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.

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.

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.

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.

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.

Sex-specific differences in age-related aortic valve calcium load: A systematic review and meta-analysis.

Aging of the aortic valve, characterized by leaflet thickening and loss of extensibility, leads to progressive changes in valve function. These age-related mechanisms have not been evaluated yet in sex-specific calcific aortic valve stenosis (CAVS) onset and progression. Recent studies reported the association between high aortic valve calcification (AVC) load and male gender in patients with CAVS while women present faster progression than men. To evaluate these age- and sex-specific differences, we performed a systematic review and meta-analysis with meta-regression. A systematic search related to AVC measured by computed tomography and gender-specific differences was conducted according to PRISMA guidelines. Seven studies, enrolling 1859 men and 1055 women, were included in the quantitative synthesis. We found a significant difference between men and women both in AVC load and density. AVC load mean difference (MD), between men and women, was 1131 ± 243 AU (p < 0.0001; I2: 96.5 %, p < 0.001), while AVC density MD was 159 ± 20 AU/cm2 (p < 0.0001) without heterogeneity among the studies (I2: 23.5, p = 0.3). Meta-regression analyses showed that AVC load MD positively correlated with age and other cardiovascular risk factors such as diabetes, hypertension, and coronary artery disease presence. Our meta-analysis shows a significant association of incremental AVC load with male gender, regardless of the individual anatomical characteristics and the cardiovascular risk factors. Further studies are needed: i) to clarify if there are different sex-related pathophysiological processes driving the development and the progression of age-related CAVS, and ii) to determine if a sex-specific therapeutic strategy should be applied for CAVS treatment and/or prevention.

Melanocortin-1 Receptor Positively Regulates Human Artery Endothelial Cell Migration.

BACKGROUND/AIMS: Melanocortin receptors (MCRs) belong to a hormonal signalling pathway with multiple homeostatic and protective actions. Microvascular and umbilical vein endothelial cells (ECs) express components of the melanocortin system, including the type 1 receptor (MC1R), playing a role in modulating inflammation and vascular tone. Since ECs exhibit a remarkable heterogeneity, we investigated whether human artery ECs express any functional MCR and whether its activation affects cell migration. METHODS: We used reverse transcription real-time PCR to examine the expression of melanocortin system components in primary human artery ECs. We assessed MC1R protein expression and activity by western blot, immunohistochemistry, cAMP production, and intracellular Ca²âº mobilization assays. We performed gap closure and scratch tests to examine cell migration after stimulation with alpha-melanocyte-stimulating hormone (α-MSH), the receptor highest-affinity natural ligand. We assessed differential time-dependent transcriptional changes in migrating cells by microarray analysis. RESULTS: We showed that human aortic ECs (HAoECs) express a functionally active MC1R. Unlike microvascular ECs, arterial cells did not express the α-MSH precursor proopiomelanocortin, nor produced the hormone. MC1R engagement with a single pulse of α-MSH accelerated HAoEC migration both in the directional migration assay and in the scratch wound healing test. This was associated with an enhancement in Ca²âº signalling and inhibition of cAMP elevation. Time-course genome-wide expression analysis in HAoECs undergoing directional migration allowed identifying dynamic co-regulation of genes involved in extracellular matrix-receptor interaction, vesicle-mediated trafficking, and metal sensing - which have all well-established influences on EC motility -, without affecting the balance between pro- and anticoagulant genes. CONCLUSION: Our work broadens the knowledge on peripherally expressed MC1R. These results indicate that the receptor is constitutively expressed by arterial ECs and provide evidence of a novel homeostatic function for MC1R, whose activation may participate in preventing/healing endothelial dysfunction or denudation in macrovascular arteries.

Does Fluoroscopy Induce DNA Oxidative Damage in Patients Undergoing Catheter Ablation?

As no studies before now have thoroughly investigated the risk associated with the exposure to low-dose ionizing radiations in patients undergoing catheter ablation (CA), we aimed to evaluate the oxidative and DNA damage in 59 CA patients (10 of whom received N-acetylcysteine (NAC) before the procedure). A burst of oxidized/reduced glutathione ratio was observed 3 hours after procedure that was diminished by NAC administration. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) concentrations, index of oxidative DNA damage, showed a peak 24 hours after CA. A positive correlation between 8-OHdG peak and fluoroscopy time and a negative correlation among 8-OHdG decrease (from the peak to 48 hours after CA) and all procedure parameters were found. Furthermore, DNA tail percentages (which reflect the extent and the number of DNA strand breaks) positively correlated with 8-OHdG concentrations. This study evaluates for the first time the kinetic of oxidative damage in patients undergoing CA procedure. Our findings raise the question of whether 8-OHdG can be used as a circulating biomarker of DNA oxidative damage induced by low-dose ionizing radiations in different clinical settings. Antioxid. Redox Signal. 28, 1137-1143.