult to know which marker or markers will signal the beginning of atherosclerosis. In fact, it is debatable exactly when atherosclerosis begins. One can argue that atherosclerosis actually starts when there is a reduction of nitric oxide (NO) concentration associated with endothelial dysfunction and slightly impaired arterial vasodilatation. However, from a practical point of view, we believe that atherosclerosis starts when foam cells, atheromas, and fibrous caps begin to appear in association with narrowing of the vessel. These events are no longer reversible by dietary modifications and life style changes. Perhaps the most important feature at this early stage is the absence of ischemia and myocyte necrosis.

Because a major function of the monocyte chemoattractant protein-1 (MCP-1) is to enable monocytes to enter the intima and because of the close association of MCP-1 with inflammation, MCP-1 may be detectable at the early stage of atherosclerosis. We believe that macrophage colony stimulator factor (M-CSF-1) may also be detectable because it is a potent monocyte activator and is responsible for promoting the expression of scavenger receptor on macrophages for the uptake of modified LDL, a critical step in the conversion of macrophages to foam cells [4].

Detection of elevated oxidized LDL (oxLDL) is conceivably a sign of early development of foam cells. Elevated oxLDL is also detectable through the entire process of atherosclerosis. Inflammation, oxidative stress, and vascular leakage occur during the stage of early risk and are intensified during the progression of atherosclerosis. The serum levels of CRP and MPO, and the urine level of microalbumin, may be further increased at subsequent stages compared to the stage of early risk. Markers related to inflammation are detectable at the early stage of atherosclerosis, including IL-6, TNF-{alpha}, plasminogen activator inhibitor-1, COX-2, fibrinogen, serum amyloid A (SAA), and lipoprotein derived phospholipase A2 (Lp-PLA2).

Inflammation does not follow a single metabolic pathway. In addition to the synthesis of CRP, fibrinogen, and SAA by hepatocytes, markers of inflammation derived from a different metabolic pathway may be detectable in the circulation. For example, the inflammation marker, Lp-PLA2, which is related to lipid peroxidation, has strong, positive association with the risk of coronary events that is not confounded by other factors [22,23]. Lp-PLA2 has shown to complement CRP, especially for healthy middle-aged men and women with LDL cholesterol (LDL-C) <130 mg/dL. Sudhir [23] reported that individuals with both Lp-PLA2 and CRP levels in the highest quartile are at the greatest risk for a CHD event, even though they have a low level of LDL-C. Lp-PLA2 may be detected later than other inflammation markers because it is not only secreted by macrophages (when atherosclerosis has begun) and because it has to wait until there is an accumulation of modified lipoprotein to bind in order to be functional. Lp-PLA2 participates in the oxidative modification of LDL by cleaving oxidized phosphatidylcholines, generating lysophosphatidylcholine, and oxidized free fatty acids [24].

We recommend measuring urine 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative stress to cellular DNA [25]. Production of reactive oxygen species (ROS) such as •O2, H2O2, and HO• occurs at the site of inflammation, which contributes to tissue damage. Presumedly, a product of oxidative DNA damage, 8-OHdG, is increased in blood leukocyte DNA at this early stage and the level of urine 8-OHdG may begin to rise.

Superoxide generated by leukocyte MPO can react directly with nitric oxide produced by endothelial cells, generating toxic peroxynitrite (ONOO–). The extent of cell damage related to the nitrosative stress caused by peroxynitrite may be reflected by the level of plasma 3-nitrotyrosine. A commercial ELISA kit is available to measure plasma nitrotyrosine [26]. However, the relationship between the circulating nitrotyrosine level and atherosclerosis has not been extensively studied.

Markers of inflammation and oxidative stress may increase from the stage of early risk to the stage of early atherosclerosis. As many products of inflammation are proinflammatory, they intensify the reactions of inflammation and oxidative stress as the disease progresses.

It is uncertain whether or not vascular endothelial growth factor (VEGF), a positive factor for angiogenesis, is detectable at this stage.

Plaque expansion.  The appearance of circulating ischemia markers and markers of cell necrosis is likely to be the major difference between the early stage of atherosclerosis and the further progression of atherosclerosis. As atherosclerosis advances, slightly elevated troponin I and ischemia-modified albumin begin to appear. The magnitude of the elevations depends on the severity of the disease. As disease progresses, narrowing of the arterial lumen will also occur, which leads to varying degrees of obstruction of blood flow to the heart muscle. As a result, ischemia leads to some degree of necrosis of the heart muscle. Troponins will be released from damaged myocytes as a consequence of cell necrosis. Troponin I appears to be a more specific marker of myocardial injury than troponin T [27,28].

