Abstract
Introduction: Leptin is an adipose tissue-derived hormone associated with cardiovascular risk factors. We examined whether leptin predicts major adverse cardiac events (MACE) in coronary artery disease (CAD) patients.
Methods: Fasting plasma leptin levels were measured in 1327 male and 619 female CAD patients. The patients were followed up for two years. The primary endpoint (MACE) was the composite of a hospitalisation for congestive heart failure (CHF) or a cardiac death. The secondary endpoint was the composite of an acute coronary syndrome (ACS) or a stroke.
Results: In regression analysis including established risk variables, high leptin levels were associated with a significantly increased risk of MACE (HR 3.37; 95%CI 1.64–6.90; p = 0.001) and ACS or stroke (HR 1.95; 95%CI 1.29–2.96; p = 0.002). Adding leptin to the risk model for MACE increased the C-index from 0.78 (95%CI 0.71–0.85) to 0.81 (0.74–0.88) and improved classification (NRI 0.36; 95%CI 0.13–0.60; p = 0.002) and discrimination of the patients (IDI 0.016; 95%CI 0.001–0.030; p = 0.031).
Conclusions: High plasma leptin levels predict short-term occurrence of CHF or cardiac death and ACS or stroke in patients with CAD independently of established risk factors. The possible harmful effects of leptin should be thoroughly investigated.
Leptin is a peptide hormone secreted mainly by adipose tissue. It has been associated with several cardiovascular risk factors.
High leptin levels predict the short-term occurrence of congestive heart failure or cardiac death and ACS or stroke in patients with CAD independently of established risk factors.
The possible detrimental effects of leptin on the cardiovascular system should be thoroughly investigated.
Key messages
Introduction
Obesity, an independent risk factor of cardiovascular disease, is associated with increased morbidity and mortality (Citation1). The exact pathophysiology of obesity-related cardiovascular disease remains unclear, but it has been suggested that leptin, a hormone strongly associated with obesity, could be a link between obesity and cardiovascular diseases (Citation2,Citation3).
Discovered in the early 1990s, leptin is a 16-kDa peptide hormone produced by the ob gene (Citation4).
It is produced primarily by the adipose tissue and it serves as a signal of adiposity for the brain to regulate energy balance (Citation5). It has since been discovered that besides adipose tissue, leptin and its receptors are expressed in other tissues as well. These include several important tissues of the cardiovascular system, such as endothelial cells (Citation6), vascular smooth muscle cells (Citation7), and cardiomyocytes (Citation8). These findings suggest that leptin may have direct effects on the cardiovascular system.
Several studies have linked leptin and the cardiovascular system. Leptin has been shown to be associated with several cardiovascular risk factors, such as blood pressure (BP) (Citation9), heart rate (Citation10), insulin resistance (Citation11), vascular dysfunction (Citation12), and inflammation (Citation13). Leptin also appears to have prothrombotic (Citation14) and profibrotic (Citation15) properties. On the other hand, leptin may promote angiogenesis and neovascularisation (Citation6). Leptin is also associated with heart failure, with both reduced and preserved ejection fraction. In one of our previous studies, we showed that high levels of circulating leptin are associated with impaired diastolic function in patients with coronary artery disease (CAD) (Citation16).
In earlier studies, circulating leptin levels have predicted CAD (Citation17) and cardiovascular events in established CAD (Citation18). High leptin levels have been associated with an increased risk of restenosis after percutaneous coronary intervention. Leptin levels have predicted first-ever myocardial infarction (Citation19), and elevated leptin levels on the first morning after acute myocardial infarction have been associated with a poorer prognosis in the long term (Citation20). Leptin has also been an independent predictor of recurrent cardiovascular events in men with earlier acute coronary syndromes (ACSs) (Citation21). In addition, high leptin levels have been associated with the incidence of stroke (Citation22). On the other hand, in a recent meta-analysis carried out by Chai et al. there was no statistically significant connection between leptin and CAD. The authors pointed out, however, that attention should be paid to high leptin levels in men (Citation23).
In conclusion, leptin has many effects on the cardiovascular system and is also associated with cardiovascular outcome. Considering our earlier observation of an association between high leptin levels and diastolic dysfunction, we investigated, whether or not baseline plasma leptin levels are associated with the risk of hospitalisation for congestive heart failure (CHF), cardiac death, ACSs or strokes in a prospective cohort of CAD patients including patients with type 2 diabetes matched with non-diabetic patients.
