Fermentation of Vaccinium floribundum Berries with Lactiplantibacillus plantarum Reduces Oxidative Stress in Endothelial Cells and Modulates Macrophages Function

Accumulating evidence suggests that high consumption of natural antioxidants promotes health by reducing oxidative stress and, thus, the risk of developing cardiovascular diseases. Similarly, fermentation of natural compounds with lactic acid bacteria (LAB), such as Lactiplantibacillus plantarum, enhances their beneficial properties as regulators of the immune, digestive, and cardiovascular system. We investigated the effects of fermentation with Lactiplantibacillus plantarum on the antioxidant and immunomodulatory effects of Pushgay berries (Vaccinium floribundum, Ericaceae family) in human umbilical vein endothelial cells (HUVECs) and macrophage cell line RAW264.7. Polyphenol content was assayed by Folin-Ciocalteu and HPLC-MS/MS analysis. The effects of berries solutions on cell viability or proliferation were assessed by WST8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt and Lactate dehydrogenase (LDH) release, Trypan blue exclusion test, and Alamar blue assay. Antioxidant activity was evaluated by a cell-based chemiluminescent probe for the detection of intracellular H2O2 production in HUVECs. Heme oxygenase-1 (HO-1) expression levels were investigated by RT-qPCR. Glutathione reductase (GR), glutathione peroxidase (Gpx), superoxide dismutase (SOD), and catalase (CAT) activities, as markers of intracellular antioxidant defense, were evaluated by spectrophotometric analysis. The immunomodulatory activity was examined in RAW 264.7 by quantification of inducible nitric oxide synthase (iNOS) and Tumor Necrosis Factor-alpha (TNF alpha) by RT-qPCR. Data showed that fermentation of Pushgay berries (i) enhances the content of quercetin aglycone, and (ii) increases their intracellular antioxidant activity, as indicated by the reduction in H2O2-induced cell death and the decrease in H2O2-induced HO-1 gene expression in HUVECs treated for 24 h with fermented berries solution (10 mu g/mL). Moreover, treatment with Pushgay berries for 72 h (10 mu g/mL) promotes cells growth in RAW 264.7, and only fermented Pushgay berries increase the expression of iNOS in the same cell line. Taken together, our results show that LAB fermentation of Pushgay berries enhances their antioxidant and immunomodulatory properties

Transcriptomic profiling of calcified aortic valves in clonal hematopoiesis of indeterminate potential carriers

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by the presence of clones of mutated blood cells without overt blood diseases. In the last few years, it has emerged that CHIP is associated with atherosclerosis and coronary calcification and that it is an independent determinant of cardiovascular mortality. Recently, CHIP has been found to occur frequently in patients with calcific aortic valve disease (CAVD) and it is associated with a poor prognosis after valve replacement. We assessed the frequency of CHIP by DNA sequencing in the blood cells of 168 CAVD patients undergoing surgical aortic valve replacement or transcatheter aortic valve implantation and investigated the effect of CHIP on 12 months survival. To investigate the pathological process of CAVD in CHIP carriers, we compared by RNA-Seq the aortic valve transcriptome of patients with or without CHIP and non-calcific controls. Transcriptomics data were validated by immunohistochemistry on formalin-embedded aortic valve samples. We confirm that CHIP is common in CAVD patients and that its presence is associated with higher mortality following valve replacement. Additionally, we show, for the first time, that CHIP is often accompanied by a broad cellular and humoral immune response in the explanted aortic valve. Our results suggest that an excessive inflammatory response in CHIP patients may be related to the onset and/or progression of CAVD and point to B cells as possible new effectors of CHIP-induced inflammation

A serum proteome signature to predict mortality in severe COVID-19 patients.

Here, we recorded serum proteome profiles of 33 severe COVID-19 patients admitted to respiratory and intensive care units because of respiratory failure. We received, for most patients, blood samples just after admission and at two more later time points. With the aim to predict treatment outcome, we focused on serum proteins different in abundance between the group of survivors and non-survivors. We observed that a small panel of about a dozen proteins were significantly different in abundance between these two groups. The four structurally and functionally related type-3 cystatins AHSG, FETUB, histidine-rich glycoprotein, and KNG1 were all more abundant in the survivors. The family of inter-α-trypsin inhibitors, ITIH1, ITIH2, ITIH3, and ITIH4, were all found to be differentially abundant in between survivors and non-survivors, whereby ITIH1 and ITIH2 were more abundant in the survivor group and ITIH3 and ITIH4 more abundant in the non-survivors. ITIH1/ITIH2 and ITIH3/ITIH4 also showed opposite trends in protein abundance during disease progression. We defined an optimal panel of nine proteins for mortality risk assessment. The prediction power of this mortality risk panel was evaluated against two recent COVID-19 serum proteomics studies on independent cohorts measured in other laboratories in different countries and observed to perform very well in predicting mortality also in these cohorts. This panel may not be unique for COVID-19 as some of the proteins in the panel have previously been annotated as mortality markers in aging and in other diseases caused by different pathogens, including bacteria

