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Background The use of alternative smoking devices, such as heat-not-burn cigarettes (HNBC), is increasing on a global scale, and their impact on health is still uncertain. Objective To investigate the effects of circulating molecules in HNBC chronic smokers on the fibrotic specification and paracrine function of cardiac stromal cells (CSCs). Methods Resident CSCs were isolated from the atrial tissue of non-smokers patients with cardiovascular diseases, and exposed to the serum pools derived from 60 young healthy subjects, stratified in exclusive HNBC smokers, traditional combustion cigarette (TCC) smokers, or non-smokers (NS) as reference. Results CSCs treated with TCC serum versus NS displayed impaired 3D growth (NS 568±24 vs. TCC 442±27 spheroids/well, p≤0,01) and reduced migration after 6h (NS 0,62±0,04 vs. TCC 0,80±0,03 normalized scratch area versus t0, p≤0,01) and 10h (NS 0,49±0,03 vs. TCC 0,36±0,03 normalized scratch area versus t0, p≤0,05), as well as significantly increased expression (IL-6, IL-8) and/or release of pro-inflammatory (e.g CRP, PAI-1, CD40L, CXCL4) and pro-fibrotic cytokines (PDGFA, MMP1). CSCs cultured with HNBC serum showed significantly increased mRNA levels of pro-fibrotic genes (PDGFA, THY1, COL1A1, p Conclusion The circulating molecules in the serum of chronic HNBC smokers induce fibrotic specification in CSCs. They also reduce the beneficial paracrine effects of stromal cells on endothelial cells and cardiomyocytes, albeit to a reduced extent for some features. These results point to a potential risk for atrial fibrosis development triggered by chronic HNBC use.

BackgroundThe use of alternative smoking devices, such as heat-not-burn cigarettes (HNBC), is increasing on a global scale, and their impact on health is still uncertain.ObjectiveTo investigate the effects of circulating molecules in HNBC chronic smokers on the fibrotic specification and paracrine function of cardiac stromal cells (CSCs).MethodsResident CSCs were isolated from the atrial tissue of patients with cardiovascular diseases, and exposed to the serum of 60 young healthy subjects, stratified in exclusive HNBC smokers, traditional combustion cigarette (TCC) smokers, or non-smokers (NS) as reference.ResultsCSCs treated with TCC serum displayed impaired 3D growth and migration, as well as increased expression and/or release of pro-inflammatory and pro-fibrotic cytokines. Cells cultured with HNBC serum showed increased mRNA levels of pro-fibrotic genes, and reduced expression of the gap junction protein CX43. Nonetheless, both TCC and HNBC sera reduced the release of angiogenic and protective factors from CSCs. In fact, their paracrine support to tube-formation by endothelial cells and to preserved cell viability of cardiomyocytes in culture was significantly impaired. Treatment with the sera of both types of smokers also increased the expression of NOX isoforms and the release of H2O2 by CSCs.ConclusionThe circulating molecules in the serum of chronic HNBC smokers induce fibrotic specification in CSCs. They also reduce the beneficial paracrine effects of stromal cells on endothelial cells and cardiomyocytes, albeit to a reduced extent for some features. These results point to a potential risk for atrial fibrosis development triggered by chronic HNBC use.CONDENSED ABSTRACTThe use of alternative smoking devices, such as heat-not-burn cigarettes (HNBC), is increasing on a global scale, and their impact on health is still uncertain. We isolated human stromal cells from the atrial tissue of patients with cardiovascular diseases, and exposed them to the serum of young healthy subjects, that are exclusive HNBC smokers. Results showed significant alterations in the phenotype of CSCs exposed to HNBC serum, suggesting a specification towards fibrosis, reduced support to parenchymal cells, and increased oxidative stress production. Data point to a potential risk for atrial fibrosis development triggered by chronic HNBC use.

Objectives Extracellular vesicles (EVs) are key biological mediators of several physiological functions within the cell microenvironment. Platelets are the most abundant source of EVs in the blood. Similarly, platelet lysate (PL), the best platelet derivative and angiogenic performer for regenerative purposes, is enriched of EVs, but their role is still too poorly discovered to be suitably exploited. Here, we explored the contribution of the EVs in PL, by investigating the angiogenic features extrapolated from that possessed by PL. Methods We tested angiogenic ability and molecular cargo in 3D bioprinted models and by RNA sequencing analysis of PL-derived EVs. Results A subset of small vesicles is highly represented in PL. The EVs do not retain aggregation ability, preserving a low redox state in human umbilical vein endothelial cells (HUVECs) and increasing the angiogenic tubularly-like structures in 3D endothelial bioprinted constructs. EVs resembled the miRNome profile of PL, mainly enriched with small RNAs and a high amount of miR-126, the most abundant angiogenic miRNA in platelets. The transfer of miR-126 by EVs in HUVEC after the in vitro inhibition of the endogenous form, restored angiogenesis, without involving VEGF as a downstream target in this system. Conclusion PL is a biological source of available EVs with angiogenic effects involving a miRNAs-based cargo. These properties can be exploited for targeted molecular/biological manipulation of PL, by potentially developing a product exclusively manufactured of EVs.

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.

