Clinica Chimica Acta 433 (2014) 34–38
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Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
The HDL anti-inflammatory function is impaired in myocardial infarction and may predict new cardiac events independent of HDL cholesterol Robin P.F. Dullaart a,⁎, Wijtske Annema b, René A. Tio c, Uwe J.F. Tietge b a b c
Department of Endocrinology, University of Groningen and University Medical Center Groningen, Groningen, The Netherlands Department of Pediatrics, University of Groningen and University Medical Center Groningen, Groningen, The Netherlands Department of Cardiology, University of Groningen and University Medical Center Groningen, Groningen, The Netherlands
a r t i c l e
i n f o
Article history: Received 4 February 2014 Received in revised form 24 February 2014 Accepted 25 February 2014 Available online 5 March 2014 Keywords: Acute coronary syndrome Atypical chest pain HDL anti-inflammatory function STEMI Non-STEMI
a b s t r a c t Background: Intrinsic functional properties of high density lipoproteins (HDL) are considered to be physiologically important for atheroprotection. We compared the HDL anti-inflammatory capacity between patients with acute myocardial infarction (MI) and patients with non-cardiac chest pain, and prospectively determined the association of new major adverse cardiovascular events (MACE) with this metric of HDL function. Methods: A prospective study was carried out in 93 patients referred for acute chest pain (65 patients with acute MI). The HDL anti-inflammatory capacity was determined as the ability to suppress tumor necrosis factor-α-induced vascular cell adhesion molecule-1 mRNA expression in endothelial cells in vitro. Results: Acute MI at admission was associated with impaired HDL anti-inflammatory capacity (p = 0.001), even after adjustment for HDL cholesterol and apolipoprotein A-I (p = 0.003). Twenty nine MACE were ascertained during a median follow-up of 1210 (910–1679) days. New MACE was associated with impaired HDL antiinflammatory capacity (hazard ratio: 1.80 (1.17–2.77) per SD change, p = 0.007) in age, sex, HDL cholesterol and apolipoprotein-AI adjusted analysis. Conclusions: The ability of HDL to attenuate endothelial inflammation is impaired in acute MI, and this metric of HDL function may serve as a predictor of new MACE, even independent of HDL cholesterol and apolipoprotein A-I. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Large population-based studies have convincingly demonstrated that high plasma levels of high density lipoprotein (HDL) cholesterol levels confer protection against atherosclerotic cardiovascular disease (CVD) [1,2]. Remarkably, pharmacological interventions aimed to raise HDL cholesterol, for example with cholesteryl ester transfer protein inhibitors or niacin, have failed to demonstrate cardiovascular benefit [3–5]. Such findings have contributed to the concept that intrinsic functional properties of HDL may be physiologically more important than HDL cholesterol mass levels to exert protection against the development or progression of atherosclerosis [3,6–11]. Among other allegedly anti-atherogenic functions, such as promoting cellular cholesterol efflux and preventing oxidative modification of low density lipoproteins (LDL), HDL particles also exert potent anti-inflammatory properties [9–11]. During the past few years evidence has accumulated that HDL function may be impaired in clinical conditions featured by increased ⁎ Corresponding author at: Department of Endocrinology, University of Groningen and University Medical Center Groningen, P.O. Box 30.001, Groningen, 9700 RB, The Netherlands. Tel.: +31 503613731; fax: +31 503619392. E-mail address: [email protected] (R.P.F. Dullaart).
http://dx.doi.org/10.1016/j.cca.2014.02.026 0009-8981/© 2014 Elsevier B.V. All rights reserved.
cardiovascular risk, although there are still considerable areas of uncertainty [6,7,9–14]. Remarkably little is known about the extent to which various measures of HDL function are altered in the context of an acute coronary syndrome. Recent cross-sectional studies have demonstrated that the ability of HDL to promote cellular cholesterol efflux, as well as the potential of HDL to inhibit LDL oxidation in vitro may be impaired during the acute phase of a myocardial infarction (MI) [15,16], but it is still uncertain whether the capacity of HDL to modify endothelial inflammation in vitro, as a metric of its anti-inflammatory capacity, is abnormal in the setting of an acute MI. In addition, no study is currently available to investigate the predictive value of a HDL function metric for the future development of new major adverse cardiovascular events (MACE). Therefore, the present study was initiated first to compare the HDL anti-inflammatory capacity between patients with acute MI and patients with non-cardiac chest pain, and second to prospectively determine the impact of this metric of HDL function on new MACE during follow-up. 2. Materials and methods 2.1. Subjects The present study represents part of a protocol which aims to determine the diagnostic yield of lipid and non-lipid biomarkers in
R.P.F. Dullaart et al. / Clinica Chimica Acta 433 (2014) 34–38
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discriminating between cardiac and non-cardiac causes of acute chest pain [17,18]. The protocol complies with the Declaration of Helsinki, and has been approved by the medical ethics committee of the University Medical Center Groningen. The protocol involves the anonymous storage of frozen plasma, for which the approval from the medical ethics committee was requested but waived, because such storage is performed as standard for additional testing in order to evaluate future clinically important diagnostic considerations. Consecutive patients referred with chest pain suspected for having an acute coronary syndrome were enrolled if they presented at weekdays between 8 a.m. and 5 p.m. Patients with MI were categorized as MI without ST-elevation (non-STEMI) and MI with ST-elevation (STEMI), as diagnosed by attending cardiologists [17,18]. The University Medical Center Groningen serves as regional referral center for STEMI patients. To be eligible other clues for a cardiac origin of chest pain had to be absent. If sequential electrocardiograms and troponin T tests were normal, patients were classified as having non-cardiac chest pain, and were discharged in case of low suspicion or if a bicycle exercise test was negative. The time frame of inclusion was between 2006 and 2008. Follow-up data were obtained from all participants with respect to subsequent MACE (cardiac mortality, MI, percutaneous coronary intervention (PCI), and/or coronary artery bypass grafting (CABG)), which was chosen as the combined end-point. Cardiac mortality was defined as sudden cardiac death and death due to acute MI or heart failure. Causes of death were obtained by linking the number of the death certificate to the primary cause of death as coded by the Central Bureau of Statistics (Voorburg/Heerlen, The Netherlands). Census date was chosen as the date of newly manifest MACE or as the date of the latest verification of the patient status which was at least 1000 days after initial presentation.
