Lack of association between cystatin C and different coronary atherosclerotic manifestations

<p>Cystatin C (CysC) is known to be related to cardiovascular disease (CVD), including the presence and severity of coronary artery disease (CAD) and future clinical events. In this study, the association between CysC levels and (1) coronary artery calcification (CAC) in asymptomatic individuals from the general population as well as (2) different subgroups of patients with suspected or definite acute myocardial infarction (MI) was investigated. CysC levels were measured in serum from asymptomatic individuals as part of a screening study for CAC using non-contrast cardiac CT scan (<i>N</i> = 1039) as well as in subgroups of hospitalized patients with a suspected MI (<i>N</i> = 769). CysC was not associated with CAC in asymptomatic individuals after adjusting for relevant risk factors. No difference in CysC levels was observed between patients with type 1 MI (1.07 mg/L) and patients with normal troponin (with or without prior CAD: 1.14 and 1.01 mg/L, respectively). However, patients with type 2 MI and patient subgroups with elevated troponin but without MI had significantly higher CysC levels (1.24, 1.23 and 1.31 mg/L), even after adjusting for other risk factors. CysC was not associated with CAC in middle-aged asymptomatic individuals from the general population. Furthermore, CysC levels were found to be significantly lower in patients with type 1 MI compared to patients with type 2 MI and patients with elevated troponins but without MI. Thus, in two independent and clinically different populations, no association between CysC and coronary atherosclerotic manifestations could be demonstrated.</p

Differences in elastin neoepitopes levels compared to patient parameters presented with p-values. n>5.

*<p>p<0.05; ** p<0.01; ^AMean of ELM release is 17,2 ng/mL for patients having hypercholesteremia and 21,3 ng/mL for patients not having it. ^BMean of ELM release is 25.8 ng/mL for patients taking Calcium channel blocker and 19,4 ng/mL for patients not taking the drug. ^CMean of ELM release is 19,6 ng/mL for patients taking thiazides and 20,6 ng/mL for patients not taking the drug. ^#Levels of mean of ELM versus smoking habit is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060936#pone-0060936-g004" target="_blank">Figure 4</a>.</p

Tissue distribution of CD68, elastin (ELN), ELM and ELM-2.

<p>ELM and ELM-2 monoclonal antibodies were also mixed with the related free peptide as a control. A) Cross section of the plaque shoulder of a carotid artery from a 77 old man B) Left lung from a patient diagnosed with IPF. All pictures were taken with 10× magnification.</p

Demographical data from the cardiovascular disease cohort.

<p>Values are mean ± standard deviation. The data were analysed using the one-way of analysis of variance (ANOVA) non-parametric Kruskal-Wallis test. * p<0.05; ** p<0.01; *** p<0.001,.^AHeart score is an assessment on risk of cardiovascular disease based on age, systolic blood pressure, total cholesterol in mmol/L, and smoking status. LDL = low density lipoprotein, HDL =  high density lipoprotein, AMI = acute myocardial infarction, CT-plusCA =  subclinical coronary calcium shown on CT scans, CT-noCa =  no coronary calcium detectable on a CT scan.</p

Summary table of the technical validation of the ELM-2 ELISA.

*<p>Percentage dilution recovery was calculated as the mean of 4 human samples diluted 1∶2 and 1∶4. **Inter- and intra-assay validation was calculated as the mean variation between 8 individual determinations of each human serum sample. ***The stability of the analyte (human serum) was calculated as the mean of three different serum samples tested after freeze/thaw for one to 4 times.</p

Biological validation of ELM and ELM-2 in the cardiovascular cohort.

<p>Human serum from patients with acute myocardial infarction (AMI) (n = 30), non-AMI (n = 30), coronary calcium shown on CT scans (CT-plusCA)(n = 30) and no coronary calcium detectable on a CT scan (n = 30). Bars indicate mean level. Groups were compared by Wilcoxon rank sum test. A)ELM-2, B) ELM and C) The Spearman correlation between ELM and ELM-2. Data are shown as mean±SD with 95% confidence intervals. ** p<0.01.</p

Diagnostic power of each biomarker for the separation of patients with AMI compared non-AMI.

<p>AUROC =  Area Under the Receiver Operating Characteristic Curve. Data are show as the AUROC, a probability of correct diagnosis by each marker. The P value indicates significance of the AUROC diagnosis compared to the null hypothesis which is an area of 0.5.</p>*<p>p<0.05, ** p<0.01, *** p<0.01.</p

Biological validation of ELM and ELM-2 in pulmonary diseases.

<p>Human serum from patients with chronic obstructive pulmonary disease (COPD)(n = 10) and idiopathic pulmonary fibrosis (IPF)(n = 29) compared with controls (n = 11). Bars indicate mean level. A) ELM-2, B) ELM (Data have been published with permission from Skjøt-Arkil et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060936#pone.0060936-SkjotArkil3" target="_blank">[45]</a>, C) The Spearman correlation between ELM and ELM-2. Groups were compared by Wilcoxon rank sum test. Data are shown as mean±1.8SD with 95% confidence intervals. *** p<0.001.</p

Correlations in the cardiovascular cohort.

<p>A) Spearman correlation between C-reactive protein and ELM, B) Spearman correlation between C-reactive protein and ELM-2. * p<0.05, C) Biological validations of ELM versus smoking habits, D) Biological validations of C-reactive protein versus smoking habits. * p<0.05; ** p<0.01; C-reactive protein was only measured in the acute myocardial infarction (AMI) and non-AMI groups.</p

Characterization of the ELM-2 monoclonal antibody.

<p>A) ELISA showing percent inhibition of the signal of the free peptide, elongated peptide and three human serum samples. The human samples were run undiluted and diluted 1∶2, 1∶4 and so forth as indicated by the dotted lines. B+C) Release of ELM-2 by MMP-9 and −12 cleavages as a function of time of human elastin from B) insoluble elastin and C) soluble elastin. The cleaved material was diluted 1∶10 in the assay.</p