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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.

This record contains raw data related to the article "Human cardiosphere-derived stromal cells exposed to SARS-CoV-2 evolve into hyper-inflammatory/pro-fibrotic phenotype and produce infective viral particles depending on the levels of ACE2 receptor expression" Abstract Aims: Patients with severe respiratory syndrome caused by SARS-CoV-2 undergo cardiac complications due to hyper-inflammatory conditions. Although the presence of the virus has been detected in the myocardium of infected patients, and infection of induced pluripotent cell-derived cardiomyocytes has been demonstrated, the reported expression of Angiotensin-Converting Enzyme-2 (ACE2) in cardiac stromal cells suggests that SARS-CoV-2 may determine cardiac injury by sustaining productive infection and increasing inflammation. Methods and results: We analysed expression of ACE2 receptor in primary human cardiac stromal cells derived from cardiospheres, using proteomics and transcriptomics before exposing them to SARS-CoV-2 in vitro. Using conventional and high sensitivity PCR methods, we measured virus release in the cellular supernatants and monitored the intracellular viral bioprocessing. We performed high-resolution imaging to show the sites of intracellular viral production and demonstrated the presence of viral particles in the cells with electron microscopy. We finally used RT-qPCR assays to detect genes linked to innate immunity and fibrotic pathways coherently regulated in cells after exposure to the virus. Conclusions: Our findings indicate that cardiac stromal cells are susceptible to SARS-CoV-2 infection and produce variable viral yields depending on the extent of cellular ACE2 receptor expression. Interestingly, these cells also evolved towards hyper-inflammatory/pro-fibrotic phenotypes independently of ACE2 levels. Thus, SARS-CoV-2 infection of myocardial stromal cells could be involved in cardiac injury and explain the high number of complications observed in severe cases of COVID-19., This work has been financed by a Grant from Regione Lombardia (POR FESR 2014-2020-LINEA 2A COVID-grant no. 1850333) granted to M.Pe. and A.A. M.Pe. and A.A. are supported by Institutional grants (Ricerca Corrente, 5 per 1000) issued by the Italian Ministry of Health. A.A. was supported also by COVID-2020-12371817 and COVID-2020-12371675 programme grants from the Italian Ministry of Health.

Aims Calcific aortic valve stenosis (CAVS) is the most frequent manifestation of aortic valve disease and the third leading cause of cardiovascular disease in the Western countries associated with significant morbidity and mortality. An active biological progression involving inflammation and oxidation leading to valve endothelial damage is considered a hallmark of the early stages of valve degeneration. However, tricuspid (TAV) and bicuspid (BAV) aortic valve deterioration are considered to differ only by shear stress. We hypothesized that endothelial cells (EC) derived from BAV and TAV patients have different miRNA expression patterns and thus distinct pathways could lead to endothelial damage in BAV than TAV patients. Methods and results We isolated ECs from patients with bicuspid or tricuspid aortic valve, which underwent surgery due to CAVS. MiRNA expression profile by PCR revealed eight upregulated miRNAs between BAV and TAV ECs. Functional analysis identified that BAV ECs presented altered cellular response to oxidative stress and DNA damage stimulus via p53 and alteration in the intrinsic apoptotic signaling pathway. GPX3 and SRXN1 mRNA were express at lower levels in BAV compared to TAV ECs, leading to an increment of DNA double-strand breaks. BAV ECs had a sustained apoptosis activation when compared to TAV ECs. This difference was exacerbated by oxidative stress stimulus leading to a reduced survival rate but completely reverted by miR-328-3p inhibition. Conclusion The present data showed molecular differences in oxidative stress susceptibility, DNA damage magnitude, and apoptosis induction between ECs derived from BAV and TAV patients.

