Platelet activation and blood extracellular vesicles: The influence of venepuncture and short blood storage.

Extracellular vesicles (EVs) as membrane-bound particles released by various cells are potential tools for diagnosis and treatment. Blood cells, particularly platelets, are the source of circulating EVs. MATERIAL: EVs were enriched with gradient ultracentrifugation and measured by nanoparticle tracking assay. A flow cytometric multiplex assay was used for cellular source determination. Activation of platelets was measured as a percentage of CD62p+/CD61+ platelets and correlated with the concentration and size of released EVs. RESULTS: In general there was no statistically significant correlation between EVs` concentration and degree of platelet activation. EVs from different cellular sources were detected. Comparing different needle thicknesses, there was a decrease in the EVs concentration for the 16G needle versus the 21G needle, but no difference was observed for EVs` size and phenotype or platelets activation. During blood storage, platelet activation increased, but there was no effect on the EVs` concentration, size, or phenotype. CONCLUSIONS: Preanalytical factors like needle thickness and storage time can affect the MVs' properties. Activation of platelets during blood collection or blood storage occurs; however, it is difficult to determine its effect on the physiological properties of EVs since the mechanisms of EVs` biogenesis and especially clearness are not precisely known.

Simple prediction of COVID-19 convalescent plasma units with high levels of neutralization antibodies.

BACKGROUND: Hyperimmune convalescent COVID-19 plasma (CCP) containing anti-SARS-CoV-2 neutralizing antibodies (NAbs) was proposed as a therapeutic option for patients early in the new coronavirus disease pandemic. The efficacy of this therapy depends on the quantity of neutralizing antibodies (NAbs) in the CCP units, with titers ≥ 1:160 being recommended. The standard neutralizing tests (NTs) used for determining appropriate CCP donors are technically demanding and expensive and take several days. We explored whether they could be replaced by high-throughput serology tests and a set of available clinical data. METHODS: Our study included 1302 CCP donors after PCR-confirmed COVID-19 infection. To predict donors with high NAb titers, we built four (4) multiple logistic regression models evaluating the relationships of demographic data, COVID-19 symptoms, results of various serological testing, the period between disease and donation, and COVID-19 vaccination status. RESULTS: The analysis of the four models showed that the chemiluminescent microparticle assay (CMIA) for the quantitative determination of IgG Abs to the RBD of the S1 subunit of the SARS-CoV-2 spike protein was enough to predict the CCP units with a high NAb titer. CCP donors with respective results > 850 BAU/ml SARS-CoV-2 IgG had a high probability of attaining sufficient NAb titers. Including additional variables such as donor demographics, clinical symptoms, or time of donation into a particular predictive model did not significantly increase its sensitivity and specificity. CONCLUSION: A simple quantitative serological determination of anti-SARS-CoV-2 antibodies alone is satisfactory for recruiting CCP donors with high titer NAbs.

ABO blood group does not influence the level of anti-SARS-CoV-2 antibodies in convalescent plasma donors.

BACKGROUND: The association of the ABO blood group with COVID-19 disease has been confirmed by several studies, with the blood group A patients being more susceptible and prone to a more severe clinical course of the disease. Additionally, several authors also addressed the association of ABO-types and the levels of anti-SARS-CoV-2 antibodies in convalescents, mostly supporting a theory that the non-O blood group convalescents present with higher levels of anti-SARS-CoV-2 antibodies. STUDY DESIGN AND METHODS: Since previous findings were based on small convalescent cohorts, we quantified the anti-SARS-CoV-2 antibody levels in a total of 3187 convalescent plasma donors with three commercial serological and one standard neutralizing antibody test. The majority of donors had undergone a mild form of the disease and the median time of sampling was 66 days after diagnosis. RESULTS: None of the antibody quantitation results showed any significant association with the ABO blood group types. The same result was evident in the subgroup of vaccinated individuals (n = 370) and the subgroups when stratified according to post-COVID-19 periods (0-60, 60-120, and 120-180 days). CONCLUSION: In conclusion, we found no evidence to confirm that the ABO blood group types influence the level of SARS-CoV-2 antibody response in COVID-19 convalescent plasma donors.

Hyperglycemia Induces Trained Immunity in Macrophages and Their Precursors and Promotes Atherosclerosis.