Abstract: 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.

Objectivesextracellular vesicles (EVs) are key biological mediators of several physiological functions within the cell microenvironment. Platelets are the most abundant source of EVs in the blood. Similarly, platelet lysate (PL), the best platelet derivative and angiogenic performer for regenerative purposes, is enriched of EVs, but their role is still too poorly discovered to be suitably exploited. Here we explored the contribution of the EVs in PL, by investigating the angiogenic features extrapolated from that possessed by PL.Methodswe tested angiogenic ability and molecular cargo in 3D bioprinted models and by RNA sequencing analysis of PL-derived EVs.Resultsa subset of small vesicles is highly represented in PL. The EVs do not retain aggregation ability, preserving a low redox state in HUVEC and increasing the angiogenic tubularly-like structures in 3D endothelial bioprinted constructs. EVs resembled the miRNome profile of PL, mainly enriched of small RNAs and a high amount of miR-126, the most abundant angiogenic miRNA in platelets. The transfer of miR-126 by EVs in HUVEC after the in vitro inhibition of the endogenous form, restored angiogenesis, without involving VEGF as downstream target in this system.ConclusionsPL is a biological source of available EVs with angiogenic effects involving a miRNAs-based cargo. These properties can be exploited for targeted molecular/biological manipulation of PL, by potentially developing a product exclusively manufactured of EVs.

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 Hsub2/subOsub2/subrelease 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 Wileyamp; Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Cardiac stromal cells (CSCs) embrace multiple phenotypes and are a contributory factor in tissue homeostasis and repair. They can be exploited as therapeutic mediators against cardiac fibrosis and remodeling, but their survival and cardioprotective properties can be decreased by microenvironmental cues. We evaluated the impact of autophagy modulation by different pharmacological/genetic approaches on the viability and phenotype of murine CSCs, which had been subjected to nutrient deprivation or hyperglycemia, in order to mimic relevant stress conditions and risk factors of cardiovascular diseases. Our results show that autophagy is activated in CSCs by nutrient deprivation, and that autophagy induction by trehalose or autophagy-related protein 7 (ATG7)-overexpression can significantly preserve CSC viability. Furthermore, autophagy induction is associated with a higher proportion of primitive, non-activated stem cell antigen 1 (Sca1)-positive cells, and with a reduced fibrotic fraction (positive for the discoidin domain-containing receptor 2, DDR2) in the CSC pool after nutrient deprivation. Hyperglycemia, on the other hand, is associated with reduced autophagic flux in CSCs, and with a significant reduction in primitive Sca1+ cells. Autophagy induction by adenoviral-mediated ATG7-overexpression maintains a cardioprotective, anti-inflammatory and pro-angiogenic paracrine profile of CSCs exposed to hyperglycemia for 1 week. Finally, autophagy induction by ATG7-overexpression during hyperglycemia can significantly preserve cell viability in CSCs, which were subsequently exposed to nutrient deprivation, reducing hyperglycemia-induced impairment of cell resistance to stress. In conclusion, our results show that autophagy stimulation preserves CSC viability and function in response to metabolic stressors, suggesting that it may boost the beneficial functions of CSCs in cardiac repair mechanisms.

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-β1 (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.

Extracellular vesicles (EVs) are key biological mediators of several physiological functions within the cell microenvironment. Platelets are the most abundant source of EVs in the blood. Similarly, platelet lysate (PL), the best platelet derivative and angiogenic performer for regenerative purposes, is enriched of EVs, but their role is still too poorly discovered to be suitably exploited. Here, we explored the contribution of the EVs in PL, by investigating the angiogenic features extrapolated from that possessed by PL.We tested angiogenic ability and molecular cargo in 3D bioprinted models and by RNA sequencing analysis of PL-derived EVs.A subset of small vesicles is highly represented in PL. The EVs do not retain aggregation ability, preserving a low redox state in human umbilical vein endothelial cells (HUVECs) and increasing the angiogenic tubularly-like structures in 3D endothelial bioprinted constructs. EVs resembled the miRNome profile of PL, mainly enriched with small RNAs and a high amount of miR-126, the most abundant angiogenic miRNA in platelets. The transfer of miR-126 by EVs in HUVEC after the in vitro inhibition of the endogenous form, restored angiogenesis, without involving VEGF as a downstream target in this system.PL is a biological source of available EVs with angiogenic effects involving a miRNAs-based cargo. These properties can be exploited for targeted molecular/biological manipulation of PL, by potentially developing a product exclusively manufactured of EVs.

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.

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.

Basic and translational research on lung biology and pathology can greatly benefit from the development of 3D in vitro models with physiological relevance. Lung organoids and lungs-on-chip allow the creation of different kinds of in vitro microenvironments, that can be useful for the elucidation of novel pathogenetic pathways, for example concerning tissue fibrosis in chronic diseases. Moreover, they represent important translational models for the identification of novel therapeutic targets, and for preliminary testing of new drugs. In this chapter, we provide a selected overview of recent studies on innovative 3D in vitro models that have enhanced our knowledge on chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), particularly concerning oxidative stress and pro-fibrotic pathogenetic mechanisms. Despite several limitations, these complex models must be considered as complementary in all respects to in vivo studies on animal models and clinical research.