This record contains raw data (powerpoint with embedded quantitative data) related to the article "Digital PCR for high sensitivity viral detection in false-negative SARS-CoV-2 patients". Abstract Patients requiring diagnostic testing for coronavirus disease 2019 (COVID-19) are routinely assessed by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) amplification of Sars-CoV-2 virus RNA extracted from oro/nasopharyngeal swabs. Despite the good specificity of the assays certified for SARS-CoV-2 molecular detection, and a theoretical sensitivity of few viral gene copies per reaction, a relatively high rate of false negatives continues to be reported. This is an important challenge in the management of patients on hospital admission and for correct monitoring of the infectivity after the acute phase. In the present report, we show that the use of digital PCR, a high sensitivity method to detect low amplicon numbers, allowed us to correctly detecting infection in swab material in a significant number of false negatives. We show that the implementation of digital PCR methods in the diagnostic assessment of COVID-19 could resolve, at least in part, this timely issue., Te present research was in part funded from Institutional resources (Ricerca 5 per mille, Italian Ministery of Health) and in part from a grant issued to MP by Regione Lombardia (POR FESR 2014���2020-LINEA 2A COVID-grant no. 1850333).

BackgroundCardiovascular complication in patients affected by novel Coronavirus respiratory disease (COVID-19) are increasingly recognized. However, although a cardiac tropism of SARS-CoV-2 for inflammatory cells in autopsy heart samples of COVID-19 patients has been reported, the presence of the virus in cardiomyocytes has not been documented yet.MethodsWe investigated for SARS-CoV-2 presence in heart tissue autopsies of 6 consecutive COVID-19 patients deceased for respiratory failure showing no signs of cardiac involvement and with no history of heart disease. Cardiac autopsy samples were analysed by digital PCR, Western blot, immunohistochemistry, immunofluorescence, RNAScope, and transmission electron microscopy assays.ResultsThe presence of SARS-CoV-2 into cardiomyocytes was invariably detected. A variable pattern of cardiomyocytes injury was observed, spanning from the absence of cell death and subcellular alterations hallmarks to the intracellular oedema and sarcomere ruptures. In addition, we found active viral transcription in cardiomyocytes, by detecting both sense and antisense SARS-CoV-2 spike RNA.ConclusionsIn this analysis of autopsy cases, the presence of SARS-CoV-2 into cardiomyocytes, determining variable patterns of intracellular involvement, has been documented. All these findings suggest the need of a cardiologic surveillance even in survived COVID-19 patients not displaying a cardiac phenotype, in order to monitor potential long-term cardiac sequelae.

Patients with severe respiratory syndrome caused by SARS-CoV-2 undergo cardiac complications due to hyper-inflammatory conditions. Although the presence of the virus has been detected in the myocardium of infected patients, and infection of cardiac cells may involve ACE2 receptor, the underlying molecular/cellular mechanisms are still uncharacterized. We analyzed expression of ACE2 receptor in primary human cardiac stromal cells using proteomic and transcriptomic methods before exposing them to SARS-CoV-2 in vitro. Using conventional and high sensitivity PCR methods, we measured virus production in the cellular supernatants and monitored the intracellular viral bioprocessing. We performed high-resolution imaging to show the sites of intracellular viral production. We finally used Q-RT-PCR assays to detect genes linked to innate immunity and fibrotic pathways coherently regulated in cells exposed to virus. Our findings indicate that human cardiac stromal cells have a susceptibility to SARS-CoV-2 infection and produce variable viral yields depending on the extent of cellular ACE2 receptor expression. Interestingly, these cells also evolved toward hyper-inflammatory/pro-fibrotic phenotypes independently of ACE2 levels, suggesting a dual cardiac damage mechanism that could account for the elevated numbers of cardiac complications in severe COVID-19 cases.

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.

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.

High levels of proprotein convertase subtilisin/kexin 9 (PCSK9) (>305 ng/ml) have been recently associated, along with insulin resistance and lipoprotein-associated phospholipase A2 activity, with aortic bioprosthesis calcification and able to predict hemodynamic valve deterioration [(1)][1].

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.

