Is circulating heat shock protein 60 a marker for susceptibility to cardiovascular disease in patients with diabetes?

SEPTEMBER 11, 2008
Brian Henderson, PhD; Jeffrey W. Stevens MD, PhD; Jacqueline A. Cooper, BSc; Andrew Steptoe, DSc; Anthony R.M. Coates, MD; Steve E. Humphries, PhD, FRCPath, MRCP; Alireza Shamaei-Tousi, PhD

Cell stress proteins, such as heat shock protein (Hsp)60, are intracellular proteins that have evolved to enable cells to cope with stress. Most of these proteins help to prevent protein denaturation or to inhibit oxidation. The prototypic cell stress protein is Hsp60, a tetradecameric protein composed of 60kDa subunits, which is synthesized in the eukaryotic cell cytoplasm, and enters into the mitochondrion, where it functions as a molecular chaperone.1 Highly homologous Hsp60 proteins are produced by both prokaryotes and eukaryotes; therefore, it was surprising to find that bacterial Hsp60s were potent immunogens in mammals.2 One explanation for this paradoxical immunogenicity is the finding that Hsp60 acts as an intercellular signaling molecule with actions similar to cytokines.3 This would only be of biological significance if Hsp60 was released from cells—a possibility that is generally discounted.1 Therefore, it came as a surprise when Hsp60 was detected in human blood in 1999.4 George Wick and colleagues have since shown a correlation between circulating Hsp60 levels and measures of atherosclerosis in a general population,5 and our group has shown an association between arterial dysfunction and the presence of Hsp60 in the blood of healthy teenagers.6 Because of the known association between diabetes and coronary heart disease, we decided to evaluate Hsp60 levels in patients with diabetes to see if they correlate with vascular pathology.

Subjects and methods
A total of 855 consecutive subjects were recruited as part of the University College London Diabetes and Cardiovascular Study (UDACS), which was designed to evaluate the association between diabetes and the risk factors involved in cardiovascular disease.7 Subjects were recruited between 2001 and 2002; 17.2% of subjects had type 1 diabetes mellitus, and 82.8% had type 2 diabetes mellitus. Subjects were classified according to the presence or absence of clinically manifest cardiovascular disease, which was defined as the presence of 1 or more of the following: coronary heart disease, peripheral vascular disease, or cerebrovascular disease. Plasma obtained from subjects was used to measure Hsp60 levels, as well as a range of relevant analytes, including glucose, glycosylated hemoglobin, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglyceride, creatinine, and C-reactive protein levels. Plasma antioxidant status was also assessed. Results were subject to multistatistical analysis because of the skewed nature of circulating Hsp60 levels.

Results
Table 1 shows the baseline clinical measurements of the subjects according to those with and without cardiovascular disease. A higher percentage of subjects with cardiovascular disease were older and were receiving aspirin and statins, resulting in a significant lowering of cholesterol levels in this group. Immunologically measurable Hsp60 levels were found in 54% of subjects, with levels ranging from low nanograms to milligrams per milliliter of plasma. The remainder of subjects had Hsp60 levels below the range of assay sensitivity. Of those with measurable Hsp60, 28% had between 1 and 1000 ng/mL, whereas 26% had >1 μg/mL, with 7% having levels > 100 μg/mL (Figure). There was no difference in the distribution of Hsp60 between patients with type 1 and type 2 diabetes mellitus. No correlation was shown between circulating Hsp60 levels and blood pressure, body mass index, weight, age, or any of the analytes measured in plasma, including total antioxidant status.

A greater percentage of subjects with cardiovascular pathology had detectable circulating Hsp60 levels compared with those without cardiovascular pathology. A greater percentage of those with myocardial infarction (MI) also had measurable Hsp60 levels compared with those without MI (70.2% vs 52%, respectively; P = .004). The odds ratio (OR) for MI was 2.29 (95% confidence interval [CI], 1.26-3.73; P = .005) for those with measurable Hsp60. The tobit regression model confirmed the association, with an OR of 12.3 (95% CI, 2.0-76.7; P = .007). As shown in Table 2, the association remained after adjusting for smoking, sex, and age.

Two other findings are of interest. The population under study included individuals from distinct ethnic groups: Oriental, South Indian, African/ Caribbean, and Caucasian. Unexpectedly, a greater percentage of the Caucasians (P = .007) had detectable Hsp60 levels, and the range of the Hsp60 levels in these subjects was significantly larger (P = .01) than in the other ethnic groups. In addition, levels of Hsp60 were significantly higher in subjects who had either never smoked or were currently nonsmokers.

Discussion
In vitro, human Hsp60 concentrations have been shown to activate human monocytes/dendritic cells8 and human vascular endothelial cells,9 but they have also been shown to inhibit aspects of lymphocyte activation,10 being biologically active in vitro at concentrations of > 1 μg/mL.9 Thus, this intracellular protein could function as a modulator of both innate and acquired immunity, and as an activator of the vasculature if it were released into the circulation at levels > 1 μg/mL.

In this study of UK diabetic subjects, Hsp60 levels were measurable in 54% of subjects, with 26% having levels of Hsp60 > 1 μg/mL. These patients had the highest levels of circulating Hsp60 that we have yet observed, with 7% having levels > 100 μg/mL. The information to date, although limited, argues that there is an association between vasculature pathology and Hsp60 levels.5,6,11 The current study of subjects with diabetes supports this hypothesis, with a significantly greater percentage of subjects with cardiovascular disease having measurable Hsp60 levels compared with subjects with diabetes without clinically manifest cardiovascular disease. The clearest relationship was between the presence of circulating Hsp60 and MI, with 70% of patients with MI exhibiting Hsp60 in the blood compared with 52% of subjects without MI. Whether the circulating Hsp60 is causal or a consequence of the release of Hsp60 from stressed cardiovascular tissue as a result of the ischemic event has not been established, and this will require additional prospective studies.

Cell stress proteins have been regarded as having a purely intracellular function. Their discovery in body fluids is therefore surprising. Perhaps more surprising is the enormous range of concentrations at which Hsp60 is found in human blood—from low nanograms/mL to milligrams/mL.5,6,12 A fundamental question is what controls the release and removal of Hsp60 from the blood. It has been established that the synthesis of cell stress proteins like Hsp60 can be substantially increased by stress. However, such increases in synthesis rarely exceed a 10-fold rise. Thus, other factors must be involved in controlling the levels of Hsp60 in the circulation.

The finding that there were significant differences in the levels of circulating Hsp60 in different ethnic groups suggests that genetics may play a role in determining the synthesis, release, and disposition of this protein. The hypothesis that the levels of circulating Hsp60 may be under strong genetic control, however, is not supported by the finding that individuals who never smoked or were currently nonsmokers had significantly higher Hsp60 levels than did current smokers. This is an unexpected finding and suggests that control over the circulating levels of Hsp60 may also involve environmental factors. How smoking influences Hsp60 levels is not known.

Conclusions
Our study of subjects with diabetes, who are known to be at higher risk for developing cardiovascular disease, has identified a relationship between circulating levels of the mitochondrial stress protein Hsp60 and cardiovascular pathology. This suggests that Hsp60 may play an unexpected role in the cardiovascular pathology associated with diabetes. In this context, it is interesting that a recent report revealed that inhibition of endoplasmic reticulum stress in diabetic mice restored normal glucose homeostasis.13  




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