This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Xu, Q. Articles by Wick, G. Search for Related Content PubMed PubMed Citation Articles by Xu, Q. Articles by Wick, G. Related Collections Pathophysiology Risk Factors Peripheral vascular disease Other diagnostic testing Carotid Stenosis

(Circulation. 2000;102:14.)
© 2000 American Heart Association, Inc.

Clinical Investigation and Reports

Serum Soluble Heat Shock Protein 60 Is Elevated in Subjects With Atherosclerosis in a General Population

Qingbo Xu, MD, PhD; Georg Schett, MD; Hannes Perschinka, MS; Manuel Mayr, MD; Georg Egger, MD; Friedrich Oberhollenzer, MD; Johann Willeit, MD; Stefan Kiechl, MD; Georg Wick, MD

From the Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck (Q.X., M.M., G.W.); the Department of Internal Medicine, University Hospital of Vienna (G.S.), Vienna, Austria; the Department of Neurology (J.W., S.K.), Institute for General and Experimental Pathology (H.P., G.W.), University of Innsbruck, Medical School, Innsbruck, Austria; and the Department of Internal Medicine, Hospital of Bruneck, Italy (G.E., F.O.).

Correspondence to Dr Qingbo Xu, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria. E-mail qingbo.xu{at}oeaw.ac.at

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Work from our laboratory has proven that increased titers of anti-heat shock protein 60 (HSP60) antibodies are associated with atherosclerosis and that HSP60-reactive T-cells are present in atherosclerotic lesions. Recent studies from others demonstrated that HSP60 directly activates endothelial cells and macrophages.

Methods and Results—To explore the possibility that HSP60 exists in the circulation, where it could exert its functions, we performed a population-based study with 826 subjects aged 40 to 79 years. The following items were measured in all participants: serum soluble HSP60 (sHSP60); anti-Escherichia coli lipopolysaccharide; anti-HSP65, anti-Chlamydia, and anti-Helicobacter pylori antibodies; and a variety of acute phase reactants (C-reactive protein, ₁-antitrypsin, and ceruloplasmin) and markers of systemic inflammation. Carotid atherosclerosis was assessed twice (1990 and 1995), and 15 other risk factors were evaluated. Our data show that levels of sHSP60 were significantly elevated in subjects with prevalent/incident carotid atherosclerosis and that these levels were correlated with common carotid artery intima/media thickness. Multiple logistic regression analysis documented these associations as independent of age, sex, and other risk factors. Interestingly, sHSP60 was also correlated with anti-lipopolysaccharide, anti-Chlamydia and anti-HSP60 antibodies, various markers of inflammation, and the presence of chronic infections. The risk of atherosclerosis associated with high sHSP60 levels was amplified when subjects had clinical and/or laboratory evidence of chronic infections.

Conclusions—Our data provide the first evidence of a strong correlation between sHSP60 and atherosclerosis, suggesting that sHSP60 may play important roles in activating vascular cells and the immune system during the development of atherosclerosis.

Key Words: heat shock proteins • chaperonin 60 • aging • infection • inflammation • atherosclerosis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
In response to stress stimuli, including high temperature, mechanical stress, infections, surgical stress, and oxidant and cytokine stimulation, cells produce high levels of heat shock protein (HSP) to protect themselves against these unfavorable conditions.^{1 2} HSPs belong to a group of 2 dozen proteins and cognates that show highly homologous sequences between different species, from bacteria to humans. They are highly expressed in cardiovascular tissues.^{2 3 4}

Recently, reports from 2 independent groups have demonstrated that both chlamydial and human HSP60 have a cytokine-like activity and induce tumor necrosis factor- and matrix metalloproteinase production in human and mouse macrophages.^{5 6} Interestingly, both chlamydial and human HSP60 induced E-selectin, intercellular adhesion molecule–1 (ICAM-1), and vascular cell adhesion molecule-1 expression and interleukin-6 production in endothelial cells.⁷ These findings suggest that HSP60 directly stimulates vascular endothelial cells, leading to an inflammatory response, which contributes to the pathophysiology of atherosclerosis.

Data from our laboratory have shown that atherosclerotic lesions can be induced in normocholesterolemic rabbits by immunization with recombinant mycobacterial HSP65.⁸ Levels of serum antibodies to HSP65 were significantly increased in clinically healthy human subjects with carotid atherosclerosis compared with those without lesions. The elevation of antibody levels was independent of age, sex, and other classic risk factors.⁹ Because HSPs show a high degree of amino acid homology between different species from prokaryotes to humans,^{1 2 3 4} an autoimmune reaction may be evoked because of the immunological cross-reaction between microbial and human HSP60.¹⁰