BACKGROUND: Cardiovascular risk in diabetes remains elevated despite glucose-lowering therapies. We hypothesized that hyperglycemia induces trained immunity in macrophages, promoting persistent proatherogenic characteristics. METHODS: Bone marrow-derived macrophages from control mice and mice with diabetes were grown in physiological glucose (5 mmol/L) and subjected to RNA sequencing (n=6), assay for transposase accessible chromatin sequencing (n=6), and chromatin immunoprecipitation sequencing (n=6) for determination of hyperglycemia-induced trained immunity. Bone marrow transplantation from mice with (n=9) or without (n=6) diabetes into (normoglycemic) Ldlr-/- mice was used to assess its functional significance in vivo. Evidence of hyperglycemia-induced trained immunity was sought in human peripheral blood mononuclear cells from patients with diabetes (n=8) compared with control subjects (n=16) and in human atherosclerotic plaque macrophages excised by laser capture microdissection. RESULTS: In macrophages, high extracellular glucose promoted proinflammatory gene expression and proatherogenic functional characteristics through glycolysis-dependent mechanisms. Bone marrow-derived macrophages from diabetic mice retained these characteristics, even when cultured in physiological glucose, indicating hyperglycemia-induced trained immunity. Bone marrow transplantation from diabetic mice into (normoglycemic) Ldlr-/- mice increased aortic root atherosclerosis, confirming a disease-relevant and persistent form of trained innate immunity. Integrated assay for transposase accessible chromatin, chromatin immunoprecipitation, and RNA sequencing analyses of hematopoietic stem cells and bone marrow-derived macrophages revealed a proinflammatory priming effect in diabetes. The pattern of open chromatin implicated transcription factor Runt-related transcription factor 1 (Runx1). Similarly, transcriptomes of atherosclerotic plaque macrophages and peripheral leukocytes in patients with type 2 diabetes were enriched for Runx1 targets, consistent with a potential role in human disease. Pharmacological inhibition of Runx1 in vitro inhibited the trained phenotype. CONCLUSIONS: Hyperglycemia-induced trained immunity may explain why targeting elevated glucose is ineffective in reducing macrovascular risk in diabetes and suggests new targets for disease prevention and therapy.

Inducibility, but not stability, of atrial fibrillation is increased by NOX2 overexpression in mice.

AIMS: Gp91-containing NADPH oxidases (NOX2) are a significant source of myocardial superoxide production. An increase in NOX2 activity accompanies atrial fibrillation (AF) induction and electrical remodelling in animal models and predicts incident AF in humans; however, a direct causal role for NOX2 in AF has not been demonstrated. Accordingly, we investigated whether myocardial NOX2 overexpression in mice (NOX2-Tg) is sufficient to generate a favourable substrate for AF and further assessed the effects of atorvastatin, an inhibitor of NOX2, on atrial superoxide production and AF susceptibility. METHODS AND RESULTS: NOX2-Tg mice showed a 2- to 2.5-fold higher atrial protein content of NOX2 compared with wild-type (WT) controls, which was associated with a significant (twofold) increase in NADPH-stimulated superoxide production (2-hydroxyethidium by HPLC) in left and right atrial tissue homogenates (P = 0.004 and P = 0.019, respectively). AF susceptibility assessed in vivo by transoesophageal atrial burst stimulation was modestly increased in NOX2-Tg compared with WT (probability of AF induction: 88% vs. 69%, respectively; P = 0.037), in the absence of significant alterations in AF duration, surface ECG parameters, and LV mass or function. Mechanistic studies did not support a role for NOX2 in promoting electrical or structural remodelling, as high-resolution optical mapping of atrial tissues showed no differences in action potential duration and conduction velocity between genotypes. In addition, we did not observe any genotype difference in markers of fibrosis and inflammation, including atrial collagen content and Col1a1, Il-1ß, Il-6, and Mcp-1 mRNA. Similarly, NOX2 overexpression did not have consistent effects on RyR2 Ca2+ leak nor did it affect PKA or CaMKII-mediated RyR2 phosphorylation. Finally, treatment with atorvastatin significantly inhibited atrial superoxide production in NOX2-Tg but had no effect on AF induction in either genotype. CONCLUSION: Together, these data indicate that while atrial NOX2 overexpression may contribute to atrial arrhythmogenesis, NOX2-derived superoxide production does not affect the electrical and structural properties of the atrial myocardium.

BH4 Increases nNOS Activity and Preserves Left Ventricular Function in Diabetes.

RATIONALE: In diabetic patients, heart failure with predominant left ventricular (LV) diastolic dysfunction is a common complication for which there is no effective treatment. Oxidation of the NOS (nitric oxide synthase) cofactor tetrahydrobiopterin (BH4) and dysfunctional NOS activity have been implicated in the pathogenesis of the diabetic vascular and cardiomyopathic phenotype. OBJECTIVE: Using mice models and human myocardial samples, we evaluated whether and by which mechanism increasing myocardial BH4 availability prevented or reversed LV dysfunction induced by diabetes. METHODS AND RESULTS: In contrast to the vascular endothelium, BH4 levels, superoxide production, and NOS activity (by liquid chromatography) did not differ in the LV myocardium of diabetic mice or in atrial tissue from diabetic patients. Nevertheless, the impairment in both cardiomyocyte relaxation and [Ca2+]i (intracellular calcium) decay and in vivo LV function (echocardiography and tissue Doppler) that developed in wild-type mice 12 weeks post-diabetes induction (streptozotocin, 42-45 mg/kg) was prevented in mGCH1-Tg (mice with elevated myocardial BH4 content secondary to trangenic overexpression of GTP-cyclohydrolase 1) and reversed in wild-type mice receiving oral BH4 supplementation from the 12th to the 18th week after diabetes induction. The protective effect of BH4 was abolished by CRISPR/Cas9-mediated knockout of nNOS (the neuronal NOS isoform) in mGCH1-Tg. In HEK (human embryonic kidney) cells, S-nitrosoglutathione led to a PKG (protein kinase G)-dependent increase in plasmalemmal density of the insulin-independent glucose transporter GLUT-1 (glucose transporter-1). In cardiomyocytes, mGCH1 overexpression induced a NO/sGC (soluble guanylate cyclase)/PKG-dependent increase in glucose uptake via GLUT-1, which was instrumental in preserving mitochondrial creatine kinase activity, oxygen consumption rate, LV energetics (by 31phosphorous magnetic resonance spectroscopy), and myocardial function. CONCLUSIONS: We uncovered a novel mechanism whereby myocardial BH4 prevents and reverses LV diastolic and systolic dysfunction associated with diabetes via an nNOS-mediated increase in insulin-independent myocardial glucose uptake and utilization. These findings highlight the potential of GCH1/BH4-based therapeutics in human diabetic cardiomyopathy. Graphic Abstract: A graphic abstract is available for this article.

