Heat shock protein 70 and antibodies to heat shock protein 60 are associated with cerebrovascular atherosclerosis

    Heat shock protein 70 and antibodies to heat shock protein 60 are associated with cerebrovascular atherosclerosis Ruˇzica Galovi´c, Zlata Flegar-Meˇstri´c, Vinko Vidjak, Mirela Matokanovi´c, Karmela Bariˇsi´c PII: DOI: Reference:

S0009-9120(15)00472-5 doi: 10.1016/j.clinbiochem.2015.10.006 CLB 9155

To appear in:

Clinical Biochemistry

Received date: Revised date: Accepted date:

31 May 2015 5 October 2015 17 October 2015

Please cite this article as: Galovi´c Ruˇzica, Flegar-Meˇstri´c Zlata, Vidjak Vinko, Matokanovi´c Mirela, Bariˇsi´c Karmela, Heat shock protein 70 and antibodies to heat shock protein 60 are associated with cerebrovascular atherosclerosis, Clinical Biochemistry (2015), doi: 10.1016/j.clinbiochem.2015.10.006

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ACCEPTED MANUSCRIPT HEAT SHOCK PROTEIN 70 AND ANTIBODIES TO HEAT SHOCK PROTEIN 60 ARE ASSOCIATED

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WITH CEREBROVASCULAR ATHEROSCLEROSIS

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Ružica Galovida, Zlata Flegar-Meštridb, Vinko Vidjakc, Mirela Matokanovidd, Karmela Barišidd

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University Hospital Centre Zagreb, Clinical Department of Laboratory Diagnostics,

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Kišpatideva 12, 10000 Zagreb, Croatia b

University Hospital Merkur, Institute of Clinical Chemistry and Laboratory Medicine, Zajceva

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19, 10000 Zagreb, Croatia c

University Hospital Merkur, Institute of Radiology, Zajceva 19, 10000 Zagreb, Croatia

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University of Zagreb, Faculty of Pharmacy and Biochemistry, Dept. of Medical Biochemistry

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and Hematology, Domagojeva 2, 10000 Zagreb, Croatia

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Corresponding author: Ružica Galovid

University Hospital Centre Zagreb Department of Laboratory Diagnostics Kišpatideva 12, 10000 Zagreb, Croatia Tel.: +385 91 2226 226 e-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract Objectives:

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Heat shock proteins (Hsps) are produced by all cells, including vascular, to ensure stress

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protection. Damaged cells release Hsps in their local environment and systemic circulation.

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The aim of this study was to investigate the involvement and prognostic utility of serum Hsp60 and Hsp70, and the respective antibodies anti-Hsp60 and anti-Hsp70 in subjects with

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advanced atherosclerosis resulting in high degree of cerebrovascular stenosis. Design and Methods:

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Ultrasound doppler examination of carotid arteries was used to discriminate between control and cerebrovascular atherosclerosis subjects. Twenty eight subjects without carotid

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obstruction were selected as controls. Fifty patients with obstruction of cerebrovascular

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blood flow were evaluated for the degree of stenosis of cerebral arteries by digital

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subtraction angiography. In parallel, serum concentrations of Hsp60, Hsp70, anti-Hsp60 and anti-Hsp70 were measured by ELISA kits.

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Results:

Anti-Hsp60 was significantly higher (P=0.003) in the atherosclerosis group than in the control group (23.62 ng/L vs. 15.28 ng/L, respectively, expressed as median). Circulating Hsp70 was lower in the atherosclerosis than in the control group (P=0.048), with respective median values of 0.00 µg/L vs. 0.22 µg/L). Concentrations of Hsp60 and anti-Hsp70 did not differ significantly between the control and atherosclerosis group. Conclusions: Higher circulating anti-Hsp60 is associated with advanced cerebrovascular atherosclerosis as a consequence of the autoimmunity part of the inflammation and bursting of 2

ACCEPTED MANUSCRIPT atherosclerosis. Higher levels of Hsp70 observed in the control group could be protective in

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the development of atherosclerotic changes.

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Keywords: cerebrovascular stenosis, atherosclerosis, heat shock proteins, antibodies

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ACCEPTED MANUSCRIPT 1.

