Classical cardiovascular risk factors

A strong relationship between baseline mean serum cholesterol levels and year-incidence of CHD was demonstrated in the Seven Countries Study⁽Reference Keys¹⁰⁾. Rates of CHD mortality have fallen in the UK over the past three decades⁽¹⁾. This fall has been attributed to the implementation of intervention strategies for risk-factor management including dietary change, statin treatment for hypercholesterolaemia, antihypertensive treatment, reduction in smoking habit and improved management of patients with established acute coronary syndrome⁽Reference Unal, Critchley and Capewell¹¹⁾. Cohort studies such as the Framingham Heart Study⁽Reference Wilson, D'Agostino, Levy, Belanger, Silbershatz and Kannel⁸⁾, the PROCAM Study⁽Reference Assmann, Cullen and Schulte⁹⁾ and the Interheart Study⁽Reference Yusuf, Hawken and Ounpuu^12, Reference Rosengren, Hawken, Ounpuu, Sliwa, Zubaid, Almahmeed, Blackett, Sitthi-amorn, Sato and Yusuf¹³⁾ have been key in identifying these and other risk factors, including physical inactivity, the post-menopausal state and psychological factors. The Framingham risk score was derived from data within a middle-class middle-aged Caucasian population and may therefore underestimate lifetime risk and risk in the elderly⁽Reference Dawber, Meadors and Moore¹⁴⁾. The applicability of the Framingham data to European populations has been questioned⁽Reference Cooper, Miller and Humphries^15, Reference Hippisley-Cox, Coupland, Vinogradova, Robson, May and Brindle¹⁶⁾. The risk scores derived from the PROCAM cohort and the QRESEARCH database in the UK⁽Reference Hippisley-Cox, Coupland, Vinogradova, Robson, May and Brindle¹⁶⁾ have attempted to address this issue.

The importance of adopting a multi-factorial approach in managing coronary risk has become widely accepted⁽Reference Jackson, Lawes, Bennett, Milne and Rodgers⁷⁾. Over the recent past several new putative risk factors have been identified, which include plasma homocysteine, lipoprotein (a)⁽Reference Stubbs, Seed, Lane, Collinson, Kendall and Noble¹⁷⁾, soluble adhesion molecules⁽Reference Ridker, Buring and Rifai¹⁸⁾, cardiac troponins⁽Reference Zethelius, Johnston and Venge¹⁹⁾, type B natriuretic peptide⁽Reference De Sutter, De Bacquer, Cuypers, Delanghe, De Buyzere, Kornitzer and De Backer²⁰⁾, C-reactive protein (CRP)⁽Reference Ridker, Hennekens, Buring and Rifai²¹⁾ and coronary Ca score⁽Reference Church, Levine, McGuire, Lamonte, FitzGerald, Cheng, Kimball, Blair, Gibbons and Nichaman²²⁾. Meticulous histopathological studies⁽Reference Stary²³⁾ and imaging studies⁽Reference Celermajer, Sorensen, Gooch, Spiegelhalter, Miller, Sullivan, Lloyd and Deanfield²⁴⁾ have led to an appreciation of the impact of risk factors early in life on atherogenesis and the notion of plaque instability in patients with advanced disease⁽Reference Fuster, Badimon, Badimon and Chesebro^25, Reference Fuster, Badimon, Badimon and Chesebro²⁶⁾.

Why the need for more risk markers?

The algorithms described earlier do not discriminate well between individuals who will and will not develop cardiovascular end points. The utility of a test may be evaluated using the c statistic, or area under the receiver operating characteristic curve (see Fig. 1), and although useful in evaluating diagnostic tests, e.g. in Down's screening, it is not the ideal tool for assessing models that predict future risk or stratify individuals into risk categories⁽Reference Cook²⁷⁾. Individual classical risk factors such as serum lipids (LDL-cholesterol, HDL-cholesterol and TAG), hypertension and smoking only have a modest impact on the c statistic, although they lead to more accurate reclassification of large proportions of patients into higher-risk or lower-risk categories. Even well-validated models of complex disease only achieve values for the c statistic that are well below the theoretical maximum of 1⁽Reference Cook²⁷⁾. Hence, the use of the c statistic alone for validating the utility of novel CVD risk factors may not be appropriate. The reported values for c using the most-widely-available risk ‘engines’ is approximately 0·7, and a number of refinements have been suggested, e.g. the inclusion of markers that reflect other aspects of susceptibility such as inflammation and insulin sensitivity⁽Reference Koenig, Lowel, Baumert and Meisinger^28, Reference Dunder, Lind, Zethelius, Berglund and Lithell²⁹⁾. The impetus for discovering new biomarkers of CVD risk therefore arises for several reasons: (a) to improve the discrimination of individuals who will or will not develop early CVD; (b) to identify patients with established subclinical disease; (c) to identify patients with established CVD who will develop recurrent events in the short or longer term; (d) to identify risk factors that may provide a target for pharmacological or lifestyle intervention.

