Neutrophil-Enriched Biomarkers and Long-Term Prognosis in Acute Coronary Syndrome: a Systematic Review and Meta-analysis.

Activated neutrophils release a range of inflammatory products that represent potential biomarkers, and there is interest in the prognostic value of these in acute coronary syndrome (ACS) patients. We conducted a systematic review to examine neutrophil-enriched biomarkers and the occurrence of major adverse cardiovascular events (MACE) in patients with ACS. We identified twenty-seven studies including 17,831 patients with ACS. The most studied biomarkers were neutrophil gelatinase-associated lipocalin (NGAL) and myeloperoxidase (MPO). Meta-analyses showed that elevated NGAL was associated with higher MACE rates (unadjusted risk ratio (RR) 1.52, 95% CI 1.12-2.06, p = 0.006) as were elevated MPO levels (unadjusted RR 1.61, 95% CI 1.22-2.13, p = 0.01). There was limited data suggesting that increased levels of calprotectin, proteinase-3 and double-stranded DNA were also associated with MACE. These results suggest that higher levels of neutrophil-enriched biomarkers may be predictive of MACE in patients with ACS, although higher-quality studies are needed to confirm these observations.

Increased levels of low density neutrophils (LDNs) in myocardial infarction.

BACKGROUND: Recent evidence suggests that neutrophils are highly plastic cells that can display heterogeneous phenotypes. Low-density neutrophils (LDNs) have been described in many inflammatory conditions, and are thought to represent an immature, hyperactivated subtype of neutrophils. Neutrophils are significantly involved in the inflammatory response to myocardial infarction (MI), although we do not know the extent to which LDNs exist, or function, in MI. This study sought to determine the frequency and phenotype of LDNs in MI patients, compared to healthy subjects (HS). METHODS: LDNs and normal-density neutrophils (NDNs) were isolated from the peripheral blood of MI subjects (n = 12) and HSs (n = 12) using density gradient centrifugation. LDNs and NDNs were analysed by flow cytometry to identify neutrophils (CD66b+CD15+CD14-CD3-CD19- cells) and examine neutrophil activation (CD11b, CD66b and CD15) and maturity (CD33 and CD16). RESULTS: We identified LDNs within the peripheral blood mononuclear cell (PBMC) fraction of blood, and this population is significantly enriched in MI patients (1.04 ± 0.75% of PBMCs), compared to HS (0.29 ± 0.24%, p = .003). Across both cohorts, LDNs express significantly higher levels of CD66b and CD15, indicating a heightened state of activation compared to NDNs. In this study, LDNs were described as CD33highCD16low, compared to CD33lowCD16high NDNs, indicating the immaturity of this neutrophil subtype. CONCLUSIONS: An increase in the frequency of hyperactivated, immature LDNs is an immunological feature of MI. We highlight a potential pathological role of LDNs in MI, which underscores the need to expand our current understanding of this subtype in MI and other cardiovascular diseases (CVDs).

Cluster analysis of extracellular matrix biomarkers predicts the development of impaired systolic function within 1 year of acute myocardial infarction.

The clinical utility of combining extracellular matrix (ECM) biomarkers to predict the development of impaired systolic function following acute myocardial infarction (AMI) remains largely undetermined. A combination of ELISA and multiplexing assays were performed to measure matrix metalloproteinase (MMP)-2, MMP-3, MMP-8, MMP-9, periostin, N-terminal type I procollagen (PINP) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in plasma samples from 120 AMI patients. All patients had an echocardiogram within 1 year of AMI, and were divided into impaired (n = 37, LVEF < 50%) and preserved (n = 83, LVEF ≥ 50%) systolic function groups. Exploratory factor analysis was performed on log-transformed biomarkers using principle axis analysis with Oblimin rotation. Cluster analysis was performed on log-transformed and normalised biomarkers using Ward's method of minimum variance and the squared Euclidean distance metric. Upon univariate analysis, current smoking, prescription of ACE inhibitors at discharge, peak hsTnT > 610 ng/L (median), MMP-8 levels, Factor 1 scores and Cluster One assignment were predictive of impaired systolic function. Upon multivariate analysis, Cluster One assignment (odds ratio [95% CI], 2.74 [1.04-7.23], p = 0.04) remained an independent predictor of systolic dysfunction in combination with clinical variables. These observations support the usefulness of combining ECM biomarkers using cluster analysis for predicting the development of impaired systolic function in AMI patients.