Study of factors involved in the calcific aortic valve disease

La stenosi della valvola aortica (AVS) è la terza causa di morte tra le malattie cardiovascolari. Non esistono terapie farmacologiche efficaci per prevenire o rallentare la progressione di questa patologia e l’unico rimedio è la sostituzione della valvola tramite chirurgia. L'infiammazione ha un ruolo fondamentale nella patogenesi di AVS, per cui, la terapia antinfiammatoria è stata proposta come possibile strategia terapeutica. La cicloossigenasi- 2 (COX-2) è un mediatore chiave dell'infiammazione. È noto che il celecoxib, inibitore di COX-2, riduce la calcificazione della valvola aortica. Tuttavia, un recente studio clinico retrospettivo ha riscontrato un’associazione tra l’uso di celecoxib e AVS. È noto che la disregolazione del pathway di Notch nelle cellule interstiziali della valvola aortica (AVICs) e nelle cellule endoteliali della valvola (VEC) è coinvolta nella patogenesi di AVS. Tuttavia, il ruolo preciso dei ligandi e recettori di Notch in AVS non è del tutto chiarito. Dati recenti mostrano che la presenza di mutazioni somatiche a carico di cellule staminali ematopoietiche, condizione nota come ematopoiesi clonale di potenziale indeterminato (CHIP), correla con un aumentato rischio di malattie cardiovascolari ma non è noto se CHIP sia associato o meno ad AVS. Questo studio si propone di: i) studiare il coinvolgimento di COX-2 e dei ligandi di Notch, Jagged1 e Dll4, nella patogenesi di AVS; ii) valutare se CHIP è associato ad un aumento dell'incidenza di AVS ed analizzare le differenze nel trascrittoma delle valvole calcifiche derivanti da pazienti CHIP e non-CHIP e da valvole non calcifiche. Per il primo scopo, è stato estratto l'RNA da valvole aortiche calcifiche e non, e sono stati quantificati mediante qRT-PCR i livelli di Jagged1, Dll4 e di COX-2. È stato inoltre indagato se l'inibizione di COX-2 influenzi la trans-differenziazione delle AVICs verso un fenotipo miofibroblastico. Inoltre, le AVICs derivanti da valvole di pazienti AVS sono state transfettate con vettori lentivirali che overesprimono Dll4, Jagged1 o i loro inibitori e la deposizione di calcio è stata analizzata mediante il saggio di Alizarina Red. Per il secondo scopo è stato estratto il DNA da campioni di sangue di pazienti sottoposti a sostituzione della valvola aortica tramite chirurgia o tramite metodo transcatetere (TAVI) e un gruppo di controllo non affetti da AVS. Il DNA è stato analizzato mediante tecniche di sequenziamento di nuova generazione (NGS). Inoltre, sono state eseguite analisi di RNA-seq da RNA estratto da valvole derivanti da pazienti affetti da mutazioni CHIP per i geni DNMT3A e TET2, valvole non calcifiche o calcifiche ma non CHIP. I risultati ottenuti hanno mostrato che: i) l'inibizione di COX-2 promuove la trans-differenziazione delle AVICs in un fenotipo miofibroblastico e aumenta i livelli di apoptosi indotta da TGF-β, contribuendo alla progressione della calcificazione; ii) i livelli di espressione di Jagged1 e di COX-2 sono più elevati nelle valvole non calcifiche rispetto a quelle AVS. Al contrario i livelli di espressione di Dll4 sono inferiori nelle valvole non calcifiche rispetto a quelle AVS. Inoltre, abbiamo osservato che i livelli di espressione di COX-2 correlano, direttamente o inversamente, rispettivamente con i livelli di Jagged1 o Dll4; iii) le analisi di Alizarina Red hanno suggerito un ruolo pro-calcifico per Jagged1 e anti-calcifico per Dll4; iv) la prevalenza di CHIP in AVS è del 30,4% e i geni più frequentemente mutati sono TET2 e DNMT3A; v) i pazienti CHIP e non-CHIP condividono l’alterata espressione di geni correlati al differenziamento osteo-condrogenico e, inoltre, nei pazienti CHIP è stato evidenziato un aumento del coinvolgimento dell’immunità adattativa. Questi risultati hanno rilevanza traslazionale poiché l'identificazione dei principali attori coinvolti in AVS può aprire la strada allo sviluppo di un approccio biologico terapeutico personalizzato.Calcific aortic valve disease (CAVD) is a degenerative, inflammatory process characterized by a progressive calcification of aortic valve (AV). It is the third leading cause of death among cardiovascular diseases (CVD) for which the only effective therapeutic solution is surgical valve replacement. Inflammation plays a key role in AV pathological remodeling; hence, anti-inflammatory therapy has been proposed as a strategy to prevent CAVD. Cyclooxygenase 2 (COX-2) is a key mediator of the inflammation, and it is the target of widely used anti-inflammatory drugs. The COX-2 inhibitor celecoxib was first shown to reduce calcification of AV. However, a recent retrospective clinical analysis found an association between CAVD and celecoxib use. Several studies have also shown that dysregulation of the Notch pathway in aortic valve interstitial cells (AVICs) and valve endothelial cells (VECs) is involved in the pathogenesis of CAVD. However, the precise mechanisms and the role of Notch components pathway in the calcific valve degeneration remain unknown. In the context of CVD, recent data show that somatic mutations leading to clonal expansion of hematopoietic stem cells, known as clonal hematopoiesis of indeterminate potential (CHIP), correlates with an increased risk of CVD such as atherosclerosis, and coronary-artery calcification. The aims of this study were i) to investigate the role of COX-2 and Notch ligands Delta-like 4 (Dll4) and Jagged1 in CAVD by establishing whether a variation in the expression levels of these molecules in specific cell type in human AVs may be linked to CAVD, ii) to assess whether CHIP is related to an increase in the incidence of CAVD and uncover the molecular mechanism by which these mutations could affect the aortic valve environment. For the first aim we extracted total RNA from surgically explanted AVs from patients without or with CAVD and we isolated human AVICs. We then quantified by qRT-PCR the levels of COX-2 and Notch ligands and we explored how COX-2 inhibition affects AVICs trans-differentiation towards a myofibroblast phenotype. In addition, we transduced AVICs in presence of osteogenic medium with lentivirus bearing Dll4, Jagged1 or their inhibitors, and calcium deposition was analyzed through Alizarine red staining. For the second aim we extracted DNA from blood samples of CAVD patients undergoing aortic valve replacement surgery or aortic transcatheter valve transplantation (TAVI) and from patients without CAVD but with severe aortic insufficiency or aneurysm of the aortic root; DNA was analyzed through new generation sequencing techniques (NGS). Then, we performed RNA-seq analyses from RNA extracted from CHIP and non-CHIP patients. We found that: i) COX-2 inhibition with celecoxib promote AVICs trans-differentiation towards a myofibroblast phenotype and increases the levels of TGF-β-induced apoptosis, leading to progression of aortic valve calcification; ii) Jagged1 and COX-2 expression was higher in non-calcific compared to CAVD samples. Conversely, Dll4 expression was lower in non-calcific vs CAVD samples. Of note, COX-2 expression correlated, directly or inversely, with Jagged1 or Dll4 levels, respectively; iii) Furthermore, Alizarin red staining suggested that inhibition of Jagged1 reduced calcium deposition. On the contrary, inhibition of Dll4 induced calcification; iv) the prevalence of CHIP in our population is 30,4% and the most frequently mutated genes are TET2 and DNMT3A; v) CHIP and non-CHIP patients shared altered expression of several genes, include genes already implicated in CAVD and related to extracellular matrix and osteo-chondrocyte transition and highlight that the involvement of adaptive immune activation is augmented in CHIP carriers and frequently involves humoral responses. These findings could be relevant since the identification of the major players involved in CAVD can pave the way for the development of biologically based tailored therapeutic approach

