Recent findings have challenged the prevailing histology- or imaging-based definition of the vulnerable plaque.
Methods
To investigate molecular characteristics associated with clinical instability of atherosclerosis, we performed a proteomics comparison of the vascular extracellular matrix and associated molecules in human carotid endarterectomy specimens from symptomatic versus asymptomatic patients. The proteomics data were integrated with gene expression profiling and an analysis of protein secretion by lipid-loaded human vascular smooth muscle cells.
Results
The molecular signature of plaques from symptomatic patients identified by proteomics and at least one of the other two approaches comprised matrix metalloproteinase-9, chitinase-3-like protein 1, S100 calcium binding protein A8, S100 calcium binding protein A9, cathepsin B, fibronectin and galectin-3-binding protein. Biomarker candidates were measured in 685 subjects of the Bruneck Study and found to be significantly associated with the progression to advanced atherosclerosis (as assessed by repeated carotid ultrasound) and the incidence of cardiovascular disease over a 10 year follow-up period. A 4-biomarker signature (matrix metalloproteinase-9, S100A8/S100A9, cathepsin D, and galectin-3-binding protein) improved risk prediction in terms of risk discrimination and classification and was successfully replicated in a second independent population (SAPHIR Study).
Conclusion
Our study highlights the strength of tissue-based proteomics for biomarker discovery.
Objective: Platelets are central to acute myocardial infarction (MI). How the platelet proteome is altered during MI is unknown. We sought to describe changes in the platelet proteome during MI and identify corresponding functional consequences. Approach and Results: Platelets from patients experiencing ST-segment–elevation MI (STEMI) before and 3 days after treatment (n=30) and matched patients with severe stable coronary artery disease before and 3 days after coronary artery bypass grafting (n=25) underwent quantitative proteomic analysis. Elevations in the proteins S100A8 and S100A9 were detected at the time of STEMI compared with stable coronary artery disease (S100A8: FC, 2.00; false discovery rate, 0.05; S100A9: FC, 2.28; false discovery rate, 0.005). During STEMI, only S100A8 mRNA and protein levels were correlated in platelets ( R =0.46, P =0.012). To determine whether de novo protein synthesis occurs, activated platelets were incubated with 13C-labeled amino acids for 24 hours and analyzed by mass spectrometry. No incorporation was confidently detected. Platelet S100A8 and S100A9 was strongly correlated with neutrophil abundance at the time of STEMI. When isolated platelets and neutrophils were coincubated under quiescent and activated conditions, release of S100A8 from neutrophils resulted in uptake of S100A8 by platelets. Neutrophils released S100A8/A9 as free heterodimer, rather than in vesicles or extracellular traps. In the community-based Bruneck study (n=338), plasma S100A8/A9 was inversely associated with platelet reactivity—an effect abrogated by aspirin. Conclusions: Leukocyte-to-platelet protein transfer may occur in a thromboinflammatory environment such as STEMI. Plasma S100A8/A9 was negatively associated with platelet reactivity. These findings highlight neutrophils as potential modifiers for thrombotic therapies in coronary artery disease.
Aims Cardiac resynchronisation therapy (CRT) is effective treatment for selected patients with heart failure (HF) but has ~30% non-response rate. We evaluated whether specific biomarkers can predict outcome. Methods A prospective single-centre pilot study of consecutive unselected patients undergoing CRT for HF between November 2013 and December 2015 evaluating cardiac extracellular matrix biomarkers and micro-ribonucleic acid (miRNA) expression before and after CRT assessing ability to predict functional response and survival. Each underwent three assessments (pre-implant, 6 weeks and 6 months postimplant) including: New York Heart Association (NYHA) class, echocardiography, electrocardiography, 6 min walk test (6MWT), Minnesota Living with Heart Failure Questionnaire (MLHFQ) and N-terminal pro-brain natriuretic peptide (NT-pro-BNP). Plasma markers of cardiac fibrosis assessed were: N-terminal pro-peptides of collagen I and III, collagen I C-terminal telopeptides (CTx) and matrix metalloproteinases (MMP-2 and MMP-9) as well as a panel of miRNAs (miRNA-21, miRNA-30d, miRNA-122, miRNA-133a, miRNA-210 and miRNA-486). Results A total of 52 patients were recruited; mean age (±SD) was 72.4±9.4 years; male=43 (82.7%), ischaemic aetiology=30 (57.7%), mean QRS duration=166.4±23.5 ms, left bundle branch block (LBBB) morphology = 39 (75.0%), mean NYHA=2.7±0.6, 6MWT=238.8±130.6 m, MLHFQ=46.4±21.3 and left ventricular ejection fraction (LVEF)=24.3%±8.0%. Mean follow-up=1.7±0.3 and 5.8±0.7 months. There were 27 (55.1%) functional responders (3 no definable 6-month response; 2 missed assessments and 1 long-term lead displacement). No marker predicted response, however, CTx and LBBB trended most towards predicting functional response. Conclusion No specific biomarkers reached significance for predicting functional response to CRT. CTx showed a trend towards predicting response and warrants further study. Trial registration number NCT02541773 .