cholesterol was measured with a homogeneous enzymatic colorimetric test (Roche/Hitachi, cat no 04713214; Roche Diagnostics GmbH, Mannheim, Germany). Non-HDL cholesterol was calculated as the difference between total cholesterol and HDL cholesterol. LDL cholesterol was calculated by the Friedewald formula. Apolipoproteins (apo) A-I and B were assayed by immunoturbidimetry (Roche/Cobas Integra Tina-quant cat no. 03032566 and 033032574, respectively, Roche Diagnostics). The HDL anti-inflammatory capacity was determined using an in vitro cell system essentially following a previously described procedure [19]. ApoB-containing lipoproteins were precipitated from plasma by adding 100 μl 36% polyethylene glycol (PEG 6000, Sigma, St. Louis, MO, USA) in 10 mM HEPES (pH = 8.0) to 200 μl plasma followed by 30 min incubation on ice. After 30 min centrifugation at 2200 g, the HDL-containing supernatant was collected, kept on ice, and used directly for the HDL anti-inflammatory assay. Human umbilical vein endothelial cells (HUVECs), provided by the Endothelial Cell Core Facility of the University of Groningen, The Netherlands, were pre-incubated for 30 min with 2% of individual apoB-depleted plasma samples or with equal amounts of phosphate buffered saline (PBS) as control [19]. Then 10 ng/ml tumor necrosis factor-α (TNF-α; R&D systems, Abingdon, UK) or PBS as control was added, and cells were incubated for an additional 8 h followed by analysis of vascular cell adhesion molecule-1 (VCAM-1) gene expression using quantitative real-time polymerase chain reaction. VCAM-1 mRNA expression was calculated relative to the average of the housekeeping gene cyclophilin. Results are expressed as fold induction over baseline in the presence of the individual subject's HDL preparations, whereby higher values indicate lower anti-inflammatory capacity. Storage of plasma at −80 °C did not influence the results of this assay (data not shown). The intra-assay coefficient of variation of this assay is 9%.
2.2. Laboratory analyses
2.3. Statistical analysis
Venous blood samples were obtained directly after admission for routine measurements. EDTA-anticoagulated samples were immediately placed on ice. The plasma samples were stored at −80 °C. Plasma cholesterol was assayed by a routine enzymatic method (Roche/Hitachi cat no 11875540; Roche Diagnostics GmbH, Mannheim, Germany). HDL
Data are given as means ± SD, medians (interquartile range) or in numbers. Continuous variables were compared by unpaired t-tests or by Mann–Whitney U-tests in case of non-parametrically distributed data. Differences in proportions of variables were determined by χ2-analysis.
Table 1 Clinical characteristics and biomarkers of myocardial damage in 65 patients with myocardial infarction (MI) and 28 patients with non-cardiac chest pain.
Age (years) Sex (M/F) Non-STEMI/STEMI Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) History of hypertension (yes/no) History of diabetes mellitus (yes/no) Previous MI (yes/no) Previous PCI (yes/no) Previous CABG (yes/no) Current smoking (yes/no) Statin treatment (yes/no) Other lipid lowering drugs (yes/no) β-blocker treatment (yes/no) Calcium antagonist treatment (yes/no/unknown) Aspirin treatment (yes/no) Other anticoagulants (yes/no) Troponin T at presentation (μg/l) Maximal troponin T (μg/l) CK at presentation (U/l) Maximal CK (U/l) CK-MB at presentation (U/l) Maximal CK-MB (U/l)
MI (n = 65)
Non-cardiac chest pain (n = 28)
p-value
68 ± 11 44/21 32/33 135 ± 25 74 ± 14 23/42 8/57 7/58 10/55 7/58 22/43 22/43 0/28 22/43 10/49/6 19/46 9/58 0.04 (0.003–17.8) 1.38 (0.14–4.82) 111 (62–192) 307 (106–999) 17 (9–26) 41 (16–173)
63 ± 11 14/14 0/0 139 ± 17 78 ± 9 7/21 2/26 4/24 2/26 2/26 3/25 10/18 1/64 10/18 2/26 7/21 4/24 0.00 (0.00–0.00) 0.00 (0.00–0.00) 29 (22–39) 37 (26–62) 2 (2–4) 3 (2–6)
0.053 0.11 b0.001 0.42 0.17 0.33 0.46 0.63 0.28 0.59 0.021 0.86 0.51 0.84 0.22 0.68 0.98 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001
Data as means ± SD or as medians (interquartile ranges). CABG, coronary artery bypass graft; CK, creatine kinase; CK-MB, MB fraction of creatine kinase; M, male; F, female; non-STEMI, myocardial infarction (MI) without ST-elevation; STEMI, MI with ST-elevation; PCI, percutaneous coronary intervention. Other anticoagulants are mainly vitamin K antagonists.
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Table 2 Plasma lipids, lipoproteins, apolipoproteins and the HDL anti-inflammatory capacity in 65 patients with myocardial infarction (MI) and in 28 patients with non-cardiac chest pain.
Total cholesterol (mmol/l) LDL cholesterol (mmol/l) Non-HDL cholesterol (mmol/l) HDL cholesterol (mmol/l) Total cholesterol/HDL cholesterol ratio Apo A-I (g/l) Apo B (g/l) Apo B/apo A-I HDL anti-inflammatory capacity (fold increase in VCAM-1 mRNA expression)
MI (n = 65)
Non-cardiac chest pain (n = 28)
p-value
5.33 ± 2.12 3.51 ± 1.51 4.14 ± 2.07 1.19 ± 0.24 4.58 ± 1.51 1.35 ± 0.24 1.07 ± 0.34 0.82 ± 0.30 13.8 (4.2–32.0)
5.19 ± 1.12 3.25 ± 0.94 3.98 ± 1.06 1.20 ± 0.25 4.39 ± 1.01 1.48 ± 0.27 1.12 ± 0.25 0.77 ± 0.18 3.5 (0.7–14.2)
0.73 0.33 0.69 0.69 0.73 0.060 0.59 0.52 0.001
Data as means ± SD or as medians (interquartile ranges). Apo, apolipoproteins; HDL, high density lipoproteins; LDL, low density lipoproteins, non-HDL cholesterol, non-high density lipoproteins; VCAM-1, vascular cell adhesion molecule-1; higher values of HDL-anti-inflammatory capacity (expressed as fold increase in VCAM-1 mRNA expression over baseline) indicate lower anti-inflammatory capacity.
