Effect of traditional or heat-not-burn cigarette smoking on circulating miRNAs in healthy subjects.

BACKGROUND: Traditional combustion cigarette (TCC) smoking is an established risk factor for several types of cancer and cardiovascular diseases. Circulating microRNAs (miRNAs) represent key molecules mediating pathogenetic mechanisms, and potential biomarkers for personalized risk assessment. TCC smoking globally changes the profile of circulating miRNAs. The use of heat-not-burn cigarettes (HNBCs) as alternative smoking devices is rising exponentially worldwide, and the circulating miRNA profile of chronic HNBC smokers is unknown. We aimed at defining the circulating miRNA profile of chronic exclusive HNBC smokers, and identifying potentially pathogenetic signatures. METHODS: Serum samples were obtained from 60 healthy young subjects, stratified in chronic HNBC smokers, TCC smokers and nonsmokers (20 subjects each). Three pooled samples per group were used for small RNA sequencing, and the fourth subgroup constituted the validation set. RESULTS: Differential expression analysis revealed 108 differentially expressed miRNAs; 72 exclusively in TCC, 10 exclusively in HNBC and 26 in both smoker groups. KEGG pathway analysis on target genes of the commonly modulated miRNAs returned cancer and cardiovascular disease associated pathways. Stringent abundance and fold-change criteria nailed down our functional bioinformatic analyses to a network where miR-25-3p and miR-221-3p are main hubs. CONCLUSION: Our results define for the first time the miRNA profile in the serum of exclusive chronic HNBC smokers and suggest a significant impact of HNBCs on circulating miRNAs.

Reduction of Cardiac Fibrosis by Interference With YAP-Dependent Transactivation.

BACKGROUND: Conversion of cardiac stromal cells into myofibroblasts is typically associated with hypoxia conditions, metabolic insults, and/or inflammation, all of which are predisposing factors to cardiac fibrosis and heart failure. We hypothesized that this conversion could be also mediated by response of these cells to mechanical cues through activation of the Hippo transcriptional pathway. The objective of the present study was to assess the role of cellular/nuclear straining forces acting in myofibroblast differentiation of cardiac stromal cells under the control of YAP (yes-associated protein) transcription factor and to validate this finding using a pharmacological agent that interferes with the interactions of the YAP/TAZ (transcriptional coactivator with PDZ-binding motif) complex with their cognate transcription factors TEADs (TEA domain transcription factors), under high-strain and profibrotic stimulation. METHODS: We employed high content imaging, 2-dimensional/3-dimensional culture, atomic force microscopy mapping, and molecular methods to prove the role of cell/nuclear straining in YAP-dependent fibrotic programming in a mouse model of ischemia-dependent cardiac fibrosis and in human-derived primitive cardiac stromal cells. We also tested treatment of cells with Verteporfin, a drug known to prevent the association of the YAP/TAZ complex with their cognate transcription factors TEADs. RESULTS: Our experiments suggested that pharmacologically targeting the YAP-dependent pathway overrides the profibrotic activation of cardiac stromal cells by mechanical cues in vitro, and that this occurs even in the presence of profibrotic signaling mediated by TGF-ß1 (transforming growth factor beta-1). In vivo administration of Verteporfin in mice with permanent cardiac ischemia reduced significantly fibrosis and morphometric remodeling but did not improve cardiac performance. CONCLUSIONS: Our study indicates that preventing molecular translation of mechanical cues in cardiac stromal cells reduces the impact of cardiac maladaptive remodeling with a positive effect on fibrosis.

Early Impairment of Paracrine and Phenotypic Features in Resident Cardiac Mesenchymal Stromal Cells after Thoracic Radiotherapy.