The plasma level of ischemia-modified albumin may reflect ischemia occurring in tissues other than the myocardium, such as from peripheral vascular disease and exercise-induced skeletal muscle ischemia [29]. Conceivably, measuring the cardiac troponin and ischemia-modified albumin levels at the same time might differentiate ischemia of heart muscle from that of the peripheral tissue.

Circulating VEGF level should become elevated, reflecting the degree of plaque expansion, ischemia, and proliferation of smooth muscle cells. Microvascular channels may develop as a result of angiogenesis (appearance of VEGF) [30].

Since inflammation and oxidative stress occur through the entire process of atherosclerosis, it is possible to detect higher levels of inflammation markers, such as CRP and Lp-PLA2, and markers of oxidative stress as the disease progresses. We speculate that elevated urine 8-OHdG will also be detected at this stage.

As markers of inflammation, Lp-PLA2 and CRP are complementary to each other. Highly elevated plasma level of Lp-PLA2 has been found in patients with angiographically proven coronary artery disease (CAD), even though LDL cholesterol levels were not increased significantly. When included in a general linear model with LDL-C and other risk factors, Lp-PLA2 appears to be an independent predictor of disease status [31].

Insulin-like growth factor-1 (IGF-1) and its binding protein have roles in the development of CVD. IGF-1, in addition to growth-promoting and metabolic effects, mediates many effects of growth hormone (GH). IGF-1 promotes cardiac growth, which improves cardiac contractility and output. Abnormal levels of IGF-1 and its binding proteins may serve as risk factors for certain cardiac disorders [32,33]. However, it is uncertain whether they provide any additional benefits over the markers listed in Table 1Go.

Stable and unstable angina.  Continued progression of atherosclerosis leads to angina pectoris. Increased inflammation largely accounts for the progression of stable angina to unstable angina. Unstable angina is a clinical syndrome that falls between stable angina and acute MI in the spectrum of CAD. In addition to increased inflammation, a major feature of unstable angina is the tendency of the fibrous cap of the atheromatous plaque to rupture. The collagen of the fibrous cap tends to be digested by proteases such as various matrix metalloproteinases (MMPs) as the result of the increased inflammatory reaction. Proteases such as MMP-2 and MMP-9 can be found in atheromatous plaques when there is inflammation associated with stable angina. The inflammation also promotes the appearance of proinflammatory cytokines such as IFN-{gamma}, TNF-{alpha}, and CD40L at this stage. The proinflammatory cytokines inhibit collagen production by macrophages, endothelial cells, and smooth muscle cells in the arterial wall; they also promote MMP expression in these cells and eventually the rupture of fibrous cap [34].

Conceivably, highly increased levels of MMP 2 and MMP 9 are associated with unstable angina. Holvoet et al [35] reported that the plasma malondialdehyde-modified low-density lipoprotein (MDA-LDL) level, not the oxLDL level, was significantly higher in patients with acute coronary syndromes than in those with stable CAD [36]. Their subsequent study [37] indicated that measuring both MDA-LDL and troponin I at the same time gave better discrimination between stable CAD and acute coronary syndromes than measuring troponin I alone. Their data suggested that oxidized LDL is a marker of coronary atherosclerosis whereas MDA-LDL is a marker of plaque instability and atherothrombosis. As a result of inflammation with unstable angina, further increased levels of CRP, troponin I, ischemia-modified albumin, VEGF, MMPs, and proinflammatory cytokines, are likely to occur [38,39].


   Major Events Associated with Atherosclerosis
Top
 Abstract
 Introduction
 Various Stages of...
 Major Events Associated with...
Importance of Measuring Multiple...
 References
 
Many events occur not only during the period of early risk but continuously during the entire process of atherosclerosis. As a consequence, circulating markers in association with these events are detectable throughout the entire process of the disease. Markers associated with various events also tend to cluster together. For example, markers for endothelial cell injury, adhesion, inflammation, and oxidative stress are detectable at almost every stage and at any given time. Only a few markers are characteristic of a certain stage. The markers associated with individual events of atherosclerosis are listed in Table 2Go.