The associations of the presence of MI at presentation with the HDL anti-inflammatory capacity, HDL cholesterol and apoA-I were determined using receiver operating characteristic (ROC) curves (presented as area under the curve with 95% confidence intervals (CI)). In addition, logistic regression analysis was performed to disclose the independent association of MI at presentation with the HDL anti-inflammatory capacity. The independent association of new MACE with the HDL anti-inflammatory capacity was analyzed using Cox proportional hazard analysis. Odds ratios and hazard ratios of the continuous variables of interest are given per SD change with 95% confidence intervals (CI). Two-sided p-values b0.05 were considered significant.
3. Results Ninety-three patients were included of whom 65 were diagnosed with acute MI (Table 1). As shown in Table 2, plasma total cholesterol, LDL cholesterol, non-HDL cholesterol, apoB, as well as HDL cholesterol and apoA-I levels were not significantly different between MI patients and patients with non-cardiac chest pain. Remarkably, the HDL antiinflammatory capacity was severely impaired in MI patients, as indicated by the higher values of TNF-α-stimulated VCAM-1 expression (p = 0.001; Table 2 and Fig. 1). The HDL anti-inflammatory capacity did not significantly differ between patients with non-cardiac chest pain and a group of 20 healthy control subjects who did not present with chest pain (3.2 (0.9–10.1) fold increase in VCAM-1 expression, data not shown, p N 0.30). Furthermore, the HDL anti-inflammatory
capacity was lower in STEMI vs. non-STEMI patients (26.4 (12.6–40.1) vs. 13.0 (1.9–19.8) fold increase in VCAM mRNA expression (p b 0.001). The area under the ROC curve of impaired HDL anti-inflammatory capacity was significant (0.736, 95% CI, 0.595–0.877, p = 0.004), whereas those of lower HDL cholesterol (0.525, 95% CI, 0.356–0.693, p = 0.76) and apoA-I (0.653, 95% CI, 0.503–0.803, p = 0.076) were not. Acute MI at presentation was also strongly associated with impaired HDL antiinflammatory capacity in age- and sex-adjusted logistic regression analysis (odds ratio per SD increment: 3.72 (95% CI, 1.66–8.33), p = 0.001). Remarkably, this association was not attenuated after adjustment for either HDL cholesterol (4.52 (95% CI, 1.89–10.78), p = 0.001), apoA-I (9.59 (95% CI, 2.44–37.73), p = 0.001) or both (8.76 (95% CI, 2.08– 36.87), p = 0.003). This association also persisted after adjustment for age, sex, previous MI, previously diagnosed diabetes, current smoking and treatment with statins, anti-hypertensives and anticoagulants (5.52 (95% CI, 1.93–15.58), p = 0.001). After a median follow-up of 1210 (910–1679) days 29 new MACE occurred in the whole study population of 93 patients. Four of these MACE occurred in the non-cardiac chest pain group. These MACE occurred N 200 days after initial admission, and were considered not to be due to a false initial diagnosis of non-cardiac chest pain. Age, sex distribution, smoking at initial presentation, as well as previously diagnosed diabetes and hypertension were not significantly different between patients who experienced new MACE compared to patients who did not (data not shown; all p N 0.10). Plasma total cholesterol, LDL cholesterol, non-HDL cholesterol and apoB levels at initial presentation were not different in patients who experienced a new MACE during follow-up compared to those who did not (data not shown; all p N 0.17). HDL cholesterol (1.19 ± 0.24 mmol/l) and apoA-I levels (1.39 ± 0.24 g/l) were also not different in those patients who experienced a new MACE compared to those patients who did not (1.20 ± 0.24 mmol/l, p = 0.92 and 1.39 ± 0.26 g/l, p = 0.97, respectively). In age-and sex-adjusted Cox proportional regression analysis new MACE was associated with impaired HDL anti-inflammatory capacity (Table 3). This association remained significant after adjustment for either HDL cholesterol, apoA-I, or both (Table 3). Conversely, neither HDL cholesterol nor apoA-I predicted new MACE taking account of the HDL anti-inflammatory capacity. In an analysis which only included those patients with verified acute MI at presentation (n = 65), the association of new MACE with the HDL anti-inflammatory capacity was also significant in age- and sex-adjusted analysis (hazard ratio per SD increment: 1.49 (95% CI, 1.02–2.17, p = 0.037). 4. Discussion
Fig. 1. Box plots showing the high density lipoprotein (HDL) anti-inflammatory capacity in 65 patients with acute myocardial infarction and in 28 patients with non-cardiac chest pain (no myocardial infarction). The HDL-anti-inflammatory capacity is expressed as fold increase in vascular cell adhesion molecule-1 mRNA expression over baseline; higher values thereby indicate lower anti-inflammatory capacity. Values are given in medians, interquartile ranges, 5 to 95% ranges and extremes. Difference between patients with and without myocardial infarction: p = 0.001 by Mann–Whitney U-test.