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 2019 Coronavirus disease (COVID-19) outbreak had detrimental effects on essential medical services such as organ and tissue donation. Lombardy, one of the most active Italian regions in organ/tissue procurement, has been strongly affected by the COVID-19 pandemic. To date, data concerning the risk of SARS-CoV-2 transmission after tissue transplantation are controversial. Here, we aimed to evaluate the presence/absence of SARS-CoV-2 in different cardiac tissues eligible for transplantation obtained from Lombard donors. We used cardiovascular tissues from eight donors potentially suitable for pulmonary valve transplantation. All donor subjects involved in the study returned negative results for the SARS-CoV-2 RNA molecular tests (quantitative real-time reverse-transcription PCR, qRT-PCR, and chip-based digital PCR) in nasopharyngeal swabs (NPS) or bronchoalveolar lavage (BAL). None of the eight donors included in this study revealed the presence of the SARS-CoV-2 viral genome. However, evaluation of the protein content of pulmonary vein wall (PVW) tissue revealed variable levels of SARS-CoV-2 nucleoprotein signal in all donors. Our study demonstrated for the first time, to the best of our knowledge, that viral nucleoprotein but not viral RNA was present in the examined tissue bank specimens, suggesting the need for caution and in-depth investigations on implantable tissue specimens collected during the COVID-19 pandemic period.

On-pump cardiac surgery may trigger inflammation and accelerate platelet cyclooxygenase-1 renewal, thereby modifying low-dose aspirin pharmacodynamics. Thirty-seven patients on standard aspirin 100 mg once-daily were studied before surgery and randomized within 36 hours postsurgery to 100 mg once-daily, 100 mg twice-daily, or 200 mg once-daily for 90 days. On day 7 postsurgery, immature and mature platelets, platelet mass, thrombopoietin, glycocalicin, leukocytes, C-reactive protein, and interleukin-6 significantly increased. Interleukin-6 significantly correlated with immature platelets. At day 7, patients randomized to 100 mg once-daily showed a significant increase in serum thromboxane (TX)B2 within the 24-hour dosing interval and urinary TXA2 metabolite (TXM) excretion. Aspirin 100 mg twice-daily lowered serum TXB2 and prevented postsurgery TXM increase (P < 0.01), without affecting prostacyclin metabolite excretion. After cardiac surgery, shortening the dosing interval, but not doubling the once-daily dose, rescues the impaired antiplatelet effect of low-dose aspirin and prevents platelet activation associated with acute inflammation and enhanced platelet turnover.

The global impact of the spectrum of valve diseases is a crucial, fast-growing, and underrecognized health problem. The most prevalent valve diseases, requiring surgical intervention, are represented by calcific and degenerative processes occurring in heart valves, in particular, aortic and mitral valve. Due to the increasing elderly population, these pathologies will gain weight in the global health burden. The two most common valve diseases are aortic valve stenosis (AVS) and mitral valve regurgitation (MR). AVS is the most commonly encountered valve disease nowadays and affects almost 5% of elderly population. In particular, AVS poses a great challenge due to the multiple comorbidities and frailty of this patient subset. MR is also a common valve pathology and has an estimated prevalence of 3% in the general population, affecting more than 176 million people worldwide. This review will focus on pathophysiological changes in both these valve diseases, starting from the description of the anatomical aspects of normal valve, highlighting all the main cellular and molecular features involved in the pathological progression and cardiac consequences. This review also evaluates the main approaches in clinical management of these valve diseases, taking into account of the main published clinical guidelines. © 2017 American Physiological Society. Compr Physiol 7:799-818, 2017.

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.

