Whole-Blood Transcriptome Unveils Altered Immune Response in Acute Myocardial Infarction Patients With Aortic Valve Sclerosis.

BACKGROUND: Aortic valve sclerosis (AVSc) presents similar pathogenetic mechanisms to coronary artery disease and is associated with short- and long-term mortality in patients with coronary artery disease. Evidence of AVSc-specific pathophysiological traits in acute myocardial infarction (AMI) is currently lacking. Thus, we aimed to identify a blood-based transcriptional signature that could differentiate AVSc from no-AVSc patients during AMI. METHODS: Whole-blood transcriptome of AVSc (n=44) and no-AVSc (n=66) patients with AMI was assessed by RNA sequencing on hospital admission. Feature selection, differential expression, and enrichment analyses were performed to identify gene expression patterns discriminating AVSc from no-AVSc and infer functional associations. Multivariable Cox regression analysis was used to estimate the hazard ratios of cardiovascular events in AVSc versus no-AVSc patients. RESULTS: This cross-sectional study identified a panel of 100 informative genes capable of distinguishing AVSc from no-AVSc patients with 94% accuracy. Further analysis revealed significant mean differences in 143 genes, of which 30 genes withstood correction for age and previous AMI or coronary interventions. Functional inference unveiled a significant association between AVSc and key biological processes, including acute inflammatory responses, type I IFN (interferon) response, platelet activation, and hemostasis. Notably, patients with AMI with AVSc exhibited a significantly higher incidence of adverse cardiovascular events during a 10-year follow-up period, with a full adjusted hazard ratio of 2.4 (95% CI, 1.3-4.5). CONCLUSIONS: Our findings shed light on the molecular mechanisms underlying AVSc and provide potential prognostic insights for patients with AMI with AVSc. During AMI, patients with AVSc showed increased type I IFN (interferon) response and earlier adverse cardiovascular outcomes. Novel pharmacological therapies aiming at limiting type I IFN response during or immediately after AMI might improve poor cardiovascular outcomes of patients with AMI with AVSc.

Efficacy of cardiometabolic drugs in reduction of epicardial adipose tissue: a systematic review and meta-analysis.

BACKGROUND: Epicardial adipose tissue (EAT) plays an important role in cardiometabolic risk. EAT is a modifiable risk factor and could be a potential therapeutic target for drugs that already show cardiovascular benefits. The aim of this study is to evaluate the effect of cardiometabolic drugs on EAT reduction. METHODS: A detailed search related to the effect on EAT reduction due to cardiometabolic drugs, such as glucagon-like peptide-1 receptor agonist (GLP-1 RA), sodium-glucose cotransporter-2 inhibitors (SGLT2-i), and statins was conducted according to PRISMA guidelines. Eighteen studies enrolling 1064 patients were included in the qualitative and quantitative analyses. RESULTS: All three analyzed drug classes, in particular GLP-1 RA, show a significant effect on EAT reduction (GLP-1 RA standardize mean difference (SMD) = - 1.005; p < 0.001; SGLT2-i SMD = - 0.552; p < 0.001, and statin SMD = - 0.195; p < 0.001). The sensitivity analysis showed that cardiometabolic drugs strongly benefit EAT thickness reduction, measured by ultrasound (overall SMD of - 0.663; 95%CI - 0.79, - 0.52; p < 0.001). Meta-regression analysis revealed younger age and higher BMI as significant effect modifiers of the association between cardiometabolic drugs and EAT reduction for both composite effect and effect on EAT thickness, (age Z: 3.99; p < 0.001 and Z: 1.97; p = 0.001, respectively; BMI Z: - 4.40; p < 0.001 and Z: - 2.85; p = 0.004, respectively). CONCLUSIONS: Cardiometabolic drugs show a significant beneficial effect on EAT reduction. GLP-1 RA was more effective than SGLT2-i, while statins had a rather mild effect. We believe that the most effective treatment with these drugs should target younger patients with high BMI.

Dysregulation of Iron Metabolism-Linked Genes at Myocardial Tissue and Cell Levels in Dilated Cardiomyopathy.