Detection of Merkel Cell Polyomavirus DNA in Serum Samples of Healthy Blood Donors

Merkel cell polyomavirus (MCPyV) has been detected in 80% of Merkel cell carcinomas (MCC). In the host, the MCPyV reservoir remains elusive. MCPyV DNA sequences were revealed in blood donor buffy coats. In this study, MCPyV DNA sequences were investigated in the sera (n = 190) of healthy blood donors. Two MCPyV DNA sequences, coding for the viral oncoprotein large T antigen (LT), were investigated using polymerase chain reaction (PCR) methods and DNA sequencing. Circulating MCPyV sequences were detected in sera with a prevalence of 2.6% (5/190), at low-DNA viral load, which is in the range of 1–4 and 1–5 copies/μl by real-time PCR and droplet digital PCR, respectively. DNA sequencing carried out in the five MCPyV-positive samples indicated that the two MCPyV LT sequences which were analyzed belong to the MKL-1 strain. Circulating MCPyV LT sequences are present in blood donor sera. MCPyV-positive samples from blood donors could represent a potential vehicle for MCPyV infection in receivers, whereas an increase in viral load may occur with multiple blood transfusions. In certain patient conditions, such as immune-depression/suppression, additional disease or old age, transfusion of MCPyV-positive samples could be an additional risk factor for MCC onset

