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(Hypertension. 1997;29:40.)
© 1997 American Heart Association, Inc.
Research Articles (Issue 1, Part 1) |
Association of Serum Antibodies to Heat-Shock Protein 65 With Borderline Hypertension
the Department of Medicine, Unit of Rheumatology (J.F.), and Department of Medicine, Unit of Cardiovascular Medicine (C.L., U. de F.), Karolinska Hospital, Stockholm; Calab (B.A.), Stockholm, Sweden; Faculty of Veterinary Science, Utrecht (the Netherlands) University (R. van der Z.); Department of Immunology, Karolinska Institutet (R.K.); and Department of Epidemiology, Institute of Environmental Medicine, Karolinska Institutet (U. de F.), Stockholm, Sweden.
Correspondence to Johan Frostegård, Department of Medicine, Division of Rheumatology, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail johanf@rheum.ks.se.
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Abstract |
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Heat-shock proteins protect cells from damage but are also often the target of immune responses in inflammation and may therefore both induce and perpetuate the chronic inflammation characterizing atherosclerosis. Hypertension is a well-established risk factor for atherosclerosis, and recently, borderline hypertension also has been related to atherosclerosis. The present study investigated the possible role of heat-shock proteins in borderline hypertension and their relation to atherosclerosis by investigating antibody titers against the 65-kD heat-shock protein (HSP65). Sixty-six men with borderline hypertension and 67 age-matched normotensive men (diastolic pressure, 85 to 94 and <80 mm Hg, respectively) were recruited from a population screening program. Titers of antibodies to HSP65 were determined by enzyme-linked immunosorbent assay. The presence of carotid atherosclerosis was determined by B-mode ultrasonography. Twenty-seven individuals had atherosclerotic plaques; 48 were smokers (more than one to two cigarettes per day). Borderline hypertensive men had higher anti-HSP65 reactivity than normotensive control subjects (P=.034). Smokers with atherosclerosis had low levels of antibodies to HSP65 compared with nonsmokers with atherosclerosis (P=.002). Furthermore, when high-risk individuals (borderline hypertension plus plaque, n=15) were compared with matched low-risk individuals (normotensive with no plaque, n=15), the high-risk men had significantly enhanced antibody titers to HSP65 (P=.041). In conclusion, we demonstrate that serum antibody titers to HSP65 are enhanced in individuals with borderline hypertension, which may indicate an ongoing immune reaction in the artery wall.
Key Words: heat-shock proteins • hypertension, borderline • atherosclerosis
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Introduction |
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Heat-shock proteins (HSPs) are a group of proteins with a highly conserved structure induced by various stress stimuli such as heat, infection, and toxic substances.1 An important function of HSPs is protection against cell damage.2 Furthermore, HSPs exert essential cell functions, including facilitating protein translocation across membranes,3 binding hormone receptors,4 and functioning as chaperones during protein folding.5 HSPs are often the target of humoral and T cell–mediated immune responses to infections and may provide a link between the immune response to infection and autoimmunity caused by T lymphocyte cross-reactivity between bacterial and human HSP65.6 Enhanced antibody titers to HSP65 have been described in several autoimmune and chronic inflammatory diseases, including rheumatoid arthritis,7 juvenile arthritis,8 type I diabetes,9 systemic sclerosis,10 and psoriasis.11
The most important risk factors for the development of atherosclerosis include hyperlipidemia, smoking, and hypertension, although the exact etiologic relationship between risk factors and the development of atherosclerosis is not known.12 Recently, BHT has also been demonstrated to be associated with increased atherosclerosis.13 Evidence has accumulated demonstrating that atherosclerosis is a chronic inflammatory process, in which monocytes and T lymphocytes play an important role. OxLDL is generally believed to be an important factor in the early development of atherosclerosis. Both T lymphocytes and monocytes show signs of activation, and monocytes differentiate into macrophages, which ingest oxLDL and develop into foam cells.14 15 OxLDL is toxic at high concentrations and has attracted attention as a possible candidate for initial damage to the endothelium, leading to atherosclerosis.16 17 However, low concentrations of oxLDL have other atherogenic properties, including activation of immune-competent cells18 19 20 and enhanced adhesive properties of endothelial cells.21 22 23 Furthermore, oxLDL induces HSP65 in monocytic cells.24 An immune reaction to HSP65 induced by oxLDL may thus be atherogenic by inducing and/or perpetuating a chronic inflammation in the artery wall.
