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
Background— We intended to identify proteins that are differentially expressed in human atherosclerotic plaques.
Methods and Results— Comparative 2-dimensional electrophoretic analysis on carotid atherosclerotic endarterectomy specimens (n=10) revealed that heat shock protein 27 (Hsp27) expression was significantly increased in the nearby normal-appearing area compared with the plaque core area from the same vessel specimen, which was further confirmed by Western blot analysis. The Hsp27 expression in the adjacent normal-appearing vessel areas was much higher than that in nonatherosclerotic reference arteries. The phosphorylation of Hsp27 showed a gradation in the degree of phosphorylation: greatest in the reference arteries, intermediate in the adjacent normal-appearing area, and lowest in plaque core area. Immunohistochemical analysis showed that the phosphorylation of Hsp27 of smooth muscle cells in the carotid endarterectomy specimens was decreased compared with that in the reference artery specimen. The mean plasma level of Hsp27 was significantly higher in patients with acute coronary syndrome (ACS) (n=27; 106.1±74.1 ng/mL) than in the normal reference subjects (n=29; 45.8±29.5 ng/mL; P<0.005). The plasma levels of Hsp27 were significantly correlated with those of heat shock protein 70 (Hsp70) (r=0.422, P<0.0005), with adjustment for ACS/reference status.
Conclusions— In the atherosclerotic lesion, Hsp27 expression is increased in the normal-appearing vessel adjacent to atherosclerotic plaque, whereas levels in the plaque itself are significantly decreased. Both plaque and adjacent artery show decreased Hsp27 phosphorylation compared with reference vessel. In ACS, plasma Hsp27 and Hsp70 are increased, and levels of Hsp27 correlate with Hsp70, C-reactive protein, and CD40L levels.
Received May 29, 2004; de novo received February 6, 2005; revision received May 21, 2006; accepted June 19, 2006.
Heat shock proteins work as “chaperones” to affect protein folding of newly synthesized or denatured proteins.1 Heat shock proteins can be expressed during ischemic heart disease as the result of such stress as hypoxia, reperfusion, and oxidative stress, and they can confer antiapoptotic effects on myocytes after ischemia/reperfusion injury.2–4 Several heat shock proteins, including Hsp60/65 and Hsp70/72, have been implicated in atherosclerosis.5 These heat shock proteins were identified by performing immunohistochemistry on atherosclerotic plaques,6–8 and their serum levels have been significantly associated with the risk of coronary artery disease.9,10
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The sequence of the heat shock proteins has been highly conserved from microorganisms to humans, and thus antibodies to bacterial heat shock proteins can interact with human heat shock proteins.11–14 Antibodies against heat shock proteins have also been reported to be associated with atherosclerotic vascular diseases.15–18
During our comparative examination of the proteome between the atheromatous plaque core area and nearby normal-appearing area in the same vessel specimens from carotid endarterectomy, we found a differential expression of heat shock protein 27 (Hsp27). Because there has not been a thorough examination of Hsp27 in atherosclerotic lesions or in the plasma of patients with coronary artery disease, we identified its localization in the atherosclerotic plaques and assessed the level of its soluble form in the plasma of patients with acute coronary syndrome (ACS).
Carotid Endarterectomy Specimen and Normal Vessel Specimen
Carotid endarterectomy specimens were obtained from 10 patients with carotid artery stenosis (age, 63 to 81 years) who underwent surgery at the Samsung Medical Center. The samples were taken from 2 different areas of each plaque: the core area and the adjacent normal-appearing area in the same vessel specimen. The tissue samples were washed with saline and were then frozen until analysis. The other 5 carotid endarterectomy specimens taken from other patients with carotid artery stenosis were embedded in optimal cutting temperature compound (OCT; Miles Laboratories) to make the frozen sections. Two renal artery specimens and 2 internal mammary artery specimens were also obtained from kidney transplantation donors and coronary artery bypass graft patients, respectively, and were used as normal reference vessels for Western blot analysis. This study was approved by the institutional review committee at our hospital, and informed consent was obtained from all the subjects.