Radiotherapy-induced cardiac toxicity and consequent diseases still represent potential severe late complications for many cancer survivors who undergo therapeutic thoracic irradiation. We aimed to assess the phenotypic and paracrine features of resident cardiac mesenchymal stromal cells (CMSCs) at early follow-up after the end of thoracic irradiation of the heart as an early sign and/or mechanism of cardiac toxicity anticipating late organ dysfunction. Resident CMSCs were isolated from a rat model of fractionated thoracic irradiation with accurate and clinically relevant heart dosimetry that developed delayed dose-dependent cardiac dysfunction after 1 year. Cells were isolated 6 and 12 weeks after the end of radiotherapy and fully characterized at the transcriptional, paracrine, and functional levels. CMSCs displayed several altered features in a dose- and time-dependent trend, with the most impaired characteristics observed in those exposed in situ to the highest radiation dose with time. In particular, altered features included impaired cell migration and 3D growth and a and significant association of transcriptomic data with GO terms related to altered cytokine and growth factor signaling. Indeed, the altered paracrine profile of CMSCs derived from the group at the highest dose at the 12-week follow-up gave significantly reduced angiogenic support to endothelial cells and polarized macrophages toward a pro-inflammatory profile. Data collected in a clinically relevant rat model of heart irradiation simulating thoracic radiotherapy suggest that early paracrine and transcriptional alterations of the cardiac stroma may represent a dose- and time-dependent biological substrate for the delayed cardiac dysfunction phenotype observed in vivo.

A Review of Therapeutic Strategies against Cardiac Fibrosis: From Classical Pharmacology to Novel Molecular, Epigenetic, and Biotechnological Approaches.

Cardiovascular diseases are the first cause of death worldwide, with a heavy social and economic impact. They include a wide range of pathological conditions, among which cardiac fibrosis represents a common pathogenetic hallmark. The fibrotic process is driven by cardiac mesenchymal stromal cells, namely fibroblasts, which become activated, proliferate, and differentiate into myofibroblasts in response to several stimuli, in the end secreting extracellular matrix proteins, and mediating cardiac tissue remodelling and stiffening. A specific therapy for the exclusive treatment of cardiac fibrosis is still lacking. Given the growing quest for reducing the burden of cardiovascular diseases, there is increasing interest in the search for new effective anti-fibrotic therapies. In this review, we will briefly summarize the limited pharmacological therapies known to act, at least in part, against cardiac fibrosis. Then we will present novel potential active molecules, molecular targets, and biotechnological approaches emerged in the last decade, as possible future therapeutic strategies for cardiac fibrosis, with a specific focus on targeting fibroblast activation and function.

Progressive stages of dysmetabolism are associated with impaired biological features of human cardiac stromal cells mediated by the oxidative state and autophagy.

Cardiac stromal cells (CSCs) are the main players in fibrosis. Dysmetabolic conditions (metabolic syndrome-MetS, and type 2 diabetes mellitus-DM2) are strong pathogenetic contributors to cardiac fibrosis. Moreover, modulation of the oxidative state (OxSt) and autophagy is a fundamental function affecting the fibrotic commitment of CSCs, that are adversely modulated in MetS/DM2. We aimed to characterize CSCs from dysmetabolic patients, and to obtain a beneficial phenotypic setback from such fibrotic commitment by modulation of OxSt and autophagy. CSCs were isolated from 38 patients, stratified as MetS, DM2, or controls. Pharmacological modulation of OxSt and autophagy was obtained by treatment with trehalose and NOX4/NOX5 inhibitors (TREiNOX). Flow-cytometry and real-time quantitative polymerase chain reaction (RT-qPCR) analyses showed significantly increased expression of myofibroblasts markers in MetS-CSCs at baseline (GATA4, ACTA2, THY1/CD90) and after starvation (COL1A1, COL3A1). MetS- and DM2-CSCs displayed a paracrine profile distinct from control cells, as evidenced by screening of 30 secreted cytokines, with a significant reduction in vascular endothelial growth factor (VEGF) and endoglin confirmed by enzyme-linked immunoassay (ELISA). DM2-CSCs showed significantly reduced support for endothelial cells in angiogenic assays, and significantly increased H2 O2 release and NOX4/5 expression levels. Autophagy impairment after starvation (reduced ATG7 and LC3-II proteins) was also detectable in DM2-CSCs. TREiNOX treatment significantly reduced ACTA2, COL1A1, COL3A1, and NOX4 expression in both DM2- and MetS-CSCs, as well as GATA4 and THY1/CD90 in DM2, all versus control cells. Moreover, TREiNOX significantly increased VEGF release by DM2-CSCs, and VEGF and endoglin release by both MetS- and DM2-CSCs, also recovering the angiogenic support to endothelial cells by DM2-CSCs. In conclusion, DM2 and MetS worsen microenvironmental conditioning by CSCs. Appropriate modulation of autophagy and OxSt in human CSCs appears to restore these features, mostly in DM2-CSCs, suggesting a novel strategy against cardiac fibrosis in dysmetabolic patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Multicellular 3D Models for the Study of Cardiac Fibrosis.

