Impaired arterial dilation and increased NOX2 generated oxidative stress in subjects with ataxia-telangiectasia mutated (ATM) kinase.

BACKGROUND: Subjects with mutations in the Ataxia-Telangiectasia mutated (ATM) gene encoding for ATM kinase have a greater predisposition to develop atherosclerosis, but the mechanism behind this phenomenon is not yet understood. NADPH oxidase type 2 may play a role in this process, leading to endothelial dysfunction and an increased susceptibility to thrombosis. The purpose of this study was to assess the redox state in individuals with ATM mutations and determine its impact on endothelial function. METHODS: In this cross-sectional study, twenty-seven children with ataxia telangiectasia (AT) (13 males and 14 females, mean age 15.1 ± 7.6 years) were compared with 27 controls (13 males and 14 females, mean age 14.6 ± 8.4 years) matched for age and gender. Additionally, 29 AT parents with heterozygous mutation of ATM (h-ATM) gene, and 29 age- and gender-matched controls were included. Endothelial function was evaluated through brachial flow-mediated dilation (FMD) and the assessment of nitric oxide (NO) bioavailability. Oxidative stress was evaluated by measuring serum activity of soluble NOX2-dp (sNOX2-dp), hydrogen peroxide (H2O2) production, and hydrogen breakdown activity (HBA). Thrombus formation was assessed through the Total Thrombus Formation Analysis System (T-TAS). RESULTS: AT children and parents with heterozygous ATM mutations exhibited significantly lower FMD, HBA, and NO bioavailability as compared to age and gender matched controls. AT children and ATM carrier of heterozygous ATM mutations had significantly higher concentrations of sNOX2-dp and H2O2 as compared to controls. Compared to the respective controls, AT children and their parents, who carried heterozygous ATM mutation, showed an accelerated thrombus growth as revealed by reduced occlusion time. Multivariable linear regression analysis revealed that sNOX2 (standardized coefficient ß: -0.296; SE: 0.044; p = 0.002) and NO bioavailability (standardized coefficient ß: 0.224; SE: 0.065; p = 0.02) emerged as the only independent predictive variables associated with FMD (R2: 0.44). CONCLUSIONS: This study demonstrates that individuals with ATM mutations experience endothelial dysfunction, increased oxidative stress, and elevated thrombus formation. These factors collectively contribute to the heightened susceptibility of these individuals to develop atherosclerosis.

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.

Recent Advances in 3D Cultures.

Methods and protocols for creating complex 3D cell culture systems have been rapidly advancing in the past decade from the perspective of biomaterials [...].

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.

Low-grade endotoxemia is associated with cardiovascular events in community-acquired pneumonia.

OBJECTIVES: Community-acquired pneumonia (CAP) is associated with low-grade endotoxemia but its relationship with cardiovascular events (CVE) has not been investigated. METHODS: We evaluated the incidence of CVE including myocardial infarction, stroke, and cardiovascular death in 523 adult patients hospitalized for CAP. Serum lipopolysaccharide (LPS) and zonulin, a marker of gut permeability, were analyzed in the cohort, that was followed-up during hospitalization and up to 43 months thereafter. RESULTS: During the hospital-stay, 55 patients experienced CVE with a progressive increase from the lowest (0.6%) to highest LPS tertile (23.6%, p < 0.001). Logistic regression analyses showed that higher LPS tertile was independently associated with CVE; LPS significantly correlated with age, hs-CRP and zonulin. In a sub-group of 23 CAP patients, blood E. coli DNA was higher in patients compared to 24 controls and correlated with LPS. During the long-term follow-up, 102 new CVE were registered; the highest tertile of LPS levels was associated with incident CVE; Cox regression analysis showed that LPS tertiles, age, history of CHD, and diabetes independently predicted CVE. CONCLUSIONS: In CAP low-grade endotoxemia is associated to short- and long-term risk of CVE. Further study is necessary to assess if lowering LPS by non-absorbable antibiotics may result in improved outcomes.

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.

Dark Chocolate Intake Positively Modulates Gut Permeability in Elite Football Athletes: A Randomized Controlled Study.

Gut barrier disruption can lead to enhanced intestinal permeability, which allows endotoxins, pathogens, and other proinflammatory substances to move through the intestinal barrier into circulation. Intense exercise over a prolonged period increases intestinal permeability, which can be further worsened by the increased production of reactive oxygen species (ROS) and pro-inflammatory cytokines. The aim of this study was to assess the degree of intestinal permeability in elite football players and to exploit the effect of cocoa polyphenols on intestinal permeability induced by intensive physical exercise. Biomarkers of intestinal permeability, such as circulating levels of zonulin, a modulator of tight junctions, occludin, a tight junction protein, and LPS translocation, were evaluated in 24 elite football players and 23 amateur athletes. Moreover, 24 elite football players were randomly assigned to either a dark chocolate (>85% cocoa) intake (n = 12) or a control group (n = 12) for 30 days in a randomized controlled trial. Biochemical analyses were performed at baseline and after 30 days of chocolate intake. Compared to amateur athletes, elite football players showed increased intestinal permeability as indicated by higher levels of zonulin, occludin, and LPS. After 30 days of dark chocolate intake, decreased intestinal permeability was found in elite athletes consuming dark chocolate. In the control group, no changes were observed. In vitro, polyphenol extracts significantly improved intestinal damage in the human intestinal mucosa cell line Caco-2. These results indicate that chronic supplementation with dark chocolate as a rich source of polyphenols positively modulates exercise-induced intestinal damage in elite football athletes.