Recently, HSPs have been implicated as an important factor in the development of atherosclerosis. Enhanced antibody titers to HSP65 have been reported to be correlated with carotid atherosclerosis,25 and immunization of rabbits with HSP65 induced aggravated atherosclerosis.26 To investigate the role of HSP65 in BHT, we studied a group of 133 middle-aged men, comparing BHT subjects with age-matched control NT subjects. We demonstrate that serum antibody titers to HSP65 are enhanced in BHT subjects. The possible implications for hypertension and atherogenesis are discussed.
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Methods |
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Study Groups
In 1985, a BP screening program was started in Åkersberga, a small community 35 km north of Stockholm. All men aged 35 to 55 years were asked by mail to visit the primary healthcare center and have their BP measured. As has been described earlier,13 193 individuals with BHT, defined as diastolic BP of 85 to 94 mm Hg, were identified. These individuals were followed up with yearly visits for 3 years. At these follow-up visits, approximately 20% of the subjects became hypertensive and 20% normotensive, with the major change (13% to 15%) occurring already at the 1-year follow-up visit. After 3 years, 81 men were still within the range for BHT on the basis of repeated measurements over the entire time period.
These 81 BHT individuals were invited to participate in the present investigation, together with 80 age-matched male control subjects from the original population who had a diastolic BP less than or equal to 80 mm Hg at the initial measurement. To obtain 80 age-matched control subjects, we asked 105 NT men to participate; 23 declined to participate and 2 had a diastolic BP above 80 mm Hg. The BP of the control subjects was measured on two occasions a few weeks apart. For participation in the study, diastolic BP had to be less than or equal to 80 mm Hg on both occasions. All BP measurements during the entire recruitment and study periods were performed by the same specially trained nurse.
The study was approved by the local Ethics Committee of Karolinska Hospital and was conducted in accordance with the Helsinki Declaration. All subjects gave their informed consent before entering the program, of which this study was a part. Of the 81 BHT and 80 NT control subjects who agreed to participate in the program, 66 in the BHT and 67 in the NT group completed all procedures of the present study. None of the subjects had any other illnesses or were using any drugs known to influence BP or metabolic variables.
Study Program
All subjects were investigated according to the same schedule. BHT men and their NT counterparts were investigated simultaneously when possible and no more than 4 weeks apart. Blood samples for analyses of basic metabolic variables and HSP were taken between 8 and 9:30 AM after subjects had fasted 8 to 12 hours. All samples were drawn after subjects had rested 15 minutes in the supine position.
Analysis of Total Serum Immunoglobulin Levels
Serum immunoglobulins—IgG, IgM, and IgA—were determined by immunoturbidimetry. Specific anti-IgG, anti-IgM, and anti-IgA reagents and calibrators were obtained from Dako. The turbidimetric reaction was measured in a Hitachi 911 analyzer by measurement of light transmission at a wavelength of 340 nm.