Ex vivo modelling systems for cardiovascular research are becoming increasingly important in reducing lab animal use and boosting personalized medicine approaches. Integrating multiple cell types in complex setups adds a higher level of significance to the models, simulating the intricate intercellular communication of the microenvironment in vivo. Cardiac fibrosis represents a key pathogenetic step in multiple cardiovascular diseases, such as ischemic and diabetic cardiomyopathies. Indeed, allowing inter-cellular interactions between cardiac stromal cells, endothelial cells, cardiomyocytes, and/or immune cells in dedicated systems could make ex vivo models of cardiac fibrosis even more relevant. Moreover, culture systems with 3D architectures further enrich the physiological significance of such in vitro models. In this review, we provide a summary of the multicellular 3D models for the study of cardiac fibrosis described in the literature, such as spontaneous microtissues, bioprinted constructs, engineered tissues, and organs-on-chip, discussing their advantages and limitations. Important discoveries on the physiopathology of cardiac fibrosis, as well as the screening of novel potential therapeutic molecules, have been reported thanks to these systems. Future developments will certainly increase their translational impact for understanding and modulating mechanisms of cardiac fibrosis even further.

Unusual Association of NF-κB Components in Tumor-Associated Macrophages (TAMs) Promotes HSPG2-Mediated Immune-Escaping Mechanism in Breast Cancer.

The cellular heterogeneity of the tumor environment of breast cancer (BC) is extremely complex and includes different actors such as neoplastic, stromal, and immunosuppressive cells, which contribute to the chemical and mechanical modification of the environment surrounding the tumor-exasperating immune-escaping mechanisms. In addition to molecular signals that make the tumor microenvironment (TME) unacceptable for the penetrance of the immune system, the physical properties of tumoral extracellular matrix (tECM) also have carved out a fundamental role in the processes of the protection of the tumor niche. Tumor-associated macrophages (TAMs), with an M2 immunosuppressive phenotype, are important determinants for the establishment of a tumor phenotype excluded from T cells. NF-κB transcription factors orchestrate innate immunity and represent the common thread between inflammation and cancer. Many studies have focused on canonical activation of NF-κB; however, activation of non-canonical signaling predicts poor survival and resistance to therapy. In this scenario, we demonstrated the existence of an unusual association of NF-κB components in TAMs that determines the deposition of HSPG2 that affects the stiffness of tECM. These results highlight a new mechanism counterbalanced between physical factors and a new perspective of mechano-pathology to be targeted to counteract immune evasion in BC.

The Microenvironment of Decellularized Extracellular Matrix from Heart Failure Myocardium Alters the Balance between Angiogenic and Fibrotic Signals from Stromal Primitive Cells.