Recent preclinical and clinical evidence suggest that immune system has a role in the progression and prognosis of Amyotrophic Lateral Sclerosis (ALS), but the identification of a clear mechanism and immune players remains to be elucidated. Here, we have investigated, in 30 and 60 days (presymptomatic) and 120 days (symptomatic) old SOD1-G93A mice, systemic, peripheral, and central innate and adaptive immune and inflammatory response, correlating it with the progression of the neurodegeneration in neuromuscular junction, sciatic nerves, and spinal cord. Surprisingly, we found a very initial (45–60 days) presence of IgG in sciatic nerves together with a gradual enhancement of A20/TNFAIP3 (protein controlling NF-kB signalling) and a concomitantly significant increase and activation of circulating mast cells (MCs) as well as MCs and macrophages in sciatic nerve and an enhancement of IL-6 and IL-10. This immunological frame coincided with a myelin aggregation. The 30–60 days old SOD1-G93A mice didn’t show real elements of neuroinflammation and neurodegeneration in spinal cord. In 120 days old mice macrophages and monocytes are widely diffused in sciatic nerves, peripheral neurodegeneration reaches the tip, high circulating levels of TNFa and IL-2 were found and spinal cord exhibits clear signs of neural damage and infiltrating immune cells. Our results underpin a clear immunological disorder at the origin of ALS axonopathy, in which MCs are involved in the initiation and sustaining of inflammatory events. These data cannot be considered a mere epiphenomenon of motor neuron degeneration and reveal new potential selective immune targets in ALS therapy.

Large scale projects such as FANTOM and ENCODE led to a revolution in our comprehension of the mammalian transcriptomes by revealing that ~53% of the produced RNAs do not encode for proteins. These transcripts, defined as noncoding RNAs (ncRNAs), constitute a heterogeneous group of molecules which can be categorized in two main classes, namely small and long, according to their length. In animals, the first-class includes Piwi-interacting RNAs (piRNAs), small interfering RNAs (siRNAs) and microRNAs (miRNAs). Among them, the best-characterized subgroup is represented by miRNAs, which are known to regulate gene expression largely at the post-transcriptional level. In contrast, long noncoding RNAs (lncRNAs) represent a more heterogeneous group of > 200 nucleotides long transcripts, that act through a variety of mechanisms at both transcriptional and posttranscriptional level. Here, we discuss how miRNAs and lncRNAs are emerging as pivotal regulators of cardiac muscle development and how the alteration of ncRNA expression was seen to disturb the physiology of all the different cell types forming the cardiac tissue. Particular emphasis is given to those species that are expressed and are known to regulate the capacity of cardiac progenitor cells (CPCs), currently used in regenerative medicine protocols, to proliferate and differentiate. Understanding how the ncRNAmediated circuitries regulate heart homeostasis is one of the research areas expected to have a high impact, improving the therapeutic efficacy of stem/progenitor-cells treatments for translation into clinical applications.

Background The role of microRNAs dysregulation in tobacco cigarette smoking–induced vascular damage still needs to be clarified. We assessed the acute effects of tobacco cigarette smoking on endothelial cell‐related circulating microRNAs in healthy subjects. In addition, we investigated the potential role of microRNAs in smoking‐dependent endothelial cell damage. Methods and Results A panel of endothelial‐related microRNAs was quantified in healthy subjects before and after smoking 1 tobacco cigarette. Serum levels of miR‐155 were found to be significantly increased shortly after smoking. We also observed a progressive and significant miR‐155 accumulation in culture media of human endothelial cells after 30 minutes and up to 4 hours of cigarette smoke condensate treatment in vitro without evidence of cell death, indicating that miR‐155 can be released by endothelial cells in response to smoking stress. Cigarette smoke condensate appeared to enhance oxidative stress and impair cell survival, angiogenesis, and NO metabolism in human endothelial cells. Notably, these effects were abrogated by miR‐155 inhibition. We also observed that miR‐155 inhibition rescued the deleterious effects of cigarette smoke condensate on endothelial‐mediated vascular relaxation and oxidative stress in isolated mouse mesenteric arteries. Finally, we found that exogenous miR‐155 overexpression mimics the effects of smoking stress by inducing the upregulation of inflammatory markers, impairing angiogenesis and reducing cell survival. These deleterious effects were associated with downregulation of vascular endothelial growth factor and endothelial NO synthetase. Conclusions Our results suggest that miR‐155 dysregulation may contribute to the deleterious vascular effects of tobacco smoking.