In heart failure, the biological and clinical connection between abnormal iron homeostasis, myocardial function, and prognosis is known; however, the expression profiles of iron-linked genes both at myocardial tissue and single-cell level are not well defined. Through publicly available bulk and single-nucleus RNA sequencing (RNA-seq) datasets of left ventricle samples from adult non-failed (NF) and dilated cardiomyopathy (DCM) subjects, we aim to evaluate the altered iron metabolism in a diseased condition, at the whole cardiac tissue and single-cell level. From the bulk RNA-seq data, we found 223 iron-linked genes expressed at the myocardial tissue level and 44 differentially expressed between DCM and NF subjects. At the single-cell level, at least 18 iron-linked expressed genes were significantly regulated in DCM when compared to NF subjects. Specifically, the iron metabolism in DCM cardiomyocytes is altered at several levels, including: (1) imbalance of Fe3+ internalization (SCARA5 down-regulation) and reduction of internal conversion from Fe3+ to Fe2+ (STEAP3 down-regulation), (2) increase of iron consumption to produce hemoglobin (HBA1/2 up-regulation), (3) higher heme synthesis and externalization (ALAS2 and ABCG2 up-regulation), (4) lower cleavage of heme to Fe2+, biliverdin and carbon monoxide (HMOX2 down-regulation), and (5) positive regulation of hepcidin (BMP6 up-regulation).

Putative Circulating MicroRNAs Are Able to Identify Patients with Mitral Valve Prolapse and Severe Regurgitation.

Mitral valve prolapse (MVP) associated with severe mitral regurgitation is a debilitating disease with no pharmacological therapies available. MicroRNAs (miRNA) represent an emerging class of circulating biomarkers that have never been evaluated in MVP human plasma. Our aim was to identify a possible miRNA signature that is able to discriminate MVP patients from healthy subjects (CTRL) and to shed light on the putative altered molecular pathways in MVP. We evaluated a plasma miRNA profile using Human MicroRNA Card A followed by real-time PCR validations. In addition, to assess the discriminative power of selected miRNAs, we implemented a machine learning analysis. MiRNA profiling and validations revealed that miR-140-3p, 150-5p, 210-3p, 451a, and 487a-3p were significantly upregulated in MVP, while miR-223-3p, 323a-3p, 340-5p, and 361-5p were significantly downregulated in MVP compared to CTRL (p ≤ 0.01). Functional analysis identified several biological processes possible linked to MVP. In addition, machine learning analysis correctly classified MVP patients from CTRL with high accuracy (0.93) and an area under the receiving operator characteristic curve (AUC) of 0.97. To the best of our knowledge, this is the first study performed on human plasma, showing a strong association between miRNAs and MVP. Thus, a circulating molecular signature could be used as a first-line, fast, and cheap screening tool for MVP identification.

Evaluation of Oxford Nanopore MinION RNA-Seq Performance for Human Primary Cells.

Transcript sequencing is a crucial tool for gaining a deep understanding of biological processes in diagnostic and clinical medicine. Given their potential to study novel complex eukaryotic transcriptomes, long-read sequencing technologies are able to overcome some limitations of short-read RNA-Seq approaches. Oxford Nanopore Technologies (ONT) offers the ability to generate long-read sequencing data in real time via portable protein nanopore USB devices. This work aimed to provide the user with the number of reads that should be sequenced, through the ONT MinION platform, to reach the desired accuracy level for a human cell RNA study. We sequenced three cDNA libraries prepared from poly-adenosine RNA of human primary cardiac fibroblasts. Since the runs were comparable, they were combined in a total dataset of 48 million reads. Synthetic datasets with different sizes were generated starting from the total and analyzed in terms of the number of identified genes and their expression levels. As expected, an improved sensitivity was obtained, increasing the sequencing depth, particularly for the non-coding genes. The reliability of expression levels was assayed by (i) comparison with PCR quantifications of selected genes and (ii) by the implementation of a user-friendly multiplexing method in a single run.

The Role of Glycemic Variability in Cardiovascular Disorders.