Cardiac adverse remodeling is characterized by biological changes that affect the composition and architecture of the extracellular matrix (ECM). The consequently disrupted signaling can interfere with the balance between cardiogenic and pro-fibrotic phenotype of resident cardiac stromal primitive cells (CPCs). The latter are important players in cardiac homeostasis and can be exploited as therapeutic cells in regenerative medicine. Our aim was to compare the effects of human decellularized native ECM from normal (dECM-NH) or failing hearts (dECM-PH) on human CPCs. CPCs were cultured on dECM sections and characterized for gene expression, immunofluorescence, and paracrine profiles. When cultured on dECM-NH, CPCs significantly upregulated cardiac commitment markers (CX43, NKX2.5), cardioprotective cytokines (bFGF, HGF), and the angiogenesis mediator, NO. When seeded on dECM-PH, instead, CPCs upregulated pro-remodeling cytokines (IGF-2, PDGF-AA, TGF-ß) and the oxidative stress molecule H2O2. Interestingly, culture on dECM-PH was associated with impaired paracrine support to angiogenesis, and increased expression of the vascular endothelial growth factor (VEGF)-sequestering decoy isoform of the KDR/VEGFR2 receptor. Our results suggest that resident CPCs exposed to the pathological microenvironment of remodeling ECM partially lose their paracrine angiogenic properties and release more pro-fibrotic cytokines. These observations shed novel insights on the crosstalk between ECM and stromal CPCs, suggesting also a cautious use of non-healthy decellularized myocardium for cardiac tissue engineering approaches.

On the Road to Regeneration: "Tools" and "Routes" Towards Efficient Cardiac Cell Therapy for Ischemic Cardiomyopathy.

PURPOSE OF REVIEW: Cardiac regenerative medicine is a field bridging together biotechnology and surgical science. In this review, we present the explored surgical roads to cell delivery and the known effects of each delivery method on cell therapy efficiency. We also list the more recent clinical trials, exploring the safety and efficacy of delivery routes used for cardiac cell therapy approaches. RECENT FINDINGS: There is no consensus in defining which way is the most suitable for the delivery of the different therapeutic cell types to the damaged heart tissue. In addition, it emerged that the "delivery issue" has not been systematically addressed in each clinical trial and for each and every cell type capable of cardiac repair. Cardiac damage occurring after an ischemic insult triggers a cascade of cellular events, eventually leading to heart failure through fibrosis and maladaptive remodelling. None of the pharmacological or medical interventions approved so far can rescue or reverse this phenomenon, and cardiovascular diseases are still the leading cause of death in the western world. Therefore, for nearly 20 years, regenerative medicine approaches have focused on cell therapy as a promising road to pursue, with numerous preclinical and clinical testing of cell-based therapies being studied and developed. Nonetheless, consistent clinical results are still missing to reach consensus on the most effective strategy for ischemic cardiomyopathy, based on patient selection, diagnosis and stage of the disease, therapeutic cell type, and delivery route.

ß-adrenergic receptors and cardiac progenitor cell biology: What is the real connection?

Resident cardiac progenitor cells (CPCs) isolated from small animal models may not always be representative of their human counterparts, especially when significant differences in isolation protocols are considered. Nonetheless, multiple evidences support an important role of ß-adrenergic signaling in human CPC survival and commitment, which will need appropriate consideration for future developments of human CPCs as regenerative medicine tools.

Beta2-adrenergic signaling affects the phenotype of human cardiac progenitor cells through EMT modulation.