The question arose regarding whether HSP60, which is normally an intracellular protein, exists in the circulation of healthy individuals, where it could directly contact the arterial wall and immune cells. The present study was designed to assess the serum levels of soluble HSP60 (sHSP60) in the general community (Bruneck study) and to investigate its association with the development of carotid atherosclerosis. We had a special focus on potential interactions with chronic infections or inflammation.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Subjects
Population recruitment was performed as part of the Bruneck Study.^{11 12 13 14} The survey area is located in the north of Italy (Bolzano province). Special features of the study design and protocol have been described in detail previously.^{11 12 13 14} In brief, in 1990, the study population was recruited as a sex- and age-stratified random sample of all inhabitants of Bruneck aged 40 to 79 years, such that 125 women and 125 men in each decade, from the 5th to 8th decades of age, were selected (n=1000). A total of 93.6% of these subjects finally participated in the study. Among these subjects, 58 died between the summer of 1990 and 1995. At the first re-evaluation of the study cohort in 1995, the follow-up rate among survivors was high (96.5%; n=826).¹⁵ These 826 subjects were the basis for the current study. All participants gave their informed consent before entering the study.

sHSP60 Assays
sHSP60 was determined as part of the 1995 evaluation.¹¹ Blood was obtained after subjects had fasted and abstained from smoking overnight.¹¹ A sandwich enzyme-linked immunosorbent assay (ELISA) was performed using 2 types of monoclonal antibodies against different epitopes of HSP60 (II-13¹⁶ and ML-30¹⁷ ). In short, 96-well microtiter plates were coated with 1 µg/mL PBS of the antibody II-13 overnight, washed with PBS, and incubated with 100 µL of human serum diluted in PBS (1:10). After washing, biotin-labeled IgG ML-30 (1 µg/mL) was added to the plates and incubated at room temperature for 1 hour. Streptavidin conjugated with horseradish peroxidase was added and developed with the substrate. Absorbance at 410 nm was measured after 30 minutes. A serum concentration (in ng/mL) of sHSP60 was calculated according to the standard curves obtained by using recombinant human HSP60 (StressGen Biotechnology Co) in the sandwich ELISA.

Chromatography and Western Blot Analysis
Monoclonal antibody II-13 was coupled to supports using the Affi-Gel HZ Immunoaffinity kit (Bio-Rad) according to the manufacturer’s instructions. sHSP60 was separately isolated from sera with higher sHSP60 levels from several subjects. Isolated proteins were electrophoresed, transferred to the membrane, probed with the antibody, and visualized with an enhanced chemiluminescent kit (Amersham Co).¹⁸

Assays of Anti-Lipopolysaccharide, Anti-HSP65, Anti-Chlamydia, and Anti-Helicobacter pylori Antibodies
The procedure used for determining anti-lipopolysaccharide (LPS) antibodies was similar to that described elsewhere.¹⁹ In short, microtiter plates were coated with 1 µg/mL polylysine in PBS (Sigma) at 37°C for 1 hour; they were then washed and incubated with Escherichia coli LPS (Sigma) overnight. After washing, the plates were incubated with 100 µL of human serum diluted in PBS (1:10 to 1:5120). A serum dilution was considered positive for antibodies against LPS if the optical density absorbance at 410 exceeded 0.200. Serum anti-HSP65 antibodies were measured using the ELISA, as described previously.⁹ Anti-Chlamydia and anti-H. pylori antibodies were determined using commercial kits (Medac) according to the manufacturer’s instructions.¹⁸

Determination of Carotid Atherosclerosis
The ultrasound protocol involves scanning the internal (bulbous and distal segments) and common carotid arteries (proximal and distal segments) on both sides with a 10-MHz imaging probe and a 5-MHz Doppler.^{11 12 13 14} Atherosclerotic lesions were defined by the following 2 ultrasound criteria: (1) wall surface (protrusions into the lumen or roughness of the arterial boundary) and (2) wall texture (echogenicity). A sensitive and reproducible atherosclerosis score was calculated by the addition of all plaque diameters. The accuracy of this procedure was established previously.¹¹ Incident atherosclerosis was defined as the occurrence of new plaques in previously normal segments (1990 to 1995). Intima/media thickness was also documented, and it was correlated with both the atherosclerosis scores (r=0.64) and the 5-year changes in the scores (r=0.48).

Clinical History and Examination
The average number of cigarettes smoked per day and the pack-years smoked (used as a measure of cumulative exposure) were noted for each smoker and ex-smoker. Regular alcohol consumption was assessed in terms of grams/day. Systolic and diastolic blood pressures were taken with a standard mercury sphygmomanometer after 10 minutes of rest while the subject was in a sitting position. The values used in the current analysis were means of 3 measurements taken by the same investigator at 1-hour intervals. Hypertension was defined by a blood pressure 160/95 or the current use of antihypertensive drugs. A standardized oral glucose tolerance test (75 g of glucose in 10% solution) was performed in all subjects except those with well-established diabetes mellitus. Diabetes mellitus was diagnosed when fasting glucose levels exceeded 7.8 mmol/L (140 mg/dL) and/or a 2-hour value was >11.1 mmol/L (200 mg/dL) (World Health Organization criteria). The body mass index and waist-to-hip ratio were used as obesity indices. Subjects with chronic infections (n=268) were identified by extensive clinical and laboratory screening, as described elsewhere.²⁰