Introduction

In the pathogenesis of atherosclerosis, both inflammatory and immune components seem to

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be involved [1,2]. The inflammatory component of atherosclerosis might involve immune

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reactivity to heat shock proteins [2]. As a part of atherogenesis, vascular cells and tissues are

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exposed to various unfavourable conditions such are mechanical injury, oxidized LDL, inflammation and cytokine stimulation. Under these conditions, heat shock proteins (Hsps)

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are produced by the affected cells to ensure their protection [3]. Members of Hsp70 family and Hsp60 family assist in correct folding of newly synthesized or stress denatured proteins,

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refolding of misfolded and aggregated proteins, membrane translocation of organellar and secretory proteins, and control of the activity of regulatory proteins [4,5]. Antibodies against

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microbial Hsp60 family were previously linked to atherosclerosis due to their cross-reacting

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abilities [6,7], but autoantibodies against Hsp60 developed by other, non-infectious

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inducers, are found to be even stronger contributors to endothelial cell damage [8,9]. Both Hsps and their antibodies are in certain concentrations normally present in peripheral

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circulation, but they are abundantly released during arterial cell damage and destruction. Several studies have indicated possible association of Hsp60, Hsp70, anti-Hsp60 and antiHsp70 with the pathogenesis, progression, severity and prognosis of atherosclerosis or cardiovascular diseases [8-14]. Literature data regarding serum levels of Hsp60, Hsp70 and respective antibodies are variable depending on type and stage (acute phase, chronicity) of vascular disease. Here, we tried to elucidate an additional segment of this disease by examining serum levels of Hsp60, Hsp70 and respective antibodies in atherosclerotic patients with established stenosis of cerebral arteries.

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ACCEPTED MANUSCRIPT 2.

Subjects and Methods

2.1 Subjects

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The study group included 50 patients, 15 women and 35 men, with symptoms of

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cerebrovascular insufficiency and stenosis of carotid artery of more than 50% of the lumen.

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The presence of manifest coronary atherosclerosis was excluded by physical and electrocardiographic examination. After duplex ultrasound examination, the degree of

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carotid artery stenosis at angiography was measured according to standard method [15]. The control group consisted of 28 people, 15 women and 13 men, with normal doppler

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examination of carotid arteries and excluded coronary atherosclerosis by physical and

not under therapy with statins.

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electrocardiographic examination. They did not have a history of diabetes mellitus and were

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All participants were interviewed, data on diabetes mellitus, drug consummation and

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smoking were recorded. Smokers were defined as those reporting daily smoking. Obesity was defined in terms of patient’s body mass index (BMI) and the patients with a BMI≥25

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were considered overweight [16]. Informed consent was obtained from all subjects according to the guidelines of the Ethics Committee of the Merkur University Hospital. 2.2 Methods

Serum concentrations of Hsp60, Hsp70, anti-Hsp60 and anti-Hsp70 were determined from sera which were fresh frozen on -80°C, using commercially available ELISA kits (Stressgen Assay Designs, USA: Hsp60 ELISA Kit, Hsp70 High Sensitivity EIA Kit, Anti-Human Hsp60 (total) ELISA Kit, and Anti-Human Hsp70 (total) ELISA Kit). All measurements concerning metabolic status (total cholesterol, HDL and LDL cholesterol, triacylglycerol) and C-reactive protein (CRP) were performed using routine clinical laboratory 5

ACCEPTED MANUSCRIPT methods accredited according to ISO 15189, using fresh sera. Subjects with CRP values above reference interval (>10 mg/L) were not included in any of the study groups because of

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the possible presence of an active inflammatory disease [16].

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Data sets were described using non-parametric presentation. Significance of the differences

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between the groups was determined using Mann-Whitney U-test. The Χ2 significance test was used to analyze qualitative variables. Correlations were analyzed by Spearman

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correlation coefficient. All P values presented are two-tailed, and P values below 0.050 were considered to reflect statistically significant differences. Analyses were performed using

Results

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3.

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MedCalc Software 11.4.4 (Mariakerke, Belgium).