Background: Studies on the association between aortic valve sclerosis (AVSc) and diabetes in patients with coronary artery disease (CAD) are lacking.In this retrospective observational study, we evaluated the association of AVSc with diabetes and to assess the risk of all-cause mortality in patients referred to isolated coronary artery bypass surgery (CABG). Methods: Echocardiograms obtained at the baseline from 2’327 patients undergoing CABG between 2006 and 2016 in a single center were assessed. The follow-up was carried out through a regional registry. Hazard ratios (HR) were adjusted for clinical factors [i.e., sex, age, previous myocardial infarction, ejection fraction (EF), dyslipidemia, and kidney function]. Results: Overall, AVSc was present in 1’116 (39.7%) and diabetes in 840 patients (29.9%). Diabetic patients showed a significant higher AVSc prevalence compared to nondiabetic patients (44 vs. 38%, respectively; p=0.005).In 5 years follow-up, 479 deaths (20.6%) for any cause were registered. Independent predictors of all-cause mortality were age (p Conclusions: Our findings indicate that: 1) AVSc is associated with diabetes; 2) AVSc and diabetes are independently associated with all-cause mortality in patients undergoing CABG; and 3) patients with both clinical conditions have the highest mortality rate. These data improve risk stratification in patients undergoing surgical myocardial revascularization. Disclosure V.A. Myasoedova: None. S. Genovese: Advisory Panel; Self; Novartis Pharmaceuticals Corporation, Sanofi. Consultant; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Hikma Pharmaceuticals. Speaker's Bureau; Self; Eli Lilly and Company, Merck Sharp & Dohme Corp., Mundipharma, Novo Nordisk A/S, Takeda Pharmaceutical Company Limited. L. Cavallotti: None. A. Bonomi: None. P. Gripari: None. J. Campodonico: None. V. Valerio: None. M. Rondinelli: Consultant; Self; Sanofi. Speaker's Bureau; Self; Eli Lilly and Company, Merck Sharp & Dohme Corp., Takeda Pharmaceutical Company Limited. P. Songia: None. M. Pepi: None. F. Alamanni: None. P. Poggio: None. Funding Fondazione Gigi e Pupa Ferrari ONLUS

Aortic valve sclerosis (AVSc) is characterized by non-uniform thickening of the leaflets without hemodynamic changes. Endothelial dysfunction, also caused by dysregulation of glutathione homeostasis expressed as ratio between its reduced (GSH) and its oxidised form (GSSG), could represent one of the pathogenic triggers of AVSc. We prospectively enrolled 58 patients with overt atherosclerosis and requiring coronary artery bypass grafting (CABG). The incidence of AVSc in the studied population was 50%. The two groups (No-AVSc and AVSc) had similar clinical characteristics. Pre-operatively, AVSc group showed significantly lower GSH/GSSG ratio than No-AVSc group (p = 0.02). Asymmetric dimethylarginine (ADMA) concentration was significantly higher in AVSc patients compared to No-AVSc patients (p &lt, 0.0001). Explanted sclerotic aortic valves presented a significantly increased protein glutathionylation (Pr-SSG) than No-AVSc ones (p = 0.01). In vitro, inhibition of glutathione reductase caused &beta, actin glutathionylation, activation of histone 2AX, upregulation of &alpha, 2 smooth muscle actin (ACTA2), downregulation of platelet and endothelial cell adhesion molecule 1 (PECAM1) and cadherin 5 (CDH5). In this study, we showed for the first time that the dysregulation of glutathione homeostasis is associated with AVSc. We found that Pr-SSG is increased in AVSc leaflets and it could lead to EndMT via DNA damage. Further studies are warranted to elucidate the causal role of Pr-SSG in aortic valve degeneration.

Circulating microRNAs (miRNAs) are considered as reliable candidates for biomarker discovery. RNA-Sequencing has become the most suitable technique to accurately quantify the miRNAome. However, RNA-Sequencing relies on several technical passages before reaching the final-end. HTG EdgeSeq technology, thanks to the abrogation of RNA extraction step, allows productivity enhancement by reducing the number of hands-on steps, the time for sample preparation and, therefore, the costs. We found a strong correlation between qPCR and dPCR with HTG (Pearson’s coefficient of 0.93 and 0.94, respectively). In conclusion, we showed that HTG EdgeSeq, performed on human plasma specimens without RNA extraction, is reliable, allows the simultaneous screening of more than 2,000 miRNAs, and thus, it could be applied to biomarker discovery in large cohorts.

Aims Mitral valve prolapse (MVP) has a prevalence of 3% in the general population, affecting > 176 million people worldwide. Despite this, little is known about the molecular and cellular mechanisms involved in the progression of MVP and surgical intervention is the only available option. In this study we investigated the role of osteoprotegerin (OPG) during endothelial to mesenchymal transition (EndMT) in MVP. Methods and results VECs and VICs were isolated from posterior mitral valve leaflets of patients undergoing mitral valve repair (n = 25). Plasma was collected from 57 subjects (29 controls and 28 MVP patients). Overexpression of OPG during EndMT followed by autocrine effects characterised by reactive oxygen species increment and accelerated migration was documented. In addition, OPG increased VIC proliferation. Finally, OPG plasma levels were significantly higher in MVP patients compared to control subjects and the area under the ROC curve was 0.92. Conclusion EndMT has been recognised as a possible pathological mechanism for MVP. For the first time, we report the involvement of OPG in cellular and molecular changes in MVP isolated cells. In addition, we detected elevated circulating OPG levels in MVP patients when compared to controls, which supports the hypothesis that OPG is involved in MVP development and progression.