The identification of the mechanisms predisposing to stroke may improve its preventive and therapeutic strategies in patients with essential hypertension. The role of macroautophagy/autophagy in the development of hypertension-related stroke needs to be clarified. We hypothesized that a defective autophagy may favor hypertension-related spontaneous stroke by promoting mitochondrial dysfunction. We studied autophagy in the stroke-prone spontaneously hypertensive (SHRSP) rat, which represents a clinically relevant model of stroke associated with high blood pressure. We assessed autophagy, mitophagy and NAD+:NADH levels in brains of SHRSP and stroke-resistant SHR fed with high salt diet. Vascular smooth muscle cells silenced for the mitochondrial complex I subunit Ndufc2 gene (NADH:ubiquinone oxidoreductase subunit C2) and cerebral endothelial cells isolated from SHRSP were also used to assess autophagy/mitophagy and mitochondrial function in response to high salt levels. We found a reduction of autophagy in brains of high salt-fed SHRSP. Autophagy impairment was associated with NDUFC2 downregulation, mitochondrial dysfunction and NAD+ depletion. Restoration of NAD+ levels by nicotinamide administration reactivated autophagy and reduced stroke development in SHRSP. A selective reactivation of autophagy/mitophagy by Tat-Beclin 1 also reduced stroke occurrence, restored autophagy/mitophagy and improved mitochondrial function. Endothelial progenitor cells (EPCs) from subjects homozygous for the thymine allele variant at NDUFC2/rs11237379, which is associated with NDUFC2 deficiency and increased stroke risk, displayed an impairment of autophagy and increased senescence in response to high salt levels. EPC senescence was rescued by Tat-Beclin 1. Pharmacological activation of autophagy may represent a novel therapeutic strategy to reduce stroke occurrence in hypertension. 10 VSMCs: aortic vascular smooth muscle cells; COX4I1/COX IV: cytochrome c oxidase subunit 4I1; ECs: endothelial cells; EPCs: endothelial progenitor cells; JD: Japanese-style diet; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NAD: nicotinamide adenine dinucleotide; NDUFC2: NADH:ubiquinone oxidoreductase subunit C2; NMN: nicotinamide mononucleotide; RD: regular diet; SHRSP: stroke-prone spontaneously hypertensive rat; SHRSR: stroke-resistant spontaneously hypertensive rat.

Type 2 diabetes (T2D) is a major metabolic disease and a key epigenetic risk factor for the development of additional clinical complications. Among them, periodontitis (PD), a severe inflammatory disease ascribable to a dysregulated physiology and composition of the oral microbiota, represents one of the most relevant complications. Periodontitis can impact the structure of the tooth and likely the stem and progenitor cell pool, which actively contributes to the periodontal microenvironment and homeostasis. Modifications of the oral plaque play a key role in the etiopathogenesis of PD caused by T2D. However, to what extent the biology of the progenitor pool is affected has still to be elucidated. In this short report, we aimed to explore the biological effects of oral plaque derived from T2D patients with PD in comparison to non-diabetic patients with PD. Oral plaque samples were isolated from T2D and non-diabetic subjects with PD. Dental pulp stem cells (DPSCs), derived from the premolar tooth, were conditioned for 21 days with oral plaque samples and tested for their clonogenic ability. Cultures were also induced to differentiate towards the osteogenic lineage, and ALP and osteocalcin gene expression levels were evaluated by real-time qPCR. Results have shown that the number of clones generated by DPSCs exposed to T2D oral plaque was significantly lower compared to controls (ctl). The multivariate analysis confirmed that the decreased clonogenesis was significantly correlated only with T2D diagnosis. Moreover, the effect of T2D oral plaque was specific to DPSCs. Indicators of osteogenic differentiation were not significantly affected. This study provides a new biological insight into the effects ascribable to T2D in PD.

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. 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.

Basic and translational research on lung biology has discovered multiple progenitor cell types, specialized or facultative, responsible for turnover, renewal, and repair. Isolation of populations of resident lung progenitor cells (LPCs) has been described by multiple protocols, and some have been successfully applied to healthy human lung tissue. We aimed at understanding how different cell culture conditions may affect, in vitro, the phenotype of LPCs to create an ideal niche-like microenvironment. The influence of different substrates (i.e., fibronectin, gelatin, laminin) and the impact of a three-dimensional/two-dimensional (3D/2D) culture switch on the biology of LPCs isolated as lung spheroids (LSs) from normal adult human lung biopsy specimens were investigated. We applied a spheroid culture system as the selective/inductive step for progenitor cell culture, as described in many biological systems. The data showed a niche-like proepithelial microenvironment inside the LS, highly sensitive to the 3D culture system and significantly affecting the phenotype of adult LPCs more than culture substrate. LSs favor epithelial phenotypes and LPC maintenance and contain cells more responsive to specific commitment stimuli than 2D monolayer cultures, while secreting a distinctive set of paracrine factors. We have shown for the first time, to our knowledge, how culture as 3D LSs can affect LPC epithelial phenotype and produce strong paracrine signals with a distinctive secretomic profile compared with 2D monolayer conditions. These findings suggest novel approaches to maintain ex vivo LPCs for basic and translational studies.

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-&beta, ) 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.