Examining variation and temporal dynamics of extracellular matrix biomarkers following acute myocardial infarction.

Aim: This study investigated an optimal extracellular matrix (ECM) biomarker panel for measurement in acute myocardial infarction (AMI). Materials & methods: Blood samples were collected from 12 healthy volunteers, and from 23 patients during hospital admission (day 1-3) and 6 months following AMI. Protein assays measured: FGFb, MMP-2, -3, -8, -9, osteopontin, periostin, PINP, TGF-ß1, TIMP-1, -4 and VEGF. Results: When compared with healthy levels, seven ECM biomarkers were significantly altered in AMI patients, and six of these biomarkers displayed stable expression during hospital admission. Clinical characteristics and baseline cardiac function were not well correlated with ECM biomarkers. Conclusion: We suggest, MMP-2, MMP-3, MMP-8, MMP-9, periostin, PINP and TIMP-1 may be useful ECM biomarkers for future studies in AMI patients.

Plain language summary The cardiac extracellular matrix (ECM) maintains the structural integrity of the heart, and can be measured in human circulation using ECM biomarkers. Understanding the levels of variation and temporal dynamics that exist in ECM biomarkers following a heart attack is important for establishing an optimal biomarker panel that may be useful in heart research. A single blood sample was collected from 12 healthy volunteers. Multiple bloods samples were collected from 23 heart attack patients during hospital admission (day 1­3) and 6 months post heart attack. About 12 ECM biomarkers were measured from these blood samples. When compared with healthy levels, seven ECM biomarkers were significantly altered in heart attack patients, and six of these biomarkers displayed stable expression during hospital admission. Variability existed within biomarker levels and this was not well described by traditional heart attack risk factors, such as age or heart attack size. Thus, the source of biomarker variability remains unknown in this study. Overall, we suggest these seven ECM biomarkers may be of interest for future studies in the setting of heart attack research.

A combined biomarker approach for characterising extracellular matrix profiles in acute myocardial infarction.

Extracellular matrix (ECM) biomarkers are useful for measuring underlying molecular activity associated with cardiac repair following acute myocardial infarction (AMI). The aim of this study was to conduct exploratory factor analysis (EFA) to examine the interrelationships between ECM biomarkers, and cluster analysis to identify if distinct ECM profiles could distinguish patient risk in AMI. Ten ECM biomarkers were measured from plasma in 140 AMI patients: MMP-2, -3, -8, -9, periostin, procollagen I N-Terminal propeptide, osteopontin, TGF-ß1, TIMP-1 and -4. EFA grouped eight ECM biomarkers into a two-factor solution, which comprised three biomarkers in Factor 1 and five biomarkers in Factor 2. Notably, ECM biomarkers were not separated based on biological function. Cluster analysis grouped AMI patients into three distinct clusters. Cluster One (n = 54) had increased levels of MMP-8, MMP-9, and TGF-B1. Cluster Two (n = 43) had elevated levels of MMP-2, MMP-3, osteopontin, periostin and TIMP-1, and increased high-sensitivity troponin T and GRACE scores. Cluster Three (n = 43) had decreased levels of ECM biomarkers. Circulating ECM biomarkers demonstrated collinearity and entwined biological functions based on EFA analysis. Using cluster analysis, patients with similar clinical presentations could be separated into distinct ECM profiles that were associated with differential patient risk. Clinical significance remains to be determined.

Association between inflammatory cytokines and long-term adverse outcomes in acute coronary syndromes: A systematic review.

