The Role Of Heat Shock Protein 60 And 70 Antigen And Antibody In Coronary Heart Disease

Coronary heart disease (CHD) is a multifactorial disease and autoimmunity is considered as one of the most important mechanisms. Heat shock proteins (HSPs) is identified as a possible autoantigenic determinant and present as self-antigens to the immune system, resulting in the production of autoantibodies, which may play a significant role in the development of atherosclerosis. Our labortary found that the increase of anti-Hsp70 was associated with the independent risk of increased electrocardiography abnormality, which suggested that anti-Hsp70 might be involved in possibly cardiovascular diseases. But until now, little is known about the exact role of HSPs and their auto-antibodies in the aetiology and pathogenesis of CHD. The prevalence of CHD and the circulating levels of HSPs differ widely among different race or ethnic populations. It can be seen that genetic variations might have an important influence on production of antigen and antibody. Work from our laboratory has demonstrated that single nucleotide polymorphism (SNP) of rs1043618 in hsp70-1 gene is associated with the risk of CHD. However, so far there no reports whether Hsp60 and hsp70 polymorphisms may change Hsp60 and Hsp70 levels, resulting in have an effect on the susceptivity of CHD.Therefore, it is intriguing for us to investigate the possible association among anti-Hsp60, Hsp60, anti-Hsp70 and CHD, and especially focus on the possible joint effects of Hsp60, anti-Hsp60 and other traditional risk factors on CHD in a large case-control study, and further explore the reasons for the relationship by evaluate the effect of Hsp60 and hsp70 polymorphisms on anti-Hsp60, Hsp60 and anti-Hsp70 levels in CHD and control subjects, as well as the influence of acute myocardial infarction (AMI) on circulating anti-Hsp60, Hsp60 and anti-Hsp70 levels in a prospective observational study and their potential impact on pathological process associated with myocardial damage. The total study may be divided to the following three parts:Part I Association of anti-Hsp60 and Hsp60 with CHDIn this part we mainly investigate the association between circulating anti-Hsp60 and Hsp60 and the risk of CHD in a Chinese Han population, and joint effects of anti-Hsp60 and Hsp60 as well as other risk factors for CHD on risk of CHD, in addition, we examined the effect of Hsp60 polymorphisms on anti-Hsp60 and Hsp60 levels in CHD and control subjects.The case-control study was composed of 1003 CHD patients and 1003 age (±5?years) and sex-matched healthy controls. Patients between 40 and 79 years old who were hospitalized were consecutively recruited from three hospitals (Tongji Hospital, Union Hospital, and Wugang Hospital) in Wuhan between May 2004 and October 2006. The inclusion criterion was the presence of significant clinical symptoms, showed a stenoses≥50% in at least 1 major coronary artery by coronary angiography or diagnosed as having CHD based on World Health Organization criteria. Controls were selected randomly from healthy subjects matched for residing regions in the same city. Plasma Hsp60 and anti-Hsp60 levels were analysed using established enzyme linked immunosorbent assay (ELISA). By resequencing and using HapMap PhaseⅠChinese data of hsp60 gene we selected 4 tagSNPs, named rs788016, rs2340690, rs2565163 and rs2305560, and their frequencies was determined using Taqman technique. The results indicated that levels of anti-Hsp60 were significantly higher in CHD patients than in controls [41.56(29.76-59.53) vs. 35.85(25.96-52.13) U/ml, p=0.000]. However, anti-Hsp60 levels did not differ significantly among myocardial infarction (MI), unstable angina (UA) and stable angina (SA) patients (P >0.05). Higher plasma anti-Hsp60 levels in subjects with hypertension and diabetes compared with those without hypertension and diabetes among controls (P <0.05). Anti-Hsp60 levels were significantly reduced in CHD cases who reported using medications (aspirin and/or statins) than those who did not use these medications. Increasing levels of anti-Hsp60 were significantly associated with higher risk of CHD (P for trend=4*10-5) and with increasing severity