Relations And Mechanisms Of Uric Acid And Cardiovascular Disease Research

Backgrounds Epidemiological studies found that the prevalence of hyperuricemia increased markedly around the world in the past two decades, as the living conditions were improved and food consumed changed worldwide. Substantial amounts of studies suggested that the increase of serum uric acid levels in human beings was related with cardiovascular/cerebrovascular diseases, but it is not clear yet whether the increase of serum uric acid levels or hyperuricemia is an independent risk factor for them, or it is just a marker of the morbidities. Furthermore, there are still lots of debates regarding the effects of uric acid on the vascular endothelial dysfunction, which has been recognized as an important character of cardiovascular/cerebrovascular diseases and plays a key role in their pathogenesis.Objectives 1)To explore the relationship between serum uric acid levels or hyperuricemia and cardiovascular/cerebrovascular diseases in an epidemiological study; 2)To explore the effects of uric acid on the vascular endothelial function and their possible pathogenesis in in vitro studies.Contents 1)The cross-sectional study investigating the relationship between serum uric acid levels or hyperuricemia and cardiovascular/cerebrovascular diseases in groups of individuals who had their yearly health examinations at Peking Union Medical College (PUMC) Hospital from September to November 2005; 2)The in vitro studies investigating the effects of uric acid on the apoptosis, proliferation, and viability of the human umbilical vein endothelial cells(HUVECs); 3)The in vitro studies investigating the effects of uric acid on gene expression of the endothelial nitric oxide synthase (eNOS) and production of nitric oxide by eNOS in HUVECs; 4)The in vitro studies investigating the effects of uric acid on production of reactive oxygen species (ROS) in HUVECs.Methods 1)In the cross-sectional study, the subjects' demographic data, smoking status, drinking habits, average alcohol intake per week, dietary habits, exercises, menstrual history for women, past medical histories, blood pressure, anthropometric indices and serum uric acid, glucose, creatinine, total cholesterol, triglyceride, high-density lipoprotein cholesterol(HDL-C) and low-density lipoprotein cholesterol(LDL-C) after fasting for 8 hours were collected by questionnaires, physical examination, and biochemical assay. The serum uric acid levels (quartiles) or hyperuricemia (≥7mg/dl for men,≥6mg/dl for women) are defined as the predictor variables, the cardiovascular/cerebrovascular diseases as the outcome variables, and other variables related with both the serum uric acid levels or hyperuricemia and the cardiovascular/cerebrovascular diseases as confounding variables. The cardiovascular/cerebrovascular diseases have two definitions ("the loose definition" and "the strict definition") based on the severity of morbidities and the reliability of subjects' report. The loose definition of cardiovascular/cerebrovascular diseases is based on subjects' report of a physician's diagnosis, while the strict definition is based on the report of severe cardiovascular/cerebrovascular events including acute coronary syndrome, myocardial infarction, coronary artery bypass graft, coronary vascular interventions, hemorrhagic stroke, ischemic stroke, and transient ischemic attack, etc. Missing variable analysis is performed to analyze variables that have missing values >5%. The distributions of serum uric acid levels or hyperuricemia and cardiovascular/cerebrovascular diseases in subjects are described by univariate analysis. The relationship between the serum uric acid levels or hyperuricemia and cardiovascular/cerebrovascular diseases is explored by the bivariate analysis, and further adjusted for confounding variables by multivariate logistic regression models. 2)In in vitro studies, HUVECs were isolated from umbilical cords of healthy newborn babies delivered at PUMC Hospital, cultured in endothelial cell medium (ECM), and verified by identification of von Willebrand Factor (vWF) antigen expressed in cytoplasm of the cells. The effects of uric acid on the apoptosis, proliferation and viability of HUVECs were evaluated by Annexin V-FITC | PI staining, CFDA-SE fluorescent probing, and CellTiter-Glo viability system respectively. 