Neuropeptide Y (NPY), a powerful neurotransmitter of the central nervous system, is a key regulator of angiogenesis and biology of adipose depots. Intriguingly, its peripheral vascular and angiogenic powerful activity is strictly associated to platelets, which are source of clinical hemoderivates, such as platelet lysate (PL), routinely employed in several clinical applications as wound healing, and to preserve ex vivo the progenitor properties of the adipose stromal cells pool. So far, the presence of NPY in PL and its biological effects on the adipose stromal cell fraction (ASCs) have never been investigated. Here, we aimed to identify endogenous sources of NPY such as PL-based preparations and to investigate which biological properties PL-derived NPY is able to exert on ASCs. The results show that PL contains a high amount of NPY, which is in part also excreted by ASCs when stimulated with PL. The protein levels of the three main NPY subtype receptors (Y1, Y2, Y5) are unaltered by stimulation of ASCs with PL, but their inhibition through selective pharmacological antagonists, considerably enhances migration, and a parallel reduction of angiogenic features of ASCs including decrease in VEGF mRNA and intracellular calcium levels, both downstream targets of NPY. The expression of VEGF and NPY is enhanced within the sites of neovascularisation of difficult wounds in patients after treatment with leuco-platelet concentrates. Our data highlight the presence of NPY in PL preparations and its peripheral effects on adipose progenitors.

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. 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.

Electronic cigarettes (E-cigarettes) have become popular as substitutes for conventional tobacco cigarettes or to aid quitting, but little is known about the potential risks to cardiovascular health for smokers and nonsmokers. We sought to compare the impact of E-cigarettes with conventional cigarettes on platelet function in healthy adult smokers and nonsmokers. A crossover single-blind study in 40 healthy participants (20 smokers, 20 nonsmokers, matched for age and sex) was conducted. Each participant smoked a conventional cigarette then returned 1 week later to vape a study E-cigarette with the same nominal nicotine content. Blood samples were drawn shortly before and 5 minutes after each episode and analyzed for platelet aggregation, soluble CD40-ligand (sCD40L) and soluble P-selectin (sP-selectin). At baseline, smokers had significantly higher levels of sCD40L and sP-selectin (all p ≤0.01) than nonsmokers. Within 5 minutes of using either a conventional cigarette or E-cigarette, changes in the levels of sCD40L, sP-selectin, and platelet aggregation (all p ≤0.01) were detectable in both smokers and nonsmokers. In smokers, there were no significant changes in sCD40L and sP-selectin but there was a significant increase in platelet aggregation. In nonsmokers, there was a significant increase in all markers of platelet activation following both cigarette and E-cigarette use. Both traditional and E-cigarettes have short-term effects on platelet activation, although in nonsmokers the use of E-cigarettes had a less important impact. In conclusion, we provide the first comparison data of the acute impact of Tobacco-cigarette and E-cigarette smoking on the platelet function in smokers and nonsmokers.

Philadelphia negative myeloproliferative neopl 28 asms (MPNs) are clonal haematological diseases characterized by excessive production of mature blood cells. Exome sequencing of patient samples have showed a relatively low degree genomic complexity for these diseases1. The majority of MPN patients carry somatic mutations in the JAK2 gene, with the JAK2V617F missense mutation being the most common in poly33 cythemia vera (PV, 95%) and essential thrombocythemia (ET, 60%) 2.

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). β2-Adrenergic signaling significantly affects stem/progenitor cells activation and mobilization in multiple tissues, and crosstalk between β2-adrenergic signaling and EMT processes has been reported. In the present study, we aimed at investigating the biological response of CSs to β2-adrenergic stimuli, focusing on EMT modulation in the 3D culture system of CSs. We treated human CSs and CS-derived cells (CDCs) with the β2-blocker butoxamine (BUT), using either untreated or β2 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, β2 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.

Objectives Extra virgin olive oil (EVOO) supplementation is associated with a significant reduction in cardiovascular disease but the underlying mechanism is still unclear. Methods In platelets that were taken from healthy subjects (n = 5), agonist-induced hydrogen peroxide (H 2 O 2 ) production and NADPH oxidase 2 (NOX2) activation in the presence of or without catalase, which catabolizes H 2 O 2 , were investigated. Platelet H 2 O 2 production, NOX2 activation, EVOO vitamin E, and total polyphenols as well as EVOO's ability to scavenge H 2 O 2 were also measured. Results Platelet NOX2 activation and H 2 O 2 production were significantly inhibited in catalase-treated platelets and platelets that were incubated with five different EVOOs. The EVOO content of vitamin E was 53 to 223 mg/kg and total polyphenols 145 to 392 mg/L Gallic acid equivalent. EVOOs quenched in vitro H 2 O 2 by 39 to 62%, which is an effect that is significantly correlated with vitamin E and total polyphenol concentrations ( R = 0.688; P R = 0.541; P Conclusions This in vitro study provides the first evidence that EVOO downregulates platelet H 2 O 2 and in turn NOX2 activity via H 2 O 2 scavenging.

Background Although adipose stromal cells (ASCs) retain the ability to transdifferentiate at low rate towards the cardiac lineage, the potential mechanisms underlying such process have still to be elucidated. Methods Since chromatin state modifications are involved in several processes regulating the cellular cell fate commitment, we aimed at evaluating the role of histone protein acetylation in the cardiovascular-like transdifferentiation of ASCs. Results We found a clear increase of histone 3 acetylation status paralleled by a significant upregulation of cardiac TnI gene expression, in ASCs treated with the conditioned medium of primary cardiomyocyte cell cultures for 72h. This result suggests that histone acetylation contributes to the transdifferentiation of ASCs towards the cardiac lineage. In order to directly test this hypothesis, ASCs cultured with regular medium were treated with SAHA, a pan histone deacetylase inhibitor. We found that SAHA enhanced the cardiac permissive state of ASCs, increasing both mRNA and protein expression of cardiovascular genes, particularly cTnI. This suggests that histone acetylation induction is sufficient to promote cardiovascular transdifferentiation. Conclusions The control of ASC fate by epigenetic regulators might be an interesting tool to boost both cardiac commitment and regenerative capacities of ASCs.