Two-Dimensional Electrophoresis and Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry
The specimens were suspended in sample buffer containing 7 mol/L urea, 2 mol/L thiourea, 4% (wt/vol) CHAPS, 40 mmol/L Tris, 0.1 mol/L dithiothreitol, and protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). The suspensions were sonicated for &30 seconds and then centrifuged at 100 000g for 45 minutes. Two-dimensional electrophoresis was performed as previously described.19 One milligram of the total protein was used for each electrophoresis. Aliquots of the specimen proteins in sample buffer were applied to immobilized nonlinear gradient strips (pH 3 to 10) (Amersham Pharmacia Biotech, Uppsala, Sweden), and the isoelectric focusing was performed for 90 000 volt-hours. The second dimension was resolved on 9% to 16% linear gradient SDS polyacrylamide gels (200×250×1.0 mm) at 15 mA per gel constant current for &12 hours until the dye front reached the bottom of the gel. After this, the proteins were fixed in 40% methanol and 5% phosphoric acid for 12 hours, and the gels were then stained with Coomassie blue G250 for 24 hours. Gels were destained with H2O and scanned with a BioRad GS-800 densitometer; the data were next converted into electronic files, which were then analyzed with Melanie III computer software (GenBio, Geneva, Switzerland).
For mass spectrometry fingerprinting, the protein spots were cut from the gels, destained, and treated with trypsin as previously described.20 Aliquots of the peptide mixtures from the trypsin treatments were applied to a target disk and allowed to air dry. The spectra were obtained with the use of a Voyager-DE STR matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (PerSeptive Biosystems, Framingham, Mass). The protein database searches were performed with ProFound (http://188.8.131.52/profound_bin/webprofound.exe) with the use of monoisotopic peaks. Mass tolerance was first allowed within 50 ppm, and then recalibration was performed with the protein lists obtained at 20 ppm.
Western Blot and Immunohistochemical Analysis
Specimen proteins were transferred onto polyvinylidene difluoride membranes, and the blots were then probed with specific antibodies and finally developed with the use of enhanced chemiluminescence reagents (ECL, Amersham, Del). For the immunohistochemical analysis, the specimens embedded in OCT were sectioned at 5-μm thickness and then stained with the use of the LSAB kit (DAKO) according to the manufacturer’s specifications. Monoclonal anti-Hsp27 and anti–phospho-Hsp27 (Ser82) antibodies were purchased from Cell Signaling Technology, and monoclonal antibodies to CD68 and smooth muscle cell (SMC) α-actin were purchased from DAKO. Monoclonal antibodies against the inducible forms of Hsp70 and Hsp60 were purchased from StressGen Biotechnologies. Other antibodies used in this study were purchased from Santa Cruz Biotechnology, Inc.
For the ACS group, 27 patients (age, 30 to 80 years) with acute myocardial infarction or unstable angina, who came to the emergency department and were then admitted to the coronary care unit of Samsung Medical Center, were entered into the study (male:female ratio, 18:9; age, 58.9±9.3 years). The blood samplings were done within 24 hours from presentation to the emergency department and before their coronary angiography. All patients in this study had their coronary stenosis proven by angiography. For the normal reference group, 29 sex- and age-matched healthy volunteers were recruited (male:female ratio, 19:10; age, 56.1±6.3 years). For the chronic stable angina (CSA) group, the patients with coronary artery disease who had been treated with antianginal medications, including statins, for >1 year, who had their risk factors adequately modified, and who had also been free of chest pain for >1 year were included in the study (17 male patients; age, 61.4±7.3 years). None of the reference subjects had any of the major risk factors for cardiovascular disease, and none were taking any medication. The risk group subjects who had ≥1 of the risk factors for coronary artery disease but who had no overt coronary artery disease (n=31; male:female ratio, 20:11; age, 59.2±6.5 years) were also enrolled in the study. We obtained informed consent from all the subjects before any blood was sampled.
Enzyme-Linked Immunosorbent Assay
Plasma samples obtained from all study subjects were aliquoted and stored at −80°C until analysis. The plasma levels of Hsp27 were measured with the use of Oncogene Hsp27 enzyme-linked immunosorbent assay (ELISA) kits (catalog No. QIA119) according to the manufacturer’s directions. Briefly, the plasma was diluted 1:10 in assay buffer and then incubated for 1 hour at room temperature in ELISA wells that were precoated with anti-Hsp27 monoclonal antibody. The plates were washed and then were incubated with rabbit anti-human Hsp27 polyclonal IgG for an additional hour at room temperature. After washing was performed, peroxidase-conjugated goat anti-rabbit IgG was added to each well and then incubated overnight at 4°C. The enzymatic activity was assessed by the addition of 100 μL tetramethylbenzidine substrate solution to each well. After the reaction had been stopped by the addition of sulfuric acid, the absorbance was measured at 405 nm with a spectrophotometer. Each assay included positive controls with a known amount of human recombinant Hsp27 and a negative control (no human recombinant Hsp27), which were run in parallel. The plasma levels of adiponectin and CD40L were also determined by ELISA kits from R&D Systems, and ELISA kits from StressGen (StressXpress Hsp70 ELISA Kit) were used to measure the Hsp70 plasma levels.
