Cardiovascular disease biomarkers derived from circulating cell-free DNA methylation.

Acute coronary syndrome (ACS) remains a major cause of worldwide mortality. The syndrome occurs when blood flow to the heart muscle is decreased or blocked, causing muscle tissues to die or malfunction. There are three main types of ACS: Non-ST-elevation myocardial infarction, ST-elevation myocardial infarction, and unstable angina. The treatment depends on the type of ACS, and this is decided by a combination of clinical findings, such as electrocardiogram and plasma biomarkers. Circulating cell-free DNA (ccfDNA) is proposed as an additional marker for ACS since the damaged tissues can release DNA to the bloodstream. We used ccfDNA methylation profiles for differentiating between the ACS types and provided computational tools to repeat similar analysis for other diseases. We leveraged cell type specificity of DNA methylation to deconvolute the ccfDNA cell types of origin and to find methylation-based biomarkers that stratify patients. We identified hundreds of methylation markers associated with ACS types and validated them in an independent cohort. Many such markers were associated with genes involved in cardiovascular conditions and inflammation. ccfDNA methylation showed promise as a non-invasive diagnostic for acute coronary events. These methods are not limited to acute events, and may be used for chronic cardiovascular diseases as well.

Rapid Inflammasome Activation Is Attenuated in Post-Myocardial Infarction Monocytes.

Inflammasomes are crucial gatekeepers of the immune response, but their maladaptive activation associates with inflammatory pathologies. Besides canonical activation, monocytes can trigger non-transcriptional or rapid inflammasome activation that has not been well defined in the context of acute myocardial infarction (AMI). Rapid transcription-independent inflammasome activation induced by simultaneous TLR priming and triggering stimulus was measured by caspase-1 (CASP1) activity and interleukin release. Both classical and intermediate monocytes from healthy donors exhibited robust CASP1 activation, but only classical monocytes produced high mature interleukin-18 (IL18) release. We also recruited a limited number of coronary artery disease (CAD, n=31) and AMI (n=29) patients to evaluate their inflammasome function and expression profiles. Surprisingly, monocyte subpopulations isolated from blood collected during percutaneous coronary intervention (PCI) from AMI patients presented diminished CASP1 activity and abrogated IL18 release despite increased NLRP3 gene expression. This unexpected attenuated rapid inflammasome activation was accompanied by a significant increase of TNFAIP3 and IRAKM expression. Moreover, TNFAIP3 protein levels of circulating monocytes showed positive correlation with high sensitive troponin T (hsTnT), implying an association between TNFAIP3 upregulation and the severity of tissue injury. We suggest this monocyte attenuation to be a protective phenotype aftermath following a very early inflammatory wave in the ischemic area. Damage-associated molecular patterns (DAMPs) or other signals trigger a transitory negative feedback loop within newly recruited circulating monocytes as a mechanism to reduce post-injury tissue damage.

The activity of catechol-O-methyltransferase (COMT) is not impaired by high doses of epigallocatechin-3-gallate (EGCG) in vivo.

Catechol-O-methyltransferase (COMT) inactivates many endogenous and exogenous compounds by O-methylation. Therefore, it represents a major enzyme of the metabolic pathway with important biological functions in hormonal and drug metabolism. The tea catechin epigallocatechin-3-gallate (EGCG) is known to inhibit COMT enzymatic activity in vitro. Based on beneficial in vitro results, EGCG is extensively used in human intervention studies in a variety of human diseases. Owing to its low bioavailability, rather high doses of EGCG are frequently applied that may impair COMT activity in vivo. Enzymatic activities of four functional COMT single-nucleotide polymorphisms (SNPs) were determined in red blood cells (RBCs) in 24 healthy human volunteers (14 women, 10 men). The subjects were supplemented with 750 mg of EGCG and EGCG plasma levels and COMT enzyme activities in erythrocytes were measured before and 2 h after intervention. The homozygous Val→Met substitution in the SNP rs4680 resulted in significantly decreased COMT activity. Enzymatic COMT activities in RBCs were also affected by the other three COMT polymorphisms. EGCG plasma levels significantly increased after intervention. They were not influenced by any of the COMT SNPs and different enzyme activities. Ingestion of 750 mg EGCG did not result in impairment of COMT activity. However, COMT activity was significantly increased by 24% after EGCG consumption. These results indicate that supplementation with a high dose of EGCG does not impair the activity of COMT. Consequently, it may not interfere with COMT-mediated metabolism and elimination of exogenous and endogenous COMT substrates.

Long-term up-regulation of eNOS and improvement of endothelial function by inhibition of the ubiquitin-proteasome pathway.

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells. Endothelial nitric oxide synthase (eNOS) is the key enzyme of vascular homeostasis involved in the pathophysiology of several cardiovascular diseases. The aim of our study was to investigate whether eNOS expression and activity are regulated by the proteasome. Bovine pulmonary artery endothelial cells (CPAE cells) were treated with the proteasome inhibitor MG132. MG132 (50-250 nmol/L) dose-dependently increased mRNA and protein levels of eNOS. Comparable results were obtained with other specific proteasome inhibitors, whereas the nonproteasomal calpain and cathepsin inhibitor ALLM had no effect. Efficacy of proteasome inhibition was evidenced by accumulation of poly-ubiquitinylated proteins and by measuring proteasomal activity in cell extracts. Cycloheximide prevented up-regulation of eNOS protein, indicating that post-translational stabilization of eNOS is not involved. eNOS activity was increased up to 2.8-fold (MG132 100 nmol/L, 48 h). Incubation of rat aortic rings with MG132 significantly enhanced endothelial-dependent vasorelaxation. Single MG132 treatment (100 nmol/L) induced long-term effects in CPAE cells, with increases of eNOS protein and activity for up to 10 days. Our results indicate that low-dose proteasome inhibition enhances eNOS expression and activity, and improves endothelial function.

Inhibition of the ubiquitin-proteasome pathway induces differential heat-shock protein response in cardiomyocytes and renders early cardiac protection.

The effects of proteasome inhibition (PI) on heat-shock protein (HSP) expression in cardiomyocytes were investigated. Neonatal rat cardiomyocytes were incubated with MG132 (0.1-10 microM) for 1 h. Induction of various HSPs was determined by real-time PCR and Western blotting. PI induced a 2- to 3-fold increase in HSP27, HSP60, and HSP90, and a 18-fold increase in HSP70 mRNA expression, whereas HSP40 levels were unaffected. Western blotting revealed increased protein expression for HSP70 after PI. Similar results were obtained with MG262. HSP induction correlated with enhanced survival of neonatal cardiomyocytes after sublethal heat stress in XTT testing. In papillary muscles, pretreatment with MG132 (10 microM, 90 min) was associated with enhanced recovery of the contractile parameters after a 40-min hypoxia. In these proof-of-principle experiments, we show that PI induces differential heat-shock response in cardiomyocytes, accompanied by enhanced cell survival and functional recovery after various forms of stress.