Material and methods
Subjects
The present work is a sub-study of the ARTEMIS study (Cardiovascular Complications in Type II Diabetes Study; registered at ClinicalTrials.gov, Record 1539/31/06, Identifier NCT01426685). The study population initially consisted of 1946 CAD patients (1327 (68%) men and 619 (32%) women) of whom 834 (43%) also had type 2 diabetes. The diabetic and non-diabetic groups were matched by sex, age, history of myocardial infarction, and treatment strategy. A flow chart of the screening and matching procedure can be found in a previous article from the same study group (Citation24). The purpose of the ARTEMIS study is to examine the prognostic value of traditional and novel risk markers in patients with stable CAD, and the main endpoint is sudden cardiac death during the five-year follow-up period. The primary endpoint of the present two-year sub-study was a major adverse cardiac event (MACE) defined as a hospitalisation for CHF or a cardiac death. The secondary endpoint was defined as an ACS or a stroke.
The subjects in the study were recruited from a series of patients who had come in for coronary angiography at the Division of Cardiology, Oulu University Hospital. Most patients had undergone percutaneous coronary intervention or coronary artery bypass graft surgery. A little less than half of the patients had suffered an ACS event before entering the study, but not during the previous three months. Patients aged less than 18 or more than 85 years, of NYHA or CCS class IV, with significant valvular disease, a pacemaker, an implantable cardioverter-defibrillator (ICD) or plans of ICD implantation, or with end-stage renal failure requiring dialysis were excluded from the study, as were patients with life expectancy of less than a year or who were deemed unfit for participation. The study conforms to the Declaration of Helsinki and was approved by the Ethics Committee of the Northern Ostrobothnia Hospital District. Informed consent was obtained from the patients.
Examinations
Upon entering the study, the patients underwent extensive risk profiling for cardiovascular diseases. This examination included clinical examination, body measurements, electrocardiography, echocardiography, an exercise stress test, Holter screening, and biomarker analyses. The patients were also interviewed about their history and characteristics such as smoking and angina pectoris symptoms assessed by using Canadian Cardiovascular Society (CCS) grading of angina pectoris.
Body weight was measured by using a digital scale. Body mass index (BMI) was calculated by dividing weight (kg) by height squared (m2). Waist circumference was measured with a tape measure during light expiration halfway between the lower rib margin and the iliac crest and rounded to the nearest 0.5 cm. Arterial BP was measured with an oscillometric device (Dinamap® Model 18465X, Criticon Ltd., Ascot, UK) from the right arm in a sitting position after an overnight fast and a 10- to 15-min rest. BP was measured three times at 1-min intervals, and the mean of the last two measurements was used in the analyses.
Venous blood samples were drawn into EDTA tubes after an overnight fast, using standardised methods. After fasting blood samples had been obtained, the subjects with no history of diabetes underwent an oral glucose tolerance test. Diabetes, impaired glucose tolerance and impaired fasting glucose were diagnosed according to the definition of the World Health Organisation (Citation25). Capillary glucose was measured by the glucose oxidase method with OneTouch® UltraEasy® test strips (FastDraw™, LifeScan, Switzerland). Plasma leptin levels were measured by using commercial enzyme-linked immunosorbent assay kits (BioVendor; Cat no. RD 191001100). Levels of high-sensitivity C-reactive protein were determined by immunonephelometry (BN ProSpec® and CardioPhase®, Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). Plasma lipids (total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and triglycerides), creatinine, glycated haemoglobin (HbA1c), and urine albumin-to-creatinine ratio (ACR) were analysed at Oulu University Hospital laboratory, using standardised methods.
Two-dimensional and M-mode echocardiography were performed by three cardiologists utilising a General Electric Vivid 7 ultrasound machine. The examinations were performed according to American Society of Echocardiography (ASE) guidelines. The echocardiographic parameters chosen for the present study were left ventricular ejection fraction (LVEF) in two-dimensional mode, left ventricular end-diastolic diameter (LVEDD), left atrial diameter (LA), ratio of peak early diastolic mitral velocity to tissue Doppler-derived peak early diastolic mitral annular velocity (E/E′), ratio of peak early diastolic mitral velocity to peak late diastolic mitral velocity (E/A), and left ventricular mass index (LVMI). Left ventricular (LV) mass was determined by using the formula recommended by the ASE (LV mass =0.8 × (1.04 ((LVIDd + PWTd + SWTd)3 − (LVIDd)3)) + 0.6 g). Left ventricular mass index (LVMI) was determined by dividing LV mass by body surface area (BSA).
