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Astrocytes are essential players in development and functions, being particularly relevant as regulators of brain energy metabolism, ionic homeostasis and synaptic transmission. They are also the major source of l-serine in the brain, which is synthesized from the glycolytic intermediate 3-phosphoglycerate through the phosphorylated pathway. l-Serine is the precursor of the two main co-agonists of the N-methyl-d-aspartate receptor, glycine and d-serine. Strikingly, dysfunctions in both l- and d-serine metabolism are associated with neurological and psychiatric disorders. Here, we exploited a differentiation protocol, based on the generation of human mature astrocytes from neural stem cells, and investigated the modification of the proteomic and metabolomic profile during the differentiation process. We show that differentiated astrocytes are more similar to mature rather than to reactive ones, and that axogenesis and pyrimidine metabolism increase up to 30 days along with the folate cycle and sphingolipid metabolism. Consistent with the proliferation and cellular maturation processes that are taking place, also the intracellular levels of l-serine, glycine, threonine, l- and d-aspartate (which level is unexpectedly higher than that of d-serine) show the same biosynthetic time course. A significant utilization of l-serine from the medium is apparent while glycine is first consumed and then released with a peak at 30 days, parallel to its intracellular level. These results underline how metabolism changes during astrocyte differentiation, highlight that d-serine synthesis is restricted in differentiated astrocytes and provide a valuable model for developing potential novel therapeutic approaches to address brain diseases, especially the ones related to serine metabolism alterations.

Before entering human clinical studies to evaluate their safety and effectiveness, new drugs and novel medical treatments are subject to extensive animal testing that are expensive and time-consuming. By contrast, advanced technologies enable the development of animal-free models that allow the efficacy of innovative therapies to be studied without sacrificing animals, while providing helpful information and details. We report on the powerful combination of 3D bioprinting (3DB) and photo-thermal therapy (PTT) applications. To this end, we realize a 3DB construct consisting of glioblastoma U87-MG cells in a 3D geometry, incorporating biomimetic keratin-coated gold nanoparticles (Ker-AuNPs) as a photo-thermal agent. The resulting plasmonic 3DB structures exhibit a homogeneous cell distribution throughout the entire volume while promoting the localization of Ker-AuNPs within the cells. A 3D immunofluorescence assay and transmission electron microscopy (TEM) confirm the uniform distribution of fluorescent-labeled Ker-AuNPs in the volume and their capability to enter the cells. Laser-assisted (λ = 532 nm) PTT experiments demonstrate the extraordinary ability of Ker-AuNPs to generate heating, producing the highest temperature rise of about 16 °C in less than 2 min.

Health visitors (HVs) and environmental health officers (EHOs) are the healthcare workers (HCWs) who, in the Italian National Health Service, mainly operate in the prevention departments of local health authorities, guaranteeing the territorial activities specifically declared with the respective professional profiles. During the SARS-CoV-2 pandemic, it was necessary to reallocate all HCWs supporting Hygiene and Public Health Services involved on the front lines of the emergency, in order to perform preventive activities and to take immediate action to fight the spread of the virus. By means of an IT survey consisting of three sections, this study investigated how 960 HVs and EHOs dealt with this reallocation, with the shifting in service assignment, and with the perceived level of fatigue and pressure, through the application of skills acquired from university training. The synergy among the preventive health professions, the ability to work in a multi-professional team, and the complementary training of HCWs represent the main strengths for overcoming future public health challenges, aimed at protecting human health.

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.

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.

Radiological imaging is currently employed as the most effective technique for screening, diagnosis, and follow up of patients with breast cancer (BC), the most common type of tumor in women worldwide. However, the introduction of the omics sciences such as metabolomics, proteomics, and molecular genomics, have optimized the therapeutic path for patients and implementing novel information parallel to the mutational asset targetable by specific clinical treatments. Parallel to the “omics” clusters, radiological imaging has been gradually employed to generate a specific omics cluster termed “radiomics”. Radiomics is a novel advanced approach to imaging, extracting quantitative, and ideally, reproducible data from radiological images using sophisticated mathematical analysis, including disease-specific patterns, that could not be detected by the human eye. Along with radiomics, radiogenomics, defined as the integration of “radiology” and “genomics”, is an emerging field exploring the relationship between specific features extracted from radiological images and genetic or molecular traits of a particular disease to construct adequate predictive models. Accordingly, radiological characteristics of the tissue are supposed to mimic a defined genotype and phenotype and to better explore the heterogeneity and the dynamic evolution of the tumor over the time. Despite such improvements, we are still far from achieving approved and standardized protocols in clinical practice. Nevertheless, what can we learn by this emerging multidisciplinary clinical approach? This minireview provides a focused overview on the significance of radiomics integrated by RNA sequencing in BC. We will also discuss advances and future challenges of such radiomics-based approach.