Diabetes mellitus (DM) is one of the most common and costly disorders that affect humans around the world. Recently, clinicians and scientists have focused their studies on the effects of glycemic variability (GV), which is especially associated with cardiovascular diseases. In healthy subjects, glycemia is a very stable parameter, while in poorly controlled DM patients, it oscillates greatly throughout the day and between days. Clinically, GV could be measured by different parameters, but there are no guidelines on standardized assessment. Nonetheless, DM patients with high GV experience worse cardiovascular disease outcomes. In vitro and in vivo studies showed that high GV causes several detrimental effects, such as increased oxidative stress, inflammation, and apoptosis linked to endothelial dysfunction. However, the evidence that treating GV is beneficial is still scanty. Clinical trials aiming to improve the diagnostic and prognostic accuracy of GV measurements correlated with cardiovascular outcomes are needed. The present review aims to evaluate the clinical link between high GV and cardiovascular diseases, taking into account the underlined biological mechanisms. A clear view of this challenge may be useful to standardize the clinical evaluation and to better identify treatments and strategies to counteract this DM aspect.

Cyclophilin A inhibition as potential treatment of human aortic valve calcification.

Aortic valve stenosis (AS) is a pathological condition that affects about 3% of the population, representing the most common valve disease. The main clinical feature of AS is represented by the impaired leaflet motility, due to calcification, which leads to the left ventricular outflow tract obstruction during systole. The formation and accumulation of calcium nodules are driven by valve interstitial cells (VICs). Unfortunately, to date, the in vitro and in vivo studies were not sufficient to fully recapitulate all the pathological pathways involved in AS development, as well as to define a specific and effective pharmacological treatment for AS patients. Cyclophilin A (CyPA), the most important immunophilin and endogenous ligand of cyclosporine A (CsA), is strongly involved in several detrimental cardiovascular processes, such as calcification. To date, there are no data on the CyPA role in VIC-mediated calcification process of AS. Here, we aimed to identify the role of CyPA in AS by studying VIC calcification, in vitro. In this study, we found that (i) CyPA is up-regulated in stenotic valves of AS patients, (ii) pro-calcifying medium promotes CyPA secretion by VICs, (iii) in vitro treatment of VICs with exogenous CyPA strongly stimulates calcium deposition, and (iv) exogenous CyPA inhibition mediated by CsA analogue MM284 abolished in vitro calcium potential. Thus, CyPA represents a biological target that may act as a novel candidate in the detrimental AS development and its inhibition may provide a novel pharmacological approach for AS treatment.

Integration of enzymatic data in Bacillus subtilis genome-scale metabolic model improves phenotype predictions and enables in silico design of poly-γ-glutamic acid production strains.

BACKGROUND: Genome-scale metabolic models (GEMs) allow predicting metabolic phenotypes from limited data on uptake and secretion fluxes by defining the space of all the feasible solutions and excluding physio-chemically and biologically unfeasible behaviors. The integration of additional biological information in genome-scale models, e.g., transcriptomic or proteomic profiles, has the potential to improve phenotype prediction accuracy. This is particularly important for metabolic engineering applications where more accurate model predictions can translate to more reliable model-based strain design. RESULTS: Here we present a GEM with Enzymatic Constraints using Kinetic and Omics data (GECKO) model of Bacillus subtilis, which uses publicly available proteomic data and enzyme kinetic parameters for central carbon (CC) metabolic reactions to constrain the flux solution space. This model allows more accurate prediction of the flux distribution and growth rate of wild-type and single-gene/operon deletion strains compared to a standard genome-scale metabolic model. The flux prediction error decreased by 43% and 36% for wild-type and mutants respectively. The model additionally increased the number of correctly predicted essential genes in CC pathways by 2.5-fold and significantly decreased flux variability in more than 80% of the reactions with variable flux. Finally, the model was used to find new gene deletion targets to optimize the flux toward the biosynthesis of poly-γ-glutamic acid (γ-PGA) polymer in engineered B. subtilis. We implemented the single-reaction deletion targets identified by the model experimentally and showed that the new strains have a twofold higher γ-PGA concentration and production rate compared to the ancestral strain. CONCLUSIONS: This work confirms that integration of enzyme constraints is a powerful tool to improve existing genome-scale models, and demonstrates the successful use of enzyme-constrained models in B. subtilis metabolic engineering. We expect that the new model can be used to guide future metabolic engineering efforts in the important industrial production host B. subtilis.

A BioBrick™-Compatible Vector for Allelic Replacement Using the XylE Gene as Selection Marker.