In this study, we review current knowledge regarding molecular pathways activation and their possible mechanisms in the perioperative period of coronary artery bypass surgery (CABG). We also highlight the role of off-pump CABG as a possible way to better understand these biological changes.We show that, after both on-pump and off-pump CABG, there is a marked and protracted activation of several molecular pathways indicating increased inflammatory status, haemostasis activation, as well as increased oxidative stress and unfavourable endothelial milieu. These changes persist for days and even weeks after surgery. Interestingly, a relatively limited number of these pathways show a more pronounced activation in case of cardiopulmonary bypass use, and these markers are mainly associated with oxidative stress activation; on the contrary, the vast majority of the pathways has a similar course both in on and off-pump procedures. Surgical stress accounts for more protracted and marked molecular pathway perturbations overall, being the effect of cardiopulmonary, if any, limited to the very early hours after surgery. The near future of the translational research in coronary bypass surgery is to develop therapeutic strategies aimed at reducing this response, that is largely unrelated to cardiopulmonary bypass use, in order to reduce perioperative complications and to speed up patients' recovery.

Calcific aortic valve disease (CAVD) is the most common valvular disorder in the elderly, with the incidence of 3% in general population of Western countries. The initial phase of CAVD is characterized by leaflet thickening and possible spotty calcification (i.e. aortic valve sclerosis (AVSc)), while advanced stages have leaflets structure degeneration (i.e. aortic valve stenosis (AS)). The pathological cellular and molecular mechanisms, involved in CAVD, are extracellular matrix degradation, aberrant matrix deposition, fibrosis, mineralization, inflammation, lipid accumulation, and neo-angiogenesis. CAVD clinical risk shares considerable overlap with those of atherosclerosis and they include hypertension, smoking habits, and hyperlipidemia. Unfortunately, surgical aortic valve replacement and transcatheter aortic valve implantation are the only available treatments when the disease become severe and symptoms occur. Indeed, no approved pharmacological approach is available for CAVD patients. In this review, we describe the current literature evidence on possible future therapeutic targets for this debilitating and fatal disease such as PCSK9, P2Y2 receptor, cadherin 11, and DDP-4.

Background: Aortic valve calcification (AVC) is one of the most common form of heart valve disease and affects 3% of the population. No pharmacological treatments have been identified yet; however, proprotein convertase subtisilin kexin type 9 (PCSK9) inhibitors have been proposed to reduce AVC progression. We aimed to evaluate the role of PCSK9 in the aortic valve calcification. Methods: We use PCSK9 -/- and PCSK9 +/+ (WT) mice littermates (n=3-4 per group, 12 month-old female, normal diet). When aortic valves were excised, total calcium content was measured and valve interstitial cells (VIC) were isolated. Calcification assays were performed on aortic VICs, left untreated or treated with β-glycerophosphate and ascorbic acid (βGAA) for seven days on plastic dishes. MinION RNA-Seq was used to evaluate the differential expression patterns in the two groups. Results: Total calcium content of aortic valve from PCSK9 -/- mice was significantly lower than WT (1.5±0.7 vs 10.1±0.5 ng of calcium/μg of total protein, respectively; p=0.0002). Calcium quantification revealed that PCSK9 -/- VICs (untreated) were able to calcify at a less extent than WT (4.4±0.6 vs 8.0±0.5 ng/μg, respectively; p=0.0001). The βGAA treatment induced calcification in both cell types; however, a significant (p=0.01) lower calcification rate was noted in PCSK9 -/- VICs (41.9±3.6 ng/μg) compared to WT (55.9±3.5 ng/μg). RNA-Seq of VICs from WT and PCSK9 -/- mice revealed that 363 genes were differentially expressed more than two-fold (adjusted p value-/- VICs upregulated genes coordinate p38 cascade and cytoskeleton modifications, while downregulated ones control apoptotic signalling pathway, cell proliferation and adhesion, and oxidative stress. Conclusion: These preliminary results show for the first time that PCSK9 could play a direct role in AVC and thus, therapies against PCSK9 may be beneficial to patients affected by calcific aortic valve disease.