The present study shows first that the HDL anti-inflammatory capacity is impaired in acute MI, and second that prospectively determined new MACE is associated with this metric of HDL function. Remarkably, this association of adverse cardiac outcome with impaired HDL anti-inflammatory capacity was independent of HDL cholesterol
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Table 3 Relationship of the high density lipoprotein anti-inflammatory capacity with 29 subsequent major adverse cardiovascular events by Cox proportional regression analysis during follow-up of 93 patients (65 patients with acute myocardial infarction and 28 patients with non-cardiac chest pain at presentation). Model 1
HDL anti-inflammatory capacity HDL cholesterol
Model 2
Model 3
Model 4
Hazard ratio (95% CI)
p-value
Hazard ratio (95% CI)
p-value
Hazard ratio (95% CI)
p-value
Hazard ratio (95% CI)
p-value
1.54 (1.09–2.18)
0.015
1.81 (1.18–2.77) 0.83 (0.42–1.61)
0.007
1.78 (1.18–2.69)
0.006
1.80 (1.17–2.77) 0.92 (0.37–2.25) 0.87 (0.37–1.98)
0.007
0.57
Apolipoprotein A-I
0.82 (0.44–1.52)
0.53
0.85 0.73
For each variable hazard ratios are given per 1 SD increment with 95% confidence intervals (CI). All models are adjusted for age and sex. HDL, high density lipoproteins. Model 1: the HDL anti-inflammatory capacity adjusted for age and sex only; model 2: the HDL anti-inflammatory capacity additionally adjusted for HDL cholesterol; model 3: the HDL antiinflammatory capacity additionally adjusted for apolipoprotein A-I; model 4: the HDL anti-inflammatory capacity additionally adjusted for both HDL cholesterol and apolipoprotein A-I.
and apoA-I. Our findings, therefore, lend support to the emerging concept of HDL function being more relevant for cardioprotection than levels of HDL cholesterol and apoA-I. The current observation with respect to impaired anti-inflammatory function of HDL from acute MI patients complements recent studies in which either the ability of HDL to promote cellular cholesterol efflux or the anti-oxidative activity of HDL was found to be diminished during the acute phase of a coronary syndrome [15,16]. To our knowledge, the prospective analysis described in this report is the first to suggest that a metric of HDL function obtained at presentation of MI is a statistically significant determinant of adverse cardiac outcome. Thus far, only the predictive value of the ability of HDL to promote cellular cholesterol efflux has been evaluated in prospective studies. In a prospective nested case–control study among subjects without a coronary event at baseline, cholesterol efflux did not predict the future development of MACE [13]. Another study unexpectedly documented that adverse cardiac outcome was associated with a higher cholesterol efflux rate [14]. However, none of these studies were carried out among patients presenting with an acute coronary syndrome. The assay system that we used to document HDL's antiinflammatory function, i.e. the ability of HDL to suppress TNF-αinduced VCAM-1 expression in HUVECs, was brought about by the notion that endothelial inflammation is conceivably implicated in the pathogenesis of (instable) atherosclerotic lesions [11]. In another study, we used the ability of HDL to suppress TNF-α induced monocyte chemoattractant protein 1 (MCP-1/CCL-2) gene expression as read out, but the assay conditions were otherwise similar [20]. In that study, which was carried out in diabetic subjects, we additionally documented cholesterol efflux and the anti-oxidative capacity of HDL, and found no significant relationships between these 3 metrics of HDL function [20]. It seems, therefore, plausible that various HDL function metrics may yield complementary information regarding allegedly atheroprotective effects attributed to HDL, although it is a limitation that we did not document other metrics of HDL function in the current study. Several other methodological considerations and limitations of our study need to be discussed. First, this study was performed in a referral center for STEMI patients, explaining the large proportion of STEMI patients in our cohort [17]. Second in the present study, HDL cholesterol was not different between subjects with and without MI, whereas the difference in apoA-I was close to statistical significance. In comparison, previous reports from our group that were carried out in somewhat larger populations showed significantly lower HDL cholesterol and apoA-I levels in acute MI patients [17,18], as expected [21].Third, the number of new MACE during follow-up was rather limited. We only adjusted for age, sex, HDL cholesterol and apoA-I in Cox-regression analysis in order to circumvent overestimation or underestimation in the regression coefficients as much as possible [22]. Thus, effect modification of other clinical variables cannot be excluded, and we interpret our finding that adverse cardiac outcome is associated with impaired HDL anti-inflammatory capacity to be preliminary. Fourth, it should be
emphasized that there is currently no consensus with respect to the isolation procedure of HDL when assaying functional properties of HDL. We decided not to use ultracentrifugation, because not only it is difficult to isolate HDL from small volumes of plasma, but more importantly because high shear forces and ionic strengths may result in considerable changes in the composition and the protein cargo of the HDL particles [10]. In conclusion, this study demonstrates that the ability of HDL to attenuate endothelial inflammation is impaired in patients presenting with acute MI. Although more work is warranted to determine the contribution of various metrics of HDL function to improved cardiovascular risk classification, our current study raises the possibility that the antiinflammatory capacity of HDL may have the potential to serve as a predictive biomarker for cardiovascular outcome even independent of HDL-C or apoA-I mass.
Conflict of interest The authors report no financial or other conflicts of interest. Acknowledgments The analytical help of L.D. Dikkeschei, PhD, for apolipoprotein assay is greatly appreciated. UJFT is supported by a grant from The Netherlands Organization for Scientific Research (VIDI Grant 917-56358). This study was investigator driven.
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[10] Triolo M, Annema W, Dullaart RPF, Tietge UJF. Assessing the functional properties of high-density lipoproteins-an emerging concept in cardiovascular research. Biomark Med 2013;7:457–72. [11] Rye KA, Barter PJ. Cardioprotective functions of HDLs. J Lipid Res 2014:168–79. [12] Khera AV, Cuchel M, de la Llera-Moya M, et al. Cholesterol efflux capacity, highdensity lipoprotein function, and atherosclerosis. N Engl J Med 2011;364:127–35. [13] de Vries R, Groen AK, Dullaart RPF. Cholesterol efflux capacity and atherosclerosis. N Engl J Med 2011;14(364):1473–4. [14] Li XM, Tang WH, Mosior MK, et al. Paradoxical association of enhanced cholesterol efflux with increased incident cardiovascular risks. Arterioscler Thromb Vasc Biol 2013;33:1696–705. [15] Hafiane A, Jabor B, Ruel I, Ling J, Genest J. High-density lipoprotein mediated cellular cholesterol efflux in acute coronary syndromes. Am J Cardiol 2014;113:249–55. [16] Patel PJ, Khera AV, Jafri K, Wilensky RL, Rader DJ. The anti-oxidative capacity of highdensity lipoprotein is reduced in acute coronary syndrome but not in stable coronary artery disease. J Am Coll Cardiol 2011;58:2068–75. [17] Willemsen HM, van der Horst IC, Nieuwland W, et al. The diagnostic value of soluble CD163 in patients presenting with chest pain. Clin Biochem 2009;42:1662–6.