BACKGROUND: Circular plasmid-mediated homologous recombination is commonly used for marker-less allelic replacement, exploiting the endogenous recombination machinery of the host. Common limitations of existing methods include high false positive rates due to mutations in counter-selection genes, and limited applicability to specific strains or growth media. Finally, solutions compatible with physical standards, such as the BioBrick™, are not currently available, although they proved to be successful in the design of other replicative or integrative plasmids. FINDINGS: We illustrate pBBknock, a novel BioBrick™-compatible vector for allelic replacement in Escherichia coli. It includes a temperature-sensitive replication origin and enables marker-less genome engineering via two homologous recombination events. Chloramphenicol resistance allows positive selection of clones after the first event, whereas a colorimetric assay based on the xylE gene provides a simple way to screen clones in which the second recombination event occurs. Here we successfully use pBBknock to delete the lactate dehydrogenase gene in E. coli W, a popular host used in metabolic engineering. CONCLUSIONS: Compared with other plasmid-based solutions, pBBknock has a broader application range, not being limited to specific strains or media. We expect that pBBknock will represent a versatile solution both for practitioners, also among the iGEM competition teams, and for research laboratories that use BioBrick™-based assembly procedures.

Non-stenotic fibro-calcific aortic valve as a predictor of myocardial infarction recurrence.

BACKGROUND: Patients with acute myocardial infarction (AMI) are at increased risk of recurrent cardiovascular events. Non-stenotic aortic valve fibro-calcific remodeling (AVSc), reflecting systemic damage, may serve as a new marker of risk. OBJECTIVES: To stratify subgroups of AMI patients with specific probabilities of recurrent AMI and to evaluate the importance of AVSc in this setting. METHODS: Consecutive AMI patients (n = 2530) were admitted at Centro Cardiologico Monzino (2010-2019) and followed up for 5 years. Patients were divided into study (n = 1070) and test (n = 966) cohorts. Topological data analysis (TDA) was used to stratify patient subgroups, while Kaplan-Meier and Cox regressions analyses were used to evaluate the significance of baseline characteristics. RESULTS: TDA identified 11 subgroups of AMI patients with specific baseline characteristics. Two subgroups showed the highest rate of reinfarction after 5 years from the indexed AMI with a combined hazard ratio (HR) of 3.8 (95%CI: 2.7-5.4) compared to the other subgroups. This was confirmed in the test cohort (HR = 3.1; 95%CI: 2.2-4.3). These two subgroups were mostly men, with hypertension and dyslipidemia, who exhibit higher prevalence of AVSc, higher levels of high-sensitive c-reactive protein and creatinine. In the year-by-year analysis, AVSc, adjusted for all confounders, showed an independent association with the increased risk of reinfarction (odds ratio of ∼2 at all time-points), in both the study and the test cohorts (all p < 0.01). CONCLUSIONS: AVSc is a crucial variable for identifying AMI patients at high risk of recurrent AMI and its presence should be considered when assessing the management of AMI patients. The inclusion of AVSc in risk stratification models may improve the accuracy of predicting the likelihood of recurrent AMI, leading to more personalized treatment decisions.

We wanted to understand the factors that make some acute myocardial infarction (AMI) patients more likely to experience recurrent infarction after leaving the hospital. Specifically, we asked whether a heart valve condition called non-stenotic aortic valve fibro-calcific remodeling (AVSc) could be a crucial factor. Our study used advanced data analysis techniques, including topological data analysis (TDA), to explore this question. We unveil that AVSc is indeed a significant predictor of recurrent infarction in AMI patients. Our findings suggest that the presence of aortic valve remodeling should be taken into account when assessing the risk of recurrent AMI and managing these patients.

Anti-Inflammation and Anti-Oxidation: The Key to Unlocking the Cardiovascular Potential of SGLT2 Inhibitors and GLP1 Receptor Agonists.