Large-animal models for Duchenne muscular dystrophy (DMD) are crucial for the evaluation of diagnostic procedures and treatment strategies. Pigs cloned from male cells lacking DMD exon 52 (DMDΔ52) exhibit molecular, clinical and pathological hallmarks of DMD, but die before sexual maturity and cannot be propagated by breeding. Therefore, we generated female DMD+/− carriers. A single founder animal had 11 litters with 29 DMDY/−, 34 DMD+/− as well as 36 male and 29 female wild-type offspring. Breeding with F1 and F2 DMD+/− carriers resulted in an additional 114 DMDY/− piglets. With intensive neonatal management, the majority survived for 3-4 months, providing statistically relevant cohorts for experimental studies. Pathological investigations and proteome studies of skeletal muscles and myocardium confirmed the resemblance to human disease mechanisms. Importantly, DMDY/− pigs displayed progressive myocardial fibrosis and increased expression of connexin-43, associated with significantly reduced left ventricular ejection fraction, at 3 months. Furthermore, behavioral tests provided evidence for impaired cognitive ability. Our breeding cohort of DMDΔ52 pigs and standardized tissue repositories provide important resources for studying DMD disease mechanisms and for testing novel treatment strategies., Editor's choice: A genetically tailored pig model that develops the hallmarks of Duchenne muscular dystrophy in an accelerated mode provides a new resource for testing targeted therapies.

Large animal models for Duchenne muscular dystrophy (DMD) are crucial for preclinical evaluation of novel diagnostic procedures and treatment strategies. Pigs cloned from male cells lackingDMDexon 52 (DMDΔ52) resemble molecular, clinical and pathological hallmarks of DMD, but cannot be propagated by breeding due to death before sexual maturity. Therefore, femaleDMD+/-carriers were generated. A single founder animal had 11 litters with 29DMDY/-, 34DMD+/-as well as 36 male and 29 female wild-type (WT) offspring. Breeding with F1 and F2DMD+/-carriers resulted in additional 114DMDY/-piglets. The majority of them survived for 3-4 months, providing large cohorts for experimental studies. Pathological investigations and proteome studies of skeletal muscles and myocardium confirmed the resemblance of human disease mechanisms. Importantly,DMDY/-pigs reveal progressive fibrosis of myocardium and increased expression of connexin-43, associated with significantly reduced left ventricular fractional shortening and ejection fraction already at age 3 months. Furthermore, behavioral tests provided evidence for impaired cognitive ability ofDMDY/-pigs. Our breeding cohort ofDMDΔ52 pigs and standardized tissue repositories fromDMDY/-pigs,DMD+/-carriers, and WT littermate controls provide important resources for studying DMD disease mechanisms and for testing novel diagnostic procedures and treatment strategies.

The Cdkn2a locus is one of the most studied tumor suppressor loci in the context of several cancer types. However, in the last years, its expression has also been linked to terminal differentiation and the activation of the senescence program in different cellular subtypes. Knock-out (KO) of the entire locus enhances the capability of stem cells to proliferate in some tissues and respond to severe physiological and non-physiological damages in different organs, including the heart. Emery–Dreifuss muscular dystrophy (EDMD) is characterized by severe contractures and muscle loss at the level of skeletal muscles of the elbows, ankles and neck, and by dilated cardiomyopathy. We have recently demonstrated, using the LMNA Δ8–11 murine model of Emery–Dreifuss muscular dystrophy (EDMD), that dystrophic muscle stem cells prematurely express non-lineage-specific genes early on during postnatal growth, leading to rapid exhaustion of the muscle stem cell pool. Knock-out of the Cdkn2a locus in EDMD dystrophic mice partially restores muscle stem cell properties. In the present study, we describe the cardiac phenotype of the LMNA Δ8–11 mouse model and functionally characterize the effects of KO of the Cdkn2a locus on heart functions and life expectancy.