Other Laboratory Assays
Total and HDL cholesterol were determined enzymatically (CHOD-PAP and GOD-PAP method, Merck; coefficient variation [CV], 2.2% to 2.4%), lipoprotein(a) concentrations were determined with ELISA (Immuno; CV, 3.5% to 6.3%), and apolipoproteins were determined with a nephelometric fixed-time method (apolipoprotein AI: CV, 5.7%; apolipoprotein B: CV, 2.4%).^{21 22} LDL cholesterol was calculated with the Friedewald formula and corrected for lipoprotein(a) cholesterol.²¹ Fibrinogen was assayed according to the method of Clauss.²³ Blood leukocyte counts were expressed as cellsx10⁹/L. Endotoxin levels were measured with commercial ELISA kits.²⁰ Serum-soluble ICAM-1 was determined with commercial ELISA kits (Boehringer Ingelhein Co). All other parameters, including microalbuminuria, ₁-antitrypsin, ceruloplasmin, C-reactive proteins, and antithrombin III, were assessed by standard assays.^{20 24}

Statistical Analysis
Strength and type of association between sHSP60 concentration and 5-year progression of carotid atherosclerosis (changes in the atherosclerosis score, size of lesions, or intima/media thickness) were assessed using standard linear regression analysis. Linear regression models were supplemented by logistic regression analyses that used prevalent or incident atherosclerosis as dichotomized outcome variables. The test procedure was based on maximum likelihood estimators, and the accuracy of fit of each model was assessed by the test of Hosmer and Lemeshow.²⁵ For ease of presentation, all multivariate regression models were adjusted for the same set of established and putative vascular risk factors.

	Results

Top Abstract Introduction Methods Results Discussion References

 
In 33% of study subjects (n=281), serum sHSP60 was not detectable; in others, serum levels ranged up to 11 000 ng/mL. Five percent of subjects had concentrations >1000 ng/mL; these high concentrations produce a maximal activation of macrophages and endothelial cells in vitro.^{5 6 7} The distribution of sHSP60 levels in the general community of Bruneck is shown in Figure 1A. Western blot analysis revealed that sHSP60 was present in the circulation as 60-kDa proteins (Figure 2).

View larger version (30K): [in this window] [in a new window]   Figure 1. Distribution of sHSP60 in the general community (A) and association with incident carotid atherosclerosis (B). ORs were derived from logistic regression analysis of incident carotid atherosclerosis on quintiles of sHSP60 concentrations, age, sex, and baseline atherosclerosis (reference category, sHSP104 ng/mL).

View larger version (70K): [in this window] [in a new window]   Figure 2. Chromatography and Western blot analysis. Monoclonal antibody II-13 was coupled to supports using an immunoglobulin-coupling kit. sHSP60 was isolated separately from 3 sera with higher sHSP60 levels, as determined by ELISA. Isolated proteins were electrophoresed, transferred to the membrane, probed with the antibody, and visualized with the enhanced chemiluminescent kit. S indicates subject.

Table 1 depicts means and SDs of selected demographic characteristics and risk factors according to levels of sHSP60. The probability value for differences in risk factor levels between low sHSP60 (first to fourth quintile) and high sHSP60 (fifth quintile; levels >280 ng/mL) were derived from the t test (unadjusted) or from ANOVA (adjusted for age and sex). Data indicate that high sHSP60 was significantly correlated with markers of infection and inflammation, including anti-LPS antibodies, anti-HSP65 and anti-Chlamydia antibodies, concentrations of circulating endotoxin, ₁-antitrypsin, C-reactive protein, ceruloplasmin, and the presence of chronic infections, as ascertained by clinical criteria (P<0.05 to P<0.001); however, high sHSP60 levels were not correlated with classic risk factors for atherosclerosis, except LDL cholesterol (apolipoprotein B) (Table 1).

View this table: [in this window] [in a new window]   Table 1. Comparison of Infectious and Inflammatory Markers and Risk Factors for Atherosclerosis Between Low and High Levels of sHSP60

Log_e-transformed serum concentrations of sHSP60 showed a significant association with prevalent carotid atherosclerosis (odds ratios [ORs], 1.28 and 1.24 per 1-SD unit increase in sHSP60; P<0.05) and the common carotid artery intima/media thickness (P<0.05). When sHSP60 levels were subdivided into 5 equally-spaced categories (quintiles), the increase in atherosclerosis risk was mainly confined to the top category (fifth quintile, >280 ng/mL) (Figure 1B). Table 2 depicts the associations between high sHSP60 and the various ultrasound measures of atherosclerosis extent and progression.