of CHD as assessed by number of diseased vessels by angiography (OR, 3.67, 95% CI, 1.56 to 8.64; P=0.003) after multivariate adjustment for traditional CHD risk factors. There were strong joint effects of high levels of anti-Hsp60 and hypertension (OR, 5.17, 95% CI, 3.95 to 6.75; P=2.8*10-26), and diabetes (OR, 6.49, 95% CI, 4.52 to 9.33; P=5.9*10-24) on CHD risk and simultaneous occurrence of high anti-Hsp60 level, hypertension, and diabetes conferred a dramatically higher risk of CHD (OR, 20.99, 95% CI, 12.50 to 35.24; P=6.2*10-30) in multivariate analyses.In addition, our results found that circulating Hsp60 was detectable in 407 of the 1003 control individuals (40.6%) and 479 of the 1003 CHD patients (47.8%), but there no difference in the prevalence of Hsp60 seropositivity between controls and CHD. The proportion of subjects in the control and CHD groups with Hsp60 levels >1000ng/mL was 1.2% and 7.0% respectively (P<0.0001). Hsp60 levels in patients with CHD were significantly higher than those in controls [171.4(92.78-319.67) vs.128.7 (60.05-250.17) ng/ml, P =3.2*10-11], and were related to CHD (OR, 4.14, 95% CI, 2.88 to 5.95, P<0.0001). Increasing levels of Hsp60 were significantly associated with higher risk of CHD (P for trend<0.0001) after multivariate adjustment for traditional CHD risk factors. However, there was no association between plasma Hsp60 levels and the severity of CHD, as assessed by the number of diseased vessels. There were a joint effect of Hsp60 (>median, 160.24ng/ml) and anti-Hsp60 (>median, 38.42U/mL), with individuals having high levels of both being at greater risk of CHD (OR, 2.30, 95% CI, 1.44 to 3.67; P<0.0001) than those with low levels of both. Stronger additive effects on the risk of CHD were found between Hsp60 (>1000ng/mL) and anti-Hsp60 (>median, 38.42U/mL), reaching an OR of 14.04 (95% CI, 3.11 to 63.40; P<0.0001). The simultaneous presence of high Hsp60 and anti-Hsp60 antibody levels, current smoking, hypertension and diabetes exhibited a cumulative association with CHD. Individuals who had any four or more of the five factors had an OR of 38.61 for CHD (95% CI, 14.85 to 100.41; P<0.0001), compared with individuals having none of these factors.Four SNPs of hsp60 gene, including rs788016, rs2340690, rs2565163 and rs2305560 were not associated with plasma Hsp60 and anti-Hsp60 levels in CHD and controls (P>0.05). Females carrying the genotypes GG or AG of hsp60 SNP rs788016 had significantly higher plasma anti-Hsp60 levels compared with those with genotype AA in controls (P<0.05).In summary, this part results indicated that anti-Hsp60 is independently associated with CHD risk and a combination of high anti-Hsp60, hypertension, and diabetes is particularly detrimental for CHD risk. Furthermore, elevated Hsp60 levels are associated with an increased risk of CHD, and that Hsp60 and anti-Hsp60 antibody levels combine to increase this risk. This risk is further heightened with the co-presence of smoking, hypertension and diabetes. Rs788016, rs2340690, rs2565163 and rs2305560 four SNPs of hsp60 gene had no significant influence on plasma Hsp60 and anti-Hsp60 levels among different genotypes in CHD and controls. Part II Association of anti-Hsp70 with CHDIn this part our aim was to investigate if there was essential association between anti-Hsp70 and CHD, and further explore the reasons for this relationship by evaluate the effect of hsp70-1 polymorphisms on anti-Hsp70 levels.417 patients with angiographically confirmed CHD and 417 age and sex-matched healthy subjects were investigated. All CHD patients from 40 to 79 years old who were hospitalized at three hospitals between May 2006 and Sep 2007 were consecutively recruited for the present study. All patients with significant clinical symptoms or typical ECG abnormalities, and enzymatic abnormalities underwent coronary angiography and showed stenoses≥50% in at least 1 major coronary artery. Plasma anti-Hsp70 was measured using anti-Human Hsp70 (total) ELISA kit and 3 tagSNPs, rs1008438, rs1043618 in hsp70-1 and rs2227956 in hsp70-hom, was determined using Taqman technique.The results indicated that anti-Hsp70 was detectable in all individuals assessed and median levels were significantly