Background: Calcific aortic valve disease (CAVD) is one of the most common form of heart valve disease and affects 3% of the population. The initial phase, called aortic valve sclerosis (AVSc), is linked to inflammation and oxidative stress with direct consequences on endothelial cells. Thus, we aimed to evaluate the effects of protein glutathionylation caused by oxidative damage in endothelial cells. Methods: We analysed blood samples from 58 subjects, 29 with AVSc and 29 without (no-AVSc). The levels of oxidized (GSSG) and reduced (GSH) forms of glutathione were measured by a liquid chromatography-tandem mass spectrometry method and their ratio (GSH/GSSG) represent a recognized an oxidative stress status index. 2-AAPA, a glutathione reductase inhibitor, was used to increase GSSG levels. Human umbilical vein endothelial cells (HUVEC) were employed to create a model of protein glutathionylation. Valve endothelial cells (VEC) were isolated from pig and human aortic valves and were used to test and to validate this model. Results: Subjects with AVSc had a circulating GSH/GSSG ratio lower than no-AVSc (17.0±1.7 vs 24.4±2.6, respectively; p=0.02), indicating an increased systemic oxidative stress status in AVSc. Protein glutathionylation was increased more than 9-fold in human specimens from AVSc compared to no-AVSc subjects (p=0.03). In HUVECs, 2-AAPA significantly reduced the GSH/GSSG ratio (p=0.003) and induced an increment in protein glutathionylation of 1.91±0.17 fold compared to untreated cells (p=0.006). In pig VECs, 2-AAPA caused a 2-fold increment in reactive oxygen species production and an activation of the histone H2AX. In human VECs, protein glutathionylation caused an upregulation of smooth muscle actin (+0.7±0.16; p=0.004) and a downregulation of vascular endothelial cadherin (-0.4±0.02; p Conclusion: These preliminary results show for the first time that protein glutathionylation could play a direct role in CAVD development via oxidative DNA damage. Restoring the proper balance of GSH and GSSG may be beneficial to reduce the endothelial damage occurring in patients affected by CAVD.

Aims The association between aortic valve sclerosis (AVSc) and cardiovascular (CV) events is not consistent among different studies. We have performed a meta-analysis evaluating the association between AVSc and fatal and/or non-fatal CV and cerebrovascular events. Methods and results A systematic search was performed in the electronic databases (PubMed, Web of Science, Scopus, EMBASE). Studies evaluating coronary artery disease (CAD), stroke and CV mortality in AVSc patients and controls were included. Differences among cases and controls were expressed as Odds Ratio (OR) with pertinent 95% Confidence Intervals (CI). Thirty-one studies on 10,537 AVSc patients and 25,005 controls were included in the final analysis. The absolute risk of CAD was 45.8% (95% CI: 32.9–59.3) in AVSc patients and 29.4% (95% CI: 21.8–38.5) in controls with an OR of 2.02 (95% CI: 1.67–2.44) and an attributable risk of 35.8%. Moreover, stroke was reported in 11.8% (95% CI: 4.4–27.7) of AVSc patients and 7.9% (95% CI: 2.5–22.7) of controls (OR: 1.41, 95% CI: 1.16–1.71) with an attributable risk of 33.0%. CV mortality was 6.2% (95% CI: 2.7–13.5) in AVSc patients and 2.0% (95% CI: 0.5–7.9) in controls (OR: 2.70, 95% CI: 1.45–5.01), with an attributable risk of 67.7%. Results were confirmed when pooling together ORs for CAD, stroke and CV mortality obtained by means of multivariate analysis. Conclusions AVSc is associated with CAD, stroke and CV mortality. Taken together, these data suggest that patients with AVSc may benefit from a stricter CV risk monitoring and that AVSc screening may be included in the frame of CV risk stratification protocols.