Plasma α-Tocopherol Measurement
The plasma α-tocopherol concentration was measured by high-performance liquid chromatography (HPLC) with the use of a reverse-phase column (NovaPak C18, 8×100-mm Radial-Pak Cartridge, Waters), as described previously.21 We used the Shimadzu HPLC system equipped with an autoinjector and a fluorescence detector at an excitation wavelength of 292 nm and an emission wavelength of 324 nm. α-Tocopherol acetate (Sigma-Aldrich Co, St Louis, Mo) was used as an internal standard, and α-tocopherol was used as an external standard.
Comparisons of continuous variables between ≥3 groups were performed by Kruskal-Wallis test or ANOVA test according to the normality of data distribution. The difference of continuous variables between 2 groups was identified with the Mann-Whitney test because of their non-normal distribution. Discrete variables were expressed as percentages and compared by Fisher exact test. Spearman correlation analysis and Pearson correlation analysis were used to analyze the interrelationship between variables. Probability values <0.05 were considered significant for all tests. All statistical analyses were performed with SPSS software (version 10.0).
The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written.
Expression of Hsp27 in Human Carotid Atherosclerotic Plaques
Proteome analysis was performed with the use of high-resolution 2D electrophoresis with human carotid endarterectomy specimens that were sampled from 2 regions: the atherosclerotic plaque core area and paired adjacent normal-appearing vessel area. More than 2000 protein spots were detected with SYPRO Ruby staining of proteins from the endarterectomy specimens; the representative diagrams for 2D electrophoresis maps of the 2 regions are shown in Figure 1. By computer-assisted comparative analysis of the respective spot patterns of the paired samples, we were able to detect &200 protein spots that showed a different expression. Each of the 21 spots that were upregulated or downregulated in the plaque area was selected, and they were subjected to protein identification by MALDI-TOF-MS analysis after trypsin digestion. We identified 17 plaque-associated proteins (Table 1), of which 4 proteins, including proapolipoprotein, ferritin light chain protein, and fibrinogen β-chain protein, were upregulated and 13 proteins, including vimentin, Hsp27, actin-binding protein (SM22-α), and β-tropomyosin, were downregulated at the atherosclerotic plaque core areas.
We were interested in the pattern of the decreased Hsp27 expression (spots indicated by arrows on representative 2D electrophoresis maps shown in Figure 1A and 1B). After separation of protein extracts by 2D electrophoresis, Western blot analysis revealed some additional anti-Hsp27 antibody–reactive spots other than the spot that had been identified previously as Hsp27 during MALDI-TOF-MS analysis (Figure 1C and 1D). Another MALDI-TOF-MS analysis on the spots responsive to anti-Hsp27 antibody in Figure 1C and 1D revealed that those spots were well matched with the peptide mapping for human Hsp27 (Figure 2) and that the spots numbered 1, 2, 5, and 8 in Figure 2 involved phosphorylations at the 15th and/or 82nd serine residue (data not shown).
In Figure 3, Western blot analysis showed that Hsp27 expression was significantly increased in the nearby normal-appearing area compared with the plaque core area from the same vessel specimen. Hsp27 expression in the adjacent normal-appearing vessel areas was much higher than those in nonatherosclerotic reference arteries from donor renal artery and internal mammary arteries. In the nearby normal-appearing areas, contrary to the higher Hsp27 expression, the phosphorylation of Hsp27 was decreased, which makes the ratios of phospho-Hsp27 to total Hsp27 expression much lower than those in reference artery specimens. The phospho-Hsp27 was hardly detectable at the atherosclerotic plaque core areas. On the other hand, the expression of Hsp70 was higher in both the plaque core areas and adjacent normal-appearing areas of carotid endarterectomy specimens compared with those in the reference artery specimens. There were no differences in the levels of β-actin among the specimen samples in this study.
Although immunohistochemical analysis revealed that the expression of Hsp27 overlapped with the SMC marker (α-SM actin; data not shown) in the reference renal artery and carotid endarterectomy specimen (Figure 4), the phosphorylation of Hsp27 (phospho-Ser82 Hsp27) in those cells was detected to a much lower degree in the atherosclerotic plaque specimen than in the reference artery specimen (Figure 4F versus 4C). On the contrary, the macrophage-infiltrated region (CD68 positive; data not shown) was colocalized with staining for Hsp27 phosphorylation (Figure 4F).