Analysis of Antibodies to HSP
Mycobacterium bovis BCG hsp65 was expressed from a recombinant Escherichia coli K12 strain harboring plasmid pRIB130027 and was purified as described.28 Lysates from nontransfected E coli were not detected by serum, as opposed to transfected E coli, as determined by Western blot (data not shown). Antibody titers to HSP65 were determined by enzyme-linked immunosorbent assay (ELISA). Microtiter wells (Dynatech Billingshurst) were coated with 1 µg/mL HSP65 in phosphate-buffered saline (PBS, pH 74), 100 µL per well, at 4°C overnight. The wells were washed with 0.02% PBS-Tween and blocked with 1% bovine serum albumin in PBS for 1 hour at room temperature. After washing, the wells were incubated for 1 hour at 37°C with serum samples at dilutions of 1:10, 1:100, and 1:1000. After washing, the bound IgG was determined with alkaline phosphatase–conjugated rabbit anti–human IgG (Dako) and the substrate p-Nitrophenylphosphate (Sigma Chemical Co). The reaction was quantified at 405/490 nm in a Dynatech MR7000 microplate reader. Results are expressed as OD values corrected for background absorbance of serum-free wells. At the 1:10 dilution, all sera showed maximal OD values, and at the 1:1000 dilution, all sera showed background values very near zero. At the 1:100 dilution, differences between sera were observed, so values from this dilution were used for comparison between investigated risk groups and control subjects.
Analysis of Plasma Lipoprotein and Insulin Levels
For determinations of very-low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein cholesterol and triglycerides, blood was allowed to clot for 2 hours at room temperature, and serum was then recovered after centrifugation at 5000g. The major plasma lipoproteins were determined by a combination of preparative ultracentrifugation followed by lipid analyses in the lipoprotein fractions as previously described.29 Venous blood samples for determination of blood glucose (Kodak Ectachem) and plasma insulin (radioimmunoassay, Kabi Pharmacia) were taken.
BP Measurements
An identical procedure was followed at each occasion during the entire recruitment period. All BP measurements were performed with a mercury sphygmomanometer. The cuff was adjusted according to the circumference of the arm and placed at heart level. BP was recorded as the mean of two measurements taken after subjects had rested 5 minutes in the supine position. Systolic and diastolic BPs were defined according to Korotkoff phases I and V, respectively. The same specially trained nurse performed the measurements at each occasion.
Carotid Ultrasound
The right and left carotid arteries were examined with a duplex scanner (Acuson 128XP/5) using a 7.0-MHz linear array transducer. The subjects were investigated while in the supine position with the head slightly turned from the sonographer, as described earlier.13 Plaque was defined as localized intimal-medial thickening, with a thickness greater than 1 mm and a 100% increase in thickness compared with normal, adjacent wall segments. Plaque occurrence was scored as present or absent. The cutoff point of 1 mm was based on results from a pilot study in newly diagnosed, untreated hypertensive men and control subjects without other cardiovascular risk factors recruited from the same population screening as in the present study. In the pilot study, none of the participants had intimal-medial thicknesses exceeding 1 mm.30 Plaque was screened for in the common, internal, and external carotid arteries on both sides.
Body Stature
All subjects were weighed without clothing other than underwear, using the same scale (Delta 707, SECA). Height was measured with a special ruler fixed to the wall. Waist circumference was measured at the level of the umbilicus, and the hips were measured at the level of the greatest circumference. Body mass index was calculated as weight (kilograms) divided by height (meters squared).
Statistical Methods
Variables were tested for skewness. For skewed variables, nonparametric tests were used for comparisons between groups (Mann-Whitney U test), whereas Student's t test was used for normally distributed variables. Categorical variables were compared with the 
2 test. Spearman rank correlation coefficients were calculated for estimation of interrelations between HSP, metabolic variables, and BP levels. Stepwise multiple regression was performed with a value of F=4 to enter. The significance level was put at a value of P<.05. Values in the text are given as mean±SD.
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Characteristics of Cases and Control Subjects
Basic characteristics of the two study groups are presented in Table 1. The two groups were well matched for age. BP levels in the two groups were clearly different from each other, being 125/75 mm Hg in the NT group and 141/89 in the BHT group. The BHT group had a significantly higher body mass index, with a somewhat more pronounced central obesity, as evidenced by a slightly but significantly higher waist-hip ratio.
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As presented earlier,31 the BHT men had a significantly altered metabolic profile, with fasting hyperinsulinemia and dyslipoproteinemia (Table 1). In the BHT group, 26% of the subjects had plaque on one or both sides. The corresponding figure for the NT group was 16% (17 versus 10 subjects, P=NS).