Human cardiac progenitor cells (CPCs) offer great promises to cardiac cell therapy for heart failure. Many in vivo studies have shown their therapeutic benefits, paving the way for clinical translation. The 3D model of cardiospheres (CSs) represents a unique niche-like in vitro microenvironment, which includes CPCs and supporting cells. CSs have been shown to form through a process mediated by epithelial-to-mesenchymal transition (EMT). ß2-Adrenergic signaling significantly affects stem/progenitor cells activation and mobilization in multiple tissues, and crosstalk between ß2-adrenergic signaling and EMT processes has been reported. In the present study, we aimed at investigating the biological response of CSs to ß2-adrenergic stimuli, focusing on EMT modulation in the 3D culture system of CSs. We treated human CSs and CS-derived cells (CDCs) with the ß2-blocker butoxamine (BUT), using either untreated or ß2 agonist (clenbuterol) treated CDCs as control. BUT-treated CS-forming cells displayed increased migration capacity and a significant increase in their CS-forming ability, consistently associated with increased expression of EMT-related genes, such as Snai1. Moreover, long-term BUT-treated CDCs contained a lower percentage of CD90+ cells, and this feature has been previously correlated with higher cardiogenic and therapeutic potential of the CDCs population. In addition, long-term BUT-treated CDCs had an increased ratio of collagen-III/collagen-I gene expression levels, and showed decreased release of inflammatory cytokines, overall supporting a less fibrosis-prone phenotype. In conclusion, ß2 adrenergic receptor block positively affected the stemness vs commitment balance within CSs through the modulation of type1-EMT (so called "developmental"). These results further highlight type-1 EMT to be a key process affecting the features of resident cardiac progenitor cells, and mediating their response to the microenvironment.

The Biological Mechanisms of Action of Cardiac Progenitor Cell Therapy.

PURPOSE OF REVIEW: Cell therapy for cardiovascular diseases is regarded as a rapidly growing field within regenerative medicine. Different cellular populations enriched for cardiac progenitor cells (CPCs), or derivate a-cellular products, are currently under preclinical and clinical evaluation. Here, we have reviewed the described mechanisms whereby resident post-natal CPCs, isolated in different ways, act as a therapeutic product on the damaged myocardium. RECENT FINDINGS: Several biological mechanisms of action have been described which can explain the multiple therapeutic effects of CPC treatment observed on cardiac function and remodelling. These mechanisms span from direct cardiovascular differentiation, through induction of resident progenitor proliferation, to paracrine effects on cardiac and non-cardiac cells mediated by exosomes and non-coding RNAs. All the reported mechanisms of action support an integrated view including cardiomyogenesis, cardioprotection, and anti-fibrotic effects. Moreover, future developments of CPC therapy approaches may support cell-free strategies, exploiting effective pleiotropic cell-derived products, such as exosomes.

JAK2V617F homozygosity arises commonly and recurrently in PV and ET, but PV is characterized by expansion of a dominant homozygous subclone.

Subclones homozygous for JAK2V617F are more common in polycythemia vera (PV) than essential thrombocythemia (ET), but their prevalence and significance remain unclear. The JAK2 mutation status of 6495 BFU-E, grown in low erythropoietin conditions, was determined in 77 patients with PV or ET. Homozygous-mutant colonies were common in patients with JAK2V617F-positive PV and were surprisingly prevalent in JAK2V617F-positive ET and JAK2 exon 12-mutated PV. Using microsatellite PCR to map loss-of-heterozygosity breakpoints within individual colonies, we demonstrate that recurrent acquisition of JAK2V617F homozygosity occurs frequently in both PV and ET. PV was distinguished from ET by expansion of a dominant homozygous subclone, the selective advantage of which is likely to reflect additional genetic or epigenetic lesions. Our results suggest a model in which development of a dominant JAK2V617F-homzygous subclone drives erythrocytosis in many PV patients, with alternative mechanisms operating in those with small or undetectable homozygous-mutant clones.

[Selection of heparin-sensitive dengue virus variants in BHK-21 cells]. / Selección de variantes de virus dengue sensibles a la heparina en células BHK-21.

Several studies have shown that adaptation of various viruses to grow in certain cell lines of vertebrates, leads to the selection of virus variants that bind heparan sulfate (HS) with high affinity. In this study we investigated the susceptibility of strains of dengue virus (DENV) to oversulfated heparin an analogue of HS after passages in BHK-21 cells. Field isolates of the four serotypes of DENV with a limited number of passes in mosquito cells C6/36HT were serially passaged eight times in BHK-21 cells. The adaptation of the DENV to the cell culture selected viral variants with an increased replicative capacity in BHK-21 cells and an increased susceptibility to heparin compared with the original not adapted strains, with a more significant inhibition of the infectivity in DENV-2 and DENV-4.The E protein of the adapted strains showed changes in the amino acid sequence, particularly at the position K204R to DENV-1, N67K to DENV-2, K308R and V452A for DENV-3 and E327G to DENV-4. These substitutions implicated a gain of basic residues that increased the net positive charge of the protein. These results suggest that adaptation of DENV strains to BHK-21 cells implies changes in the envelope protein, changes associated to the protein reactivity with heparin, the inhibitory effectiveness of this compound varying depending on the viral strain. In addition, these results suggest that the HS can play an important role in the infectivity of the DENV strains adapted to vertebrate cell culture, but not in the infectivity of non-adapted DENV isolates.