Fig. 1. Receiver operator characteristics curves for men (a) and women (b) for the Framingham risk equation, blood pressure and cholesterol ratio (total cholesterol (TC)/HDL-cholesterol (HDL)). (■–■), Framingham 1991; (□–□), blood pressure; (), age; (△–△), TC/HDL. The values shown are the risk cut-off points for each risk factor or the risk equation. (From Milne et al.⁽Reference Milne, Gamble, Whitlock and Jackson¹¹⁷⁾.)

Insights from the pathogenesis of atherosclerosis

According to the ‘response to injury’ hypothesis a subtle form of endothelial injury is the initiating event in atherogenesis, and a number of risk factors could be responsible including smoking, hypertension, hyperlipidaemia and infection⁽Reference Ross³⁾. Leucocyte recruitment to the injured endothelium is mediated by pairs of adhesion molecules that are up regulated early in atherogenesis⁽Reference Nakashima, Raines, Plump, Breslow and Ross³⁰⁾. Monocytes and T lymphocytes subsequently accumulate within the sub-endothelial space⁽Reference Katsuda, Boyd, Fligner, Ross and Gown³¹⁾. The monocytes are converted to lipid-laden foam cells within the artery wall, giving rise to a lesion termed the fatty streak⁽Reference Masuda and Ross³²⁾, while T-cells may become activated and are initially involved in a T-helper 1-type response associated with the release of pro-inflammatory cytokines⁽Reference Hansson³³⁾. The existence of a alternative ‘scavenger receptor’ pathway that allows the unregulated uptake of cholesterol in the form of modified LDL has been proposed⁽Reference Brown and Goldstein³⁴⁾. Cells of the arterial wall can cause LDL oxidation and the release of toxic LDL oxidation products⁽Reference Witztum and Steinberg³⁵⁾. These products may in turn react with apoB-100 within LDL particles causing cross-linking, altered antigen expression and subsequent recognition and uptake by scavenger receptors⁽Reference Steinberg, Parthasarathy, Carew, Khoo and Witztum³⁶⁾. LDL modification also appears to lead to an autoimmune response, and auto-antibodies to oxidised LDL have been found in plasma of patients with atherosclerosis⁽Reference Palinski, Ylaherttuala, Rosenfeld, Steinberg and Witztum³⁷⁾. The formation of immune complexes of oxidatively-modified LDL and antibody may also allow the uptake of modified LDL by macrophages via their Fc receptors⁽Reference LopesVirella, Binzafar, Rackley, Takei, Lavia and Virella³⁸⁾. Oxidatively-modified LDL causes an induction of endothelial cell tissue factor and colony-stimulating factor gene expression and impairs the biological activity of NO⁽Reference Rubbo, Trostchansky, Botti and Batthyany^39–Reference Clinton, Underwood, Hayes, Sherman, Kufe and Libby⁴¹⁾.

The conversion of the fatty streak into a fibrous plaque necessitates the recruitment and proliferation of vascular smooth-muscle cells⁽Reference Stary, Chandler, Dinsmore, Fuster, Glagov, Insull, Rosenfeld, Schwartz, Wagner and Wissler⁴²⁾. This process is driven by the synergistic interplay of several growth factors⁽Reference Ross³⁾. The mature plaque is characterised by a fibrous cap composed of smooth-muscle cells and extracellular matrix that overlies a pool of lipid, cholesterol crystals and inflammatory cells⁽Reference Stary, Chandler, Dinsmore, Fuster, Glagov, Insull, Rosenfeld, Schwartz, Wagner and Wissler⁴²⁾.