lower in CHD than in control [260.35(190.78-391.01) ug/ml vs. 297.93(212.60-430.14) ug/ml, P=0.010]. Anti-Hsp70 levels were significantly lower in MI and UA [255.32(185.03-391.40)ug/ml, 251.31(179.61-339.33) ug/ml, respectively] in comparison to SA [295.56(220.12-434.17) ug/ml]. However, anti-Hsp70 levels did not differ significantly between MI and UA. Aspirin and/or statins treatment had no influence on anti-Hsp70 levels in CHD patients. After adjustment to traditional CHD risk factors, high anti-Hsp70 level (>median, 277.08ug/ml) was found to be significantly decreased risk of CHD about 40% (OR= 0.61, 95%CI, 0.44 to 0.85, P=0.003). There was a trend for subjects with increased levels of anti-Hsp70 to have a graded decreased risk of CHD (P for trend=0.002), and had a decreased risk of CHD severity, as assessed by number of diseased vessels, however the trend failed to achieve statistical significance (P for trend=0.129). Furthermore, we observed that no association between anti-hsp70 antibody levels and other classical risk factors for CHD, including age, sex, smoking statue, BMI, cholesterol, triglyceride levels, hypertension and diabetes. Patients carrying the genotypes CC of hsp70-1 SNP rs1043618 and rs1008438 had significantly lower plasma anti-Hsp70 levels compared with those with genotype GG or AA (P=0.033 and P =0.019, respectively).In conclusion, our study shows that high levels of anti-Hsp70 were independently associated with a relatively lower CHD risk, to some extent, related to the lower severity in a Chinese population, it compatible with the lower anti-Hsp70 level in relation to the alleles were at an elevated risk of CHD. In addition, the lower levels of anti-Hsp70 in both MI and UA in comparison to SA. It is postulated from our data that anti-Hsp70 is maybe more friend than foe in CHD, under certain circumstance, protect against the disease. However, it needs to be confirmed further in larger prospective studies.Part III Dynamic changes of Hsp60, anti-Hsp60 and anti-Hsp70 in patients with AMIThe above results demonstrated that the presence of Hsp60, anti-Hsp60 and anti-Hsp70 play completely different role in CHD. Therefore, it is intriguing for us to explore the influence of AMI on circulating Hsp60, anti-Hsp60 and anti-Hsp70 levels in a prospective observational study and their potential impact on pathological events associated with myocardial damage.The prospective study was conducted in 20 patients with AMI who were consecutively admitted (Oct. 2007 to Feb 2008) to Wugang Hospital within 12 h of the onset of symptoms. The diagnosis of AMI was based on the following criteria: 1) chest pain lasting longer than 20 min; 2) development of pathologic Q waves on the ECG or ST segment elevation or depression; and 3) elevation of biochemical markers of myocardial necrosis. Blood samples were drawn immediately after admission and at 2, 3 and 7 days thereafter. Plasma Hsp60, anti-Hsp60 and anti-Hsp70 levels were analysed using ELISA, and serum cardiac troponin I (cTnI), creatine kinase MB (CK-MB) and myoglobin were assayed using standard laboratory procedures.The results indicated that plasma Hsp60 levels were elevated significantly on the day of arrival (117.47 ng/mL) and the following day (124.41 ng/mL) in comparison to those on day 7 (0.00 ng/mL) after the onset of AMI (P=0.011 and P =0.026, respectively), and the number of patients in which circulating Hsp60 was detectable gradually reduced at the four time points. However, the change of anti-Hsp60 levels was not statistically significant at different time points (all P>0.05). Anti-Hsp70 was detectable in all of the 20 patients with AMI at the four time points and it gradually increased and reached a relative peak value on day 7. Anti-Hsp70 levels on day 7 (294.58ug/ml) were significantly elevated in comparison to those on the day of arrival (212.65ug/ml) and the following day (249.81ug/ml) after the onset of AMI (P=0.005 and P =0.048, respectively).The markers of myocardial necrosis cTnI and CK-MB rapidly increased and reached a relative peak value on day 2 and decreased significantly to very lower or normal levels on day 7. Myoglobin levels peaked immediately after the onset of AMI, and then gradually