Endothelial function after ST-elevation myocardial infarction in patients with high levels of high-sensitivity CRP and Lp-PLA2: A substudy of the RESPONSE randomized trial

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Endothelial function after ST-elevation myocardial infarction in patients

with high levels of high-sensitivity CRP and Lp-PLA

2

: A substudy of the

RESPONSE randomized trial

☆

,

☆☆

Jasveen J. Kandhai-Ragunath

a

, Bjorn de Wagenaar

b

, Cees Doelman

c

, Jan van Es

a

, Harald T. Jørstad

d

,

Ron J.G. Peters

d

_{, Carine J.M. Doggen}

e

_{, Clemens von Birgelen}

a,e,

⁎

a

Department of Cardiology, Thoraxcentrum Twente, MST, Enschede, Netherlands

b

MESA, Institute for Nanotechnology, University of Twente, Enschede, Netherlands

c_{Medlon Laboratory Diagnostics, Medisch Spectrum Twente, Enschede, Netherlands} d

Department of Cardiology, Academisch Medisch Centrum, Amsterdam, Netherlands

e

Health Technology and Services Research, MIRA– Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, Netherlands

a b s t r a c t

a r t i c l e i n f o

Article history: Received 29 August 2016

Received in revised form 23 December 2016 Accepted 28 December 2016

Keywords:

Endothelial function (dysfunction) ST-elevation myocardial infarction (STEMI) Lipoprotein-associated phospholipase A2(Lp-PLA2)

High-sensitive C-reactive protein (hs-CRP)

Background: The combination of high levels of high-sensitive C-reactive protein (hs-CRP) and lipoprotein-associated phospholipase-A2(Lp-PLA2) was recently shown to correlate with increased cardiovascular risk.

En-dothelial dysfunction is also known to be a risk factor for cardiovascular events.

Aim: To test among patients with previous ST-elevation myocardial infarction (STEMI) the hypothesis that high levels of both hs-CRP and Lp-PLA2may be associated with impaired endothelium-dependent vasodilatation.

Methods: In this substudy of the RESPONSE randomized trial, we used reactive hyperemia peripheral artery to-nometry (RH-PAT) 4 to 6 weeks after STEMI and primary percutaneous coronary intervention (PPCI) to non-invasively assess endothelial function (RH-PAT indexb1.67 identiﬁed endothelial dysfunction). Reliable mea-surements of RH-PAT, hs-CRP, and Lp-PLA2were obtained in 68 patients, who were classiﬁed as high-risk if levels

of both hs-CRP and Lp-PLA2were in the upper tertile (≥3.84 mg/L and N239 μg/L, respectively).

Results: Patients were 57.4 ± 9.7 years and 53 (77.9%) were men. 11 (16%) patients were classiﬁed as high-risk and 57 (84%) as low-to-intermediate-risk. The RH-PAT index was 1.68 ± 0.22 in high-risk and 1.95 ± 0.63 in low-to-intermediate-risk patients (p = 0.17). Endothelial dysfunction was present in 8 (72.7%) high-risk and 26 (45.6%) low-to-intermediate-risk patients (p = 0.09). Framingham risk score, NT-proBNP andﬁbrinogen levels were higher in high-risk patients (p≤ 0.03).

Conclusion: In this population of patients with recent STEMI and PPCI, we observed between patients with high hs-CRP and Lp-PLA levels and all other patients no more than numerical differences in endothelial function that did not reach a statistical signiﬁcance. Nevertheless, further research in larger study populations may be warranted.

1. Introduction

While common cardiovascular risk factors at best provide a reason-able prediction of cardiovascular events[1], the addition of biomarkers of systemic in_{ﬂammation, such as high-sensitive C-reactive protein} (hs-CRP), can improve risk prediction[2]. A recently published study showed that an aggregate risk score including hs-CRP greatly enhanced the risk prediction of death and myocardial infarction in patients with

suspected or known coronary artery disease [4]. In addition,

lipoprotein-associated phospholipase A2(Lp-PLA2), a speciﬁc marker for vascular inﬂammation, was found to predict future cardiovascular events independently of hs-CRP[5–7], suggesting an additive role for risk prediction. This is supported by the fact that in a population-based study, the combination of elevated levels of hs-CRP and Lp-PLA2 correlated with increased cardiovascular risk[8].