Unstable plaques, characterised by a large lipid pool, thin fibrous cap and large numbers of inflammatory cells located in the shoulder region of the plaque⁽Reference Lendon, Davies, Born and Richardson^43, Reference Richardson, Davies and Born⁴⁴⁾, are thought to be particularly prone to fissuring and rupture. Activated macrophages within the plaque are a rich source of matrix metalloproteinases that have the ability to degrade extracellular matrix⁽Reference Nikkari, O'Brien, Ferguson, Hatsukami, Welgus, Alpers and Clowes⁴⁵⁾, leading to localised regions of de-endothelialisation that may subsequently lead to focal thrombosis and plaque rupture. In most cases of fatal myocardial infarction at least one major coronary artery is narrowed by >70%, and this narrowing is usually associated with thrombi and plaque fissuring⁽Reference Qiao and Fishbein⁴⁶⁾. Some plaques may become stabilised through the development of a thick fibrous cap. Although such lesions may be sufficiently prominent to cause luminal narrowing, and hence angina or claudication, they are less likely to rupture and are therefore considered to be relatively safe⁽Reference Richardson, Davies and Born⁴⁴⁾. Moreover, in many cases the formation of these lesions is associated with arterial remodelling⁽Reference Glagov, Weisenberg, Zarins, Stankunavicius and Kolettis⁴⁷⁾ in which there is a compensatory increase in arterial diameter as the atherosclerotic lesion encroaches on the lumen, thereby maintaining luminal diameter.

Reversal of endothelial dysfunction by risk factor intervention

In patients with familial hypercholesterolaemia apheresis (extracorporeal removal of LDL) is associated with a rapid improvement in endothelial responsiveness in the brachial artery⁽Reference Tamai, Matsuoka, Itabe, Wada, Kohno and Imaizumi⁸⁶⁾. Treatment with hydroxymethylglutaryl-CoA reductase inhibitors such as lovastatin and pravastatin, which lower LDL, has also been shown to have a favourable effect on vasomotor tone⁽Reference Jarvisalo, Toikka, Vasankari, Mikkola, Viikari, Hartiala and Raitakari^87, Reference Dupuis, Tardif, Cernacek and Theroux⁸⁸⁾. Recent studies have demonstrated beneficial effects of the antidiabetic glitazone drugs on endothelial function in patients with diabetes⁽Reference Martens, Visseren, de Koning and Rabelink⁸⁹⁾, and oestrogen-replacement therapy can improve endothelial function in healthy post-menopausal women, although not if they already have CVD⁽Reference Yeboah, Reboussin, Waters, Kowalchuk and Herrington^90, Reference McCrohon, Adams, McCredie, Robinson, Pike, Abbey, Keech and Celermajer⁹¹⁾. Antioxidants such as vitamin E, C and probucol appear to improve endothelial dysfunction⁽Reference Gilligan, Sack, Guetta, Casino, Quyyumi, Rader, Panza and Cannon^92, Reference Jiang, Li, Li and Deng⁹³⁾. Epidemiological studies support the benefits of dietary antioxidants, although the results of clinical intervention studies are inconsistent. It appears that some of the discrepancies in these studies may be associated with the complex mechanisms of action of the antioxidants⁽Reference Bruckdorfer and Stanner⁹⁴⁾.

Markers of instability of coronary artery lesions

Careful morphological and immunocytochemical studies have revealed the complexity of the atherogenic process and the sequence of cellular events that generally take place over decades. However, the clinical end points, marked by myocardial infarct or stroke, happen abruptly and are the result of an acute occlusive event⁽Reference Fuster, Badimon, Badimon and Chesebro^25, Reference Fuster, Badimon, Badimon and Chesebro²⁶⁾. This factor has led to the concept of the unstable plaque, a lesion characterised by a large lipid pool and thin fibrous cap⁽Reference Lendon, Davies, Born and Richardson^95, Reference Richardson, Davies and Born⁹⁶⁾ and having a high inflammatory cell and metalloproteinase content⁽Reference Libby and Ridker²⁾. It has been proposed that systemic markers of inflammation such as CRP, IL-6 and lipoprotein-associated phospholipase A₂ may reflect or contribute to the local pro-inflammatory milieu⁽Reference Koenig and Khuseyinova⁹⁷⁾. The nature of the plaque may also contribute to the outcome following percutaneous coronary interventions such as angioplasty⁽Reference Saadeddin, Habbab, Sobki and Ferns⁹⁸⁾.