returned to normal levels on day 7. Furthermore, Hsp60 in plasma correlated positively with the levels of cTnI (r = 0.391, P<0.05) and CK-MB (r = 0.205, P< 0.05) measured at the all time points, illustrating the more rapid release of Hsp60 than cTnI and CK-MB. Anti-Hsp70 correlated negatively with the levels of myoglobin (r=-0.230, p<0.05), but no correlation with cTnI and CK-MB was detected.To summarize, the novel findings in the prospective study were that Hsp60 is rapidly released after AMI and positive correlated to cTnI and CK-MB. It suggests that determination of plasma Hsp60 levels might serve as a diagnostic marker for CHD. Anti-Hsp70 levels were significantly decreased in patients with AMI on the day of arrival and the following day after the onset of AMI. It is postulated that anti-Hsp70 is maybe have more protective than deleterious role in CHD. However, the sample size in the prospective study is too small to make any definite conclusions, and there is a need for further investigations to help verification. In conclusion, we investigated the associations among circulating Hsp60, anti-Hsp60 and anti-Hsp70 and CHD, and especially focus on the joint effects of Hsp60, anti-Hsp60 and other risk factors of CHD. Furthermore, we evaluated the effect of hsp60 and hsp70 polymorphisms on anti-Hsp60, Hsp60 and anti-Hsp70 levels, as well as the influence of AMI on anti-Hsp60, Hsp60 and anti-Hsp70 levels, in order to explore the reason for change of Hsp60, anti-Hsp60 and anti-Hsp70 in CHD. Our results suggested1. Hsp60 and anti-Hsp60 levels were significantly higher in CHD patients than in controls and independently associated with higher CHD risk. Furthermore, increasing levels of anti-Hsp60 were significantly associated with increasing severity of CHD as assessed by number of diseased vessels by angiography after multivariate adjustment for traditional CHD risk factors.2. High levels of Hsp60 and anti-Hsp60 combine to increase CHD risk. This risk is further heightened with the co-presence of smoking, hypertension and diabetes.3. Rs788016, rs2340690, rs2565163 and rs2305560 four tagSNPs of hsp60 gene had no significant influence on Hsp60 and anti-Hsp60 levels among different genotypes in CHD and controls.4. Anti-Hsp70 levels were significantly lower in CHD than in control, and also significantly decreased in MI and UA in comparison to SA. High levels of anti-Hsp70 were independently associated with relatively lower CHD risk, to some extent, related to the lower severity.5. Patients with CHD carrying the genotypes CC of hsp70-1 SNP rs1043618 and rs1008438 had significantly lower plasma anti-Hsp70 levels compared with those with genotype GG or AA.6. Hsp60 is rapidly released after AMI and positive correlated to cTnI and CK-MB. However, anti-Hsp70 levels were significantly decreased in patients with AMI on the day of arrival and the following day after the onset of AMI. It demonstrated that Hsp60 and anti-Hsp70 play deleterious and protective role in CHD respectively. Noteworthy strengths of this study are its large sample size, age and sex-matched strictly design, which minimizes potential bias. More importantly, the large sample size allowed us to examine the combined effects of elevated Hsp60 and anti-Hsp60 levels and other risk factors on CHD risk. In addition, this is the first study showing that the effect of hsp60 and hsp70 polymorphisms on Hsp60, anti-Hsp60 and anti-Hsp70 levels. Finally, we provided the first evidence about dynamic change of Hsp60, anti-Hsp60 and anti-Hsp70 at different time points in AMI in a prospective study.However, there are several potential limitations in the present study. Firstly, case-control design limits the causal interpretation of the relationship among Hsp60, anti-Hsp60 and anti-Hsp70 and CHD risk. Secondly, the sample size in the prospective study is too small to make any definite conclusions. Thus, our results need to be confirmed in large prospective studies. In addition, protective role of anti-Hsp70 in CHD needs for further investigations to help verification. Finally, further analyze the association between near SNPs and antigen and antibody of Hsp60 and Hsp70 levels to explore the possible mechanism of SNPs on CHD risk.