Exposure to serum from exclusive heated tobacco product smokers induces mTOR activation and fibrotic features in human cardiac stromal cells.

Chronic smokers have increased risk of fibrosis-related atrial fibrillation. The use of heated-tobacco products (HTPs) is increasing exponentially, and their health impact is still uncertain. We aim to investigate the effects of circulating molecules in exclusive HTP chronic smokers on the fibrotic behavior of human atrial cardiac stromal cells (CSCs). CSCs were isolated from atrial tissue of elective cardiac surgery patients, and exposed to serum lots from young healthy subjects, stratified in exclusive HTP smokers, tobacco combustion cigarette (TCC) smokers, or nonsmokers (NS). CSCs treated with TCC serum displayed impaired migration and increased expression of pro-inflammatory cytokines. Cells cultured with HTP serum showed increased levels of pro-fibrotic markers, and reduced expression of connexin-43. Both TCC and HTP sera increased collagen release and reduced secretion of angiogenic protective factors from CSCs, compared to NS serum. Paracrine support to tube-formation by endothelial cells and to viability of cardiomyocytes was significantly impaired. Treatment with sera of both smokers groups impaired H2O2/NO release balance by CSCs and reduced early phosphorylation of several pathways compared to NS serum, leading to mTOR activation. Cotreatment with rapamycin was able to reduce mTOR phosphorylation and differentiation into aSMA-positive myofibroblasts in CSCs exposed to TCC and HTP sera. In conclusion, the circulating molecules in the serum of chronic exclusive HTP smokers induce fibrotic behavior in CSCs through activation of the mTOR pathway, and reduce their beneficial paracrine effects on endothelial cells and cardiomyocytes. These results point to a potential risk for cardiac fibrosis in chronic HTP users.

Clonal analyses reveal associations of JAK2V617F homozygosity with hematologic features, age and gender in polycythemia vera and essential thrombocythemia.

Subclones homozygous for JAK2V617F are more common and larger in patients with polycythemia vera compared to essential thrombocythemia, but their role in determining phenotype remains unclear. We genotyped 4564 erythroid colonies from 59 patients with polycythemia vera or essential thrombocythemia to investigate whether the proportion of JAK2V617F -homozygous precursors, compared to heterozygous precursors, is associated with clinical or demographic features. In polycythemia vera, a higher proportion of homozygous-mutant precursors was associated with more extreme blood counts at diagnosis, consistent with a causal role for homozygosity in polycythemia vera pathogenesis. Larger numbers of homozygous-mutant colonies were associated with older age, and with male gender in polycythemia vera but female gender in essential thrombocythemia. These results suggest that age promotes development or expansion of homozygous-mutant clones and that gender modulates the phenotypic consequences of JAK2V617F homozygosity, thus providing a potential explanation for the long-standing observations of a preponderance of men with polycythemia vera but of women with essential thrombocythemia.