Agri-Food Waste from Apple, Pear, and Sugar Beet as a Source of Protective Bioactive Molecules for Endothelial Dysfunction and Its Major Complications

Endothelial damage is recognized as the initial step that precedes several cardiovascular diseases (CVD), such as atherosclerosis, hypertension, and coronary artery disease. It has been demonstrated that the best treatment for CVD is prevention, and, in the frame of a healthy lifestyle, the consumption of vegetables, rich in bioactive molecules, appears effective at reducing the risk of CVD. In this context, the large amount of agri-food industry waste, considered a global problem due to its environmental and economic impact, represents an unexplored source of bioactive compounds. This review provides a summary regarding the possible exploitation of waste or by-products derived by the processing of three traditional Italian crops—apple, pear, and sugar beet—as a source of bioactive molecules to protect endothelial function. Particular attention has been given to the bioactive chemical profile of these pomaces and their efficacy in various pathological conditions related to endothelial dysfunction. The waste matrices of apple, pear, and sugar beet crops can represent promising starting material for producing “upcycled” products with functional applications, such as the prevention of endothelial dysfunction linked to cardiovascular diseases

The Use of Nutraceuticals to Counteract Atherosclerosis: The Role of the Notch Pathway

Despite the currently available pharmacotherapies, today, thirty percent of worldwide deaths are due to cardiovascular diseases (CVDs), whose primary cause is atherosclerosis, an inflammatory disorder characterized by the buildup of lipid deposits on the inside of arteries. Multiple cellular signaling pathways have been shown to be involved in the processes underlying atherosclerosis, and evidence has been accumulating for the crucial role of Notch receptors in regulating the functions of the diverse cell types involved in atherosclerosis onset and progression. Several classes of nutraceuticals have potential benefits for the prevention and treatment of atherosclerosis and CVDs, some of which could in part be due to their ability to modulate the Notch pathway. In this review, we summarize the current state of knowledge on the role of Notch in vascular health and its modulation by nutraceuticals for the prevention of atherosclerosis and/or treatment of related CVDs

Well-Known and Novel Players in Endothelial Dysfunction: Updates on a Notch(ed) Landscape

Endothelial dysfunction characterizes every aspect of the so-called cardiovascular continuum, a series of events ranging from hypertension to the development of atherosclerosis and, finally, to coronary heart disease, thrombus formation, myocardial infarction, and heart failure. Endothelial dysfunction is the main prognostic factor for the progression of vascular disorders, which responds to drug intervention and lifestyle changes. Virtually all of the drugs used to prevent cardiovascular disorders, such as long-used and new antilipidemic agents and inhibitors of angiotensin enzyme (ACEi), exert an important effect on the endothelium. Endothelial dysfunction is a central feature of coronavirus disease -19 (COVID-19), and it is now clear that life-risk complications of the disease are prompted by alterations of the endothelium induced by viral infection. As a consequence, the progression of COVID-19 is worse in the subjects in whom endothelial dysfunction is already present, such as elderly, diabetic, obese, and hypertensive patients. Importantly, circulating biomarkers of endothelial activation and injury predict the severity and mortality of the disease and can be used to evaluate the efficacy of treatments. The purpose of this review is to provide updates on endothelial function by discussing its clinical relevance in the cardiovascular continuum, the latest insights from molecular and cellular biology, and their implications for clinical practice, with a focus on new actors, such as the Notch signaling and emerging therapies for cardiovascular disease

The Notch pathway: a novel therapeutic target for cardiovascular diseases?

Introduction: The Notch pathway is involved in determining cell fate during development and postnatally in continuously renewing tissues, such as the endothelium, the epithelium, and in the stem cells pool. The dysregulation of the Notch pathway is one of the causes of limited response, or resistance, to available cancer treatments and novel therapeutic strategies based on Notch inhibition are being investigated in preclinical and clinical studies in oncology. A large body of evidence now shows that the dysregulation of the Notch pathway is also involved in the pathophysiology of cardiovascular diseases (CVDs). Areas covered: This review discusses the molecular mechanisms involving Notch which underlie heart failure, aortic valve calcification, and aortic aneurysm. Expert opinion: Despite the existence of preventive, pharmacological and surgical interventions approaches, CVDs are the first causes of mortality worldwide. The Notch pathway is becoming increasingly recognized as being involved in heart failure, aortic aneurysm and aortic valve calcification, which are among the most common global causes of mortality due to CVDs. As already shown in cancer, the dissection of the biological processes and molecular mechanisms involving Notch should pave the way for new strategies to prevent and cure these diseases