Type 2 diabetes mellitus (T2DM) is a prevalent and complex metabolic disorder associated with various complications, including cardiovascular diseases. Sodium-glucose co-transporter 2 inhibitors (SGLT2i) and glucagon-like peptide 1 receptor agonists (GLP1-RA) have emerged as novel therapeutic agents for T2DM, primarily aiming to reduce blood glucose levels. However, recent investigations have unveiled their multifaceted effects, extending beyond their glucose-lowering effect. SGLT2i operate by inhibiting the SGLT2 receptor in the kidneys, facilitating the excretion of glucose through urine, leading to reduced blood glucose levels, while GLP1-RA mimic the action of the GLP1 hormone, stimulating glucose-dependent insulin secretion from pancreatic islets. Both SGLT2i and GLP1-RA have shown remarkable benefits in reducing major cardiovascular events in patients with and without T2DM. This comprehensive review explores the expanding horizons of SGLT2i and GLP1-RA in improving cardiovascular health. It delves into the latest research, highlighting the effects of these drugs on heart physiology and metabolism. By elucidating their diverse mechanisms of action and emerging evidence, this review aims to recapitulate the potential of SGLT2i and GLP1-RA as therapeutic options for cardiovascular health beyond their traditional role in managing T2DM.

Multimodality imaging and transcriptomics to phenotype mitral valve dystrophy in a unique knock-in Filamin-A rat model.

AIMS: Degenerative mitral valve dystrophy (MVD) leading to mitral valve prolapse is the most frequent form of MV disease, and there is currently no pharmacological treatment available. The limited understanding of the pathophysiological mechanisms leading to MVD limits our ability to identify therapeutic targets. This study aimed to reveal the main pathophysiological pathways involved in MVD via the multimodality imaging and transcriptomic analysis of the new and unique knock-in (KI) rat model for the FilaminA-P637Q (FlnA-P637Q) mutation associated-MVD. METHODS AND RESULTS: Wild-type (WT) and KI rats were evaluated morphologically, functionally, and histologically between 3-week-old and 3-to-6-month-old based on Doppler echocardiography, 3D micro-computed tomography (microCT), and standard histology. RNA-sequencing and Assay for Transposase-Accessible Chromatin (ATAC-seq) were performed on 3-week-old WT and KI mitral valves and valvular cells, respectively, to highlight the main signalling pathways associated with MVD. Echocardiographic exploration confirmed MV elongation (2.0 ± 0.1 mm vs. 1.8 ± 0.1, P = 0.001), as well as MV thickening and prolapse in KI animals compared to WT at 3 weeks. 3D MV volume quantified by microCT was significantly increased in KI animals (+58% vs. WT, P = 0.02). Histological analyses revealed a myxomatous remodelling in KI MV characterized by proteoglycans accumulation. A persistent phenotype was observed in adult KI rats. Signalling pathways related to extracellular matrix homeostasis, response to molecular stress, epithelial cell migration, endothelial to mesenchymal transition, chemotaxis and immune cell migration, were identified based on RNA-seq analysis. ATAC-seq analysis points to the critical role of transforming growth factor-ß and inflammation in the disease. CONCLUSION: The KI FlnA-P637Q rat model mimics human myxomatous MVD, offering a unique opportunity to decipher pathophysiological mechanisms related to this disease. Extracellular matrix organization, epithelial cell migration, response to mechanical stress, and a central contribution of immune cells are highlighted as the main signalling pathways leading to myxomatous MVD. Our findings pave the road to decipher underlying molecular mechanisms and the specific role of distinct cell populations in this context.

Purinergic Receptor P2Y2 Stimulation Averts Aortic Valve Interstitial Cell Calcification and Myofibroblastic Activation.

Rationale-Calcific aortic valve stenosis (CAVS) is a pathological condition of the aortic valve with a prevalence of 3% in the general population. It is characterized by massive rearrangement of the extracellular matrix, mostly due to the accumulation of fibro-calcific deposits driven by valve interstitial cells (VIC), and no pharmacological treatment is currently available. The aim of this study was to evaluate the effects of P2Y2 receptor (P2RY2) activation on fibro-calcific remodeling of CAVS. Methods-We employed human primary VICs isolated from CAVS leaflets treated with 2-thiouridine-5'-triphosphate (2ThioUTP, 10 µM), an agonist of P2RY2. The calcification was induced by inorganic phosphate (2 mM) and ascorbic acid (50 µg/mL) for 7 or 14 days, while the 2ThioUTP was administered starting from the seventh day. 2ThioUTP was chronically administered for 5 days to evaluate myofibroblastic activation. Results-P2RY2 activation, under continuous or interrupted pro-calcific stimuli, led to a significant inhibition of VIC calcification potential (p < 0.01). Moreover, 2ThioUTP treatment was able to significantly reduce pro-fibrotic gene expression (p < 0.05), as well as that of protein α-smooth muscle actin (p = 0.004). Conclusions-Our data suggest that P2RY2 activation should be further investigated as a pharmacological target for the prevention of CAVS progression, acting on both calcification and myofibroblastic activation.