View this table: [in this window] [in a new window]   Table 2. Association of sHSP60 with Carotid Atherosclerosis

Analyses of incident atherosclerosis and changes in the atherosclerosis score over time were performed in light of a long-term intraindividual consistency of sHSP60 levels (n=100; correlation coefficient, r=0.42; P<0.0001) between sHSP60 levels from 2 independent samplings at a 5-year interval; the coefficient =0.44 indicates substantial accordance in the classification of subjects with respect to their sHSP60 level (quintiles 1 through 4 versus quintile 5). To exclude the possible confounding effects of vascular risk factors, multiple logistic regression models were adjusted for the potential effects of age, sex, smoking, alcohol consumption, hypertension, LDL and HDL cholesterol, ferritin concentrations, microalbuminuria, and hypothyreosis (Table 2). The significant association between sHSP60 and atherosclerosis applied equally to men and women, smokers and nonsmokers, and various age groups. Finally, analyses were virtually unchanged when systolic or diastolic blood pressure was substituted for hypertension (yes versus no) or apolipoproteins (AI and B) for cholesterol fractions (LDL and HDL).

A large number of epidemiological studies reported an association between atherosclerosis and certain persistent bacterial and viral infections, including Chlamydia pneumoniae and herpesviruses.^{26 27 28} Surprisingly, we found that anti-E. coli LPS antibody titers were negatively correlated with incident carotid atherosclerosis (OR, 0.85 for a 1-titer increase in anti-LPS antibodies; P=0.03) (Figure 3), intima/media thickness (regression coefficient, -0.017; P=0.011), and 5-year changes in the atherosclerosis score (regression coefficient, -0.076; P=0.02) and that these associations were independent of age, sex, and other vascular risk predictors. Notably, low titers of anti-LPS antibodies and high levels of sHSP60 seemed to synergistically affect atherosclerosis risk (Figure 4). ORs of incident atherosclerosis were 5.1 (model adjusted for age, sex, and baseline atherosclerosis) or 6.6 (multivariate adjustment) when both conditions coexisted in a single individual.

View larger version (51K): [in this window] [in a new window]   Figure 3. Association of anti-LPS antibody titers with incident carotid atherosclerosis. ORs were derived from logistic regression analysis of incident carotid atherosclerosis on LPS antibody titer (log), age, sex, and baseline atherosclerosis (reference category, titer 5).

View larger version (29K): [in this window] [in a new window]   Figure 4. Association of sHSP60 and anti-LPS antibodies with the risk of carotid atherosclerosis. ORs were derived from logistic regression analysis of incident carotid atherosclerosis on LPS antibodies/sHSP60 categories, age, sex, and baseline atherosclerosis (A) plus cigarette smoking, hypothyreosis, hypertension, HDL and LDL cholesterol, microalbuminuria, ferritin, and alcohol consumption (B). *P<0.05; **P<0.01 (reference category, high anti-LPS antibodies [Ab]/low sHSP60).

Accumulating evidence supports the view that atherosclerosis is a chronic inflammatory disease and that infection plays an important role in this scenario.²⁹ In this population-based prospective study, we demonstrated that markers of inflammation (C-reactive protein or soluble ICAM-1) and the presence of chronic infections (as ascertained by clinical criteria) are strongly associated with the development of atherosclerosis in the carotid arteries. The data shown in Figure 5 indicate that the predictive significance of sHSP60 for atherosclerosis progression grows with laboratory and/or clinical evidence of chronic infection/inflammation.

View larger version (16K): [in this window] [in a new window]   Figure 5. Risk of carotid atherosclerosis according to baseline serum concentrations of sHSP60, chronic infection, and the combination of both parameters. ORs were derived from logistic regression analysis of incident carotid atherosclerosis on sHSP60 categories and chronic infection.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Data from our laboratories showed an association between serum anti-HSP65 antibodies and carotid atherosclerosis⁹ and the presence of T-lymphocytes specifically responding to HSP60/65 in atherosclerotic lesions.³⁰ Other groups reported increased antibody titers in patients with atherosclerosis or coronary heart diseases,^{31 32} which declined after angioplasty or myocardial infarction.^{33 34} Recently, novel findings demonstrated that HSP60, like cytokines, stimulates the release of tumor necrosis factor- from macrophages and the expression of E-selectin, ICAM-1, and vascular cell adhesion molecule-1 by endothelial cells; this could lead to inflammatory responses in the arterial wall and eventually to the development of atherosclerosis.^{5 6 7} In the present population-based study, we provide the first evidence of an elevation of a soluble form of circulating HSP60 in subjects with atherosclerosis. The findings provide solid evidence to explain how HSP60 contacts vascular and immune cells and, thus, could significantly enhance our understanding of the role of HSP60 in the pathobiology of atherosclerosis.