Adult cardiac progenitor cells (CPCs), isolated as cardiosphere-derived cells (CDCs), represent promising candidates for cardiac regenerative therapy. CDCs can be expanded in vitro manyfolds without losing their differentiation potential, reaching numbers that are appropriate for clinical applications. Since mechanisms of successful CDC survival and engraftment in the damaged myocardium are still critical and unresolved issues, we aimed at deciphering possible key factors capable of bolstering CDC function. In particular, the response and the phenotype of CDCs exposed to low concentrations of the multifunctional cytokine tumor necrosis factor α (TNF-α), known to be capable of activating cell survival pathways, have been investigated. Furthermore, differential biological responses of CDCs from male and female donors, in terms of cell cycle progression and cell spreading, have also been assessed. The results obtained indicate that (i) the intracellular signaling activated in our experimental conditions is most likely due to the prosurvival and proliferative signaling of TNF-α receptor 2 and that (ii) cells from female patients appear more responsive to TNF-α treatment in terms of cell cycle progression and migration ability. In conclusion, the present report highlights the hypothesis that TNF-stimulated CDCs isolated from females may represent a promising candidate for cardiac regenerative therapy applications.

Human resident cardiac progenitor cells (CPCs) isolated as cardiosphere-derived cells (CDCs) are under clinical evaluation as a therapeutic product for cardiac regenerative medicine. Unfortunately, limited engraftment and differentiation potential of transplanted cells significantly hamper therapeutic success. Moreover, maladaptive remodelling of the extracellular matrix (ECM) during heart failure progression provides impaired biological and mechanical signals to cardiac cells, including CPCs. In this study, we aimed at investigating the differential effect on the phenotype of human CDCs of cardiac fibroblast-derived ECM substrates from healthy or diseased hearts, named, respectively, normal or pathological cardiogel (CG-N/P). After 7 days of culture, results show increased levels of cardiogenic gene expression (NKX2.5, CX43) on both decellularized cardiogels compared to control, while the proportion and staining patterns of GATA4, OCT4, NKX2.5, ACTA1, VIM, and CD90-positive CPCs were not affected, as assessed by immunofluorescence microscopy and flow cytometry analyses. Nonetheless, CDCs cultured on CG-N secreted significantly higher levels of osteopontin, FGF6, FGF7, NT-3, IGFBP4, and TIMP-2 compared to those cultured on CG-P, suggesting overall a reduced trophic and antiremodelling paracrine profile of CDCs when in contact with ECM from pathological cardiac fibroblasts. These results provide novel insights into the bidirectional interplay between cardiac ECM and CPCs, potentially affecting CPC biology and regenerative potential.

Oxidative states exert a significant influence on a wide range of biological and molecular processes and functions. When their balance is shifted towards enhanced amounts of free radicals, pathological phenomena can occur, as the generation of reactive oxygen species (ROS) in tissue microenvironment or in the systemic circulation can be detrimental. Epidemic chronic diseases of western societies, such as cardiovascular disease, obesity, and diabetes correlate with the imbalance of redox homeostasis. Current advances in our understanding of epigenetics have revealed a parallel scenario showing the influence of oxidative stress as a major regulator of epigenetic gene regulation via modification of DNA methylation, histones, and microRNAs. This has provided both the biological link and a potential molecular explanation between oxidative stress and cardiovascular/metabolic phenomena. Accordingly, in this review, we will provide current insights on the physiological and pathological impact of changes in oxidative states on cardiovascular disorders, by specifically focusing on the influence of epigenetic regulation. A special emphasis will highlight the effect on epigenetic regulation of human’s current life habits, external and environmental factors, including food intake, tobacco, air pollution, and antioxidant-based approaches. Additionally, the strategy to quantify oxidative states in humans in order to determine which biological marker could best match a subject’s profile will be discussed.

Global aging is a hallmark of our century. The natural multifactorial process resulting in aging involves structural and functional changes, affecting molecules, cells, and tissues. As the western population is getting older, we are witnessing an increase in the burden of cardiovascular events, some of which are known to be directly linked to cellular senescence and dysfunction. In this review, we will focus on the description of a few circulating molecules, which have been correlated to life span, aging, and cardiovascular homeostasis. We will review the current literature concerning the circulating levels and related signaling pathways of selected proteins (insulin-like growth factor 1, growth and differentiation factor-11, and PAI-1) and microRNAs of interest (miR-34a, miR-146a, miR-21), whose bloodstream levels have been associated to aging in different organisms. In particular, we will also discuss their potential role in the biology and senescence of cardiovascular regenerative cell types, such as endothelial progenitor cells, mesenchymal stromal cells, and cardiac progenitor cells.

