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.

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. Endothelial 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-PLA2 may be associated with impaired endothelium-dependent vasodilatation.


METHODS
In this substudy of the RESPONSE randomized trial, we used reactive hyperemia peripheral artery tonometry (RH-PAT) 4 to 6weeks after STEMI and primary percutaneous coronary intervention (PPCI) to non-invasively assess endothelial function (RH-PAT index <1.67 identified endothelial dysfunction). Reliable measurements of RH-PAT, hs-CRP, and Lp-PLA2 were obtained in 68 patients, who were classified as high-risk if levels of both hs-CRP and Lp-PLA2 were in the upper tertile (≥3.84mg/L and >239μg/L, respectively).


RESULTS
Patients were 57.4±9.7years 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 and fibrinogen 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.


Introduction
While common cardiovascular risk factors at best provide a reasonable prediction of cardiovascular events [1], the addition of biomarkers of systemic inflammation, 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 A 2 (Lp-PLA 2 ), a specific marker for vascular inflammation, was found to predict future cardiovascular events independently of hs-CRP [5][6][7], suggesting an additive role for risk prediction. This is supported by the fact that in a populationbased study, the combination of elevated levels of hs-CRP and Lp-PLA 2 correlated with increased cardiovascular risk [8]. Abbreviations: hs-CRP, high-sensitive C-reactive protein; Lp-PLA2, lipoproteinassociated phospholipase A 2 ; PCI, percutaneous coronary intervention; PPCI, primary percutaneous coronary intervention; RH-PAT, reactive hyperemia peripheral artery tonometry; 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 multicenter trial, funded by AstraZeneca. There is no financial or other conflict of interest other than stated; there were no other sponsors than indicated. ⁎

Cardiovascular Revascularization Medicine
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-PLA 2 might 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 cardiovascular events [10,11], we tested in a local substudy of the RESPONSE (Randomised Evaluation of Secondary Prevention by Outpatient Nurse SpEcialists) trial [14] the hypothesis that elevated levels of both hs-CRP and Lp-PLA 2 may be associated with reduced endothelial function in patients with recent STEMI.

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 endothelial 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 participants at Thoraxcentrum Twente, who were treated for STEMI and underwent RH-PAT measurements, 71 had analyzable RH-PAT registrations 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 reported [12]. In brief, patients had to be 18 to 80 years, without surgery or additional PCI being planned within 8 weeks from PPCI, without congestive 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][14][15]. All patients were seen in the outpatient clinic and the research department of Thoraxcentrum Twente, where non-invasive assessment of the endothelial function was performed 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 investigation in human beings and were approved by accredited Medical Ethical Committees in Amsterdam and Twente, the Netherlands.

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/m 2 ); 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 diabetes 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 determined to sketch a patient risk profile.

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, triglycerides, 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-PLA 2 enzyme-linked immunosorbent assay kit (third generation PLAC™ Test; diaDexus Inc., South San Francisco, CA) was used to determine the lipoprotein-associated phospholipase A 2 mass. The inter-assay precision was determined by measuring two controls of known concentration (low and high) in 10 separate assays. The coefficient of variation on all 10 plates was 7% and 6%, respectively. 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-PLA 2 tertiles were 190 μg/L and 239 μg/L comparable to AHA/CDC guidelines [17] and ARIC study [8].

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 and finger plethysmographic probes (Itamar Medical, Caesarea, Israel), both at baseline and during ischemia-induced hyperemia. All measurements were performed in the early morning in a dedicated 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][19][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 endothelium and thus vasodilatation, which was recorded by the sensors in the finger 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 hyperemic finger and the baseline amplitude was calculated. 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. demonstrated that the maximum hyperemic response can be expected 90 to 120 s after cuff deflation [20]. Therefore, in the present study, the reactive 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 deflation, divided by the baseline pulse wave analysis (mean baseline measurements for 3.5 min), and normalized to the concurrent measurements of the control arm. Based on previously reported data, endothelial function was divided into two groups: endothelial dysfunction (RH-PAT b1.67) and normal endothelial function (RH-PAT ≥1.67) [21].

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-value b0.05 was considered statistically significant. Analyses were performed with SPSS (version 21.0).

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%).

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-PLA 2 tertiles were 190 μg/L and 239 μg/L. The thresholds of the upper tertiles of Lp-PLA 2 and hs-CRP were used to classify patients into a high-risk group versus a lowto-intermediate-risk group. A total of 11 patients with Lp-PLA 2 and 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 between 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. Although the Framingham Risk Score is not designed to be used in this subset of patients, it does, however, provide an insight in the cardiovascular risk. The risk score is as predicted higher in the biomarker highrisk group (14.2 ± 6.8 vs. 10.0 ± 5.4, p b 0.05). Among the laboratory parameters (Table 2), the following showed higher levels in the highrisk patient group: fibrinogen (4.84 ± 0.87 g/L vs. 3.71 ± 0.75 g/L, p b 0.001) and NT-proBNP (93.83 ± 106.54 vs. 44.29 ± 48.44 pmol/L, p b 0.05). The separation in three biomarker-based risk groups did not made any difference.

Discussion
Traditional risk factor-based identification 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 predict 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-PLA 2 , measured 30 days after an acute event, predicted recurrent cardiovascular events [6]. The combined prognostic value of hs-CRP and Lp-PLA 2 , 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 particularly high risk of subsequent adverse events and death [22], improved prediction of individuals with the highest event risk would be particularly valuable among such patients.
Endothelial dysfunction is considered to be a legitimate surrogate marker of cardiovascular risk [23][24][25]30]. In addition, the RH-PAT method 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-PLA 2 had a worse endothelial function, as determined by RH-PAT. The STEMI and the primary PCI induce systemic endothelial dysfunction and an inflammatory response, which peaks at 1 to 2 days after the acute event before it gradually decreases, and the process of "myocardial repair" may be greatly finished after 3 weeks [27,28]. In order to avoid disturbance of endothelial function and inflammatory marker measurements, we performed it after 4 to 6 weeks from STEMI.
Data from other studies on the relation between biomarkers and endothelial 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-PLA 2 were found to be useful to distinguish high-risk patients from patients with moderate or low event risk [29]. This supports the findings of the large Atherosclerosis Risk in Communities (ARIC) study, which showed that the levels of Lp-PLA 2 and CRP may be complementary beyond traditional risk factors in identifying middle-aged men and women at increased risk for ischemic stroke or incident coronary heart disease [7][8].
Our current study showed a non-significantly (p = 0.09) lower endothelial 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 subjects with endothelial dysfunction might be higher among patients with elevation of both biomarkers of inflammation, but the absence of statistical 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 recent 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.

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]. However, this accounts for both study groups, and there was no significant between-group difference in medical treatment. The present substudy of the RESPONSE trial is limited by its relatively small sample size, and its findings 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.

Conclusions
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 dissimilarities in endothelial function that did not reach a statistical significance. Nevertheless, further research in larger patient populations may be warranted.