BACKGROUND: Inflammatory cytokines are involved in the pathophysiology of acute coronary syndromes (ACS) and have been associated with major adverse cardiovascular events (MACE). We systematically reviewed studies investigating the ability of multiple cytokines to predict MACE in ACS patients with follow-up of at least one year. METHODS: A Medical Subject Heading search criteria was applied on Ovid Medline(R), EMBASE, EMBASE Classic and Cochrane Library to systematically identify relevant studies published between 1945 and 2017 that had an observational study design or were randomised controlled trials. Studies were excluded if only one cytokine was analysed, follow-up period was less than one year, subjects were non-human, or blood samples were taken more than 10 days from symptom onset. RESULTS: Ten observational studies met the inclusion criteria. Six had acceptable internal validity when evaluated for quality. The studies were varied in terms of study methods (time of blood collection, study population, cytokines assessed, MACE definition, follow-up length) and result reporting, so a meta-analysis could not be conducted. Six of the studies found significant associations between individual cytokines and MACE. Four studies measured the combined effects of multiple cytokines to predict MACE, and all had statistically significant results. CONCLUSION: A combination of multiple cytokines had a better association with MACE than individual cytokines. It appears promising for future studies to determine the optimal multi-marker methodology and confirm its predictive value.

White blood cell subtypes as predictors of adverse cardiac events.

BACKGROUND: White blood cell (WBC) subtypes have been associated with major adverse cardiovascular events (MACEs) in acute myocardial infarction (AMI). More recently, combining neutrophil and lymphocyte counts or lymphocyte and monocyte counts into a ratio has found to be promising for predicting MACE. This study aimed to confirm the association between MACE and the following WBC subtypes: neutrophils, lymphocytes, monocytes, neutrophil-lymphocyte ratio (NLR) and lymphocyte-monocyte ratio (LMR). METHODS: In a cohort of 860 AMI patients, we collected levels of WBC subtypes from the earliest blood tests recorded prior to angiography. Data on baseline demographics and one-year outcomes were also collected. RESULTS: At one year, 130 patients (15.1%) developed MACE. NLR and LMR were significantly associated with MACE on univariate analysis (P = 0.006 and 0.005, respectively). However, when combined into a multivariate model with age, hypertension, prior myocardial infarction and Type 2 diabetes, neither NLR nor LMR had significant associations (odds ratio = 1.058 and 0.966, P = 0.069 and 0.612, respectively). CONCLUSION: As NLR and LMR were correlated with MACE only on univariate analysis, we do not believe that they are predictive enough to be used alone in a clinical setting. Further studies are required to assess the prognostic ability of these ratios in combination with other inflammatory markers.

An IL-6-IL-8 score derived from principal component analysis is predictive of adverse outcome in acute myocardial infarction.

INTRODUCTION: Many studies have shown that elevated biomarkers of inflammation following acute myocardial infarction (AMI) are associated with major adverse cardiovascular events (MACE). However, the optimal way of measuring the complex inflammatory response following AMI has not been determined. In this study we explore the use of principal component analysis (PCA) utilising multiple inflammatory cytokines to generate a combined cytokine score that may be predictive of MACE post-AMI. METHODS: Thirteen inflammatory cytokines were measured in plasma of 317 AMI patients, drawn 48-72 h following symptom onset. Patients were followed-up for one year to determine the incidence of MACE. PCA was used to generate a combined score using six cytokines that were detectable in the majority of patients (IL-1ß, -6, -8, and -10; MCP-1; and RANTES), and using a subset of cytokines that were associated with MACE on univariate analysis. Multivariate models using baseline characteristics, elevated individual cytokines and PCA-derived scores determined independent predictors of MACE. RESULTS: IL-6 and IL-8 were significantly associated with MACE on univariate analysis and were combined using PCA into an IL-6-IL-8 score. The combined cytokine score and IL-6-IL-8 PCA-derived score were both significantly associated with MACE on univariate analysis. In multivariate models IL-6-IL-8 scores (OR = 2.77, p = 0.007) and IL-6 levels (OR = 2.18, p = 0.035) were found to be independent predictors of MACE. CONCLUSION: An IL-6-IL-8 score derived from PCA was found to independently predict MACE at one year and was a stronger predictor than any individual cytokine, which suggests this may be an appropriate strategy to quantify inflammation post-AMI. Further investigation is required to determine the optimal set of cytokines to measure in this context.

Immunoglobulin G levels predicts risk of recurrent adverse cardiovascular events in myocardial infarction patients.