Respiratory diseases represent a major healthcare burden worldwide. Lung transplantation (LTx) is the "gold standard" for end-stage patients, strongly limited by shortage of available/suitable donor lungs. Normothermic ex vivo lung perfusion (EVLP) has significantly increased the number of lungs suitable for transplantation. Steen solution is used for EVLP, but the mechanisms involved in its beneficial properties remain to be clarified. We investigated the effects of Steen solution in an in vitro protocol of cold starvation and normothermic recovery on human lung spheroids, named pneumospheres (PSs), containing epithelial/basal cells, and on endothelial human umbilical vein endothelial cells (HUVEC). Steen solution significantly preserved the viability of PSs, reduced reactive oxygen species (ROS) release by PSs and HUVECs, decreased NADPH-oxidase (NOX) activity in PSs, and reduced inflammatory cytokines expression levels in HUVECs. Steen solution was able to specifically reduce NADPH oxidase 2 (NOX2) isoform activation, particularly in PSs, as detected by soluble-NOX2 peptide and p47-phosphorylation. Interestingly, a specific NOX2 inhibitor could partly mimic the pro-survival effect of Steen on PSs. We provide the first evidence that Steen solution can preserve lung epithelial/progenitor cells viability partially through NOX2 downregulation, and exert antioxidant effects on parenchymal cells, with consequent ROS reduction. These results suggest that NOX2 inhibition might be an additional strategy to reduce cellular damage during LTx procedures.

The first step in microRNA (miRNA) biogenesis occurs in the nucleus and is mediated by the Microprocessor complex containing the RNase III-like enzyme Drosha and its cofactor DGCR8. Here we show that the 5'-->3' exonuclease Xrn2 associates with independently transcribed miRNAs and, in combination with Drosha processing, attenuates transcription in downstream regions. We suggest that, after Drosha cleavage, a torpedo-like mechanism acts on nascent long precursor miRNAs, whereby Xrn2 exonuclease degrades the RNA polymerase II-associated transcripts inducing its release from the template. While involved in primary transcript termination, this attenuation effect does not restrict clustered miRNA expression, which, in the majority of cases, is separated by short spacers. We also show that transcripts originating from a miRNA promoter are retained on the chromatin template and are more efficiently processed than those produced from mRNA or snRNA Pol II-dependent promoters. These data imply that coupling between transcription and processing promotes efficient expression of independently transcribed miRNAs.

In recent years, exosomes have attracted increasing scientific interest and are no longer considered just as containers for cell waste, but as important mediators of intercellular communication. Among many biomedical research topics, a possible direct role of exosomes in the regenerative medicine field has been underlined in recent studies, including those regarding the so called "paracrine hypothesis". In this perspective, a therapeutic role and/or use of exosomes for tissue regeneration seems to be plausible. However, the majority of the cells isolated and cultured in vitro are exposed to an exogenous exosomes source because of the wide use of foetal bovine serum as cell culture supplement. Bovine serum has been gradually considered as a major biological stimulus, but with still unknown outcome. In this review, we present the state of the art about the role of exosomes in regenerative medicine, particularly for the cardiovascular system. We also analyse the most commonly used exosome isolation techniques that, since their discovery, have undergone continuous development to reach the highest degree of scalability for future clinical translation.

Senescence exerts a great impact on both biological and functional properties of circulating endothelial progenitor cells (EPCs), especially in cardiovascular diseases where the physiological process of aging is accelerated upon clinical administration of certain drugs such as doxorubicin. EPC impairment contributes to doxorubicin-induced cardiotoxicity. Doxorubicin accelerates EPC aging, although mechanisms underlying this phenomenon remain to be fully clarified. Here we investigated if Nox2 activity is able to modulate the premature senescence induced in vitro by doxorubicin in human EPCs. Results showed that in conditioned media obtained from late EPC cultures, the levels of interleukin-6, isoprostanes and nitric oxide bioavailability were increased and reduced respectively after 3h of doxorubicin treatment. These derangements returned to physiological levels when cells were co-treated with apocynin or gp91ds-tat (antioxidant and specific Nox2 inhibitors, respectively). Accordingly, Nox2 activity resulted to be activated by doxorubicin. Importantly, we found that Nox2 inhibition reduced doxorubicin-induced EPC senescence, as indicated by a lower percentage of β-gal positive EPCs. In conclusion, Nox2 activity efficiently contributes to the mechanism of oxidative stress-induced increase in premature aging conferred by doxorubicin. The importance of modulation of Nox2 in human EPCs could reveal a useful tool to restore EPC physiological function and properties.

JAK2 exon 12 mutations are associated with more marked and isolated erythrocytosis than JAK2V617F. We analyzed expression profiles of JAK2 exon 12-mutant and wild-type erythroid colonies from patients with polycythemia vera (PV). Exon 12 mutations were associated with interferon-target gene upregulation, STAT1 activation and additional gene expression changes that were quantitatively and qualitatively similar to those in JAK2V617F-heterozygous cells from essential thrombocythemia (ET) patients. These results demonstrate that JAK2 exon 12-mutated PV does not reflect attenuated STAT1 signaling, and that transcriptional consequences of JAK2 mutations are remarkably similar in JAK2 exon 12-mutated PV and JAK2V617F-positive ET.