Red Flags, Prognostic Impact, and Management of Patients With Cardiac Amyloidosis and Aortic Valve Stenosis: A Systematic Review and Meta-Analysis.

Background: Cardiac amyloidosis (CA) has been recently recognized as a condition frequently associated with aortic stenosis (AS). The aim of this study was to evaluate: the main characteristics of patients with AS with and without CA, the impact of CA on patients with AS mortality, and the effect of different treatment strategies on outcomes of patients with AS with concomitant CA. Materials and Methods: A detailed search related to CA in patients with AS and outcomes was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Seventeen studies enrolling 1,988 subjects (1,658 AS alone and 330 AS with CA) were included in the qualitative and quantitative analysis of main patients with AS characteristics with and without CA, difference in mortality, and treatment strategy. Results: The prevalence of CA resulted in a mean of 15.4% and it was even higher in patients with AS over 80 years old (18.2%). Patients with the dual diagnosis were more often males, had lower body mass index (BMI), were more prone to have low flow, low gradient with reduced left ventricular ejection fraction AS phenotype, had higher E/A and E/e', and greater interventricular septum hypertrophy. Lower Sokolow-Lyon index, higher QRS duration, higher prevalence of right bundle branch block, higher levels of N-terminal pro-brain natriuretic peptide, and high-sensitivity troponin T were significantly associated with CA in patients with AS. Higher overall mortality in the 178 patients with AS + CA in comparison to 1,220 patients with AS alone was observed [odds ratio (OR) 2.25, p = 0.004]. Meta-regression analysis showed that younger age and diabetes were associated with overall mortality in patients with CS with CA (Z-value -3.0, p = 0.003 and Z-value 2.5, p = 0.013, respectively). Finally, patients who underwent surgical aortic valve replacement (SAVR) or transcatheter aortic valve implantation (TAVI) had a similar overall mortality risk, but lower than medication-treated only patients. Conclusion: Results from our meta-analysis suggest that several specific clinical, electrocardiographic, and echocardiographic features can be considered "red flags" of CA in patients with AS. CA negatively affects the outcome of patients with AS. Patients with concomitant CA and AS benefit from SAVR or TAVI.

Enduring Reactive Oxygen Species Emission Causes Aberrant Protein S-Glutathionylation Transitioning Human Aortic Valve Cells from a Sclerotic to a Stenotic Phenotype.

Aims: During calcific aortic valve stenosis (CAVS) progression, oxidative stress and endothelial dysfunction mark the initial pathogenic steps with a parallel dysregulation of the antioxidant systems. Here, we tested whether oxidation-induced protein S-glutathionylation (P-SSG) accounts for a phenotypic switch in human aortic valvular tissue, eventually leading to calcium deposition. Next, we tested whether countering this reactive oxygen species (ROS) surge would prevent these perturbations. Results: We employed state-of-the-art technologies, such as electron paramagnetic resonance (EPR), liquid chromatography-tandem mass spectrometry, imaging flow-cytometry, and live-cell imaging on human excised aortic valves and primary valve endothelial cells (VECs). We observed that a net rise in EPR-detected ROS emission marked the transition from fibrotic to calcific in human CAVS specimens, coupled to a progressive increment in P-SSG deposition. In human VECs (hVECs), treatment with 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid triggered highly oxidizing conditions prompting P-SSG accumulation, damaging mitochondria, and inducing endothelial nitric oxide synthase uncoupling. All the events conjured up in morphing these cells from their native endothelial phenotype into a damaged calcification-inducing one. As proof of principle, the use of the antioxidant N-acetyl-L-cysteine prevented these alterations. Innovation: Borne as a compensatory system to face excessive oxidative burden, with time, P-SSG contributes to the morphing of hVECs from their innate phenotype into a damaged one, paving the way to calcium deposition. Conclusion: Our data suggest that, in the human aortic valve, unremitted ROS emission along with a P-SSG build-up occurs and accounts, at least in part, for the morphological/functional changes leading to CAVS. Antioxid. Redox Signal. 37, 1051-1071.