[18] Dullaart RPF, van Pelt LJ, Kwakernaak AJ, Dikkeschei BD, van der Horst IC, Tio RA. Plasma lipoprotein-associated phospholipase A2 mass is elevated in STEMI compared to non-STEMI patients but does not discriminate between myocardial infarction and non-cardiac chest pain. Clin Chim Acta 2013;424C:136–40. [19] Nijstad N, de Boer JF, Lagor WR, et al. Overexpression of apolipoprotein O does not impact on plasma HDL levels or functionality in human apolipoprotein A-I transgenic mice. Biochim Biophys Acta 1811;2011:294–9. [20] Triolo M, Annema W, de Boer JF, Tietge UJ, Dullaart RPF. Simvastatin and bezafibrate increase cholesterol efflux in men with type 2 diabetes. Eur J Clin Invest Dec 11 2013. http://dx.doi.org/10.1111/eci.12226. [21] Acharjee S, Roe MT, Amsterdam EA, Holmes DN, Boden WE. Relation of admission high-density lipoprotein cholesterol level and in-hospital mortality in patients with acute non-ST segment elevation myocardial infarction (from the National Cardiovascular Data Registry). Am J Cardiol 2013;112:1057–62. [22] Peduzzi P, Concato J, Kemper E, Holford TR, Feinstein AR. A simulation study of the number of events per variable in logistic regression analysis. J Clin Epidemiol 1996;49:1373–9.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
The HDL anti-inflammatory function is impaired in myocardial infarction and may predict new cardiac events independent of HDL cholesterol Robin P.F. Dullaart a,⁎, Wijtske Annema b, René A. Tio c, Uwe J.F. Tietge b a b c
Department of Endocrinology, University of Groningen and University Medical Center Groningen, Groningen, The Netherlands Department of Pediatrics, University of Groningen and University Medical Center Groningen, Groningen, The Netherlands Department of Cardiology, University of Groningen and University Medical Center Groningen, Groningen, The Netherlands
a r t i c l e
i n f o
Article history: Received 4 February 2014 Received in revised form 24 February 2014 Accepted 25 February 2014 Available online 5 March 2014 Keywords: Acute coronary syndrome Atypical chest pain HDL anti-inflammatory function STEMI Non-STEMI
a b s t r a c t Background: Intrinsic functional properties of high density lipoproteins (HDL) are considered to be physiologically important for atheroprotection. We compared the HDL anti-inflammatory capacity between patients with acute myocardial infarction (MI) and patients with non-cardiac chest pain, and prospectively determined the association of new major adverse cardiovascular events (MACE) with this metric of HDL function. Methods: A prospective study was carried out in 93 patients referred for acute chest pain (65 patients with acute MI). The HDL anti-inflammatory capacity was determined as the ability to suppress tumor necrosis factor-α-induced vascular cell adhesion molecule-1 mRNA expression in endothelial cells in vitro. Results: Acute MI at admission was associated with impaired HDL anti-inflammatory capacity (p = 0.001), even after adjustment for HDL cholesterol and apolipoprotein A-I (p = 0.003). Twenty nine MACE were ascertained during a median follow-up of 1210 (910–1679) days. New MACE was associated with impaired HDL antiinflammatory capacity (hazard ratio: 1.80 (1.17–2.77) per SD change, p = 0.007) in age, sex, HDL cholesterol and apolipoprotein-AI adjusted analysis. Conclusions: The ability of HDL to attenuate endothelial inflammation is impaired in acute MI, and this metric of HDL function may serve as a predictor of new MACE, even independent of HDL cholesterol and apolipoprotein A-I. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Large population-based studies have convincingly demonstrated that high plasma levels of high density lipoprotein (HDL) cholesterol levels confer protection against atherosclerotic cardiovascular disease (CVD) [1,2]. Remarkably, pharmacological interventions aimed to raise HDL cholesterol, for example with cholesteryl ester transfer protein inhibitors or niacin, have failed to demonstrate cardiovascular benefit [3–5]. Such findings have contributed to the concept that intrinsic functional properties of HDL may be physiologically more important than HDL cholesterol mass levels to exert protection against the development or progression of atherosclerosis [3,6–11]. Among other allegedly anti-atherogenic functions, such as promoting cellular cholesterol efflux and preventing oxidative modification of low density lipoproteins (LDL), HDL particles also exert potent anti-inflammatory properties [9–11]. During the past few years evidence has accumulated that HDL function may be impaired in clinical conditions featured by increased ⁎ Corresponding author at: Department of Endocrinology, University of Groningen and University Medical Center Groningen, P.O. Box 30.001, Groningen, 9700 RB, The Netherlands. Tel.: +31 503613731; fax: +31 503619392. E-mail address: [email protected] (R.P.F. Dullaart).
http://dx.doi.org/10.1016/j.cca.2014.02.026 0009-8981/© 2014 Elsevier B.V. All rights reserved.