Abbreviations: hs-CRP, high-sensitive C-reactive protein; Lp-PLA2, lipoprotein-associated phospholipase A2; PCI, percutaneous coronary intervention; PPCI, primary

per-cutaneous coronary intervention; RH-PAT, reactive hyperemia peripheral artery tonome-try; STEMI, ST-elevation myocardial infarction.

☆ Potential conﬂicts of interest: The research department of Thoraxcentrum Twente has received research grants funded by AstraZeneca, and CvB has received lecture fees from AstraZeneca. RJGP is a consultant to AstraZeneca. All other authors declare that they have no conﬂict of interest.

☆☆ Funding: This investigator-initiated, non-funded observational study was performed in a single-center STEMI patient population that was also enrolled in the RESPONSE multi-center trial, funded by AstraZeneca. There is noﬁnancial or other conﬂict of interest other than stated; there were no other sponsors than indicated.

⁎ Corresponding author at: Thoraxcentrum Twente, Department of Cardiology, Haaksbergerstraat 55, 7513ER Enschede, Netherlands. Tel.: +31 53 487 2105; fax: +31 53 487 2107.

E-mail address:c.vonbirgelen@mst.nl(C. von Birgelen).

http://dx.doi.org/10.1016/j.carrev.2016.12.019

Contents lists available atScienceDirect

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Patients who survive an ST-elevation myocardial infarction (STEMI) are at high risk of future recurrent cardiovascular events[9]. Within this patient population, the combination of elevated levels of hs-CRP and Lp-PLA2might be able to identify subjects with a particularly high risk. As endothelial dysfunction (i.e. impaired endothelium-dependent arterial vasodilatation) is known to be an independent risk factor of future car-diovascular events [10,11], we tested in a local substudy of the RESPONSE (Randomised Evaluation of Secondary Prevention by Outpa-tient Nurse SpEcialists) trial[14]the hypothesis that elevated levels of both hs-CRP and Lp-PLA2may be associated with reduced endothelial function in patients with recent STEMI.

2. Methods

2.1. Study population and study design

This prospective cohort study was performed in STEMI patients of the RESPONSE trial[12], who underwent treatment by PPCI for acute STEMI (≤12 h after symptom onset) and non-invasive assessment of en-dothelial function with the RH-PAT method after 4 to 6 weeks at Thoraxcentrum Twente in Enschede. PPCIs were performed between October 2007 and December 2008. Of a total of 75 RESPONSE trial par-ticipants at Thoraxcentrum Twente, who were treated for STEMI and underwent RH-PAT measurements, 71 had analyzable RH-PAT registra-tions and 68 patients had laboratory measurements of hs-CRP and LpPLA2. This resulted in a study population of 68 patients.

Details of the randomized RESPONSE trial have previously been re-ported[12]. In brief, patients had to be 18 to 80 years, without surgery or additional PCI being planned within 8 weeks from PPCI, without con-gestive heart failure New York Heart Association (NYHA) class III or IV, and with a life expectancy of at least 2 years.

As inﬂammation and repair processes of the infarcted myocardium might have disturbed endothelial function measurements during the ﬁrst weeks after the STEMI and endothelial dysfunction had not been fully recovered under medication, endothelial function was assessed 4 to 6 weeks after the PPCI[13–15]. All patients were seen in the outpa-tient clinic and the research department of Thoraxcentrum Twente, where non-invasive assessment of the endothelial function was per-formed according to strict rules in a dedicated laboratory[16].

All patients provided written informed consent for both participation in the RESPONSE multicenter trial and the present single-center substudy. Trial and substudy complied with the Declaration of Helsinki for investi-gation in human beings and were approved by accredited Medical Ethical Committees in Amsterdam and Twente, the Netherlands.