Anti-HSP Titers
In the material as a whole, anti-HSP levels were significantly higher in the BHT group than the NT group (Table 2). There was no difference between individuals with plaque (n=27) and those without (n=106).
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When the subjects with established vascular changes irrespective of BP status (ie, BHT or NT men with plaque present in the carotids) and their age-matched partners were selected (n=27+27), the BHT group again exhibited significantly higher levels of anti-HSP titers (Table 2). In addition, smokers had significantly lower levels of anti-HSP titers compared with nonsmokers (0.28±0.20 versus 0.58±0.41, P=.0023). When the high-risk subjects (BHT men with plaque) and age-matched low-risk subjects (NT men without plaque, n=15+15) were compared, the same pattern remained, with significantly higher anti-HSP levels in the BHT subjects (Table 2) and lower HSP levels in smokers (0.31±0.22 versus 0.55±0.34, P=.036).
In the subjects without plaque, anti-HSP levels did not differ significantly between BHT and NT subjects (Table 2). There were also no differences between smokers and nonsmokers (0.53±0.38 versus 0.50±0.40).
To exclude the possibility that differences in antibody titers simply reflected enhanced total antibody titers, we determined IgA, IgG, and IgM levels. There was no difference between the BHT and NT groups (IgA, 0.91±0.46 versus 0.99±0.64; IgG, 9.71±1.86 versus 9.76±2.31; IgM, 2.25±0.81 versus 2.1±0.88, respectively).
Relations Between HSP, BP Levels, Plaque Occurrence, and Metabolic Variables
In the material as a whole and the two groups separately, there were no significant correlations between HSP and BP levels. There were also no significant correlations between HSP and intimal-medial thickness or metabolic variables (ie, lipoproteins, insulin, body mass index, and waist-hip ratio).
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Discussion |
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The main finding of this study is that BHT was significantly associated with enhanced titers to HSP65. Total antibody concentration showed no difference between control and BHT subjects, indicating that the results obtained are specific. Individuals with BHT and the presence of carotid atherosclerosis had significantly enhanced antibody titers to HSP65 compared with age-matched NT control subjects without carotid atherosclerosis, but HSP65 titers in all individuals with atherosclerosis, irrespective of BP status, did not differ from those in subjects without carotid plaque.
The statistical association between hypertension and atherosclerosis is well documented,12 13 and hypertension may also be etiologically related to atherosclerosis, as indicated by experiments with animals in which hypertension per se induced atherosclerosis.32 33 The distribution of atherosclerosis in the vascular system is limited to areas exposed to mechanical stress such as turbulent flow.34 35 Enhanced expression of HSP70, another member of the HSP family, has been detected in both cultured cells and organs from hypertensive experimental animals.36 37 Recent findings indicate that HSP65 plays an important role in the development of atherosclerosis. Recently, HSP65 has been implicated as an important factor in the development of atherosclerosis, since titers of antibodies to HSP65 correlate with atherosclerosis in individuals older than 60 years.25 Immunization of rabbits with hsp65 induced enhanced atherosclerosis.26 Furthermore, HSP65 expression in the vascular system has been demonstrated in endothelial cells at critical hemodynamically stressed sites.38 Since HSP65 is also induced by stress, including mechanical stress, an intriguing possibility is that HSP65 expression may be further enhanced by hypertension in these vascular segments, leading to an exaggerated immune reaction to HSP65, prompted by continuously enhanced expression of HSP65. Our findings are therefore likely to indicate an ongoing immune reaction in the artery wall, which may represent early changes related to atherosclerosis.
Antibodies to HSP65 have recently been demonstrated to be cytotoxic to stressed endothelial cells.39 These antibodies may thus also be atherogenic by inducing injury to the endothelium. Taken together, recent findings indicate that an enhanced expression of HSP65 is associated with the distribution of atherosclerosis in the vascular system.