Proteins of the HSP60 family were previously considered to be located intracellularly in the mitochondria, where they facilitate protein translocation and act as chaperones protecting proteins from harmful enzymatic attacks during folding.¹ Accumulating evidence now points to an additional surface location of HSP60 proteins on eukaryotic^{35 36} and even prokaryotic cells, such as H. pylori.³⁷

In the present study, we found that sHSP60 was present in the human circulation, which tempts us to hypothesize about various possibilities resulting in HSP60 releases into the blood. First, infectious agents may be the major cause of sHSP60 release from themselves and human cells. For example, during their life cycle, chlamydiae undergo both phases of chronic, persistent, nonlytic infection, in which they remain viable but do not replicate, and phases of lytic infection.³⁸ During these lytic phases, the host cells release both their own HSP60, which is produced during the previous chronic phase of infection, and also the chlamydial HSP60, which has been produced in the host cell in response to the infection and to previous noninfectious stimuli.³⁹ Support for this notion is the fact that sHSP60 levels are significantly correlated with anti-Chlamydia antibodies and that both chlamydial and human HSP60s exist at high levels in human atherosclerotic lesions.^{5 40} Second, sHSP60 release could be from the dead cells of tissues suffering from chronic inflammation and atheroma, because several observations support the occurrence of cell death within atheroma.^{41 42} Our data demonstrate the correlation of sHSP60 concentrations with chronic infection and markers of inflammation. Therefore, HSP60 release into the circulation is an important event because it can then activate vascular cells and the immune system.

The second novel finding of the present study is that anti-LPS antibody titers are negatively correlated with atherosclerosis, which further supports the impact of infections. E. coli infections may result in the release of endotoxin-LPS into the circulation. LPS may stimulate the body to produce specific antibodies that will neutralize and remove LPS from the circulation. In fact, Wiedermann et al²⁰ demonstrated that circulating endotoxin is positively correlated with atherosclerosis in this population. Thus, anti-LPS antibodies may have a protective role for the arterial wall.

In summary, we demonstrated an association between sHSP60 and atherosclerosis in the general population; this association relates to infection and inflammation. The functional role of circulating sHSP60 may involve the direct stimulation of vascular cells and the immune system, thus promoting the progression of atherosclerosis. In addition, sHSP60 could serve as a diagnostic marker for atherosclerosis risk, especially when combined with other infectious and inflammatory markers. Thus, this new approach to arterial risk assessment may be useful in the clinic.

	Acknowledgments

 
We thank Dr R.S. Gupta, Department of Biochemistry, McMaster University, Hamilton, Canada, for providing the hybridoma cell line of II-13; Dr J. Ivanyi, MRC, London, UK, for the hybridoma cell line of ML-30; Drs A. Mair, G. Rungger, and F. Spögler, Hospital of Bruneck, Italy, for help with organization; and A. Jenewein for excellent technical assistance. Supported by grants P-13099-BIO and P-12213-MED from the Austrian Science Fund. H. Perschinka is the recipient of a PhD student fellowship from the Austrian Academy of Sciences. We are grateful to the company Medac (Hamburg) for providing anti-Chlamydia and anti-H. pylori test kits.

	Footnotes

 
This article originally appeared online only with the June 6, 2000 issue of Circulation.

Received May 2, 2000; revision received May 19, 2000; accepted May 19, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Morimoto RI. Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev. 1998;12:3788–2396.[Free Full Text]
   
2. Xu Q, Wick G. The role of heat shock proteins in protection and pathophysiology of the arterial wall. Mol Med Today. 1996;2:372–379.[Medline] [Order article via Infotrieve]
   
3. Roma P, Catapano AL. Stress proteins and atherosclerosis. Atherosclerosis. 1996;127:147–154.[Medline] [Order article via Infotrieve]
   
4. Benjamin IJ, McMillan DR. Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and disease. Circ Res. 1998;83:117–132.[Abstract/Free Full Text]
   
5. Kol A, Sukhova GK, Lichtman AH, et al. Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor- and matrix metalloproteinase expression. Circulation. 1998;98:300–307.[Abstract/Free Full Text]
   
6. Chen W, Syldath U, Bellmann K, et al. Human 60-kDa heat-shock protein: a danger signal to the innate immune system. J Immunol. 1999;162:3212–3219.[Abstract/Free Full Text]
   
7. 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;103:571–577.[Medline] [Order article via Infotrieve]
   
8. Xu Q, Dietrich H, Steiner HJ, et al. Induction of atherosclerosis in normocholesterolemic rabbits by immunisation with heat shock protein 65. Arterioscler Thromb. 1992;12:789–799.[Abstract/Free Full Text]
   
9. Xu Q, Willeit J, Marosi M, et al. Association of serum antibodies to heat shock-protein 65 with carotid atherosclerosis. Lancet. 1993;341:255–259.[Medline] [Order article via Infotrieve]
   
10. Wick G, Romen M, Amberger A, et al. Atherosclerosis, autoimmunity and vascular-associated lymphoid tissue. FASEB J.. 1997;11:1199–1207.[Medline] [Order article via Infotrieve]
    
11. Willeit J, Kiechl S. Prevalence and risk factors of asymptomatic carotid artery atherosclerosis: a population-based study. Arterioscler Thromb.. 1993;13:1666–1668.
    