Primary microRNA transcripts are processed co-transcriptionally.

microRNAs (miRNAs) are generated from long primary (pri-) RNA polymerase II (Pol II)-derived transcripts by two RNase III processing reactions: Drosha cleavage of nuclear pri-miRNAs and Dicer cleavage of cytoplasmic pre-miRNAs. Here we show that Drosha cleavage occurs during transcription acting on both independently transcribed and intron-encoded miRNAs. We also show that both 5'-3' and 3'-5' exonucleases associate with the sites where co-transcriptional Drosha cleavage occurs, promoting intron degradation before splicing. We finally demonstrate that miRNAs can also derive from 3' flanking transcripts of Pol II genes. Our results demonstrate that multiple miRNA-containing transcripts are co-transcriptionally cleaved during their synthesis and suggest that exonucleolytic degradation from Drosha cleavage sites in pre-mRNAs may influence the splicing and maturation of numerous mRNAs.

Modified Risk Tobacco Products and Cardiovascular Repair: Still Very "Smoky".

Smoking habits represent a cardiovascular risk factor with a tremendous impact on health. Other than damaging differentiated and functional cells of the cardiovascular system, they also negatively affect reparative mechanisms, such as those involved in cardiac fibrosis and in endothelial progenitor cell (EPC) activation. In recent years, alternative smoking devices, dubbed modified tobacco risk products (MRPs), have been introduced, but their precise impact on human health is still under evaluation. Also, they have not been characterized yet about the possible negative effects on cardiovascular reparative and regenerative cells, such as EPCs or pluripotent stem cells. In this perspective, we critically review the still scarce available data on the effects of MRPs on molecular and cellular mechanisms of cardiovascular repair and regeneration.

Metabolomic NMR analysis and organoleptic perceptions of pomegranate wines: Influence of cultivar and yeast on the product characteristics.

Pomegranate (Punica granatum L.) fruits are a historical agricultural product of the Mediterranean basin that became increasingly popular in the latest years for being rich in antioxidants and other micronutrients, and are extensively commercialized as fruits, juice, jams and, in some Eastern countries, as a fermented alcoholic beverage. In this work, four different pomegranate wines specifically designed using combinations of two cultivars (Jolly Red and Smith) and two yeast starters with markedly different characteristics (Saccharomyces cerevisiae Clos and Saccharomyces cerevisiae ex-bayanus EC1118) were analyzed. The chemical characterization of the wines together with the originating unfermented juices was performed by 1H NMR spectroscopy metabolomic analysis. The full spectra were used for unsupervised and supervised statistical multivariate analysis (MVA), namely Principal Component Analysis (PCA), Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA), and sparse PCA (SPCA). The MVA of the wines showed a clear discrimination between the cultivars, and a smaller, yet significant, discrimination between the yeasts used. In particular, a higher content of citrate and gallate was observed for the Smith cv. and, on the contrary, a statistically significant higher content of fructose, malate, glycerol, 2,3 butanediol, trigonelline, aromatic amino acids and 4-hydrophenylacetate was observed in Jolly Red pomegranate wines samples. Significant interaction among the pomegranate cultivar and the fermenting yeast was also observed. Sensorial analysis was performed by a panel of testing experts. MVA of tasting data showed that the cultivar significantly affected the organoleptic parameters considered, while the yeast had a minor impact. Correlation analysis between NMR-detected metabolites and organoleptic descriptors identified several potential sensorially-active molecules as those significantly impacting the characteristics of the pomegranate wines.

The dynamic facets of the cardiac stroma: from classical markers to omics and translational perspectives.

Cardiac stromal cells have been long underestimated in their functions in homeostasis and repair. Recent evidence has changed this perspective in that many more players and facets than just "cardiac fibroblasts" have entered the field. Single cell transcriptomic studies on cardiac interstitial cells have shed light on the phenotypic plasticity of the stroma, whose transcriptional profile is dynamically regulated in homeostatic conditions and in response to external stimuli. Different populations and/or functional states that appear in homeostasis and pathology have been described, particularly increasing the complexity of studying the cardiac response to injury. In this review, we outline current phenotypical and molecular markers, and the approaches developed for identifying and classifying cardiac stromal cells. Significant advances in our understanding of cardiac stromal populations will provide a deeper knowledge on myocardial functional cellular components, as well as a platform for future developments of novel therapeutic strategies to counteract cardiac fibrosis and adverse cardiac remodeling.