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The general characteristics of the study groups are presented in Table 1. The groups differ

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significantly regarding age, but could be considered as age relevant because the influence of age on the main tested parameters was excluded by correlation testing (Hsp60, Hsp70, anti-

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Hsp60, anti-Hsp70; P>0.050 for all of the correlation coefficients, results presented in Table 2). As expected, the groups differ significantly regarding statin therapy, as well as diabetes mellitus, but those were included in the selection criteria for the groups. Statin therapy and diabetes mellitus did not influence main tested parameters (Table 2). The groups were not different regarding other general characteristics (gender, obesity, and smoking habits). In the present study, Hsp60 levels were not different between the tested groups, but the difference was observed for anti-Hsp60, which was markedly higher in the cerebrovascular atherosclerosis group than in the control group (Table 3). Circulating Hsp70 was lower in the cerebrovascular atherosclerosis group than in the control group (Table 3). There was no 6

ACCEPTED MANUSCRIPT significant difference in anti-Hsp70 levels. Circulating Hsp60 was detected in 68% of all subjects (74% in patients with cerebrovascular atherosclerosis and 54% of the control

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subjects), and Hsp70 in 40% of all subjects (32% in patients with cerebrovascular

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atherosclerosis and 60% of the control subjects). Anti-Hsp60 and anti-Hsp70 antibodies were

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detected in all of the subjects. Serum levels of Hsp60, Hsp70, anti-Hsp60 and anti-Hsp70 were independent from age, diabetes and statin therapy (Table 2). Distributions of

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proportions across the quartiles were examined for Hsps and respective antibodies. There was no difference in distribution of proportions for Hsp60. The proportions of subjects of the

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tested groups were differently distributed regarding anti-Hsp60 antibody (Χ2=26.5, P<0.0001). Higher proportions of subjects from the control group could be found below the

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median value, in the first and the second quartile, whereas the proportion of the

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cerebrovascular atherosclerosis patients dominates above the median value, within the third

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and the fourth quartile (Figure 1). The proportions of subjects of the tested groups were also differently distributed across the quartiles of whole population regarding Hsp70 (Χ2=12.1,

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P=0.0005), concerning the third quartile of the whole subject population as a layout (Figure 2). No differences in distribution of proportions across the quartiles were found for antiHsp70.

Comparing the results obtained for the metabolic risk factors (total cholesterol, HDL and LDL cholesterol, triacylglycerol) between the group of patients with cerebrovascular atherosclerosis and the control group, significant differences were found for total cholesterol, HDL cholesterol and LDL cholesterol; all were lower in the group of patients with cerebrovascular atherosclerosis than in the control group (Table 1). Most of the patients included in the cerebrovascular atherosclerosis group (74%) were on statin therapy, 7

ACCEPTED MANUSCRIPT previously recognized as having potential risk for vascular diseases because of their lipid status. Therefore, lower levels of total and LDL cholesterol in the cerebrovascular

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atherosclerosis group of patients could be attributed to statin therapy. Although generally

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expected to be slightly increased from its basic level due to the statin therapy [17], levels of

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HDL cholesterol were still lower in cerebrovascular atherosclerosis than in the control group,

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confirming lower HDL cholesterol as a well known risk factor.

Discussion

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In its nature, atherosclerosis is an inflammatory disease [18] that can have cardiovascular, peripheral or cerebrovascular localization [19]. Inflammatory component of atherosclerosis,

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including Hsps as participants in inflammation and innate immune response has been widely

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investigated. Hirono et al. [20] explained possible role of Hsp60 in atherogenesis at the

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cellular level. They observed proliferation of vascular smooth muscle cells exposed to Chlamydia pneumoniae and overexpression of Hsp60 in these cells, proposing that

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overexpression of endogenous Hsp60 may be a central intracellular event responsible for the mitogenic effects induced by infectious agents and other atherogenic risk factors, thus stimulating vascular smooth muscle cells proliferation and contributing to the lesion formation through the induction of Hsp60. Protective role of Hsp70 was proved by Johnson et al. [21] on stressed aortic cells in vitro. Wendling et al. [22] proposed that Hsp70 could serve as an anti-inflammatory agent by inducing regulatory T-cells reactivity to self-Hsp70 epitopes thus down-regulating inflammatory disease by inducing Th2 cells to produce antiinflammatory cytokines.