Extraction-Free Absolute Quantification of Circulating miRNAs by Chip-Based Digital PCR.

Circulating microRNAs (miRNA) have been proposed as specific biomarkers for several diseases. Quantitative Real-Time PCR (RT-qPCR) is the gold standard technique currently used to evaluate miRNAs expression from different sources. In the last few years, digital PCR (dPCR) emerged as a complementary and accurate detection method. When dealing with gene expression, the first and most delicate step is nucleic-acid isolation. However, all currently available protocols for RNA extraction suffer from the variable loss of RNA species due to the chemicals and number of steps involved, from sample lysis to nucleic acid elution. Here, we evaluated a new process for the detection of circulating miRNAs, consisting of sample lysis followed by direct evaluation by dPCR in plasma from healthy donors and in the cardiovascular setting. Our results showed that dPCR is able to detect, with high accuracy, low-copy-number as well as highly expressed miRNAs in human plasma samples without the need for RNA extraction. Moreover, we assessed a known myocardial infarction-related miR-133a in acute myocardial infarct patients vs. healthy subjects. In conclusion, our results show the suitability of the extraction-free quantification of circulating miRNAs as disease markers by direct dPCR.

Sex-Specific Cell Types and Molecular Pathways Indicate Fibro-Calcific Aortic Valve Stenosis.

Background: Aortic stenosis (AS) is the most common valve disorder characterized by fibro-calcific remodeling of leaflets. Recent evidence indicated that there is a sex-related difference in AS development and progression. Fibrotic remodeling is peculiar in women's aortic valves, while men's leaflets are more calcified. Our study aimed to assess aortic valve fibrosis (AVF) in a severe AS cohort using non-invasive diagnostic tools and determine whether sex-specific pathological pathways and cell types are associated with severe AS. Materials and Methods: We have included 28 men and 28 women matched for age with severe AS who underwent echocardiography and cardiac contrast-enhanced computed tomography (CT) before intervention. The calcium and fibrosis volumes were assessed and quantified using the ImageJ thresholding method, indexed calcium and fibrosis volume were calculated by dividing the volume by the aortic annular area. For a deeper understanding of molecular mechanisms characterizing AS disorder, differentially expressed genes and functional inferences between women and men's aortic valves were carried out on a publicly available microarray-based gene expression dataset (GSE102249). Cell types enrichment analysis in stenotic aortic valve tissues was used to reconstruct the sex-specific cellular composition of stenotic aortic valves. Results: In agreement with the literature, our CT quantifications showed that women had significantly lower aortic valve calcium content compared to men, while fibrotic tissue composition was significantly higher in women than men. The expression profiles of human stenotic aortic valves confirm sex-dependent processes. Pro-fibrotic processes were prevalent in women, while pro-inflammatory ones, linked to the immune response system, were enhanced in men. Cell-type enrichment analysis showed that mesenchymal cells were over-represented in AS valves of women, whereas signatures for monocytes, macrophages, T and B cells were enriched men ones. Conclusions: Our data provide the basis that the fibro-calcific process of the aortic valve is sex-specific, both at gene expression and cell type level. The quantification of aortic valve fibrosis by CT could make it possible to perform population-based studies and non-invasive assessment of novel therapies to reduce or halt sex-related calcific aortic valve stenosis (CAVS) progression, acting in an optimal window of opportunity early in the course of the disease.

Aortic Valve Sclerosis as an Important Predictor of Long-Term Mortality in Patients With Carotid Atheromatous Plaque Requiring Carotid Endarterectomy.