cardiovascular risk, although there are still considerable areas of uncertainty [6,7,9–14]. Remarkably little is known about the extent to which various measures of HDL function are altered in the context of an acute coronary syndrome. Recent cross-sectional studies have demonstrated that the ability of HDL to promote cellular cholesterol efflux, as well as the potential of HDL to inhibit LDL oxidation in vitro may be impaired during the acute phase of a myocardial infarction (MI) [15,16], but it is still uncertain whether the capacity of HDL to modify endothelial inflammation in vitro, as a metric of its anti-inflammatory capacity, is abnormal in the setting of an acute MI. In addition, no study is currently available to investigate the predictive value of a HDL function metric for the future development of new major adverse cardiovascular events (MACE). Therefore, the present study was initiated first to compare the HDL anti-inflammatory capacity between patients with acute MI and patients with non-cardiac chest pain, and second to prospectively determine the impact of this metric of HDL function on new MACE during follow-up. 2. Materials and methods 2.1. Subjects The present study represents part of a protocol which aims to determine the diagnostic yield of lipid and non-lipid biomarkers in
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discriminating between cardiac and non-cardiac causes of acute chest pain [17,18]. The protocol complies with the Declaration of Helsinki, and has been approved by the medical ethics committee of the University Medical Center Groningen. The protocol involves the anonymous storage of frozen plasma, for which the approval from the medical ethics committee was requested but waived, because such storage is performed as standard for additional testing in order to evaluate future clinically important diagnostic considerations. Consecutive patients referred with chest pain suspected for having an acute coronary syndrome were enrolled if they presented at weekdays between 8 a.m. and 5 p.m. Patients with MI were categorized as MI without ST-elevation (non-STEMI) and MI with ST-elevation (STEMI), as diagnosed by attending cardiologists [17,18]. The University Medical Center Groningen serves as regional referral center for STEMI patients. To be eligible other clues for a cardiac origin of chest pain had to be absent. If sequential electrocardiograms and troponin T tests were normal, patients were classified as having non-cardiac chest pain, and were discharged in case of low suspicion or if a bicycle exercise test was negative. The time frame of inclusion was between 2006 and 2008. Follow-up data were obtained from all participants with respect to subsequent MACE (cardiac mortality, MI, percutaneous coronary intervention (PCI), and/or coronary artery bypass grafting (CABG)), which was chosen as the combined end-point. Cardiac mortality was defined as sudden cardiac death and death due to acute MI or heart failure. Causes of death were obtained by linking the number of the death certificate to the primary cause of death as coded by the Central Bureau of Statistics (Voorburg/Heerlen, The Netherlands). Census date was chosen as the date of newly manifest MACE or as the date of the latest verification of the patient status which was at least 1000 days after initial presentation.
cholesterol was measured with a homogeneous enzymatic colorimetric test (Roche/Hitachi, cat no 04713214; Roche Diagnostics GmbH, Mannheim, Germany). Non-HDL cholesterol was calculated as the difference between total cholesterol and HDL cholesterol. LDL cholesterol was calculated by the Friedewald formula. Apolipoproteins (apo) A-I and B were assayed by immunoturbidimetry (Roche/Cobas Integra Tina-quant cat no. 03032566 and 033032574, respectively, Roche Diagnostics). The HDL anti-inflammatory capacity was determined using an in vitro cell system essentially following a previously described procedure [19]. ApoB-containing lipoproteins were precipitated from plasma by adding 100 μl 36% polyethylene glycol (PEG 6000, Sigma, St. Louis, MO, USA) in 10 mM HEPES (pH = 8.0) to 200 μl plasma followed by 30 min incubation on ice. After 30 min centrifugation at 2200 g, the HDL-containing supernatant was collected, kept on ice, and used directly for the HDL anti-inflammatory assay. Human umbilical vein endothelial cells (HUVECs), provided by the Endothelial Cell Core Facility of the University of Groningen, The Netherlands, were pre-incubated for 30 min with 2% of individual apoB-depleted plasma samples or with equal amounts of phosphate buffered saline (PBS) as control [19]. Then 10 ng/ml tumor necrosis factor-α (TNF-α; R&D systems, Abingdon, UK) or PBS as control was added, and cells were incubated for an additional 8 h followed by analysis of vascular cell adhesion molecule-1 (VCAM-1) gene expression using quantitative real-time polymerase chain reaction. VCAM-1 mRNA expression was calculated relative to the average of the housekeeping gene cyclophilin. Results are expressed as fold induction over baseline in the presence of the individual subject's HDL preparations, whereby higher values indicate lower anti-inflammatory capacity. Storage of plasma at −80 °C did not influence the results of this assay (data not shown). The intra-assay coefficient of variation of this assay is 9%.
2.2. Laboratory analyses
2.3. Statistical analysis
Venous blood samples were obtained directly after admission for routine measurements. EDTA-anticoagulated samples were immediately placed on ice. The plasma samples were stored at −80 °C. Plasma cholesterol was assayed by a routine enzymatic method (Roche/Hitachi cat no 11875540; Roche Diagnostics GmbH, Mannheim, Germany). HDL
Data are given as means ± SD, medians (interquartile range) or in numbers. Continuous variables were compared by unpaired t-tests or by Mann–Whitney U-tests in case of non-parametrically distributed data. Differences in proportions of variables were determined by χ2-analysis.
Table 1 Clinical characteristics and biomarkers of myocardial damage in 65 patients with myocardial infarction (MI) and 28 patients with non-cardiac chest pain.
Age (years) Sex (M/F) Non-STEMI/STEMI Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) History of hypertension (yes/no) History of diabetes mellitus (yes/no) Previous MI (yes/no) Previous PCI (yes/no) Previous CABG (yes/no) Current smoking (yes/no) Statin treatment (yes/no) Other lipid lowering drugs (yes/no) β-blocker treatment (yes/no) Calcium antagonist treatment (yes/no/unknown) Aspirin treatment (yes/no) Other anticoagulants (yes/no) Troponin T at presentation (μg/l) Maximal troponin T (μg/l) CK at presentation (U/l) Maximal CK (U/l) CK-MB at presentation (U/l) Maximal CK-MB (U/l)
MI (n = 65)
Non-cardiac chest pain (n = 28)
p-value
68 ± 11 44/21 32/33 135 ± 25 74 ± 14 23/42 8/57 7/58 10/55 7/58 22/43 22/43 0/28 22/43 10/49/6 19/46 9/58 0.04 (0.003–17.8) 1.38 (0.14–4.82) 111 (62–192) 307 (106–999) 17 (9–26) 41 (16–173)
63 ± 11 14/14 0/0 139 ± 17 78 ± 9 7/21 2/26 4/24 2/26 2/26 3/25 10/18 1/64 10/18 2/26 7/21 4/24 0.00 (0.00–0.00) 0.00 (0.00–0.00) 29 (22–39) 37 (26–62) 2 (2–4) 3 (2–6)
0.053 0.11 b0.001 0.42 0.17 0.33 0.46 0.63 0.28 0.59 0.021 0.86 0.51 0.84 0.22 0.68 0.98 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001
Data as means ± SD or as medians (interquartile ranges). CABG, coronary artery bypass graft; CK, creatine kinase; CK-MB, MB fraction of creatine kinase; M, male; F, female; non-STEMI, myocardial infarction (MI) without ST-elevation; STEMI, MI with ST-elevation; PCI, percutaneous coronary intervention. Other anticoagulants are mainly vitamin K antagonists.