Non-coding RNAs (ncRNAs), including microRNAs (miRNAs), circular RNAs (circRNA) and long non-coding RNAs (lncRNA), have been implicated as novel cardiac biomarkers.
Objective
To compare the tissue-specificity and release kinetics of ncRNAs and protein biomarkers after induced myocardial injury by transcoronary ablation of septal hypertrophy (TASH).
Methods and results
Upon screening the relative abundance of 109 circRNA and 21 lncRNAs in human cardiac tissue, 12 circRNAs and 11 lncRNAs were selected for further analyses. Human myocardial tissue was spiked into plasma from healthy individuals and the expression levels of ncRNAs were compared to a panel of miRNAs, including muscle- (miR-1, miR-133a) and cardiac-enriched miRNAs (miR-208a, miR-208b, miR-499). Curve fitting analyses of each of the three ncRNA classes with the highest R2 values revealed no significant differences in the regression coefficients compared with high sensitive troponin T and I (hs-cTnT, hs-cTnI) and cardiac myosin-binding protein C (cMyC). At low spike-in concentrations, however, significantly higher regression coefficients were observed for all ncRNA species (Mann Whitney test: miRNAs vs. proteins p<0.0001, fold-change 2.6; circRNAs vs proteins p=0.0028, fold change 2.8; lncRNAs vs. proteins p=0.0028, fold-change 1.6). To assess whether ncRNAs allow earlier detection of myocardial injury, circulating ncRNAs were quantified in patients undergoing TASH before and at 1 hour, 8 hours, 24 hours after the procedure. Unlike cardiac proteins, miR-1 and miR-133a showed the steepest rise within the first hour after cardiac injury. These muscle-enriched miRNAs were also more readily detectable in plasma than the cardiac-specific miRNAs, miR-208b and miR-499. In contrast, cardiac circRNAs remained undetectable in plasma even after myocardial injury. Putative cardiac lncRNAs, including the Long Intergenic Non-Coding RNA Predicting Cardiac Remodelling And Survival (LIPCAR) were abundant in plasma but failed to show significant changes after TASH, refuting a predominant cardiac origin. Finally, the stability of ncRNAs was assessed in plasma. Degradation of cardiac/muscle-enriched miRNAs was observed in plasma left at room temperature for 1 hour and reduced by treatment with RNAse inhibitors. As expected, circRNAs were less susceptible to degradation. Mitochondrial-derived lncRNAs such as LIPCAR increased after 1 hour of incubation in plasma, while non-mitochondrial lncRNAs showed a degradation pattern similar to miRNAs.
Conclusions
Our results demonstrate that circulating heart-associated ncRNAs may enhance early detection of myocardial injury. All three ncRNA classes demonstrated superior release kinetics in vitro compared with established cardiac protein biomarkers. At early time points after TASH, however, a higher sensitivity was only observed for muscle-enriched miRNAs, but not for circRNAs or lncRNAs.
Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder, characterized by cardiomyocyte hypertrophy, cardiomyocyte disarray and fibrosis, which has a prevalence of ~1: 200–500 and predisposes individuals to heart failure and sudden death. The mechanisms through which diverse HCM-causing mutations cause cardiac dysfunction remain mostly unknown and their identification may reveal new therapeutic avenues. MicroRNAs (miRNAs) have emerged as critical regulators of gene expression and disease phenotype in various pathologies. We explored whether miRNAs could play a role in HCM pathogenesis and offer potential therapeutic targets. Using high-throughput miRNA expression profiling and qPCR analysis in two distinct mouse models of HCM, we found that miR-199a-3p expression levels are upregulated in mutant mice compared to age- and treatment-matched wild-type mice. We also found that miR-199a-3p expression is enriched in cardiac non-myocytes compared to cardiomyocytes. When we expressed miR-199a-3p mimic in cultured murine primary cardiac fibroblasts and analyzed the conditioned media by proteomics, we found that several extracellular matrix (ECM) proteins (e.g., TSP2, FBLN3, COL11A1, LYOX) were differentially secreted (data are available via ProteomeXchange with identifier PXD042904). We confirmed our proteomics findings by qPCR analysis of selected mRNAs and demonstrated that miR-199a-3p mimic expression in cardiac fibroblasts drives upregulation of ECM gene expression, including Tsp2, Fbln3, Pcoc1, Col1a1 and Col3a1. To examine the role of miR-199a-3p in vivo, we inhibited its function using lock-nucleic acid (LNA)-based inhibitors (antimiR-199a-3p) in an HCM mouse model. Our results revealed that progression of cardiac fibrosis is attenuated when miR-199a-3p function is inhibited in mild-to-moderate HCM. Finally, guided by computational target prediction algorithms, we identified mRNAs Cd151 and Itga3 as direct targets of miR-199a-3p and have shown that miR-199a-3p mimic expression negatively regulates AKT activation in cardiac fibroblasts. Altogether, our results suggest that miR-199a-3p may contribute to cardiac fibrosis in HCM through its actions in cardiac fibroblasts. Thus, inhibition of miR-199a-3p in mild-to-moderate HCM may offer therapeutic benefit in combination with complementary approaches that target the primary defect in cardiac myocytes.
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