Β-blockers (BB) are a primary treatment for chronic heart disease (CHD), resulting in prognostic and symptomatic benefits. Cardiac cell therapy represents a promising regenerative treatment and, for autologous cell therapy, the patients clinical history may correlate with the biology of resident progenitors and the quality of the final cell product. This study aimed at uncovering correlations between clinical records of biopsy-donor CHD patients undergoing cardiac surgery and the corresponding yield and phenotype of cardiospheres (CSs) and CS-derived cells (CDCs), which are a clinically relevant population for cell therapy, containing progenitors. We describe a statistically significant association between BB therapy and improved CSs yield and CDCs phenotype. We show that BB-CDCs have a reduced fibrotic-like CD90 + subpopulation, with reduced expression of collagen-I and increased expression of cardiac genes, compared to CDCs from non-BB donors. Moreover BB-CDCs had a distinctive microRNA expression profile, consistent with reduced fibrotic features (miR-21, miR-29a/b/c downregulation), and enhanced regenerative potential (miR-1, miR-133, miR-101 upregulation) compared to non-BB. In vitro adrenergic pharmacological treatments confirmed cytoprotective and anti-fibrotic effects of β1-blocker on CDCs. This study shows anti-fibrotic and pro-commitment effects of BB treatment on endogenous cardiac reparative cells, and suggests adjuvant roles of β-blockers in cell therapy applications.

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.

We welcome the paper by Wever-Pinzon et al. [(1)][1] who investigated the longitudinal effects of left ventricular assist device (LVAD) unloading on cardiac function. Notably, subjects who experienced cardiac recovery more frequently took beta-blockers (BBs), which have an established

Retinoic acid (RA), the major bioactive metabolite of retinol or vitamin A, induces a spectrum of pleiotropic effects in cell growth and differentiation that are relevant for embryonic development and adult physiology. The RA activity is mediated primarily by members of the retinoic acid receptor (RAR) subfamily, namely RARα, RARβ and RARγ, which belong to the nuclear receptor (NR) superfamily of transcription factors. RARs form heterodimers with members of the retinoid X receptor (RXR) subfamily and act as ligand-regulated transcription factors through binding specific RA response elements (RAREs) located in target genes promoters. RARs also have non-genomic effects and activate kinase signaling pathways, which fine-tune the transcription of the RA target genes. The disruption of RA signaling pathways is thought to underlie the etiology of a number of hematological and non-hematological malignancies, including leukemias, skin cancer, head/neck cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, liver cancer, glioblastoma and neuroblastoma. Of note, RA and its derivatives (retinoids) are employed as potential chemotherapeutic or chemopreventive agents because of their differentiation, anti-proliferative, pro-apoptotic, and anti-oxidant effects. In humans, retinoids reverse premalignant epithelial lesions, induce the differentiation of myeloid normal and leukemic cells, and prevent lung, liver, and breast cancer. Here, we provide an overview of the biochemical and molecular mechanisms that regulate the RA and retinoid signaling pathways. Moreover, mechanisms through which deregulation of RA signaling pathways ultimately impact on cancer are examined. Finally, the therapeutic effects of retinoids are reported.

The epithelial-to-mesenchymal transition (EMT) is an essential trans-differentiation process, which plays a critical role in embryonic development, wound healing, tissue regeneration, organ fibrosis, and cancer progression. It is the fundamental mechanism by which epithelial cells lose many of their characteristics while acquiring features typical of mesenchymal cells, such as migratory capacity and invasiveness. Depending on the contest, EMT is complemented and balanced by the reverse process, the mesenchymal-to-epithelial transition (MET). In the saving economy of the living organisms, the same (Ying-Yang) tool is integrated as a physiological strategy in embryonic development, as well as in the course of reparative or disease processes, prominently fibrosis, tumor invasion and metastasis. These mechanisms and their related signaling (e.g., TGF-β and BMPs) have been effectively studied in vitro by tissue-derived cell spheroids models. These three-dimensional (3D) cell culture systems, whose phenotype has been shown to be strongly dependent on TGF-β-regulated EMT/MET processes, present the advantage of recapitulating in vitro the hypoxic in vivo micro-environment of tissue stem cell niches and their formation. These spheroids, therefore, nicely reproduce the finely regulated Ying-Yang equilibrium, which, together with other mechanisms, can be determinant in cell fate decisions in many pathophysiological scenarios, such as differentiation, fibrosis, regeneration, and oncogenesis. In this review, current progress in the knowledge of signaling pathways affecting EMT/MET and stemness regulation will be outlined by comparing data obtained from cellular spheroids systems, as ex vivo niches of stem cells derived from normal and tumoral tissues. The mechanistic correspondence in vivo and the possible pharmacological perspective will be also explored, focusing especially on the TGF-β-related networks, as well as others, such as SNAI1, PTEN, and EGR1. This latter, in particular, for its ability to convey multiple types of stimuli into relevant changes of the cell transcriptional program, can be regarded as a heterogeneous "stress-sensor" for EMT-related inducers (growth factor, hypoxia, mechano-stress), and thus as a therapeutic target.