3)The effects of uric acid on the relative gene expression of eNOS at both mRNA and protein levels in HUVECs were determined by RT-PCR, Real-Time PCR and Western Blotting. The intracellular and extracellular nitrite levels of HUVECs were measured by the Griess reaction, which corresponds to the nitric oxide produced by eNOS. 4)The effects of uric acid on the intracellular ROS production in HUVECs were determined by H2DCF-DA fluorescent probing. The production of nitric oxide and reactive oxygen species in HUVEC incubated with uric acid was compared to analyze the effect of uric acid on the vascular endothelial function in vitro.Results 1)In the cross-sectional study, HDL-C and LDL-C have missing values over 5% and they are not missing at completely random. The prevalences of hyperuricemia in men and women are 13.7% and 6.0% respectively. The serum uric acid levels are very low in women with menses and increase in older women of menopause, while in men the serum uric acid levels are relatively high across all age groups, with highest levels in men of 20-50 years old, and the prevalences of hyperuricemia in different age groups of men appear in the shape of inverted "V", the highest prevalence being in men of 50-60 years old. The bivariate analysis finds that hyperuricemia is associated with cardiovascular/cerebrovascular diseases significantly in women, and higher serum uric acid levels were also associated with increased odds of cardiovascular/cerebrovascular diseases significantly, even within the normal ranges. However, there is no significant relationship between the increase of serum uric acid levels or hyperuricemia with the cardiovascular/cerebrovascular diseases in men, although the odds ratios of cardiovascular/cerebrovascular diseases is over 1 in subjects with hyperuricemia compared with those whose serum uric acid levels were normal. The bivariate analysis also finds that multiple risk factors of cardiovascular/cerebrovascular diseases including age, ethnicity, education, menopause for women, smoking, alcohol, hypertension, diabetes mellitus, dyslipidemia, obesity, renal dysfunction, fasting plasma glucose levels, total cholesterols, triglycerides, HDL-C and LDL-C are also related with the increase of serum uric acid levels or hyperuricemia. They are confounding variables for the analysis. After adjustment of confounding variables by multivariate logistic regression models, the odds ratios of cardiovascular diseases by the loose definition are 4.0(95%CI 1.4-11.1) and 3.3(95%CI 1.2-8.9) in the 2^nd quartile (3.6-4.1mg/dl) and 4^th quartile (≥4.9mg/dl) of serum uric acid levels in women without adjustment of HDL-C and LDL-C. Further adjustment of these two variables does not alter the results significantly: 3.5(95%CI 1.2-9.8) and 3.0(95%CI 1.1-8.1) in the 2^nd and 4^th quartiles of serum uric acid levels in women. Although the association between hyperuricemia and the cardiovascular diseases by the strict definition is not statistically significant, with OR 7.6(95%CI 0.9-65.7) without adjustment for HDL-C/LDL-C and OR 13.7(95%CI 0.8-231.3) after adjustment, there may exist independent associations between them as the p values are approaching the level of significance (p≈0.07), which may be validated by enlarging the sample size. The adjustment of confounding variables by multivariate logistic regression models does not change the results in men by the bivariate analysis substantially. 2)The in vitro studies find that after incubation with uric acid (0, 5, 10, 20, 30mg/dl) for 24h, 48h, and 72h, there is no significant difference in apoptosis of HUVECs among different incubation groups; the proliferation and total viability of HUVECs do not change significantly after incubation in different concentrations of uric acid for 24h and 48h, but the proliferation and total viability indices decrease as the concentration of uric acid increases after incubation for 72h. 3)The relative gene expression of eNOS at both mRNA and protein levels in HUVECs and the production of nitrite in culture medium increase as the concentration of uric acid increases after incubation for 24h, 48h, and 72h. 4)The production of ROS in HUVECs decreases as the concentration of uric acid increases after incubation for 24h, 48h, and 72h.Conclusion 1)For the epidemiological study, the increase of serum uric acid levels or hyperuricemia may be an independent association factor with cardiovascular diseases in women, but does not find the association in men. We suspect the high serum uric acid levels and prevalence of hyperuricemia in younger male subjects may have blurred the association that is observed in women. 2)For the in vitro studies, uric acid does not have significant role in the apoptosis and viability of HUVECs, but it may suppress the proliferation of HUVECs after incubation for 72h, the significance of which is still unknown. 3)Uric acid improves the gene expression of eNOS (most in coupled status) and production of nitric oxide in HUVECs. 4)Uric acid does not increase the uncoupled eNOS, and decrease the production of ROS in HUVECs. In summary, in the simple in vitro culture environment uric acid improves the oxidative stress of HUVECs and is a protective factor for vascular endothelial function.