Follow-up
Two years after entering the study, the subjects were contacted by mail or telephone or both and they responded to a survey. Data on hospitalisation were obtained from electronic patient record systems. Data on causes of death were obtained from available death certificates and autopsy reports.
Statistical analyses
The data were analysed using IBM® SPSS® Statistics for Windows, Version 21 software (IBM Corp., NY) and R Statistics (3.1.2, The R Foundation for Statistical Computing, Vienna, Austria). To determine differences between the sexes, chi-square tests, analysis of variance, or independent-samples Mann–Whitney U tests were used. Receiver operating characteristic (ROC) curves were used to determine the optimal cut-off value for plasma leptin. The cut-off point was set to the value that provided the maximum sum of specificity and sensitivity and at least a sensitivity of 20% above the median value (≥14.1 ng/ml for MACE and ≥9.9 ng/ml for ACS or stroke). Because of the highly skewed distribution of plasma leptin levels, they were also divided into deciles to assess the prognostic significance of leptin as a continuous risk marker.
The prognostic significance of leptin levels was determined according to the recommendations of the American Heart Association (Citation26). Hazard ratios (HRs) and 95% confidence intervals (CIs) related to plasma leptin (separately for optimal cut-off values and deciles) were determined by univariate, and, subsequently, multivariate Cox regression analysis. The covariates chosen for the analyses were age, sex, BMI, LVEF, and presence of diabetes.
The incremental prognostic significance related to plasma leptin was assessed by the C-index, the integrated discrimination index (IDI), and the continuous net reclassification index (NRI). These analyses were based on predicted risks that were calculated with and without plasma leptin (optimal cut-off point) and rounded to the closest integer (%). Values of p < 0.05 were considered statistically significant.
Results
The characteristics of the patients are presented in . Male patients were younger than female patients, their waist circumference was greater and systolic BP lower. There were more current smokers among men than among women. Male current and ex-smokers had also smoked longer than female current and ex-smokers. Male patients had suffered myocardial infarction more often than female patients and a history of revascularisation was more common in male than in female patients. Male patients appeared to be less symptomatic than female patients when assessed by the CCS score. The use of diuretics was less common among male than female patients. In male patients, LVEF was lower, LVEDD was higher, LA was higher, E/E′ was lower, and LVMI was higher than in female patients. Also, E/A was slightly higher in male than in female patients. Total, HDL and LDL cholesterol levels as well as triglyceride levels were lower in male than in female patients. Creatinine clearance was greater in male than in female patients. Finally, leptin levels were significantly higher in female than in male patients, as expected.
Table 1. Characteristics of the patients.
During the two-year follow-up period, 51 patients (42 male and 9 female) experienced MACE and 155 patients (97 male and 58 female) encountered an ACS or stroke. High leptin levels were not associated with an increased risk of MACE in the female population, and the number of female patients who experienced MACE was low. Thus, male and female patients were analysed as a single group. Plasma leptin predicted MACE significantly in both univariate and multivariate Cox regression analyses, with a 3.4-fold increased risk (multivariate analysis) at the optimal cut-off point (, ). Plasma leptin also predicted ACS or stroke in both univariate and multivariate Cox regression analyses ().
Figure 1. Kaplan–Meier curve re. MACE-free survival in the two-year follow-up period in low- (<14.1 ng/ml) and high-level (≥14.1 ng/ml) leptin groups.
Table 2. Univariate and multivariate Cox regression analyses.
Regarding MACE, the optimal cut-off point yielded sensitivity of 71%, specificity of 61%, and positive and negative predictive values of 5% and 99%, respectively. For ACS or stroke, the optimal cut-off point yielded sensitivity of 64%, specificity of 49%, and positive and negative predictive values of 10% and 94%, respectively.
Based on the findings in the Cox regression analyses, we also performed discrimination and reclassification analyses of the risk models for MACE and ACS or stroke. The addition of leptin (optimal cut-off point ≥14.1 ng/ml) to the established risk model (age, sex, BMI, LVEF, and presence of diabetes) for MACE increased the C-index from 0.78 (95% CI 0.71–0.85) to 0.81 (95% CI 0.74–0.88), significantly improved the classification (NRI 0.36; 95% CI 0.13–0.60; p = 0.002) and discrimination of the patients (IDI 0.016; 95% CI: 0.001–0.030; p = 0.031).
Regarding ACS or stroke, adding leptin (optimal cut-off point ≥9.9 ng/ml) to the established risk model (age, sex, BMI, LVEF, and presence of diabetes) increased the C-index from 0.59 (95% CI 0.54–0.64) to 0.62 (95% CI 0.57–0.67), significantly improved the classification (NRI 0.31; 95% CI 0.15–0.46; p < 0.001) and discrimination of the patients (IDI 0.01; 95% CI 0.00–0.01; p = 0.002).