12. Kiechl S, Willeit J, Egger G, et al. Body iron stores and the risk of carotid atherosclerosis: prospective results from the Bruneck study. Circulation.. 1997;96:3300–3307.[Abstract/Free Full Text]
    
13. Kiechl S, Willeit J. The natural course of atherosclerosis, part I: incidence and progression. Arterioscler Thromb Vasc Biol.. 1999;19:1484–1490.[Abstract/Free Full Text]
    
14. Kiechl S, Willeit J. The natural course of atherosclerosis, part II: vascular remodeling. Arterioscler Thromb Vasc Biol.. 1999;19:1491–1498.[Abstract/Free Full Text]
    
15. Xu Q, Kiechl S, Mayr M, et al. Association of serum antibodies to heat shock-protein 65 with carotid atherosclerosis: clinical significance determined in a follow-up study. Circulation. 1999;100:1169–1174.[Abstract/Free Full Text]
    
16. Singh B, Gupta RS. Expression of human 60-kD heat shock protein (HSP 60 or P1) in Escherichia coli and the development and characterization corresponding monoclonal antibodies. DNA Cell Biol.. 1992;11:489–496.[Medline] [Order article via Infotrieve]
    
17. Evans DJ, Norton P, Ivanyi J. Distribution in tissue sections of the human GroEL stress protein homologue. APMIS. 1990;98:437–441.[Medline] [Order article via Infotrieve]
    
18. Mayr M, Metzler B, Kiechl S, et al. Endothelial cytotoxicity mediated by serum antibodies to heat shock proteins of E. coli and Chlamydia pneumoniae: immune reactions to heat shock proteins as a possible link between infections and atherosclerosis. Circulation. 1999;99:1560–1566.[Abstract/Free Full Text]
    
19. Takahashi K, Fukada M, Kawai M, et al. Detection of lipopolysaccharide (LPS) and identification of its serotype by an enzyme-linked immunosorbent assay (ELISA) using poly-L-lysine. J Immunol Methods. 1992;153:67–71.[Medline] [Order article via Infotrieve]
    
20. Wiedermann CJ, Kiechl S, Dunzendorfer S, et al. Association of endotoxemia with carotid atherosclerosis and cardiovascular disease: prospective results from the Bruneck Study. J Am Coll Cardiol.. 1999;34:1975–1981.[Abstract/Free Full Text]
    
21. Siedel J, Hagele EO, Ziegenhorn J, et al. Reagent for the enzymatic determination of serum total cholesterol with improved lipolytic efficiency. Clin Chem.. 1983;29:1075–1080.[Abstract/Free Full Text]
    
22. Kronenberg F, Kronenberg MF, Kiechl S, et al. Role of lipoprotein(a) and apolipoprotein(a) phenotype in atherogenesis: prospective results from the Bruneck study. Circulation. 1999;100:1154–1160.[Abstract/Free Full Text]
    
23. Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol. 1957;17:237–246.[Medline] [Order article via Infotrieve]
    
24. Bonora E, Kiechl S, Willeit J, et al. Plasma glucose within the normal range is not associated with carotid atherosclerosis: prospective results in subjects with normal glucose tolerance from the Bruneck Study. Diabetes Care.. 1999;22:1339–1346.[Abstract]
    
25. Hosmer DW, Lemeshow S. Applied Logistic Regression. New York: Wiley & Sons; 1988.
    
26. Saikku P, Leinonen M, Mattila K, et al. Serological evidence of an association of a novel chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction. Lancet. 1988;2:983–986.[Medline] [Order article via Infotrieve]
    
27. Thom DH, Wang SP, Grayston T, et al. Chlamydia pneumoniae strain TWAR antibody and angiographically demonstrated coronary artery disease. Arterioscler Thromb.. 1991;11:547–551.[Abstract/Free Full Text]
    
28. Hajjar DP, Fabricant CG, Minick CR, et al. Virus-induced atherosclerosis. Am J Pathol.. 1986;122:62–70.[Abstract]
    
29. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med.. 1999;340:115–126.[Free Full Text]
    
30. Xu Q, Kleindienst R, Waitz W, et al. Increased expression of heat shock protein 65 coincides with a population of infiltrating T lymphocytes in atherosclerotic lesions of rabbits specifically responding to heat shock protein 65. J Clin Invest.. 1993;91:2693–2702.
    