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  		Table 2. Soluble markers associated with various major events of atherosclerosis.
 
Endothelium dysfunction.  Reduced plasma level of NO is probably the most important sign of injury to endothelial cells in the inner lining of the artery. Reduced NO production is usually associated with impaired vascular relaxation [3]. The plasma level of NO can now be quantified by an ELISA.

Several other circulating markers have also been shown to be associated with various aspects of endothelial dysfunction. Appearance of plasma endothelin-1 (a vasoconstrictor peptide) plays a potential role in the development of microalbuminuria in diabetic nephropathy [40]. The von Willebrand factor (vWF), mainly synthesized by endothelial cells involved in platelet adhesion to the injured vessel wall, is also increased in response to endothelial cell injury [41]. The soluble ectodomain of thrombomodulin, a transmembrane protein expressed in endothelial cells, has been proposed as a marker reflecting endothelial dysfunction [41,42].

Expression of adhesion molecules.  Expression of adhesion molecules is considered to be the most critical event following endothelial dysfunction in order to recruit leukocytes to the injured endothelium and to initiate the leukocyte-mediated inflammatory reaction [4]. All of the adhesion markers listed in Table 2Go are detectable in the circulation even though they are expressed at various sites and differ in specificity and sensitivity.

Inflammatory reaction.  Inflammation has emerged as perhaps the most important risk factor for CVD [2]. As listed in Table 2Go, markers related to inflammation can be divided into proinflammatory cytokines (IL-6, TNF-{alpha}, IL-ß), inflammation markers associated with lipid peroxidation and prostaglandin synthesis (Lp-PLA2, COX-2, MCP-1) [23], and inflammation markers synthesized by hepatocytes (CRP, SAA, and fibrinogen). Proinflammatory cytokines provide a systemic stimulus that leads to hepatic synthesis of inflammatory markers such as CRP, SAA, and fibrinogen. There may be benefit from measurement of multiple inflammatory markers including the proinflammatory cytokines. For example, Cesari et al [43] showed that high incidence of cardiovascular events in the elderly was linked with 3 markers of inflammation (ie, IL-6, TNF-{alpha}, and CRP).

Soluble CD40 ligand (sCD40L), expressed by activated platelets, a transmembrane protein structurally related to TNF-{alpha}, may contribute to the inflammatory response of the vessel wall by inducing endothelial cells to secrete chemokines and to express adhesion molecules.

As mentioned above, circulating Lp-PLA2 may reflect a specific metabolic pathway of inflammation in atherogenesis [23]. The circulating Lp-PLA2 level has been shown to complement the CRP level. Lp-PLA2 belongs to the phospholipase A2 superfamily of enzymes that hydrolyze phospholipids.

MCP-1 is produced as a result of inflammation reaction and plays a causal role in the recruitment of leukocytes into the atheroma. Measurement of circulating MCP-1 has been shown to be useful in predicting the risk of atherogenesis [24].

Although fibrinogen, SAA, and CRP are all synthesized by hepatocytes upon proinflammatory cytokine stimulation, the SAA level was found to complement CRP for the prediction of cardiovascular events [44]. SAA and CRP levels both increased about 1000-fold in response to inflammation; the increase of serum fibrinogen level was only 50%. Unlike CRP and SAA, fibrinogen is related to the clotting system. Fibrinogen also has been found to be an independent risk factor for cardiovascular disease [45]. Not included in Table 2Go is IL-18, which has been reported as a marker for chronic inflammation.

Vascular permeability.  Microalbuminuria is a marker not only for diabetic nephropathy but also for predicting the risk of cardiovascular disease in the general population [14]. Microalbuminuria has been proposed to be associated with increased endothelial permeability. Vascular endothelial growth factor (VEGF), an angiogenesis factor, is another marker for vascular permeability. In a study of a general population, Asselbergs et al [46] found that subjects with microalbuminuria had significantly higher plasma levels of VEGF. Increased plasma VEGF level appears to develop before the appearance of microalbuminuria.