2.2. Patient characteristics and follow-up

The following information was collected through interviews during visits in our outpatient clinic 4 to 6 weeks after STEMI: age; sex; body mass index (kg/m2_{); arterial hypertension (blood pressure}

N140/ 90 mmHg or treatment with anti-hypertensive medication); history of smoking (previous or current smoker); history of previous myocardial infarction or percutaneous coronary intervention (PCI); presence of di-abetes mellitus (patient history and/or treatment with insulin or oral anti-glycemic agents); and history of hyperlipidemia or treatment with lipid-lowering drugs. The Framingham Risk Score (10-year risk prediction for fatal and non-fatal coronary heart disease) was deter-mined to sketch a patient risk proﬁle.

2.3. Laboratory assessment of the biochemical markers

Venous blood samples were drawn after a minimum of 8 h of fasting. The laboratory markers total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycer-ides, glucose, hemoglobin A1c, creatinine,ﬁbrinogen, apolipoprotein A1 & B100, troponin I and N-terminal pro-B-type natriuretic peptide

(NT-proBNP) were assessed according to manufacturer instructions. After centrifugation, plasma and serum aliquots were stored at−80 °C.

A commercially available Lp-PLA2 enzyme-linked immunosorbent

assay kit (third generation PLAC™ Test; diaDexus Inc., South San Francisco, CA) was used to determine the lipoprotein-associated phos-pholipase A2mass. The inter-assay precision was determined by measur-ing two controls of known concentration (low and high) in 10 separate assays. The coefﬁcient of variation on all 10 plates was 7% and 6%, respec-tively. The hs-CRP concentrations were determined by nephelometry using a BN ProSpec® system (Siemens). The detection limit was 0.23 mg/L and the mean inter-assay coefﬁcient of variation was 2.8%.

The thresholds of the hs-CRP tertiles were determined at 1.03 mg/L and 3.84 mg/L; the thresholds of the Lp-PLA2tertiles were 190μg/L and 239μg/L comparable to AHA/CDC guidelines[17]and ARIC study[8]. 2.4. Assessment of endothelial function

Endothelial function was evaluated with the RH-PAT method. The ﬁnger pulse wave amplitude was assessed with the EndoPAT-2000 sensing device andﬁnger plethysmographic probes (Itamar Medical, Caesarea, Israel), both at baseline and during ischemia-induced hyper-emia. All measurements were performed in the early morning in a ded-icated laboratory after fasting for at least 8 h. Patients also had to refrain from caffeine consumption, smoking, and vasoactive medications. At least 15 min prior to testing, blood pressure was measured and a blood sample was drawn in the control arm. Before any measurement, patients had an acclimatization period of 20 min in a quiet room, lying in a hospital bed at an ambient temperature of 21 to 23 °C.

The RH-PAT method has previously been reported in detail[18-20]. In brief, measurements were performed by the use of probes on index fingers of both the study and control arm. Baseline measurements were recorded for 5 min prior to inducing ischemia by inflating a blood pressure cuff on the upper arm of the study arm for 5 min to supra-systolic pressures. This led to NO release from functional endo-thelium and thus vasodilatation, which was recorded by the sensors in thefinger cuff through beat-to-beat finger pulse wave analysis[19,21]. Following the release of the blood pressure cuff, the ratio of the pulse amplitude of the hyperemicfinger and the baseline amplitude was cal-culated. Subsequently, that ratio was divided by the corresponding ratio, obtained in the control arm, to calculate the RH-PAT index (high values indicate good endothelial function)[19,21]. Hamburg et al. dem-onstrated that the maximum hyperemic response can be expected 90 to 120 s after cuff deflation[20]. Therefore, in the present study, the reac-tive RH-PAT index was calculated as the ratio of the mean hyperemic pulse wave analysis over a period of 30 s, beginning at 90 s after cuff de-flation, divided by the baseline pulse wave analysis (mean baseline measurements for 3.5 min), and normalized to the concurrent measure-ments of the control arm. Based on previously reported data, endotheli-al function was divided into two groups: endotheliendotheli-al dysfunction (RH-PATb1.67) and normal endothelial function (RH-PAT ≥1.67)[21]. 2.5. Statistical analysis