Increased Plasma Levels of Hsp27 in ACS Patients
The clinical characteristics and lipid profiles of each study group are summarized in Table 2. There were no significant differences between the ACS patients and the normal reference subjects for the serum levels of total cholesterol, triglyceride, and LDL, whereas the serum levels of HDL in patients with ACS were significantly lower than those in the reference subjects (P<0.001). There were significant differences for serum total cholesterol and LDL levels between the ACS and CSA groups (P<0.005 for both variables) and between the reference and CSA groups (P<0.001 for both variables).
The serum levels of C-reactive protein (CRP) in the ACS group (1.341±2.382 mg/dL) were significantly higher than levels in both the reference group (0.101±0.096 mg/dL; P<0.05) and the CSA group (0.088±0.056 mg/dL; P<0.05) (Figure 5A). The plasma α-tocopherol concentrations in the ACS group (29.5±7.8 μmol/L; P<0.05 versus control) and the CSA group (33.7±10.1 μmol/L; P<0.05 versus control) were significantly lower than those in the reference subjects (42.8±14.7 μmol/L) (Figure 5B). The levels of plasma Hsp70 were significantly higher in patients with ACS than in reference subjects (13.84±9.64 versus 8.21±7.00 ng/mL; P<0.005) (Figure 5C), whereas the plasma levels of adiponectin in patients with ACS were significantly lower than the levels in the reference subjects (3.10±2.18 versus 5.25±3.99 μg/mL; P<0.05). The mean values for plasma CD40L were increased by almost 2-fold in patients with ACS compared with those in the reference group (2.07±1.92 versus 1.22±1.45 ng/mL), but the differences did not reach statistical significance.
Between all groups, the plasma levels of Hsp27 (106.1±74.1 mg/mL) were significantly higher in ACS patients than in the healthy reference subjects (45.8±29.5 mg/mL; P<0.005) (Figure 6). As shown in Table 2, when the healthy reference group was compared with the group with risk factors for coronary artery disease, the plasma levels of Hsp27 were not significantly different between the 2 groups (reference group versus risk group, 45.8±29.5 versus 46.5±29.3 ng/mL). Although the difference in the Hsp27 levels between the ACS and CSA groups was not statistically significant (P=0.066), marginal significance was found between them when the statistical analysis was limited to 3 groups involving the reference, ACS, and CSA groups (P<0.05).
Correlations of Plasma Hsp27 Levels With Other Variables
When statistical analysis was performed in the whole group of combined ACS and reference groups, the plasma levels of Hsp27 were significantly correlated with the plasma levels of Hsp70 (r=0.506, P<0.0001), serum levels of CRP (r=0.234, P<0.05), plasma levels of CD40L (r=0.417, P<0.005), and serum levels of total cholesterol (r=0.254, P<0.05). When we used Spearman partial correlation analysis to adjust for ACS/reference group status, these results of positive correlations remained significant except for between Hsp27 and CRP. The partial correlation coefficient was 0.422 for between Hsp27 and Hsp70 (P<0.005), 0.351 for between Hsp27 and CD40L (P<0.01), and 0.094 for between Hsp27 and CRP (P=0.494). The correlations between plasma levels of Hsp27 with the other serum or plasma factors such as triglyceride, HDL, LDL, α-tocopherol, and adiponectin were not significant. The correlation between the plasma Hsp70 levels and the serum CRP levels was not significant (r=0.178, P=0.093). In our study groups, age, gender, smoking status, and the presence of diabetes mellitus or hypertension were not associated with the plasma Hsp27 levels.
In contrast to Hsp60/65 and Hsp70/72, Hsp27 has not been much studied for its relationship with atherosclerosis. Hsp27 is highly expressed in the heart,22 and it has been previously studied in myocardial protection models.23 Hsp27 is induced by oxidative stress, and it is known to have an antiapoptotic effect.24–26
Recently, Martin-Ventura et al27 showed the decreased release of Hsp27 from buffer-soaked atherosclerotic plaques compared with those from normal reference arteries and concluded that these results might be associated with the observed low level of plasma Hsp27 in patients with carotid stenosis. However, the release of a certain protein from a specimen might differ according to the applied buffer system, and such a small area of diseased vessel of carotid arteries could not explain the systemically lowered level of plasma Hsp27, the reverse of our findings in the present study. In contrast to Martin-Ventura et al, who compared the patterns of protein secretion between carotid endarterectomy specimens and healthy reference arteries, we compared the proteome of the atheromatous plaque core areas and the adjacent normal-appearing areas in human carotid endarterectomy specimens. We found that Hsp27 expression was lower in the plaque core areas than in the nearby normal-appearing areas. These results were confirmed by Western blotting with identical results.