31. Gruber R, Lederer S, Bechtel U, et al. Increased antibody titers against myobacterial heat-shock protein 65 in patients with vasculitis and arteriosclerosis. Int Arch Allergy Immunol.. 1996;110:95–98.[Medline] [Order article via Infotrieve]
    
32. Mikherjee M, De Benedictis C, Jewitt D, et al. Association of antibodies to heat-shock protein-65 with percutaneous transluminal coronary angioplasty and subsequent restenosis. Thromb Haemost.. 1996;75:258–260.[Medline] [Order article via Infotrieve]
    
33. Hoppichler F, Lechleitner M, Tragweger C, et al. Changes of serum antibodies to heat-shock protein 65 in coronary heart disease and acute myocardial infarction. Atherosclerosis. 1996;126:333–338.[Medline] [Order article via Infotrieve]
    
34. Birnie DH, Holme ER, McKay IC, et al. Association between antibodies to heat shock protein 65 and coronary atherosclerosis: possible mechanism of action of Helicobacter pylori and other bacterial infections in increasing cardiovascular risk. Eur Heart J.. 1998;19:387–394.[Abstract/Free Full Text]
    
35. Xu Q, Schett G, Seitz CS, et al. Surface staining and cytotoxic activity of heat shock protein 60 antibody on stressed aortic endothelial cells. Circ Res. 1994;75:1078–1085.[Abstract/Free Full Text]
    
36. Soltys BJ, Gupta RS. Immunoelectron microscopic localization of the 60-kDa heat shock chaperonin protein (Hsp60) in mammalian cells. Exp Cell Res.. 1996;222:16–27.[Medline] [Order article via Infotrieve]
    
37. Yamaguchi H, Osaki T, Taguchi H, et al. Flow cytometric analysis of the heat shock protein 60 expressed on the cell surface of Helicobacter pylori. J Med Microbiol.. 1996;45:270–277.
    
38. Beatty WL, Morrison RP, Byrne GI. Persistent chlamydiae: from cell culture to a paradigm for chlamydial pathogenesis. Microbiol Rev.. 1994;58:686–699.[Abstract/Free Full Text]
    
39. Young RA, Elliott TJ. Stress proteins, infection, and immune surveillance. Cell. 1989;59:5–8.[Medline] [Order article via Infotrieve]
    
40. Kleindienst R, Xu Q, Willeit J, et al. Immunology of atherosclerosis: demonstration of heat shock protein 60 expression and T-lymphocytes bearing /ß or / receptor in human atherosclerotic lesions. Am J Pathol.. 1993;142:1927–1937.[Abstract]
    
41. Kockx MM. Apoptosis in the atherosclerotic plaque: quantitative and qualitative aspects. Arterioscler Thromb Vasc Biol.. 1998;18:1519–1522.[Abstract/Free Full Text]
    
42. Haunstetter A, Izumo S. Apoptosis: basic mechanisms and implications for cardiovascular disease. Circ Res. 1998;82:1111–1129.[Free Full Text]
    

This article has been cited by other articles:

				H. Loppnow, K. Werdan, and M. Buerke Invited review: Vascular cells contribute to atherosclerosis by cytokine- and innate-immunity-related inflammatory mechanisms Innate Immunity, April 1, 2008; 14(2): 63 - 87. [Abstract] [PDF]

				I. Kardys, N. Rifai, O. Meilhac, J.-B. Michel, J. L. Martin-Ventura, J. E. Buring, P. Libby, and P. M Ridker Plasma Concentration of Heat Shock Protein 27 and Risk of Cardiovascular Disease: A Prospective, Nested Case-Control Study Clin. Chem., January 1, 2008; 54(1): 139 - 146. [Abstract] [Full Text] [PDF]

				K. Udvarnoki, L. Cervenak, K. Uray, F. Hudecz, I. Kacskovics, R. Spallek, M. Singh, G. Fust, and Z. Prohaszka Antibodies against C-Reactive Protein Cross-React with 60-Kilodalton Heat Shock Proteins Clin. Vaccine Immunol., April 1, 2007; 14(4): 335 - 341. [Abstract] [Full Text] [PDF]

				A. Shamaei-Tousi, J. P. Halcox, and B. Henderson Stressing the obvious? Cell stress and cell stress proteins in cardiovascular disease Cardiovasc Res, April 1, 2007; 74(1): 19 - 28. [Abstract] [Full Text] [PDF]

				J. Kim, A. Ogai, S. Nakatani, K. Hashimura, H. Kanzaki, K. Komamura, M. Asakura, H. Asanuma, S. Kitamura, H. Tomoike, et al. Impact of Blockade of Histamine H2 Receptors on Chronic Heart Failure Revealed by Retrospective and Prospective Randomized Studies J. Am. Coll. Cardiol., October 3, 2006; 48(7): 1378 - 1384. [Abstract] [Full Text] [PDF]

				G. Wick The Heat Is on: Heat-Shock Proteins and Atherosclerosis Circulation, August 29, 2006; 114(9): 870 - 872. [Full Text] [PDF]

				H. K. Park, E.-C. Park, S. W. Bae, M. Y. Park, S. W. Kim, H. S. Yoo, M. Tudev, Y. H. Ko, Y.-H. Choi, S. Kim, et al. Expression of Heat Shock Protein 27 in Human Atherosclerotic Plaques and Increased Plasma Level of Heat Shock Protein 27 in Patients With Acute Coronary Syndrome Circulation, August 29, 2006; 114(9): 886 - 893. [Abstract] [Full Text] [PDF]