Alterations in the DMD gene, which codes for the protein dystrophin, cause forms of dystrophinopathies such as Duchenne muscular dystrophy, an X-linked disease. Cardiomyopathy linked to DMD mutations is becoming the leading cause of death in patients with dystrophinopathy. Since phenotypic pathophysiological mechanisms are not fully understood, the improvement and development of new disease models, considering their relative advantages and disadvantages, is essential. The application of genetic engineering approaches on induced pluripotent stem cells, such as gene-editing technology, enables the development of physiologically relevant human cell models for in vitro dystrophinopathy studies. The combination of induced pluripotent stem cells-derived cardiovascular cell types and 3D bioprinting technologies hold great promise for the study of dystrophin-linked cardiomyopathy. This combined approach enables the assessment of responses to physical or chemical stimuli, and the influence of pharmaceutical approaches. The critical objective of in vitro microphysiological systems is to more accurately reproduce the microenvironment observed in vivo. Ground-breaking methodology involving the connection of multiple microphysiological systems comprised of different tissues would represent a move toward precision body-on-chip disease modelling could lead to a critical expansion in what is known about inter-organ responses to disease and novel therapies that have the potential to replace animal models. In this review, we will focus on the generation, development, and application of current cellular, animal, and potential for bio-printed models, in the study of the pathophysiological mechanisms underlying dystrophin-linked cardiomyopathy in the direction of personalized medicine.

Tensions between different religious beliefs are often on the agenda with their ferocious degenerations, discriminations, and radicalizations: “universalistic” visions clash with the need of every credo to be alternative to the others, and so in contrast. This ambiguity is contained in the word “religion” itself. In most European languages two meanings can be associated to this word, through its Latin roots: first, to tie, knotting together human and divine (religare: to connect), and second to collect around a mode (a rite), chosen to guarantee and verify an identity (religere: to distinguish). Also, spaces hosting rites live on this ambiguity: they are dedicated to gather the broadest and inclusive community, and at the same time they become the sign of difference and limit for every different behavior, in an idea of sacred as secluded and forbidden. Contemporary society amplifies these problems: the mobility of things, ideas, and people intensify potential tensions, and at the same time it opens to places and possibilities to verify new ways of living the religious experience. For this reason, the need of finding new open configurations for every place of worship is accompanied by the urgency of experimenting with sharing places for religious practices in a situation of diversity. We think above all to places of culture and higher education, or infrastructures of transports, but also to community based on the communion of the religious experience. Architecture, especially in a declination careful to people and its gestures, can answer this question through places able to synthesize undeniable specificities and possible sharing. We propose a dissertation developing in parallel theoretical questions, historical case studies, and design evaluation carried on through the university design didactics.

Extracellular vesicles (EVs) have become a key tool in the biotechnological landscape due to their well-documented ability to mediate intercellular communication. This feature has been explored and is under constant investigation by researchers, who have demonstrated the important role of EVs in several research fields ranging from oncology to immunology and diagnostics to regenerative medicine. Unfortunately, there are still some limitations to overcome before clinical application, including the inability to confine the EVs to strategically defined sites of interest to avoid side effects. In this study, for the first time, EV application is supported by 3D bioprinting technology to develop a new strategy for applying the angiogenic cargo of human umbilical vein endothelial cell-derived EVs in regenerative medicine. EVs, derived from human endothelial cells and grown under different stressed conditions, were collected and used as bioadditives for the formulation of advanced bioinks. After in vivo subcutaneous implantation, we demonstrated that the bioprinted 3D structures, loaded with EVs, supported the formation of a new functional vasculature in situ, consisting of blood-perfused microvessels recapitulating the printed pattern. The results obtained in this study favour the development of new therapeutic approaches for critical clinical conditions, such as the need for prompt revascularization of ischaemic tissues, which represent the fundamental substrate for advanced regenerative medicine applications.