The reference artery specimens in our study showed a much higher ratio of phospho-Hsp27 to total Hsp27 expression compared with the nearby normal-appearing areas of the carotid plaque, and the plaque core areas showed markedly decreased expression of both Hsp27 and phospho-Hsp27 (Figure 3), suggesting that the nearby normal-appearing area of the atherosclerotic plaque is not a normal area free from atherosclerosis. Immunohistochemical analysis (Figure 4) showed that the phospho-Hsp27 stain in the SMCs was much lower in atherosclerotic plaque specimens than in normal artery specimens. Because many protective roles of Hsp27 are expressed by its phosphorylated form, those cells showing a lower ratio of phospho-Hsp27 to total Hsp27 might be more vulnerable to oxidative stress and inflammation. Hsp27 of the macrophages in atherosclerotic legions was observed to be phosphorylated on the 82nd serine residue, which might be involved in the oxidative defense mechanism24,26 and/or cell migration in atherosclerotic plaque.28,29
We also found that the plasma levels of Hsp27 were significantly increased in patients with ACS compared with the healthy reference group (Figure 6). The risk factors for coronary artery disease may not be related to the increased plasma Hsp27 because the plasma Hsp27 levels of a group of subjects with risk factors for coronary artery disease but no symptomatic coronary stenosis were not different compared with those of the healthy reference group (Figure 6). The levels of plasma Hsp70 showed much stronger correlation with the levels of plasma Hsp27 (r=0.422, P<0.005) than any other variables in the ACS groups and reference group. It has been reported previously that the plasma Hsp70 levels were lower in patients at the time of diagnosis of coronary artery diseases by coronary angiography and that the patients with lower Hsp70 levels had a higher risk of coronary artery disease than the patients with higher Hsp70 levels.10 However, contrary to the association between plasma Hsp60 and coronary artery disease, which has been supported by multiple studies, the inverse relationship between the plasma Hsp70 level and the risk of coronary artery disease is not a settled issue. Moreover, some methodological aspects in the report by Zhu et al10 are questionable because they evaluated the plasma Hsp70 level by the extrapolation method. In addition, it has been reported recently that the inflammatory status determines the serum level of Hsp70,30 which might support our results.
Although further research is needed to clarify the roles of Hsp27 and its phosphorylation in the pathogenesis and progression of atherosclerosis, these findings suggest that Hsp27 may increase in the earlier stage of atherosclerosis and the decreased potency of phosphorylating Hsp27 of the SMCs may be an important factor in the progression of atherosclerosis. We also observed increased levels of plasma Hsp27 in ACS patients, which may represent vulnerable or complicated plaque and an associated increase in systemic inflammatory or oxidative stress.
The authors thank Dr Seonwoo Kim at Samsung Medical Center for critical comments on the statistical analysis and Min Gyoung Kang for assistance in patient care and obtaining blood samples.
Sources of Funding
This work was supported by Korea Science and Engineering Foundation grant R01-2000-000-00090-0, an Industry-Academia Cooperation research grant from the Korean Society of Circulation (sponsored by Boehringer Ingelheim Korea) in 2002 (to Dr J.E. Park), and the Korea Science and Engineering Foundation through Protein Network Research Center at Yonsei University (to Dr J.-B. Yoon).
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In our study we found differentially expressed proteins in atherosclerotic lesions by performing 2-dimensional gel electrophoresis on human carotid endarterectomy specimens. We divided the atherosclerotic plaque lesion into core area and nearby normal-appearing area, and we used the internal mammary artery and renal artery graft as reference arteries. Among the differentially expressed proteins, we were interested in heat shock protein 27 (Hsp27), which showed markedly decreased expression in the atherosclerotic core lesion compared with the nearby normal-appearing lesion. The phosphorylation of Hsp27 showed a gradation in the degree of phosphorylation: greatest in the reference arteries, intermediate in the adjacent normal-appearing area, and lowest in the plaque core area. Immunohistochemical analysis showed that Hsp27 expression was observed mainly in the smooth muscle cells. Heat shock protein 70 (Hsp70) did not show any significant difference in the degree of expression between the plaque core area and nearby normal-appearing area. The mean plasma level of Hsp27 was significantly higher in patients with acute coronary syndrome than in the normal reference subjects. The plasma levels of Hsp27 were significantly correlated with those of Hsp70. Therefore, it can be suggested that Hsp27 may increase in the earlier stages of atherosclerosis and that the decreased potency of phosphorylating Hsp27 on the smooth muscle cells may be an important factor in the progression of atherosclerosis. Increased plasma levels of Hsp27 may also represent the vulnerable or complicated plaque and associated increase in systemic inflammatory or oxidative stress.
↵*The first 3 authors contributed equally to this work.