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ACCEPTED MANUSCRIPT Several studies have reported possible association of heat shock proteins and/or antibodies to heat shock proteins with cardiovascular diseases [8-14, 23-25]. In their studies, Pockley et

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al. found that the increased level of serum Hsp60 was associated with early stage of

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cardiovascular disease that could be involved in induction/progression of atherosclerotic

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lesions and hypertension, while low circulating levels of Hsp70 could predict the development of atherosclerosis in patients with established hypertension, with possibility

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that Hsp70 protects against or modifies the progression of atherosclerosis of carotid arteries [10,11,13]. The protective role of increased Hsp70 in coronary artery disease was also

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confirmed by Zhu et al. [14]. Differently, Zhang et al. found that increased Hsp70 and decreased anti-Hsp70 are independently associated with an increased severity of coronary

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heart disease [12]. Increased serum levels of antibodies against Hsp60 demonstrated

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significant association with the presence and severity of coronary artery disease [8].

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In this study, we wanted to find whether the proposed influences of Hsp60, Hsp70 and respective antibodies could be attributed to advanced atherosclerosis presented with

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cerebrovascular stenosis, which is a widespread disease with stroke as a serious consequence. The study group consisted of patients with more than 50% stenosis of cerebral arteries, corresponding to moderate to high classification of cerebrovascular stenosis [26]. Although both patients and control group subjects had no clinical manifestations of atherosclerotic disease in other vascular beds, general atherosclerosis at certain level could not be completely excluded. The findings in our study, performed on the patients with established atherosclerosis where we did not find the difference in serum concentrations of Hsp60 from the control group, do not presume that Hsp60 could not possibly be involved in early pathogenesis of vascular 9

ACCEPTED MANUSCRIPT disorders, as proposed by Pockley et al [11]. It could be expected that Hsp60 is intensively released in the circulation in response to cardiovascular injury, as proved by Schett et al. [23]

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in the case of myocardial ischemia and injury, causing immunological response in the early

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stages of atherosclerosis development [24,25]. As the consequence of such an immunologic

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reactivity, anti-Hsp60 antibodies could possibly also be present in later stages of vascular diseases, as supported by our result for elevated serum anti-Hsp60 in the later stage of

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cerebrovascular disease presented in this study.

We found lower levels of circulating Hsp70 in patients with cerebrovascular atherosclerosis

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than in the control group. This finding is in line with extensive investigation of the protective and predictive role of Hsp70 in atherosclerosis of carotid arteries performed by Pockley et al.

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[10,11,13]. Available literature findings describe involvement of increased autoantibodies

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against Hsp70 in the progression of atherosclerosis [27]. In our study, circulating levels of

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anti-Hsp70 antibodies did not differ from the control group, suggesting that autoimmune response to Hsp70 did not participate in the pathogenesis of atherosclerosis within the

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group of affected subjects. 4.1 Conclusions

As there are varieties of atherosclerotic disease, in different manifestations and especially in different stages diverse results could be found for Hsp60, Hsp70, anti-Hsp60 and anti-Hsp70 [8,10-14,23,24]. It could be concluded that heat shock proteins and related autoantibodies play important role in this disease with complex pathogenesis. This study showed that serum concentrations of anti-Hsp60 and Hsp70 differ in patients with advanced atherosclerosis from those not having the disease, with the possibility to serve as potential prognostic markers. 10

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Acknowledgments

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This study was supported by the Ministry of Science, Technology and Sports of the Republic

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of Croatia.

Conflict of Interests

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The authors declare that there is no conflict of interest regarding the publication of this

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paper.

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isolated aortic cells from normal and atherosclerotic cynomolgus macaques. Atherosclerosis 1990;84:111-119.

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mycobacterial heat shock protein (hsp) 70 sequence prevents adjuvant arthritis upon nasal administration and induces IL-10-producing T cells that cross-react with the

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mammalian self-hsp70 homologue. J Immunol 2000;164:5560-5565.

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injury leads to a release of heat shock protein (hsp) 60 and a suppression of the antihsp65 immune response. Cardiovasc Res 1999;42:685–695.

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ACCEPTED MANUSCRIPT 27. Leng X, Zhan R, Wang Y, Liu X, Gong J, Gao X, Wu L, Wang L, Zhao Y, Wang X, Zhang Z, Pang W, Qian L: Anti-heat shock protein 70 autoantibody epitope changes and BD091

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promotes atherosclerosis in rats. Cell Stress Chaperones 2010;15:947–958.

