Circulating biomarkers have been used in cardiovascular medicine as predictors of incident or prevalent disease. Examples include C-reactive protein as a marker for inflammation in atherosclerosis, von Willebrand factor as a marker for endothelial damage, and cholesterol and its subtypes as markers for atherosclerosis. Although easy and attractive targets to measure, increased biomarker levels with any disease entity simply delineate an association and do not necessarily define a cause-and-effect relationship.

Heat shock protein (Hsp)60, an intracellular protein, is produced in response to stress, such as high temperature, mechanical stress, and oxidant and cytokine stress, and is meant to prevent protein denaturation or oxidation, or both. It may be expressed on the cell surface and released into the circulation after cell denaturation.

In the cross-sectional study discussed in this issue, the authors examined the circulating levels of Hsp60 biomarker in subjects with diabetes mellitus with and without known cardiovascular disease. They found increased levels of Hsp60 in those with a history of cardiovascular disease compared with those without. No other associations were found, including correlations with C-reactive protein or cholesterol subtypes. Levels of Hsp60 were lower in smokers. This finding remains unexplained.

Although 70% of subjects with prior myocardial infarction (MI) had detectable circulating Hsp60 protein levels, 52% of those without MI also had detectable Hsp60 levels, and in those with any cardiovascular event versus none, the prevalence of Hsp60 became even more uniform (59% vs 51%). Thus, the marker does not appear very specific. One of the reasons for this is may be because a group definition based on events missed subjects with diabetes with subclinical atherosclerosis or those who had had silent cardiovascular events in the past. It is also of note that those with high levels of Hsp60 were much older than those with unmeasurable levels. Although age was adjusted in multivariate modeling, the groups were not truly age matched, and the influence of aging alone in producing detectable Hsp60 levels cannot be ignored.

The findings of this cross-sectional study should therefore be viewed in context; that is, Hsp60 may be another biomarker that predicts prevalent disease. Similar to this study, other cross-sectional studies have shown elevated levels of soluble Hsp60 in patients with atherosclerosis¹ and in those with borderline hypertension.² Thus, this biomarker may simply be an effect of the disease and may represent surface-expressed Hsp60^3,4 released into the circulation by stressed cells undergoing apoptosis. Nevertheless, the presence of this biomarker in as much as 52% of the population expected to have high atherosclerotic burden and, in extraordinary amounts not reported previously, does seem to indicate that the biomarker may at least reflect the severity of atherosclerotic burden. The findings of this study, however, do not shed light on whether Hsp60 is pathogenically involved in the development of atherosclerosis in patients with diabetes mellitus.

In this study, serum autoantibodies to Hsp60 were not measured. These autoantibodies may develop secondary to expression of Hsp60 on stressed endothelial cells, inciting immune-mediated development of atheroma.⁵ Alternatively, these antibodies may develop in response to infective agents, such as Chlamydia, Mycobacteria, and other bacteria that contain Hsp proteins homologous to mammalian Hsp60, and are released into the circulation during the lytic phases of their life cycle.^6,7 Hsp60, both human and chlamydial, has also been shown to enhance the production of proinflammatory cytokines, such as tumor necrosis factor and matrix metalloproteinase produced in macrophages, and to increase expression of interleukin-6, adhesion molecules such as E selectin, and intercellular adhesion molecule 1 in vascular endothelial cells,⁸ and thus may play a pathogenic role in the development of atheroma.

Prospective studies have shown that elevated Hsp60 levels have been associated with progression of carotid atherosclerosis.⁹ If the goal is to detect atherosclerosis in an asymptomatic population, it might be better to investigate the end organ in which all biomarkers may work, that is, the vessel wall and use imaging-based methods such as intima-media thickness to detect disease, rather than measuring currently known and unknown markers. However, if the goal is to evaluate whether this biomarker may help in disease treatment, one might ask whether the use of antimicrobials that largely have not been successful thus far^10,11 might be more effective for acute coronary syndrome in those with high levels of Hsp60, such as those with diabetes mellitus who have high levels of antibodies to Hsp60.

References
1. Xu Q, Schett G, Perschinka H, et al. Serum soluble heat shock protein 60 is elevated in subjects with atherosclerosis in a general population. Circulation. 2000; 102(1):14-20.

2. Pockley AG, Wu R, Lemne C, et al. Circulating heat shock protein 60 is associated with early cardiovascular disease. Hypertension. 2000; 36(2): 303-307.

3. Kirchhoff S, Gupta S, Knowlton AA. Cytosolic HSP60, apoptosis, and myocardial injury. Circulation. 2002; 105(24): 2899-2904.

4. Gupta S, Knowlton AA. Cytosolic heat shock protein 60, hypoxia, and apoptosis. Circulation. 2002; 106(21):2727-2733.

5. Schett G, Xu Q, Amberger A, et al. Autoantibodies against heat shock protein 60 mediate endothelial cytotoxicity. J Clin Invest. 1995; 96(6): 2569-2577.

6. Beatty WL, Morrison RP, Byrne GI. Persistent chlamydiae: from cell culture to a paradigm for chlamydial pathogenesis. Microbiol Rev. 1994; 58(4): 686-699.

7. Cohen IR. A heat shock protein, molecular mimicry and autoimmunity. Isr J Med Sci. 1990; 26(12):673-676.

8. Kol A, Bourcier T, Lichtman AH, et al. Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages. J Clin Invest. 1999; 103(4):571-577.

9. Xiao Q, Mandal K, Schett G, et al. Association of serum-soluble heat shock protein 60 with carotid atherosclerosis. Clinical significance determined in a follow-up study. Stroke. 2005; 36(12):2571-2576.

10. Cercek B, Shah PK, Noc M, et al, for the AZACS Investigators. Effect of short-term treatment with azithromycin on recurrent ischaemic events in patients with acute coronary syndrome in the Azithromycin in Acute Coronary Syndrome (AZACS) trial: a randomised controlled trial. Lancet. 2003; 361(9360):809-813.

11. Grayston JT, Kronmal RA, Jackson LA, et al, for the ACES Investigators. Azithromycin for the secondary prevention of coronary events. N Engl J Med. 2005; 352(16):1637-1645.