Data are presented as frequencies (%) or mean ± standard deviation (SD). We used the Fisher's exact chi-square test for categorical variables and the T-test for continuous variables to compare patients with high risk versus patients with low-to-intermediate risk. A p-valueb0.05 was considered statis-tically signiﬁcant. Analyses were performed with SPSS (version 21.0). 3. Results

3.1. Characteristics of the study population

Demographics and clinical characteristics of the 68 patients of the study population are presented in Table 1. Overall, patients were 57.4 ± 9.7 years and predominantly men (n = 53, 77.9%).

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3.2. Biochemical marker-based classiﬁcation of risk

The thresholds of the hs-CRP tertiles were determined at 1.03 mg/L and 3.84 mg/L; the thresholds of the Lp-PLA2tertiles were 190μg/L and 239μg/L. The thresholds of the upper tertiles of Lp-PLA2and hs-CRP were used to classify patients into a high-risk group versus a low-to-intermediate-risk group. A total of 11 patients with Lp-PLA2and hs-CRP levels in the highest tertile were classified as high-risk, while the remaining 57 patients were classified as low-to-intermediate-risk. Medical therapy and most cardiovascular risk factors did not differ be-tween high-risk and low-to-intermediate-risk patients (Table 2). When the patients were further subdivided in three risk groups, high-, intermediate- and low-risk group, no differences were found either. Al-though the Framingham Risk Score is not designed to be used in this subset of patients, it does, however, provide an insight in the cardiovas-cular risk. The risk score is as predicted higher in the biomarker high-risk group (14.2 ± 6.8 vs. 10.0 ± 5.4, pb 0.05). Among the laboratory parameters (Table 2), the following showed higher levels in the high-risk patient group:fibrinogen (4.84 ± 0.87 g/L vs. 3.71 ± 0.75 g/L, pb 0.001) and NT-proBNP (93.83 ± 106.54 vs. 44.29 ± 48.44 pmol/L, pb 0.05). The separation in three biomarker-based risk groups did not made any difference.

3.3. Endothelial function and biochemical marker-based classiﬁcation of risk

The RH-PAT index was non-signiﬁcantly lower (i.e. lower endotheli-al function) in high-risk patients than in low-to-intermediate-risk pa-tients (1.68 ± 0.22 vs. 1.95 ± 0.63, p = 0.17) (Fig. 1). Of the high-risk group, 8 patients (72.7%) had endothelial dysfunction (i.e. RH-PAT b1.67), whereas in the low-to-intermediate-risk group, endothelial dys-function was observed in 26 (45.6%) patients (p = 0.09).

4. Discussion

Traditional risk factor-based identi_{fication of patients who are at an} increased risk of cardiovascular events is of limited value[1]. In several studies, the systemic inflammation marker hs-CRP was shown to pre-dict the risk of secondary adverse cardiovascular events in patients with unstable angina or acute myocardial infarction[3]. Moreover, in the PROVE IT-TIMI 22 trial, the vascular inflammatory marker Lp-PLA2, measured 30 days after an acute event, predicted recurrent cardiovascu-lar events[6]. The combined prognostic value of hs-CRP and Lp-PLA2, which has previously been studied in a population-based trial[7,8], may be of value for secondary risk prediction in patients with a recently acute coronary event. As patients who survive a STEMI are at particular-ly high risk of subsequent adverse events and death[22], improved

prediction of individuals with the highest event risk would be particu-larly valuable among such patients.

Endothelial dysfunction is considered to be a legitimate surrogate marker of cardiovascular risk[23–25,30]. In addition, the RH-PAT meth-od previously found a lower endothelial function in patients with versus without coronary artery disease, suggesting a relation between RH-PAT index and cardiovascular risk[26]. Therefore, in the present study, we assessed whether patients with recent STEMI and elevated levels of both hs-CRP and Lp-PLA2had a worse endothelial function, as deter-mined by RH-PAT. The STEMI and the primary PCI induce systemic en-dothelial dysfunction and an inflammatory response, which peaks at 1 to 2 days after the acute event before it gradually decreases, and the pro-cess of“myocardial repair” may be greatly finished after 3 weeks [27,28]. In order to avoid disturbance of endothelial function and in-flammatory marker measurements, we performed it after 4 to 6 weeks from STEMI.