				B. Henderson, E. Allan, and A. R. M. Coates Stress Wars: the Direct Role of Host and Bacterial Molecular Chaperones in Bacterial Infection Infect. Immun., July 1, 2006; 74(7): 3693 - 3706. [Full Text] [PDF]

				A. Shamaei-Tousi, J. W. Stephens, R. Bin, J. A. Cooper, A. Steptoe, A. R.M. Coates, B. Henderson, and S. E. Humphries Association between plasma levels of heat shock protein 60 and cardiovascular disease in patients with diabetes mellitus Eur. Heart J., July 1, 2006; 27(13): 1565 - 1570. [Abstract] [Full Text] [PDF]

				M. Mayr, S. Kiechl, S. Tsimikas, E. Miller, J. Sheldon, J. Willeit, J. L. Witztum, and Q. Xu Oxidized Low-Density Lipoprotein Autoantibodies, Chronic Infections, and Carotid Atherosclerosis in a Population-Based Study J. Am. Coll. Cardiol., June 20, 2006; 47(12): 2436 - 2443. [Abstract] [Full Text] [PDF]

				L. Romics Jr, G. Szabo, J. C. Coffey, J. H. Wang, and H. P. Redmond The Emerging Role of Toll-Like Receptor Pathways in Surgical Diseases Arch Surg, June 1, 2006; 141(6): 595 - 601. [Abstract] [Full Text] [PDF]

				K Mandal, A R Afzal, S J D Brecker, J Poloniecki, Q Xu, and M Jahangiri Association of serum soluble heat shock protein 60 with toll-like receptor 4 polymorphism and severity of coronary artery disease. Heart, May 1, 2006; 92(5): 683 - 685. [Full Text] [PDF]

				A. Tedgui and Z. Mallat Cytokines in Atherosclerosis: Pathogenic and Regulatory Pathways Physiol Rev, April 1, 2006; 86(2): 515 - 581. [Abstract] [Full Text] [PDF]

				A. V. Ovechkin, N. Tyagi, W. E. Rodriguez, M. R. Hayden, K. S. Moshal, and S. C. Tyagi Role of matrix metalloproteinase-9 in endothelial apoptosis in chronic heart failure in mice J Appl Physiol, December 1, 2005; 99(6): 2398 - 2405. [Abstract] [Full Text] [PDF]

				Q. Xiao, K. Mandal, G. Schett, M. Mayr, G. Wick, F. Oberhollenzer, J. Willeit, S. Kiechl, and Q. Xu Association of Serum-Soluble Heat Shock Protein 60 With Carotid Atherosclerosis: Clinical Significance Determined in a Follow-Up Study Stroke, December 1, 2005; 36(12): 2571 - 2576. [Abstract] [Full Text] [PDF]

				J. P.J. Halcox, J. Deanfield, A. Shamaei-Tousi, B. Henderson, A. Steptoe, A. R.M. Coates, A. Singhal, and A. Lucas Circulating Human Heat Shock Protein 60 in the Blood of Healthy Teenagers: A Novel Determinant of Endothelial Dysfunction and Early Vascular Injury? Arterioscler. Thromb. Vasc. Biol., November 1, 2005; 25(11): e141 - e142. [Full Text] [PDF]

				J. Wang, N. Xu, X. Feng, N. Hou, J. Zhang, X. Cheng, Y. Chen, Y. Zhang, and X. Yang Targeted Disruption of Smad4 in Cardiomyocytes Results in Cardiac Hypertrophy and Heart Failure Circ. Res., October 14, 2005; 97(8): 821 - 828. [Abstract] [Full Text] [PDF]

				K Mandal, G Foteinos, M Jahangiri, and Q Xu Role of antiheat shock protein 60 autoantibodies in atherosclerosis Lupus, September 1, 2005; 14(9): 742 - 746. [Abstract] [PDF]

				A G Pockley and J Frostegard Heat shock proteins in cardiovascular disease and the prognostic value of heat shock protein related measurements Heart, September 1, 2005; 91(9): 1124 - 1126. [Abstract] [Full Text] [PDF]

				F. J. Quintana and I. R. Cohen Heat Shock Proteins as Endogenous Adjuvants in Sterile and Septic Inflammation J. Immunol., September 1, 2005; 175(5): 2777 - 2782. [Abstract] [Full Text] [PDF]

				G. Foteinos, A. R. Afzal, K. Mandal, M. Jahangiri, and Q. Xu Anti-Heat Shock Protein 60 Autoantibodies Induce Atherosclerosis in Apolipoprotein E-Deficient Mice via Endothelial Damage Circulation, August 23, 2005; 112(8): 1206 - 1213. [Abstract] [Full Text] [PDF]

U. Hagg, M. E. Johansson, J. Gronros, A. S. Naylor, I. H. Jonsdottir, G. Bergstrom, P.-A. Svensson, and L.-m. Gan Gene expression profile and aortic vessel distensibility in voluntarily exercised spontaneously hypertensive rats: potential role of heat shock proteins Physiol Genomics, August 11, 2005; 22(3): 319 - 326.