Discussion
We studied the possibility of using leptin levels for risk stratification in CAD patients. In the two-year follow-up period, adding leptin to a risk model of established cardiovascular risk factors increased the predictive power of the risk model and led to a tendency towards better reclassification of the risks of incident CHF and cardiac death as well as ACS or stroke.
In our previous analysis of the same data, we found that elevated leptin levels were associated with impaired left ventricular filling patterns (Citation16). In the present study, we tested the hypothesis that high leptin levels also have a clinical impact in the same population, using hospitalisation for heart failure and cardiac death as endpoints. Our primary hypothesis did not include vascular events such as an acute coronary event or stroke as primary endpoints, which have different pathophysiological backgrounds. We recognise, however, that heart failure may be a consequence of a previous myocardial infarction.
The prognostic power of leptin in the context of CAD has been reported in previous studies. As reviewed by Beltowski (2006), leptin has many potentially proatherogenic effects, but the association between leptin and atherosclerosis does not directly prove a causal relationship, as leptin might correlate with other proatherosclerotic factors (Citation27). In a prospective study carried out by Wolk et al. (2004), leptin was shown to be an independent predictor of cardiovascular events in patients with angiographically confirmed atherosclerosis, but diabetic patients were excluded to better examine the independent effects of leptin (Citation18). In our study, patients with glucose metabolism disorders were included, and patients with type 2 diabetes were matched with non-diabetic controls.
The definitive role of leptin in the development of cardiovascular diseases remains to be clarified. As reviewed by Ghantous et al. (Citation28), there are several studies suggesting that leptin is detrimental for the cardiovascular system, and, controversially, the results of several studies suggest that leptin is, in fact, beneficial. Obesity is a well-established risk factor for cardiovascular diseases, and circulating leptin levels are increased in obesity (Citation1). However, leptin is not just an inert marker of obesity but an active hormone with diverse effects in the cardiovascular system and other parts of the body. In the present study, we suggest a harmful effect of leptin on cardiovascular outcome independent of obesity.
Leptin resistance—a phenomenon encountered in obesity and reminiscent of insulin resistance—is presumably an important source of interference (Citation18). The most important limitation of our study is that the exact body composition of the subjects is not known. However, we adjusted the statistical analyses for BMI, which gives an accurate characterisation of body composition. Male and female patients were analysed as a single group, as there was only a small number of events in female patients. However, plasma leptin levels are significantly higher among women and the biological actions of leptin can differ between the sexes.
In the present study, the predictive power of leptin was independent not only of obesity but also of the known risk factors of cardiovascular events. However, the mechanism by which leptin causes more severe heart disease is not clear and is likely to be multifactorial. We previously reported a positive correlation between leptin levels and impaired diastolic function in the same population of patients (Citation16). In the light of our previous findings, the present findings suggest that leptin could have detrimental effects on left ventricular diastolic properties, thereby increasing the risk of CHF and subsequent cardiac death.
A possible pathway by which leptin could bring about more severe heart disease concerns myocardial fibrosis. It is likely that leptin could act as a profibrotic agent in the myocardium (Citation15). This could explain the association between high leptin levels and the composite endpoint of CHF and cardiac death. Also, it has been shown that leptin levels predict first-ever acute myocardial infarction and stroke (Citation19,Citation22). Thus, it is possible that leptin could cause a more severe CAD.
In conclusion, we have shown that leptin levels predict the occurrence of short-term CHF events or cardiac deaths and ACSs or strokes in patients with established CAD. It is uncertain whether leptin is merely a marker of increased cardiovascular risk or an active component in the development of cardiovascular diseases. Further research on the exact mechanisms by which leptin affects the cardiovascular system is warranted. In addition, circulating leptin levels could serve as an additional tool in short-term risk stratification of CAD patients. Completion of the five-year follow-up period of the ARTEMIS study will clarify whether leptin predicts cardiovascular events in the long term.
Acknowledgements
The authors wish to acknowledge the technical assistance of Ms Pirkko Huikuri, Ms Sari Kaarlenkaski, Ms Päivi Kastell and Ms Päivi Koski. This work was supported by a grant from the Finnish Funding Agency for Technology and Innovation (TEKES, Helsinki, Finland) and the Medical Research Center Oulu Doctoral Programme (MRC Oulu DP).
Disclosure statement
The authors disclose no conflicts of interest.
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