Association Of Estrogen And Heat Shock Protein 27 To Coronary Heart Disease And Their Effects On Endothelial Cell Function

BACKGROUND: Recently, coronary heart disease(CHD), increased year by year, and has become one of the major human diseases. To study the pathophysiological mechanism and control of risk factors is an important topic in medical research. It deems that the incidence of coronary heart disease risk factors in a variety of under the action of severe coronary artery atherosclerotic stenosis and occlusion, or spasm in the merger on this basis as well as thrombosis, blood vessel blockage, caused by coronary insufficiency, cardiac ischemia or infarction of a heart attack. Endothelial dysfunction, smooth muscle cell proliferation, platelet aggregation and thrombosis activation is the main mechanism by which inflammatory response throughout the entire process. Prevalence of coronary heart disease varies greatly between men and women, the incidence of premenopausal women compared to men is 1:3 1:10, while postmenopausal women and men are in line. This difference can not be fully explained by the CHD common risk factors, but be suggested it may be associated with the level of estrogen.Estrogen plays an important role in the development of cardiovascular disease. Estrogen can affect lipid metabolism, decrease serum total cholesterol (TC) as well as low-density lipoprotein cholesterol (LDL-C) levels and increase high density lipoprotein cholesterol (HDL-C). Evidences showed estrogen carris an antioxidant effect through clearing free radicals and resisting lipid peroxidation. Estrogen can improve endothelial function, reduce the release of vasoconstrictor substance, so that blood vessels expand to increase blood flow, and also stimulates the mature endothelial cells to produce nitric oxide which protects against endothelial cell apoptosis and promotes proliferation. Estrogen can also accelerates reendothelialization after blood vessel wall injury , and protects against atherosclerosis. However, the specific mechanism of endothelial protection of estrogen remains to be further studied.Vascular endothelium is the physical barrier between circulating blood and vascular smooth muscle. Endothelial dysfunction (ED) is the trigger of atherosclerosis (AS) development. Prevention or controll of endothelial dysfunction has become the new trend of coronary heart disease prevention.Heat shock protein 27 (HSP27) is an important member of small molecular weight heat shock protein family. It's upregulation under stress, protect cell function via the effect of molecular chaperone, regulating cell movement, anti-apoptosis, anti-oxidative stress and anti-inflammation.HSP27 is a recently reported potential biomarker of atherosclerosis. The serum HSP27 levels were inversely related to atherosclerotic burden. HSP27 may be a significant new therapeutic target in Atherosclerosis.Part I The Relationship between Estrogen and Blood Lipid and HSP27 Levels in Coronary Heart Disease PatientsObjective: A great number of studies showed that estrogen can play a protective role by regulating lipid metabolism in cardiovascular disease. HSP27 is a biomarker of atherosclerosis. Serum HSP27 levels were inversely related to atherosclerotic burden. HSP27 which induced by estrogen, is an ERβassociated protein, and acts as a co-repressor of estrogen signaling. So what's the relationship between serum HSP27 and estrogen levels HSP27 in coronary heart disease patients? Whether is the level of serum HSP27 associated with blood lipid levels which regulated by estrogen? Whether is HSP27 played a potential role in regulating lipids metabolism by estrogen? The study is aim to observe the correlation between estrogen and HSP27, lipid by collecting serum from coronary heart disease patients and healthy human, and testing serum levels of HSP27, estrogen and lipid content.Methods: In accordance with the study criteria, the survey covers hospitalized coronary heart disease patients and healthy examinees from February to September 2009 in Department of Cardiology of the First People's Hospital of Chenzhou City, Hunan Province. 1. The collected clinical records included age, sex, height, weight, heart rate, habits, history of diabetes, history of hypertension. 2. Using Bayer ADVIA 2400 automatic biochemical analyzer, determined serum lipid levels by enzymatic assay. 3. Using Bayer ADVIA Centaur automated chemiluminescence immunoassay-type instrument, evaluated the serum levels of estradiol (E₂) by chemiluminescence. 4.The serum HSP27 levels were analysed by ELISA assay. Results: All age, BMI, high-salt diet, smoking, alcohol consumption, the incidence of diabetes, hypertension, morbidity of the CHD group were significantly higher than those of the control group. Significant differences of serum E₂, HSP27, and lipid levels were found between the CHD group and the healthy control group. Same differences were also found between female CHD patients and male CHD patients,and between female CHD patients≤55 years old group and> 55 age group.Linear correlation analysis showed there was a significant negative correlation between E₂ and TC (r=-0.6669,P<0.05), LDL-C (r=-0.5739,P<0.05), and a significant positive correlation between E₂ and HDL-C (r=-0.7019,P<0.01), HSP27 (r=-0.7946,P<0.01). No obvious relations are found between serum HSP27 levels and lipid.Part II Effects of HSP27 on E₂ Induced Endothelial ProtectionObjectives: The first part of the experiment showed that serum HSP27 levels associated with estrogen levels in coronary heart disease, HSP27 was not involved in the effects of estrogen on regulating lipid metabolism. Then is HSP27 participated in the endothelial protective effects of estrogen? Whether can exogenous estrogen induce upregulation of HSP27 expression in human umbilical vein endothelial cells (HUVEC), with/without a dose-dependent manner? The experiment intends to observe the different expression of HSP27 by treating HUVEC with different concentrations of estradiol (E₂) and estrogen receptor antagonist tamoxifen (tamoxifen), and to observe the potential role of HSP27 on the protection effect of estrogen on endothelial cells by transfection with HSP27 siRNA.Methods: 1.Different concentrations of E₂(10^-9 mol/L ,10^-8 mol/L ,10^-7mol/L) incubated with HUVEC for 24 hours. 10^-7mol/L E₂ and 10^-6mol/L tamoxifen treated HUVEC for 24 hours. Incubation with DMEM medium alone as a negative control group, RT-PCR and Western blot assays were used to detect the HSP27 expression. 2. HUVECs were Transfected with HSP27 siRNA or mock RNA for 48 hours respectively, then incubated with 10^-7mol/L E₂ for 24 hours. Incubation with DMEM medium alone as a negative control group, RT-PCR and Western blot were applied to detect the HSP27 expression. Meanwhile, the level of NO and ET-1 in medium was detected by nitrite/ nitrate colorimetric method and ELISA. The surface expression of ICAM-1 on cells was detected by ELISA.Results: Both in transcription and post-transcription level, HSP27 expression was increased significantly in HUVEC by treating E₂ in a dose-dependent manner,with peak expression at 10^-7mol/L. 10^-6mol/L tamoxifen could inhibited the effects of 10^-7mol/L E₂. RT-PCR and Western blot detection of HSP27 mRNA and protein expression showed HSP27 siRNA sequence could down-regulate HSP27 expression. Interestingly, later transfected HSP27 siRNA for 48 hours then incubated with 10^-7mol/L E₂ for 24 hours, the effects of E₂ on HUVEC to secrete NO and ICAM-1 was significantly inhibited, but the ET-1 production was not obviously changed.CONCLUSIONS:1. Serum E₂ levels is positively correlated with HSP27, HSP27 may be associated with the effect of estrogen on cardiovascular disease .2. E₂ can induce HUVEC to produce HSP27 in a dose-dependent manner, which is associated with estrogen receptor. 3. There could be a potential role of HSP27 on E₂ induced NO and ICAM-1 produce in HUVEC.