Identification of patients with atrial fibrillation: a big data exploratory analysis of the UK Biobank.

OBJECTIVE: Atrial fibrillation (AF) is the most common cardiac arrhythmia, with an estimated prevalence of around 1.6% in the adult population. The analysis of the electrocardiogram (ECG) data acquired in the UK Biobank represents an opportunity to screen for AF in a large sub-population in the UK. The main objective of this paper is to assess ten machine-learning methods for automated detection of subjects with AF in the UK Biobank dataset. APPROACH: Six classical machine-learning methods based on support vector machines are proposed and compared with state-of-the-art techniques (including a deep-learning algorithm), and finally a combination of a classical machine-learning and deep learning approaches. Evaluation is carried out on a subset of the UK Biobank dataset, manually annotated by human experts. MAIN RESULTS: The combined classical machine-learning and deep learning method achieved an F1 score of 84.8% on the test subset, and a Cohen's kappa coefficient of 0.83, which is similar to the inter-observer agreement of two human experts. SIGNIFICANCE: The level of performance indicates that the automated detection of AF in patients whose data have been stored in a large database, such as the UK Biobank, is possible. Such automated identification of AF patients would enable further investigations aimed at identifying the different phenotypes associated with AF.

Endothelium-derived extracellular vesicles promote splenic monocyte mobilization in myocardial infarction.

Transcriptionally activated monocytes are recruited to the heart after acute myocardial infarction (AMI). After AMI in mice and humans, the number of extracellular vesicles (EVs) increased acutely. In humans, EV number correlated closely with the extent of myocardial injury. We hypothesized that EVs mediate splenic monocyte mobilization and program transcription following AMI. Some plasma EVs bear endothelial cell (EC) integrins, and both proinflammatory stimulation of ECs and AMI significantly increased VCAM-1-positive EV release. Injected EC-EVs localized to the spleen and interacted with, and mobilized, splenic monocytes in otherwise naive, healthy animals. Analysis of human plasma EV-associated miRNA showed 12 markedly enriched miRNAs after AMI; functional enrichment analyses identified 1,869 putative mRNA targets, which regulate relevant cellular functions (e.g., proliferation and cell movement). Furthermore, gene ontology termed positive chemotaxis as the most enriched pathway for the miRNA-mRNA targets. Among the identified EV miRNAs, EC-associated miRNA-126-3p and -5p were highly regulated after AMI. miRNA-126-3p and -5p regulate cell adhesion- and chemotaxis-associated genes, including the negative regulator of cell motility, plexin-B2. EC-EV exposure significantly downregulated plexin-B2 mRNA in monocytes and upregulated motility integrin ITGB2. These findings identify EVs as a possible novel signaling pathway by linking ischemic myocardium with monocyte mobilization and transcriptional activation following AMI.

Compromised redox homeostasis, altered nitroso-redox balance, and therapeutic possibilities in atrial fibrillation.

Although the initiation, development, and maintenance of atrial fibrillation (AF) have been linked to alterations in myocyte redox state, the field lacks a complete understanding of the impact these changes may have on cellular signalling, atrial electrophysiology, and disease progression. Recent studies demonstrate spatiotemporal changes in reactive oxygen species production shortly after the induction of AF in animal models with an uncoupling of nitric oxide synthase activity ensuing in the presence of long-standing persistent AF, ultimately leading to a major shift in nitroso-redox balance. However, it remains unclear which radical or non-radical species are primarily involved in the underlying mechanisms of AF or which proteins are targeted for redox modification. In most instances, only free radical oxygen species have been assessed; yet evidence from the redox signalling field suggests that non-radical species are more likely to regulate cellular processes. A wider appreciation for the distinction of these species and how both species may be involved in the development and maintenance of AF could impact treatment strategies. In this review, we summarize how redox second-messenger systems are regulated and discuss the recent evidence for alterations in redox regulation in the atrial myocardium in the presence of AF, while identifying some critical missing links. We also examine studies looking at antioxidants for the prevention and treatment of AF and propose alternative redox targets that may serve as superior therapeutic options for the treatment of AF.