The Cdkn2a locus is one of the most studied tumor suppressor loci in the context of several cancer types. However, in the last years, its expression has also been linked to terminal differentiation and the activation of the senescence program in different cellular subtypes. Knock-out (KO) of the entire locus enhances the capability of stem cells to proliferate in some tissues and respond to severe physiological and non-physiological damages in different organs, including the heart. Emery–Dreifuss muscular dystrophy (EDMD) is characterized by severe contractures and muscle loss at the level of skeletal muscles of the elbows, ankles and neck, and by dilated cardiomyopathy. We have recently demonstrated, using the LMNA Δ8–11 murine model of Emery–Dreifuss muscular dystrophy (EDMD), that dystrophic muscle stem cells prematurely express non-lineage-specific genes early on during postnatal growth, leading to rapid exhaustion of the muscle stem cell pool. Knock-out of the Cdkn2a locus in EDMD dystrophic mice partially restores muscle stem cell properties. In the present study, we describe the cardiac phenotype of the LMNA Δ8–11 mouse model and functionally characterize the effects of KO of the Cdkn2a locus on heart functions and life expectancy.

Expression of the β-myosin heavy chain (β-MHC), a major component of the cardiac contractile apparatus, is tightly regulated as even modest increases can be detrimental to heart under stress. In healthy hearts, continuous inhibition of β-adrenergic tone upregulates β-MHC expression. However, it is unknown whether the duration of the β-adrenergic inhibition and β-MHC expression are related. Here, we evaluated the effects of intermittent β-blockade on cardiac β-MHC expression. To this end, the β-blocker propranolol, at the dose of 15mg/kg, was administered once a day in mice for 14 days. This dosing schedule caused daily drug-free periods of at least 6 h as evidenced by propranolol plasma concentrations and cardiac β-adrenergic responsiveness. Under these conditions, β-MHC expression decreased by about 75% compared to controls. This effect was abrogated in mice lacking β1- but not β2-adrenergic receptors (β-AR) indicating that β-MHC expression is regulated in a β1-AR-dependent manner. In β1-AR knockout mice, the baseline β-MHC expression was fourfold higher than in wild-type mice. Also, we evaluated the impact of intermittent β-blockade on β-MHC expression in mice with systolic dysfunction, in which an increased β-MHC expression occurs. At 3 weeks after myocardial infarction, mice showed systolic dysfunction and upregulation of β-MHC expression. Intermittent β-blockade decreased β-MHC expression while attenuating cardiac dysfunction. In vitro studies showed that propranolol does not affect β-MHC expression on its own but antagonizes catecholamine effects on β-MHC expression. In conclusion, a direct relationship occurs between the duration of the β-adrenergic inhibition and β-MHC expression through the β1-AR.

The recent advances, offered by cell therapy in the regenerative medicine field, offer a revolutionary potential for the development of innovative cures to restore compromised physiological functions or organs. Adult myogenic precursors, such as myoblasts or satellite cells, possess a marked regenerative capacity, but the exploitation of this potential still encounters significant challenges in clinical application, due to low rate of proliferation in vitro, as well as a reduced self-renewal capacity. In this scenario, induced pluripotent stem cells (iPSCs) can offer not only an inexhaustible source of cells for regenerative therapeutic approaches, but also a valuable alternative for in vitro modeling of patient-specific diseases. In this study we established a reliable protocol to induce the myogenic differentiation of iPSCs, generated from pericytes and fibroblasts, exploiting skeletal muscle-derived extracellular vesicles (EVs), in combination with chemically defined factors. This genetic integration-free approach generates functional skeletal myotubes maintaining the engraftment ability in vivo. Our results demonstrate evidence that EVs can act as biological &ldquo, shuttles&rdquo, to deliver specific bioactive molecules for a successful transgene-free differentiation offering new opportunities for disease modeling and regenerative approaches.