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Table 2 Plasma lipids, lipoproteins, apolipoproteins and the HDL anti-inflammatory capacity in 65 patients with myocardial infarction (MI) and in 28 patients with non-cardiac chest pain.
Total cholesterol (mmol/l) LDL cholesterol (mmol/l) Non-HDL cholesterol (mmol/l) HDL cholesterol (mmol/l) Total cholesterol/HDL cholesterol ratio Apo A-I (g/l) Apo B (g/l) Apo B/apo A-I HDL anti-inflammatory capacity (fold increase in VCAM-1 mRNA expression)
MI (n = 65)
Non-cardiac chest pain (n = 28)
p-value
5.33 ± 2.12 3.51 ± 1.51 4.14 ± 2.07 1.19 ± 0.24 4.58 ± 1.51 1.35 ± 0.24 1.07 ± 0.34 0.82 ± 0.30 13.8 (4.2–32.0)
5.19 ± 1.12 3.25 ± 0.94 3.98 ± 1.06 1.20 ± 0.25 4.39 ± 1.01 1.48 ± 0.27 1.12 ± 0.25 0.77 ± 0.18 3.5 (0.7–14.2)
0.73 0.33 0.69 0.69 0.73 0.060 0.59 0.52 0.001
Data as means ± SD or as medians (interquartile ranges). Apo, apolipoproteins; HDL, high density lipoproteins; LDL, low density lipoproteins, non-HDL cholesterol, non-high density lipoproteins; VCAM-1, vascular cell adhesion molecule-1; higher values of HDL-anti-inflammatory capacity (expressed as fold increase in VCAM-1 mRNA expression over baseline) indicate lower anti-inflammatory capacity.
The associations of the presence of MI at presentation with the HDL anti-inflammatory capacity, HDL cholesterol and apoA-I were determined using receiver operating characteristic (ROC) curves (presented as area under the curve with 95% confidence intervals (CI)). In addition, logistic regression analysis was performed to disclose the independent association of MI at presentation with the HDL anti-inflammatory capacity. The independent association of new MACE with the HDL anti-inflammatory capacity was analyzed using Cox proportional hazard analysis. Odds ratios and hazard ratios of the continuous variables of interest are given per SD change with 95% confidence intervals (CI). Two-sided p-values b0.05 were considered significant.
3. Results Ninety-three patients were included of whom 65 were diagnosed with acute MI (Table 1). As shown in Table 2, plasma total cholesterol, LDL cholesterol, non-HDL cholesterol, apoB, as well as HDL cholesterol and apoA-I levels were not significantly different between MI patients and patients with non-cardiac chest pain. Remarkably, the HDL antiinflammatory capacity was severely impaired in MI patients, as indicated by the higher values of TNF-α-stimulated VCAM-1 expression (p = 0.001; Table 2 and Fig. 1). The HDL anti-inflammatory capacity did not significantly differ between patients with non-cardiac chest pain and a group of 20 healthy control subjects who did not present with chest pain (3.2 (0.9–10.1) fold increase in VCAM-1 expression, data not shown, p N 0.30). Furthermore, the HDL anti-inflammatory
capacity was lower in STEMI vs. non-STEMI patients (26.4 (12.6–40.1) vs. 13.0 (1.9–19.8) fold increase in VCAM mRNA expression (p b 0.001). The area under the ROC curve of impaired HDL anti-inflammatory capacity was significant (0.736, 95% CI, 0.595–0.877, p = 0.004), whereas those of lower HDL cholesterol (0.525, 95% CI, 0.356–0.693, p = 0.76) and apoA-I (0.653, 95% CI, 0.503–0.803, p = 0.076) were not. Acute MI at presentation was also strongly associated with impaired HDL antiinflammatory capacity in age- and sex-adjusted logistic regression analysis (odds ratio per SD increment: 3.72 (95% CI, 1.66–8.33), p = 0.001). Remarkably, this association was not attenuated after adjustment for either HDL cholesterol (4.52 (95% CI, 1.89–10.78), p = 0.001), apoA-I (9.59 (95% CI, 2.44–37.73), p = 0.001) or both (8.76 (95% CI, 2.08– 36.87), p = 0.003). This association also persisted after adjustment for age, sex, previous MI, previously diagnosed diabetes, current smoking and treatment with statins, anti-hypertensives and anticoagulants (5.52 (95% CI, 1.93–15.58), p = 0.001). After a median follow-up of 1210 (910–1679) days 29 new MACE occurred in the whole study population of 93 patients. Four of these MACE occurred in the non-cardiac chest pain group. These MACE occurred N 200 days after initial admission, and were considered not to be due to a false initial diagnosis of non-cardiac chest pain. Age, sex distribution, smoking at initial presentation, as well as previously diagnosed diabetes and hypertension were not significantly different between patients who experienced new MACE compared to patients who did not (data not shown; all p N 0.10). Plasma total cholesterol, LDL cholesterol, non-HDL cholesterol and apoB levels at initial presentation were not different in patients who experienced a new MACE during follow-up compared to those who did not (data not shown; all p N 0.17). HDL cholesterol (1.19 ± 0.24 mmol/l) and apoA-I levels (1.39 ± 0.24 g/l) were also not different in those patients who experienced a new MACE compared to those patients who did not (1.20 ± 0.24 mmol/l, p = 0.92 and 1.39 ± 0.26 g/l, p = 0.97, respectively). In age-and sex-adjusted Cox proportional regression analysis new MACE was associated with impaired HDL anti-inflammatory capacity (Table 3). This association remained significant after adjustment for either HDL cholesterol, apoA-I, or both (Table 3). Conversely, neither HDL cholesterol nor apoA-I predicted new MACE taking account of the HDL anti-inflammatory capacity. In an analysis which only included those patients with verified acute MI at presentation (n = 65), the association of new MACE with the HDL anti-inflammatory capacity was also significant in age- and sex-adjusted analysis (hazard ratio per SD increment: 1.49 (95% CI, 1.02–2.17, p = 0.037). 4. Discussion
Fig. 1. Box plots showing the high density lipoprotein (HDL) anti-inflammatory capacity in 65 patients with acute myocardial infarction and in 28 patients with non-cardiac chest pain (no myocardial infarction). The HDL-anti-inflammatory capacity is expressed as fold increase in vascular cell adhesion molecule-1 mRNA expression over baseline; higher values thereby indicate lower anti-inflammatory capacity. Values are given in medians, interquartile ranges, 5 to 95% ranges and extremes. Difference between patients with and without myocardial infarction: p = 0.001 by Mann–Whitney U-test.