Remote Adipose Tissue-Derived Stromal Cells of Patients with Lung Adenocarcinoma Generate a Similar Malignant Microenvironment of the Lung Stromal Counterpart.

Cancer alters both local and distant tissue by influencing the microenvironment. In this regard, the interplay with the stromal fraction is considered critical as this latter can either foster or hamper the progression of the disease. Accordingly, the modality by which tumors may alter distant niches of stromal cells is still unclear, especially at early stages. In this short report, we attempt to better understand the biology of this cross-talk. In our "autologous stromal experimental setting," we found that remote adipose tissue-derived mesenchymal stem cells (mediastinal AMSC) obtained from patients with lung adenocarcinoma sustain proliferation and clonogenic ability of A549 and human primary lung adenocarcinoma cells similarly to the autologous stromal lung counterpart (LMSC). This effect is not observed in lung benign diseases such as the hamartochondroma. This finding was validated by conditioning benign AMSC with supernatants from LAC for up to 21 days. The new reconditioned media of the stromal fraction so obtained, was able to increase cell proliferation of A549 cells at 14 and 21 days similar to that derived from AMSC of patients with lung adenocarcinoma. The secretome generated by remote AMSC revealed overlapping to the corresponding malignant microenvironment of the autologous local LMSC. Among the plethora of 80 soluble factors analyzed by arrays, a small pool of 5 upregulated molecules including IL1-ß, IL-3, MCP-1, TNF-α, and EGF, was commonly shared by both malignant-like autologous A- and L-MSC derived microenvironments vs those benign. The bioinformatics analysis revealed that these proteins were strictly and functionally interconnected to lung fibrosis and proinflammation and that miR-126, 101, 486, and let-7-g were their main targets. Accordingly, we found that in lung cancer tissues and blood samples from the same set of patients here employed, miR-126 and miR-486 displayed the highest expression levels in tissue and blood, respectively. When the miR-126-3p was silenced in A549 treated with AMSC-derived conditioned media from patients with lung adenocarcinoma, cell proliferation decreased compared to control media.

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.

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.

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.

Human platelet lysate-derived extracellular vesicles enhance angiogenesis through miR-126.

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.

Sex-Related Differences in Oxidative, Platelet, and Vascular Function in Chronic Users of Heat-not-Burn vs. Traditional Combustion Cigarettes.

Smoking is still a major cardiovascular risk factor, despite many public awareness campaigns and dedicated interventions. Recently, modified risk products (MRP), e.g., heat-not-burn cigarettes (HNBCs), have been introduced as surrogates of traditional combustion cigarettes (TCCs). Although these products are promoted as healthier than TCCs, few studies have been conducted to assess it. This work is a sex-focused sub-study of a prospective observational study in which apparently healthy chronic TCC smokers were age-matched with regular HNBC users. Blood samples were collected for biochemical assays and blood pressure and flow-mediated dilation (FMD) were measured. Out of 60 subjects, 33 (55%) were women, and 27 (45%) men, with 11 (33%) vs. 9 (33%) non-smokers, respectively, 10 (30%) vs. 10 (37%) TCC smokers, and 12 (36%) vs. 8 (30%) HNBC smokers (p = 0.946). Bivariate and multivariable analyses showed no statistically significant between-sex differences in NO, H2O2, sCD40L, sNox2-dp, sP-selectin, platelet aggregation, cotinine or FMD, overall, in non-smokers, in TCC smokers, or in HNBC smokers (all p > 0.05). HNBCs appeared safer than TCCs when focusing on Nox2-dp (p = 0.026) and sP-selectin (p = 0.050) but had similar levels of the other measured markers. In conclusion, HNBCs have similar detrimental effects on women and men's oxidative stress (H2O2: p = 0.49; sNox2-dp: p = 0.31) and platelet activation (sP-selectin: p = 0.33; platelet aggregation p = 0.87).

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.

The impact of autophagy modulation on phenotype and survival of cardiac stromal cells under metabolic stress.

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.

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.

Inhibition of miR-155 Attenuates Detrimental Vascular Effects of Tobacco Cigarette Smoking.

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 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.

Building an Artificial Cardiac Microenvironment: A Focus on the Extracellular Matrix.

The increased knowledge in cell signals and stem cell differentiation, together with the development of new technologies, such as 3D bioprinting, has made the generation of artificial tissues more feasible for in vitro studies and in vivo applications. In the human body, cell fate, function, and survival are determined by the microenvironment, a rich and complex network composed of extracellular matrix (ECM), different cell types, and soluble factors. They all interconnect and communicate, receiving and sending signals, modulating and responding to cues. In the cardiovascular field, the culture of stem cells in vitro and their differentiation into cardiac phenotypes is well established, although differentiated cardiomyocytes often lack the functional maturation and structural organization typical of the adult myocardium. The recreation of an artificial microenvironment as similar as possible to the native tissue, though, has been shown to partly overcome these limitations, and can be obtained through the proper combination of ECM molecules, different cell types, bioavailability of growth factors (GFs), as well as appropriate mechanical and geometrical stimuli. This review will focus on the role of the ECM in the regulation of cardiac differentiation, will provide new insights on the role of supporting cells in the generation of 3D artificial tissues, and will also present a selection of the latest approaches to recreate a cardiac microenvironment in vitro through 3D bioprinting approaches.

The Noncoding Side of Cardiac Differentiation and Regeneration.

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.