Reactive oxygen species (ROS) play a pivotal role in different pathologic conditions, including ischemia, diabetes, and aging. We previously showed that ROS enhance miR-200c expression, causing endothelial cell (EC) apoptosis and senescence. Herein, we dissect the interaction among miR-200c and three strictly related proteins that modulate EC function and ROS production: sirtuin 1 (SIRT1), endothelial nitric oxide synthase (eNOS), and forkhead box O1 (FOXO1). Moreover, the role of miR-200c on ROS modulation was also investigated.We demonstrated that miR-200c directly targets SIRT1, eNOS, and FOXO1; via this mechanism, miR-200c decreased NO and increased the acetylation of SIRT1 targets, that is, FOXO1 and p53. FOXO1 acetylation inhibited its transcriptional activity on target genes, that is, SIRT1 and the ROS scavengers, catalase and manganese superoxide dismutase. In keeping, miR-200c increased ROS production and induced p66Shc protein phosphorylation in Ser-36; this mechanism upregulated ROS and inhibited FOXO1 transcription, reinforcing this molecular circuitry. These in vitro results were validated in three in vivo models of oxidative stress, that is, human skin fibroblasts from old donors, femoral arteries from old mice, and a murine model of hindlimb ischemia. In all cases, miR-200c was higher versus control and its targets, that is, SIRT1, eNOS, and FOXO1, were downmodulated. In the mouse hindlimb ischemia model, anti-miR-200c treatment rescued these targets and improved limb perfusion. Innovation and Conclusion: miR-200c disrupts SIRT1/FOXO1/eNOS regulatory loop. This event promotes ROS production and decreases NO, contributing to endothelial dysfunction under conditions of increased oxidative stress such as aging and ischemia. Antioxid. Redox Signal. 27, 328-344.

In the central nervous system, oligodendrocytes are the glial element in charge of myelin formation. Obtaining an overall presence of oligodendrocyte precursor cells/oligodendrocytes. (OPCs/OLs) in culture from different sources of NSCs is an important research area, because OPCs/OLs may provide a promising therapeutic strategy for diseases affecting myelination of axons. The present study was designed to differentiate human olfactory bulb NSCs (OBNSCs) into OPCs/OLs and using expression profiling (RT-qPCR) gene, immunocytochemistry and specific protein expression to highlight molecular mechanism(s) underlying differentiation of human OBNSCs into OPCs/OLs. The differentiation of OBNSCs was characterized by a simultaneous appearance of neurons and glial cells. The differentiation medium, containing cAMP, PDGFA, T3, and all-trans-retinoic acid (RA), promotes OBNSCs to generate mostly oligodendrocytes displaying morphological changes and appearance of long cytoplasmic processes. OBNSCs showed, after five days in oligodendrocytes differentiation medium, a considerable decrease in the number of nestin positive cells, which was associated with a concomitant increase of NG2 immunoreactive cells and few O4(+)-OPCs. In addition, a significant up regulation in gene and protein expression profile of stage specific cell markers for OPCs/OLs (CNPase, Galc, NG2, MOG, OLIG1, OLIG2, MBP), neurons and astrocytes (MAP2, ?-TubulinIII, GFAP) and concomitant decrease of OBNSCs pluripotency markers (Oct4, Sox2, Nestin), was demonstrated following induction of OBNSCs differentiation. Taken together, the present study demonstrate the marked ability of a cocktail of factors containing PDGFA, T3, cAMP and ATRA, to induce OBNSCs differentiation into OPCs/OLs and shed light on the key genes and pathological pathways involved in this process. This article is protected by copyright. All rights reserved.

The limited number of resins, available for stereolithography applications, is one of the key drivers in research applied to rapid prototyping. In this work an acrylic photocrosslinkable resin based on methyl methacrylate (MMA), butyl methacrylate (BMA) and poly(ethylene glycol) dimethacrylate (PEGDA) was developed with different composition and characterized in terms of mechanical, thermal and biological behaviour. Two different systems have been developed using different amount of reagent. The influence of every components have been evaluated on the final characteristic of the resin in order to optimize the final composition for applications in bone tissue engineering. The crosslinked materials showed good mechanical properties and thermal stabilities and moreover cytotoxicity test confirms good biocompatibility with no cytotoxic effect on cells metabolism. Moreover two different treatments have been proposed, using fetal bovine serum (FBS) and methanol (MeOH), in order to improve cell recognition of the surfaces. Samples threatened with MeOH allow cell adhesion and survival, promoting spreading, elongation and fusion of C2C12 muscle myoblast cells., Graphical abstract Image 1, Highlights • Photocrosslinkable biocompatible resin for application in tissue engineering. • Surface treatment to improve materials wettability. • Myoblast spreading and elongation on photocrosslinked modified surfaces.