Background: A strong association between aortic valve sclerosis (AVSc), the earliest manifestation of calcific aortic valve disease, and atherosclerosis exists. The aim of the study was to evaluate the predictive capabilities of AVSc on long-term all-cause mortality, in patients requiring carotid endarterectomy (CEA). Methods and Results: 806 consecutive CEA patients were enrolled. Preoperative echocardiography was used to assess AVSc. Computed tomography angiography was applied for plaque characterization. Kaplan-Meier curves, Cox linear regression, and area under the receiving operator characteristic (AUC) curve analyses were used to evaluate the predictive capability of AVSc. Overall, 348 of 541 patients had AVSc (64%). Age, diabetes, and estimated glomerular filtration rate (eGFR) were associated with AVSc. In the 5-year follow-up, AVSc group had a mortality rate of 16.7% while in no-AVSc group was 7.8%. Independent predictors of all-cause mortality were age, sex, eGFR, left ventricular ejection fraction, and AVSc. After adjustments, AVSc was associated with a significant increase in all-cause mortality risk (hazard ratio, HR = 1.9; 95%CI: 1.04-3.54; p = 0.038). We stratify our cohort based on carotid atheromatous plaque-type: soft, calcified, and mixed-fibrotic. In patients with mixed-fibrotic plaques, the mortality rate of AVSc patients was 15.5% compared to 2.4% in no-AVSc patients. In this group, AVSc was associated with an increased long-term all-cause mortality risk with an adjusted HR of 12.8 (95%CI: 1.71-96.35; p = 0.013), and the AUC, combing eGFR and AVSc was 0.77 (p < 0.001). Conclusions: Our findings indicate that AVSc together with eGFR may be used to improve long-term risk stratification of patients undergoing CEA surgery.

Engineering endogenous fermentative routes in ethanologenic Escherichia coli W for bioethanol production from concentrated whey permeate.

Whey permeate (WP) is a lactose-rich waste effluent, generated during cheese manufacturing and further valorization steps, such as protein extraction. The production of ethanol by WP fermentation has been proposed to increase cost-competitiveness of dairy waste processing. In previous work, the Escherichia coli W strain was selected for its efficient growth in dairy waste and it was engineered to convert lactose into ethanol as the main fermentation product from WP and concentrated WP (CWP). To improve its performance, here the lactate dehydrogenase, fumarate reductase and pyruvate formate lyase fermentative routes were disrupted, obtaining new deletion strains. In test tubes, growth and fermentation profiles obtained in standard laboratory media and CWP showed large differences, and were affected by oxygen, medium and ethanologenic gene expression level. Among the tested strains, the one with triple deletion was superior in both high-oxygen and low-oxygen test tube fermentations, in terms of ethanol titer, rate and yield. The improved performance was due to a lower inhibition by medium acidification rather than an improved ethanol flux. The parent and triple deletion strains showed similar performance indexes in pH-controlled bioreactor experiments. However, the deletion strain showed lower base consumption and residual waste, in terms of both dry matter and chemical oxygen demand after distillation. It thus represents a step towards sustainable dairy wastewater valorization for bioenergy production by decreasing process operation costs.

Relationship Between Plasma Osteopontin and Arginine Pathway Metabolites in Patients With Overt Coronary Artery Disease.

INTRODUCTION: Osteopontin (OPN) is involved in ectopic calcification. Its circulating form is upregulated in coronary artery disease (CAD) patients. Circulating OPN levels positively correlate with oxidative stress, one of the major triggers of endothelial dysfunction. Endothelial dysfunction is, in turn, associated with reduced nitric oxide (NO) bioavailability due to the impaired arginine pathway. The aim of this study was to better understand the correlations between OPN, oxidative stress markers, and the arginine pathway metabolites. METHODS AND RESULTS: ELISA and mass spectrometry techniques have been used to evaluate circulating OPN and arginine pathway/oxidative stress metabolites, respectively, in twenty-five control subjects and thirty-three patients with overt atherosclerosis. OPN positively correlates with 2,3-dinor-8isoPGF2a levels (p = 0.02), ornithine (p = 0.01), ADMA (p = 0.001), SDMA (p = 0.03), and citrulline (p = 0.008) levels only in CAD patients. In addition, citrulline positively correlated with ADMA (p = 0.02) levels, possibly as result of other sources of citrulline biosynthetic pathways. CONCLUSION: The association between OPN and impaired arginine/NO pathway could play a role in the inhibition of endothelial NO synthase (eNOS) and/or in the arginase activation in the context of CAD patients. However, further studies are needed to verify the cause-effect relationship between OPN, oxidative stress, and arginine/NO pathway dysregulation.