Background: Patients with myocardial infarction (MI) are at an increased risk of experiencing recurrent major adverse cardiovascular events (MACE) but predicting MACE has remained challenging. Immunoglobulins are implicated in cardiovascular disease, although the predictive value of total immunoglobulin G (IgG) has not yet been evaluated in a secondary prevention setting. This study examined whether total IgG is predictive of MACE in an MI population, and how total IgG compared to the predictive value of C-reactive protein (CRP), an acute inflammatory marker. Methods: We conducted a case-control study with 40 MI subjects (cases) who experienced MACE within 1 year of their index admission. Cases were matched for age, sex, diabetes and presentation with 77 controls who did not have MACE. Pre-discharge plasma samples were analysed for total IgG and CRP. Results: We observed higher levels of total plasma IgG in MI subjects with MACE (24.9 (16.2-43.7) mg/mL) compared to controls (18.4 (9.1-37.3) mg/mL; p < 0.05). Higher levels of IgG were associated with increased risk of MACE in our MI population. MI subjects within quartiles 3 and 4 of total IgG had 6 times and 4 times, respectively, the rate of MACE compared to subjects in quartile 1. There was no difference in CRP levels between cases and controls (1.1 (0.5-3.0) vs. 1.9 (0.6-6.1) mg/mL, p = 0.10), and no relationship was observed between CRP and MACE. Conclusion: Pre-discharge IgG level was a better marker for predicting MACE post-MI than CRP, which had no predictive value in this study.

Comparison of Multiplate and VerifyNow platelet function tests in predicting clinical outcome in patients with acute coronary syndromes.

INTRODUCTION: This study examined the ability of two widely used "point of care" platelet function assays, VerifyNow and Multiplate, to predict adverse outcomes in patients with acute coronary syndromes (ACS). METHODS: We examined platelet reactivity using VerifyNow and Multiplate P2Y12 assays in patients with ACS and the relationship between platelet reactivity and both MACE (defined as a composite of death, myocardial infarction, stroke, stent thrombosis and unplanned revascularisation) and TIMI major bleeding at 1year. RESULTS: In 619 ACS patients, 65 patients (10.5%) had experienced MACE at 1year and 6 patients (1%) had TIMI major bleeding events. The two measures of platelet reactivity were only moderately correlated (Rho=0.43, p=0.0001). Both measures demonstrated a statistically significant relationship with MACE, with area under the curve for VerifyNow of 0.632 (0.001) and for Multiplate of 0.577 (p=0.04), and neither measure showed a significant relationship with bleeding. Logistic regression analysis found that only VerifyNow was a statistical predictor of MACE (p=0.01). MACE occurred in 16% of those classified as having HPR using VerifyNow compared to 7% in those without HPR (odds ratio of 2.6 (95% CI 1.5-4.4, p=0.001). In those classified as having HPR by the Multiplate assay, MACE occurred in 13% compared to 9% of those without HPR (Odds ratio 1.5 95% CI 0.9-2.5, p=0.11). CONCLUSION: The two points of care platelet function tests examined in this study were only moderately correlated. The VerifyNow assay demonstrated a stronger relationship to MACE than the Multiplate assay.

Relationship between Glutathione Peroxidase, Platelet Reactivity, and Reactive Oxygen Species in an Acute Coronary Syndrome Population.

Low activity levels of the antioxidant enzyme, glutathione peroxidase (GPx), have been implicated in adverse clinical outcomes in coronary artery disease. A potential mechanistic link for this relationship is that low GPx activity predisposes patients to thrombotic complications due to impaired reactive oxygen species (ROS) metabolism. GPx potentially regulates the bioavailability of nitric oxide (NO) - a potent platelet inhibitor - therefore indirectly affecting platelet activation. This study examined the relationship between levels of GPx activity, ROS, and platelet activation in 51 acute coronary syndrome (ACS) patients. No relationship was observed between GPx activity and platelet reactivity nor between ROS levels and platelet reactivity. However patients with low GPx activity had significantly higher ROS levels (r2=0.1, p<0.05), suggesting a differential capacity to upregulate antioxidant defence in response to oxidative stress. Low levels of GPx activity may contribute to increased clinical risk by an inability to protect against oxidant-mediated damage.