Recently, Xu et al25 detected enhanced antibody titers to HSP65 in age groups over 60 years with carotid atherosclerosis as detected by ultrasonography, but no such relationship was found at younger ages. Our subjects were between 35 and 60 years old, and we thus confirm these negative findings in the age group studied. However, ultrasonography does not easily detect the earliest stages of atherosclerosis.
Smokers with atherosclerosis and BHT had markedly decreased antibody titers to HSP65 compared with age-matched smoking NT control subjects (P=.002), but in nonsmokers, no such relationship was detected. Neither lipoprotein profile nor age, two other important risk factors for atherosclerosis, was associated with HSP65 titers. The finding that smoking was associated with decreased anti-HSP65 titers is intriguing. The relationship between atherosclerosis and smoking is well documented.35 HSPs have various functions, but one of the most important is generally believed to be protection against damage. Cells exposed to obnoxious agents, such as free radicals and oxygen stress, increase their survival if preexposed to sublethal stress, which induces the protective HSPs.1 2 According to the response-to-injury hypothesis, atherosclerosis is induced as a response to injury to the vascular endothelium,16 and thus, a decreased expression of HSP65 in endothelial cells may in principle be atherogenic because of a decreased ability to resist stress. If anti-HSP65 titers thus reflect defective stress-induced expression of HSP65, decreased levels of HSP65 antibodies may be of prognostic value in smokers. Available information about decreased HSP65 response after stress is scarce, but enhanced HSP expression may be related to decreased tumor development in experimental animals.40 Whether components in cigarette smoke contain HSP65-inhibiting agents41 is not known, but if this is the case, these findings may also indicate a mechanism that may play a role in the induction of tumors in smokers. In principle, it is also possible that smoking-induced release of HSP65 leads to the formation of immune complexes with HSP65 antibodies and a subsequent decrease of antibody titers.
Taken together, our data indicate that the role of HSP65 in atherogenesis is complex and its significance may vary among different risk factors. In BHT individuals, HSP65 may be overexpressed because of enhanced stress to the artery wall, leading to an exaggerated immune reaction to HSP65, which in turn leads to the development of atherosclerosis. On the other hand, decreased antibody titers to HSP65, as detected in the present study in smokers with atherosclerosis, may indicate low levels of HSP65 and thus increased susceptibility to injury and enhanced risk for atherosclerosis. The ultimate outcome may depend on both the balance of the immune reactions initiated and the disease stage.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by the Swedish Medical Research Council, the Swedish Heart Lung Foundation, the Foundation for Old Servants, the King Gustaf V 80th Birthday Fund, the Swedish Society of Medicine, the Ostermans Fund, The Swedish Association Against Rheumatism, the Wibergs Fund, and the Nanna Svartz Fund. We are grateful to Ulla Hellmark Augustsson for her help with BP measurements over the years and for help with blood sampling, to Anders Hamsten for help with lipoprotein fractionation, and to Suad Efendic for help with insulin analysis.
Received May 28, 1996; first decision July 5, 1996; accepted July 5, 1996.
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Q. Xu, S. Kiechl, M. Mayr, B. Metzler, G. Egger, F. Oberhollenzer, J. Willeit, and G. Wick Association of Serum Antibodies to Heat-Shock Protein 65 With Carotid Atherosclerosis : Clinical Significance Determined in a Follow-Up Study Circulation, September 14, 1999; 100(11): 1169 - 1174. [Abstract] [Full Text] [PDF]
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R. Wu, U. de Faire, C. Lemne, J. L. Witztum, and J. Frostegard Autoantibodies to OxLDL Are Decreased in Individuals With Borderline Hypertension Hypertension, January 1, 1999; 33(1): 53 - 59. [Abstract] [Full Text] [PDF]
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J. Frostegard, R. Wu, C. Gillis-Haegerstrand, C. Lemne, and U. de Faire Antibodies to Endothelial Cells in Borderline Hypertension Circulation, September 15, 1998; 98(11): 1092 - 1098. [Abstract] [Full Text] [PDF]
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