Background Aortic valve stenosis (AVS) is a multifactorial-progressive pathological process. In the past decades, many studies have focus their attention on circulating biomarkers able to identify AVS and/or to predict its progression. One of the many biomarkers studied is the fetuin A. The aim of the present meta-analysis was to evaluate the correlation between fetuin A levels and end-stage AVS. Methods and results A systematic search was performed in three electronic databases (PubMed, Web of Science and Scopus), looking for studies that compared control subjects with AVS patients and that have measured fetuin A circulating levels in both groups. The main outcome was to evaluate the difference in circulating fetuin A concentration in the two groups. Seven studies, enrolling 2283 AVS patients and 1549 controls, were included. Differences between control subjects and AVS patients were expressed as standardized mean difference (SMD) with pertinent 95% confidence intervals (95%CI) and standard deviation (SD), analysing the data using a random effect model. We found significantly lower circulating levels of fetuin A in AVS patients compared to healthy subjects (SMD: −0.96μg/mL, 95% CI: −1.62, −0.30; p =0.004). In addition, meta-regression analyses showed that several cardiovascular risk factors were significantly associated with circulating levels of fetuin A between patients affected by AVS and healthy controls. Conclusion In conclusion, our meta-analysis shows that AVS patients have significant lower circulating levels of fetuin A compared to control subjects. However, dedicated studies with large and matched cohorts are needed to validate these findings, evaluating if there is a real link or just a mere association.

Background. Mitral valve prolapse (MVP) is the most common cause of severe mitral regurgitation. Besides echocardiography, up to now there are no reliable biomarkers available for the identification of this pathology. We aim to generate a predictive model, based on circulating biomarkers, able to identify MVP patients with the highest accuracy.Methods. We analysed 43 patients who underwent mitral valve repair due to MVP and compared to 29 matched controls. We assessed the oxidative stress status measuring the oxidized and the reduced form of glutathione by liquid chromatography-tandem mass spectrometry method. Osteoprotegerin (OPG) plasma levels were measured by an enzyme-linked immunosorbent assay. The combination of these biochemical variables was used to implement several logistic regression models.Results. Oxidative stress levels and OPG concentrations were significantly higher in patients compared to control subjects (0.116±0.007versus0.053±0.013and1748±100.2versus1109±45.3 pg/mL, respectively;p<0.0001). The best regression model was able to correctly classify 62 samples out of 72 with accuracy in terms of area under the curve of 0.92.Conclusions. To the best of our knowledge, this is the first study to show a strong association between OPG and oxidative stress status in patients affected by MVP with severe regurgitation.

While the importance of cyclooxygenase (COX) in platelet function has been amply elucidated, the identification of the role of 12-lipoxygenase (12-LOX) and of its stable metabolite, 12-hydroxyeicosat r etraenoic acid (12-HETE), has not been clarified as yet. Many studies have analysed the implications of 12-LOX products in different pathological disorders but the information obtained from these works is controversial. Several analytical methods have been developed over the years to simultaneously detect eicosanoids, and specifically 12-HETE, in different biological matrices, essentially enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), high performance liquid chromatography (HPLC) and mass spectrometry coupled with both gas and liquid chromatography methods (GC– and LC–MS). This review is aimed at summarizing the up to now known physiological and clinical features of 12-HETE together with the analytical methods used for its determination, focusing on the critical issues regarding its measurement.

Mitral valve prolapse (MVP) is the most common cause of severe mitral regurgitation. It has been reported that MVP patients&mdash, candidates for mitral valve repair (MVRep)&mdash, showed an alteration in the antioxidant defense systems as well as in the L-arginine metabolic pathway. In this study, we investigate if oxidative stress and endothelial dysfunction are an MVP consequence or driving factors. Forty-five patients undergoing MVRep were evaluated before and 6 months post surgery and compared to 29 controls. Oxidized (GSSG) and reduced (GSH) forms of glutathione, and L-arginine metabolic pathway were analyzed using liquid chromatography-tandem mass spectrometry methods while osteoprotegerin (OPG) through the ELISA kit and circulating endothelial microparticles (EMP) by flow cytometry. Six-month post surgery, in MVP patients, the GSSG/GSH ratio decreased while symmetric and asymmetric dimethylarginines levels remained comparable to the baseline. Conversely, OPG levels significantly increased when compared to their baseline. Finally, pre-MVRep EMP levels were significantly higher in patients than in controls and did not change post surgery. Overall, these results highlight that MVRep completely restores the increased oxidative stress levels, as evidenced in MVP patients. Conversely, no amelioration of endothelial dysfunction was evidenced after surgery. Thus, therapies aimed to restore a proper endothelial function before and after surgical repair could benefit MVP patients.