Oxidative stress.  Reactive oxygen species (ROS) interacts with a variety of macromolecules leading to lipid peroxidation, DNA strand breakage, changes in proteins, and free thiol oxidation. Peroxidation of LDL in lipids, either initiated by free radicals or catalyzed by myeloperoxidase (MPO), can result in the generation of oxLDL. Phospholipase activity, prostaglandin synthesis, and platelet adhesion/activation are all associated with release of aldehydes, which induce oxidative modifications of LDL in the absence of lipid peroxidation and generation of MDA-LDL. Antibodies against oxLDL can be detected in blood. Numerous results from clinical studies have suggested that anti-modified LDL antibodies are risk factors for the initiation and progression of cardiovascular disease. Apparently the amount of oxLDL production in the arterial intima is a function of the concentration of circulating native LDL and the extent of oxidative stress.

In addition to ROS, active nitrogen species (peroxynitrite, ONOO–) play important roles in vascular cell dysfunction and atherogenesis. As mentioned above, nitrotyrosine can serve as a marker for nitrosative stress [26].

The majority of damage caused by inflammation is actually mediated by inflammation-derived oxidative stress. ROS associated with oxidative stress appears to be the major causative factor promoting foam cell formation. Plasma MPO and oxLDL, and urine F2-isoprostane are all markers of leukocyte-derived oxidative stress. Measuring the urinary level of 8-OHdG is useful to reflect the damage of cellular DNA by ROS [25,47].

Although the majority of the oxidative stress is derived from leukocytes attached to the injured endothelium, ROS can also be generated from hyperhomocysteinemia [48], hyperglycemia [49], excess adipose tissue (excess central fat) [50], and hypercholesterolemia [51]. For example, homocysteine is believed to exert its effects through a mechanism involving oxidative damage. Free homocysteine is capable of generating oxidative stress upon oxidation to homocystine, causing endothelial dysfunctions [52].

Ischemia.  As atherosclerosis progresses there is gradual narrowing of the blood vessel, which leads eventually to a degree of ischemia. Two markers can be used to indicate the presence of ischemia (Table 2Go). One is ischemia-modified albumin, which is sensitive but may also be elevated by ischemia derived from peripheral muscle. The other marker is troponin. Troponin is actually a marker of cell necrosis, but since it is specific to the myocytes, troponin can be used to indicate ischemia in the heart muscle. As noted above, measuring troponin in combination with ischemia-modified albumin might help to differentiate cardiac ischemia from ischemia of peripheral tissues [29].


   Importance of Measuring Multiple Markers
Top
 Abstract
 Introduction
 Various Stages of...
 Major Events Associated with...
 Importance of Measuring Multiple...
References
 
Since atherosclerosis is a very complex process, monitoring a single marker or a few markers in a clinical study could lead to erroneous conclusions. We believe that in order to identify characteristic patterns in respect to various stages of atherosclerosis for risk prediction, diagnosis, prognosis, and identification of therapeutic targets, one should measure as many markers as possible, such as by using microarray techniques. The following facts associated with atherosclerosis exemplify the complexity of the disease:

The advantages of monitoring more than one marker have been recognized in many studies. Outlined below are a few examples from the literature:


   References
Top
 Abstract
 Introduction
 Various Stages of...
 Major Events Associated with...
 Importance of Measuring Multiple...
 References
 

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  15. Wu LL, Wu JT. Hyperhomocysteinemia is a risk factor for cardiovascular disease and a marker for cancer risk: biochemical basis. Clin Chim Acta 2002;322:21–28.[Medline]
  16. Tawakol A, Omland T, Gerhard M, Wu JT, Creager MA. Hyperchromocyst(e)inemia is associated with impaired endothelium-dependent vasodilation in humans. Circulation 1997;95:1119–1121.[Abstract/Free Full Text]
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  18. Okumura K, Aso Y. High plasma homocysteine concentrations are associated with plasma concentrations of thrombomodulin in patients with type 2 diabetes and link diabetic nephropathy to macroangiopathy. Metabolism 2003;52:1517–1522.[Medline]
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  22. Packard CJ, O’Reilly DS, Caslake MJ, McMahon AD, Ford I, Cooney J, Macphee CH, Suckling KE, Krishna M, Wilkinson FE, Rumley A, Lowe GD. Lipoprotein-associated phospholipase A2 as an independent predictor of coronary heart disease. West of Scotland Coronary Prevention Study Group. NEJM 2003;43:1148–1155.
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  25. Wu LL, Chiou CC, Chang PY ,Wu JT. Urinary 8-OHdG: a DNA marker of oxidative damage and a risk factor for cancer, atherosclerosis, and diabetes. Clin Chim Acta 2004:339;1–9.[Medline]
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