Data from other studies on the relation between biomarkers and en-dothelial function following recent STEMI are scarce. However, in a study in 42 men with stable coronary artery disease and well controlled low-density lipoprotein cholesterol serum levels, hs-CRP and Lp-PLA2 were found to be useful to distinguish high-risk patients from patients with moderate or low event risk[29]. This supports thefindings of the large Atherosclerosis Risk in Communities (ARIC) study, which showed that the levels of Lp-PLA2and CRP may be complementary beyond tra-ditional risk factors in identifying middle-aged men and women at in-creased risk for ischemic stroke or incident coronary heart disease[7–8]. Our current study showed a non-signi_{ficantly (p = 0.09) lower} en-dothelial function in STEMI patients who were classified as high-risk, based on the biomarker assessment. Based on the data of the present substudy of the RESPONSE trial, we cannot exclude that the rate of sub-jects with endothelial dysfunction might be higher among patients with elevation of both biomarkers of inflammation, but the absence of statis-tical significance prevents any definite conclusion. Larger studies are warranted to further investigate this issue and to assess whether the use of additional biomarkers or patient characteristics may result in clinically useful secondary risk prediction models for patients with a re-cent STEMI. Nevertheless, one should bear in mind that all patients with a recent STEMI have an increased risk of additional cardiovascular events and that the identification of patients with the highest risk is most challenging among such a population.

4.1. Limitations

Endothelial function was measured 4 to 6 weeks after the acute event when all patients were treated with similar secondary preventive medications that included statins and frequently ACE inhibitors, which might have had a favorable effect on endothelial function [33]. Howev-er, this accounts for both study groups, and there was no signiﬁcant

Table 1

Demographics and risk factors of 68 patients classiﬁed as high-risk versus low-to-intermediate-risk. High-risk group (n = 11) Low-to-intermediate-risk group (n = 57) P Age 59.3 ± 11.8 (41.2–78.8) 57.3 ± 9.3 (38.3–76.9) 0.54 Men, n (%) 8 (72.7) 45 (78.9) 0.70

Systolic blood pressurea

125.1 ± 13.2 (103–144) 132.8 ± 15.9 (104–172) 0.15

Diastolic blood pressurea

75.5 ± 8.9 (60–92) 80.5 ± 11.7 (59–108) 0.20

Hypertension, n (%) 4 (36.4) 16 (28.1) 0.72

Hyperlipidaemia, n (%) 3 (27.3) 14 (24.6) 1.00

Diabetes, n (%) 2 (18.2) 7 (12.3) 0.63

Smoking, n (%) 6 (54.5) 21 (36.8) 0.32

Body mass indexb _{27.3 ± 2.9 (22.2–33.1)} _{28.6 ± 4.7 (21.0–51.0)} _0.39

10-year Framingham riska

14.2 ± 6.8 (1.4–20.8) 10.0 ± 5.4 (1.8–23.0) 0.03

Data are mean ± standard deviation (range) unless otherwise indicated. % indicate percentage within column.

a

Missing: 1.

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between-group difference in medical treatment. The present substudy of the RESPONSE trial is limited by its relatively small sample size, and itsﬁndings were meant to be hypothesis generating only. Nevertheless, there is an absence of any other large studies on the relation between

elevated inﬂammatory biomarkers and endothelial function in patients with a recent STEMI.

5. Conclusions

In this population of patients with recent STEMI and PPCI, we ob-served between patients with high hs-CRP and Lp-PLA levels and all other patients no more than numerical dissimilarities in endothelial function that did not reach a statistical signiﬁcance. Nevertheless, fur-ther research in larger patient populations may be warranted. Acknowledgment

The authors thank Mrs. Liefke C. van der Heijden, MD, for her assis-tance with preparing theﬁgure.