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ACCEPTED MANUSCRIPT Table 1. Anthropometric and biochemical parameters for the control and cerebrovascular atherosclerosis group of patients (>50% stenosis of cerebral arteries).

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Cerebrovascular atherosclerosis group (n=28)

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Control group

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66.5 (55-83)

<0.001*

58.0 (50-65)

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Age (years; median, range)

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(n=50)

13 (46 %)

35 (70 %)

0.073

Obesity, BMI >25 kg/m2 (n; %)

17 (61 %)

32 (64 %)

0.965

0

37 (74 %)

<0.001*

0

19 (38 %)

<0.001*

5 (18 %)

11 (22 %)

0.887

1.4 (1.1-1.7)

1.5 (1.1-2.0)

0.300

6.3 (5.9-6.8)

4.8 (3.8-5.9)

<0.001*

1.5 (1.4-2.0)

1.3 (1.1-1.5)

<0.001*

4.0 (3.4-4.4)

2.7 (2.0-3.7)

<0.001*

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Gender, male (n; %)

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Smoking (n; %)

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Diabetes mellitus (n; %)

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Statin therapy (n; %)

Triacylglycerol (mmol/L;

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median, IQRa)

Total cholesterol (mmol/L; median, IQRa) HDL-cholesterol (mmol/L; a

median, IQR ) LDL-cholesterol (mmol/L; a

median, IQR )

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ACCEPTED MANUSCRIPT CRP (mg/L; median, IQRa)

1.9 (1.2-2.7)

2.6 (1.4-4.6)

0.085

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IQR = interquartile range

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*Statistically significant difference between the control group and the cerebrovascular

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atherosclerosis group (P<0.050).

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ACCEPTED MANUSCRIPT Table 2. Results of Spearman correlation testing for relationships between age, statin therapy or diabetes mellitus and tested parameters in serum of all subjects.

P

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Hsp60

0.069

0.546

-0.088

Hsp70

-0.125

0.273

anti-Hsp60

0.150

0.210

anti-Hsp70

0.187

0.100

P

r

P

0.447

-0.158

0.169

-0.082

0.473

-0.148

0.195

0.209

0.098

0.174

0.126

0.050

0.664

-0.047

0.679

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r

Diabetes mellitus

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Statin therapy

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Age

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Test

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Significant correlations are considered with P<0.050.

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ACCEPTED MANUSCRIPT Table 3. Values of Hsp60, Hsp70, anti-Hsp60 and anti-Hsp70 in sera of control examinees and patients with cerebrovascular atherosclerosis.

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Cerebrovascular

Control group (n=28)

Median (IQRa)

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13.78 (0.00-31.84)

0.673

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Median (IQRa)

(n=50)

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Tested parameter

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atherosclerosis group

14.24 (0.00-31.81)

Hsp70 (µg/L)

0.22 (0.00-1.27)

0.00 (0.00-0.25)

0.048*

anti-Hsp60 (ng/L)

15.28 (10.05-22.48)

23.62 (17.84-34.93)

0.003*

anti-Hsp70 (ng/L)

83.49 (61.84-114.10)

83.42 (64.91-123.99)

1.000

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Hsp60 (µg/L)

IQR = interquartile range

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*Statistically significant difference between the control group and the cerebrovascular atherosclerosis group (P<0.050).

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ACCEPTED MANUSCRIPT Figure 1. Distribution of control subjects and cerebrovascular atherosclerosis subjects regarding anti-Hsp60, presented as a proportion (%) from the total number of subjects of the

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particular group across the quartiles of the whole tested population (Q1=12.35 ng/L,

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Q2=19.70 ng/L, Q3=32.81 ng/L).

Figure 2. Distribution of control subjects and cerebrovascular atherosclerosis subjects

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regarding Hsp70, presented as a proportion (%) from the total number of subjects of the

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particular group across the quartiles of the whole tested population (Q1=0.00 µg/L, Q2=0.00

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µg/L, Q3=0.77 µg/L).

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Fig. 1

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Fig. 2

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ACCEPTED MANUSCRIPT Highlights

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Heat shock proteins (Hsps) play important role in atherosclerosis. Hsps and related autoantibodies are possible prognostic markers in atherosclerosis. Anti-Hsp60 is an autoimmunity part of atherosclerotic disease. Hsp70 could be protective in the development of atherosclerotic changes.

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