Plaque inflammation and local thrombus formation are major determinants of coronary artery plaque instability⁽Reference Fuster, Badimon, Badimon and Chesebro²⁵⁾. It has been proposed that plaque disruption causes a release of cytokines from activated monocytes and macrophages at the disrupted site, and that these cytokines promote hepatic synthesis of the acute-phase proteins, CRP and serum amyloid A⁽Reference Rader⁴⁹⁾. Increased serum concentrations of these markers may provide a means of identifying patients with unstable coronary plaques, and they have been reported to be higher in patients with unstable angina and to carry prognostic importance in these subjects⁽Reference Verheggen, de Maat, Cats, Haverkate, Zwinderman, Kluft and Bruschke⁹⁹⁾. CRP levels have also been investigated for their ability to predict unfavourable outcomes and impairment of left ventricular function in patients with acute coronary necrosis or previous myocardial infarction⁽Reference Suleiman, Khatib and Agmon¹⁰⁰⁾.

Platelet activation is an important feature of thrombus formation, and hence indicators of platelet activation such as P-selectin may help to assess a patient's risk of intracoronary thrombosis. P-selectin is an adhesion molecule expressed on the surface of platelets when activated by exposure to collagen and other agonists released following plaque disruption⁽Reference Christenson and Azzazy¹⁰¹⁾. Soluble fibrin and fibrin degradation products may also indicate a recent thrombotic process or risk of an impending event⁽Reference Ridker, Hennekens, Cerskus and Stampfer¹⁰²⁾. Elevated levels of these markers are pathognomonic of procoagulant fibrinolytic activity and may help to identify patients at an increased risk of myocardial infarction-related complications⁽Reference Lee, Ewald, McKenzie and Eisenberg¹⁰³⁾. Other indicators of activation of the coagulation system or inhibition of fibrinolytic activity are also elevated in patients with unstable angina⁽Reference van der Putten, Glatz and Hermens¹⁰⁴⁾. However, the use of most of the biochemical markers of inflammation and activation of the coagulation cascade for the detection of unstable coronary artery disease is limited by their low specificity and lack of standardisation of the analytical procedures⁽Reference Morrow, Cannon, Jesse, Newby, Ravkilde, Storrow, Wu and Christenson¹⁰⁵⁾.

The rationale for autoimmune biomarkers

Monocytes and lymphocytes have both been detected within atherosclerotic lesions⁽Reference Katsuda, Boyd, Fligner, Ross and Gown¹⁰⁶⁾ and auto-antibodies directed against modified LDL and heat-shock proteins 60 and 27 have been identified in the blood of individuals with atherosclerosis⁽Reference Wick, Kleindienst, Schett, Amberger and Xu^60, Reference Ferns, Shams and Shafi^107, Reference Salonen, Ylaherttuala, Yamamoto, Butler, Korpela, Salonen, Nyyssonen, Palinski and Witztum¹⁰⁸⁾. It is proposed that LDL oxidation that occurs in the absence of adequate antioxidant protection promotes an autoimmune response that may itself lead to progressive disease. These data have contributed to the formulation of the ‘lipid-oxidation hypothesis⁽Reference Steinberg, Parthasarathy, Carew, Khoo and Witztum³⁶⁾ and provide the basis for the measurement of markers of oxidant stress in assessing coronary risk. Furthermore, autoimmune conditions such as rheumatoid arthritis⁽Reference Solomon, Karlson, Rimm, Cannuscio, Mandl, Manson, Stampfer and Curhan¹⁰⁹⁾ and systemic lupus erythematosus⁽Reference Rhew and Ramsey-Goldman¹¹⁰⁾ are associated with a substantially increased coronary risk, suggesting that a heightened state of immune activation may be pro-atherogenic.

Direct evaluation of the vasculature

Plasma biomarkers only provide an indirect assessment of vascular health and may be confounded by other co-morbidities. More direct methods of evaluating vascular dysfunction have been advocated, such as imaging and functional vascular assessment. In addition to flow-mediated dilatation (a measure of endothelial function), these methods include carotid artery intima media thickness, multislice computed tomography (for quantifying coronary artery Ca accumulation) and coronary thermography (measuring temperature differentials as an indicator of localised inflammation), and they appear to predict clinical events and may improve the discrimination of conventional risk scores⁽Reference Greenland, LaBree, Azen, Doherty and Detrano^111–Reference Casscells, Hathorn, David, Krabach, Vaughn, McAllister, Bearman and Willerson¹¹⁴⁾. These methods now require further evaluation for clinical utility. The coronary artery Ca score is an independent predictor of CHD that may be of particular value in patients at intermediate cardiovascular risk. Invasive assessments of coronary disease, such as angiography and intravascular ultrasound, are not indicated in the absence of symptomatic disease; however, ultrafast MRI with intravenous contrast now makes ‘virtual angiography’ a possibility⁽Reference Dewey and Hamm¹¹⁵⁾.