The present study shows first that the HDL anti-inflammatory capacity is impaired in acute MI, and second that prospectively determined new MACE is associated with this metric of HDL function. Remarkably, this association of adverse cardiac outcome with impaired HDL anti-inflammatory capacity was independent of HDL cholesterol
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Table 3 Relationship of the high density lipoprotein anti-inflammatory capacity with 29 subsequent major adverse cardiovascular events by Cox proportional regression analysis during follow-up of 93 patients (65 patients with acute myocardial infarction and 28 patients with non-cardiac chest pain at presentation). Model 1
HDL anti-inflammatory capacity HDL cholesterol
Model 2
Model 3
Model 4
Hazard ratio (95% CI)
p-value
Hazard ratio (95% CI)
p-value
Hazard ratio (95% CI)
p-value
Hazard ratio (95% CI)
p-value
1.54 (1.09–2.18)
0.015
1.81 (1.18–2.77) 0.83 (0.42–1.61)
0.007
1.78 (1.18–2.69)
0.006
1.80 (1.17–2.77) 0.92 (0.37–2.25) 0.87 (0.37–1.98)
0.007
0.57
Apolipoprotein A-I
0.82 (0.44–1.52)
0.53
0.85 0.73
For each variable hazard ratios are given per 1 SD increment with 95% confidence intervals (CI). All models are adjusted for age and sex. HDL, high density lipoproteins. Model 1: the HDL anti-inflammatory capacity adjusted for age and sex only; model 2: the HDL anti-inflammatory capacity additionally adjusted for HDL cholesterol; model 3: the HDL antiinflammatory capacity additionally adjusted for apolipoprotein A-I; model 4: the HDL anti-inflammatory capacity additionally adjusted for both HDL cholesterol and apolipoprotein A-I.
and apoA-I. Our findings, therefore, lend support to the emerging concept of HDL function being more relevant for cardioprotection than levels of HDL cholesterol and apoA-I. The current observation with respect to impaired anti-inflammatory function of HDL from acute MI patients complements recent studies in which either the ability of HDL to promote cellular cholesterol efflux or the anti-oxidative activity of HDL was found to be diminished during the acute phase of a coronary syndrome [15,16]. To our knowledge, the prospective analysis described in this report is the first to suggest that a metric of HDL function obtained at presentation of MI is a statistically significant determinant of adverse cardiac outcome. Thus far, only the predictive value of the ability of HDL to promote cellular cholesterol efflux has been evaluated in prospective studies. In a prospective nested case–control study among subjects without a coronary event at baseline, cholesterol efflux did not predict the future development of MACE [13]. Another study unexpectedly documented that adverse cardiac outcome was associated with a higher cholesterol efflux rate [14]. However, none of these studies were carried out among patients presenting with an acute coronary syndrome. The assay system that we used to document HDL's antiinflammatory function, i.e. the ability of HDL to suppress TNF-αinduced VCAM-1 expression in HUVECs, was brought about by the notion that endothelial inflammation is conceivably implicated in the pathogenesis of (instable) atherosclerotic lesions [11]. In another study, we used the ability of HDL to suppress TNF-α induced monocyte chemoattractant protein 1 (MCP-1/CCL-2) gene expression as read out, but the assay conditions were otherwise similar [20]. In that study, which was carried out in diabetic subjects, we additionally documented cholesterol efflux and the anti-oxidative capacity of HDL, and found no significant relationships between these 3 metrics of HDL function [20]. It seems, therefore, plausible that various HDL function metrics may yield complementary information regarding allegedly atheroprotective effects attributed to HDL, although it is a limitation that we did not document other metrics of HDL function in the current study. Several other methodological considerations and limitations of our study need to be discussed. First, this study was performed in a referral center for STEMI patients, explaining the large proportion of STEMI patients in our cohort [17]. Second in the present study, HDL cholesterol was not different between subjects with and without MI, whereas the difference in apoA-I was close to statistical significance. In comparison, previous reports from our group that were carried out in somewhat larger populations showed significantly lower HDL cholesterol and apoA-I levels in acute MI patients [17,18], as expected [21].Third, the number of new MACE during follow-up was rather limited. We only adjusted for age, sex, HDL cholesterol and apoA-I in Cox-regression analysis in order to circumvent overestimation or underestimation in the regression coefficients as much as possible [22]. Thus, effect modification of other clinical variables cannot be excluded, and we interpret our finding that adverse cardiac outcome is associated with impaired HDL anti-inflammatory capacity to be preliminary. Fourth, it should be
emphasized that there is currently no consensus with respect to the isolation procedure of HDL when assaying functional properties of HDL. We decided not to use ultracentrifugation, because not only it is difficult to isolate HDL from small volumes of plasma, but more importantly because high shear forces and ionic strengths may result in considerable changes in the composition and the protein cargo of the HDL particles [10]. In conclusion, this study demonstrates that the ability of HDL to attenuate endothelial inflammation is impaired in patients presenting with acute MI. Although more work is warranted to determine the contribution of various metrics of HDL function to improved cardiovascular risk classification, our current study raises the possibility that the antiinflammatory capacity of HDL may have the potential to serve as a predictive biomarker for cardiovascular outcome even independent of HDL-C or apoA-I mass.
Conflict of interest The authors report no financial or other conflicts of interest. Acknowledgments The analytical help of L.D. Dikkeschei, PhD, for apolipoprotein assay is greatly appreciated. UJFT is supported by a grant from The Netherlands Organization for Scientific Research (VIDI Grant 917-56358). This study was investigator driven.
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