Background Nowadays, aortic valve sclerosis (AVSc) might be considered an atherosclerosis-like process due to significant association with age, male gender and some major features of metabolic syndrome. Nonalcoholic fatty liver disease (NAFLD) has been recognized as a clinical expression of the metabolic syndrome and as a predictor of cardiovascular events. We aim, with this meta-analysis, to evaluate the correlation between NAFLD and AVSc; this finding might suggest new insights and interactions among NAFLD, AVSc and the atherosclerotic process. Methods and results A detailed search was conducted according to Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines to identify all studies providing data about the association between AVSc and NAFLD. 3 studies enrolling a total of 1172 patients with NAFLD and 1467 controls without NAFLD were included in the meta-analysis. The prevalence of AVSc was 41.3% (95% CI: 32.0%, 51.4%) in NAFLD patients and 24.6% (18.4%, 32.0%) in subjects without NAFLD with a corresponding OR of 2.28 (95% CI: 1.21, 4.28, p=0.01, I 2 : 77.6%, p=0.011). A meta-regression analysis showed that age, prevalence of male gender, hypertension, body mass index, and dyslipidemia were directly and significantly associated with the difference in the prevalence of AVSc between patients with NAFLD and those without. Conclusions In conclusion, our meta-analysis shows a significant association between NAFLD and AVSc. However, further evidence is needed to validate these findings and find out if there is a real link or just a mere association.

Background Literature studies suggested a lower prevalence of coronary artery disease ( CAD ) in bicuspid aortic valve ( BAV ) than in tricuspid aortic valve ( TAV ) patients. However, this finding has been challenged. We performed a meta‐analysis to assess whether aortic valve morphology has a different association with CAD , concomitant coronary artery bypass grafting ( CABG ), and postoperative mortality. Methods and Results Detailed search was conducted according to the PRISMA (Preferred Reporting Items for Systematic reviews and Meta‐Analyses) guideline to identify all patients with BAV or TAV and presence of CAD , concomitant myocardial surgical revascularization, and the postoperative mortality. Thirty‐one studies on 3017 BAV and 4586 TAV patients undergoing aortic valve surgery were included. BAV patients showed a lower prevalence of CAD (odds ratio [ OR ]: 0.33; 95% CI : 0.17, 0.65), concomitant CABG ( OR , 0.45; 95% CI : 0.35, 0.59), and postoperative mortality ( OR , 0.62; 95% CI : 0.40, 0.97) than TAV . However, BAV subjects were significantly younger than TAV (mean difference: −7.29; 95% CI : −11.17, −3.41) were more frequently males ( OR , 1.61; 95% CI : 1.33, 1.94) and exhibited a lower prevalence of hypertension ( OR , 0.58; 95% CI : 0.39, 0.87) and diabetes ( OR , 0.71; 95% CI : 0.54, 0.93). Interestingly, a metaregression analysis showed that younger age and lower prevalence of diabetes were associated with lower prevalence of CAD ( Z value: −3.03; P =0.002 and Z value: −3.10; P =0.002, respectively) and CABG ( Z value: −2.69; P =0.007 and Z value: −3.36; P =0.001, respectively) documented in BAV patients. Conclusions Analysis of raw data suggested an association of aortic valve morphology with prevalence of CAD , concomitant CABG , and postoperative mortality. Interestingly, the differences in age and diabetes have a profound impact on prevalence of CAD between BAV and TAV . In conclusion, our meta‐analysis suggests that the presence of CAD is independent of aortic valve morphology.