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Table 2

Laboratory parameters and medication of 68 patients classiﬁed as high-risk versus low-to-intermediate-risk. High-risk group (n = 11) Low-to-intermediate-risk group (n = 57) P Laboratory parameters Glucose (mmol/L)a _{5.65 ± 1.79 (4.38–10.74)} _{5.65 ± 1.67 (4.16–14.79)} 0.99 HbA1C, n (%)b _{6.06 ± 1.04 (5.16–9.08)} _{6.00 ± 0.84 (4.89–9.39)} 0.86 Total cholesterol (mmol/L) 3.45 ± 0.85 (2.35–4.66) 3.41 ± 0.77 (2.28–5.68) 0.86 HDL cholesterol (mmol/L) 0.97 ± 0.26 (0.66–1.51) 1.09 ± 0.24 (0.66–1.62) 0.14 LDL cholesterol (mmol/L) 1.93 ± 0.72 (0.92–3.10) 1.68 ± 0.57 (0.68–3.50) 0.21 Triglycerides (mmol/L) 1.24 ± 0.49 (0.67–2.26) 1.42 ± 0.78 (0.60–4.56) 0.47 Apo A1 (g/L)a 1.24 ± 0.15 (0.99–1.53) 1.21 ± 0.25 (0.60–1.64) 0.68 Apo B 100 (g/L)a _{0.78 ± 0.24 (0.52–1.17)} _{0.65 ± 0.20 (0.35–1.29)} 0.10 Creatinine (μmol/L) 79.46 ± 19.67 (49.80–108.10) 81.31 ± 17.72 (48.60–142.10) 0.76 Fibrinogen (g/L) 4.84 ± 0.87 (3.70–6.20) 3.71 ± 0.75 (2.30–6.40) b0.001 NT-proBNP (pmol/L) 93.83 ± 106.54 (16.66–340.2) 44.29 ± 48.44 (1.62–281.70) 0.02 hs CRP (mg/L) 6.82 ± 2.56 (3.99–13.10) 2.84 ± 3.70 (0.15–20.30) 0.001 Lp-PLA2(ng/mL) 265.00 ± 29.24 (239.0–331.0) 207.21 ± 40.27 (137.0–315.0) b0.001 Resistin (ng/mL) 13.06 ± 6.82 (6.60–25.70) 11.01 ± 6.01 (4.00–34.30) 0.30 RH-PAT 1.68 ± 0.22 (1.45–2.11) 1.95 ± 0.63 (1.10–3.63) 0.17 Medication Beta blockers, n (%)b _{8 (80.0%)} _{47 (85.5%)} _0.65 Calcium ch. blockers, n (%)b 2 (20.0%) 10 (18.2%) 1.00 ACE-inhibitors, n (%)b 6 (60.0%) 27 (49.1%) 0.73 AT II antagonists, n (%)b 1 (10.0%) 8 (14.5%) 1.00 Statins, n (%)b 10 (100.0%) 51 (92.7%) 1.00 Acetylsalicylic acid, n (%)b _{9 (90.0%)} _{53 (96.4%)} _0.40 Clopidogrel, n (%)b _{8 (80%)} _{52 (94.5%)} _0.17 Oral anticoagulation, n (%)b _{1 (10%)} _{2 (3.6%)} _0.40

Data are mean ± standard deviation (range) unless otherwise indicated.

ACE = angiotensin-converting enzyme. Apo 1 = Apolipoprotein 1. Apo B 100 = Apolipoprotein B 100. AT II = angiotensin receptor antagonist. Ch. = channel. HDL = high-density lipo-protein. LDL = low-density lipolipo-protein.

a_{Missing: 1.} b

Missing:N2.

Fig. 1. Endothelial function as measured by RH-PAT in risk stratiﬁcation groups. The mean RH-PAT index in the low-intermediate group was 1.95 ± 0.63 and in the high-risk group 1.68 ± 0.22 (p = 0.17).

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