In mammals, glucocorticoid levels rise dramatically shortly before birth and prepare the foetus for post-natal life by promoting the maturation of the lungs and other organs. However, their impact on cardiac postnatal growth and regenerative plasticity is unknown.Here, we demonstrate that exposure to endogenous glucocorticoids facilitates cell cycle exit and reduces the proliferation of neonatal cardiomyocytes. This cytostatic activity is shared by several synthetic glucocorticoid receptor (GR) agonists routinely used in clinical settings. We also observed that GR levels increase in cardiomyocytes during early post-natal development. Importantly, in vivo cardiomyocyte-specific GR ablation delayed the transition from hyperplastic (increase in cell number) to hypertrophic (increase in cell size) growth. Further, GR ablation partially impaired cardiomyocyte maturation, reducing myofibrils-mitochondria organization along with the expression of genes involved in fatty acid metabolism, mitochondrial respiration and energy transfer from mitochondria to the cytosol. Finally, we show increased cardiomyocyte proliferation in GR ablated juvenile and adult cardiomyocytes in response to myocardial infarction in vivo, thus promoting cardiac tissue regeneration.We suggest that GR antagonization could serve as a strategy for heart regeneration based on endogenous cardiomyocyte renewal.

SummaryBackground and Purpose Propranolol is a vasoactive drug which shows antiangiogenic and antitumour activities in melanoma. However, it is unknown whether these activities are dose dependent and whether there is a relationship between systemic vascular effects of propranolol and anti-melanoma activity. Experimental approach Effects of increasing doses of propranolol (10, 20, 30 and 40 mg kg-1day-1) on tumour growth were studied in B16F10 melanoma-bearing mice. Histological and biochemical analyses were used to assess propranolol effects on angiogenesis and cancer cell proliferation. Systemic vascular resistance (SVR) was evaluated by measuring cardiac output and arterial blood pressure. Key results In vitro analyses revealed that B16F10 cells express β-adrenoceptors, but neither isoproterenol, a β-adrenoceptor agonist, nor the β-blocker propranolol affect cancer cell proliferation. In vivo studies showed that the antitumour efficacy of propranolol follows a U-shaped biphasic dose-response curve. Low doses (10 and 20 mg kg-1day-1) significantly inhibit tumour growth, whereas higher doses are progressively less effective. We also found that high-dose propranolol stimulates tumour arteriogenesis whereas no effect on angiogenesis was observed at any dose. Based on these data and considering that propranolol is a vasoactive drug, we hypothesized that it causes systemic vasoconstriction or vasodilation depending on the dose and thus alters tumour perfusion and growth. Consistent with this hypothesis, we found that propranolol has a biphasic effect on SVR with low and high doses producing vasoconstriction and vasodilation, respectively. Conclusions and Implications Propranolol inhibits melanoma growth in a U-shaped biphasic manner. A direct relationship exists between SVR and anti-melanoma activity.

The Notch receptor mediates cell fate decision in multiple organs. In the current work we tested the hypothesis that Nkx2.5 is a target gene of Notch1 and raised the possibility that Notch1 regulates myocyte commitment in the adult heart. Cardiac progenitor cells (CPCs) in the niches express Notch1 receptor, and the supporting cells exhibit the Notch ligand Jagged1. The nuclear translocation of Notch1 intracellular domain (N1ICD) up-regulates Nkx2.5 in CPCs and promotes the formation of cycling myocytes in vitro. N1ICD and RBP-Jk form a protein complex, which in turn binds to the Nkx2.5 promoter initiating transcription and myocyte differentiation. In contrast, transcription factors of vascular cells are down-regulated by Jagged1 activation of the Notch1 pathway. Importantly, inhibition of Notch1 in infarcted mice impairs the commitment of resident CPCs to the myocyte lineage opposing cardiomyogenesis. These observations indicate that Notch1 favors the early specification of CPCs to the myocyte phenotype but maintains the newly formed cells in a highly proliferative state. Dividing Nkx2.5-positive myocytes correspond to transit amplifying cells, which condition the replicative capacity of the heart. In conclusion, Notch1 may have critical implications in the control of heart homeostasis and its adaptation to pathologic states.

Mechanistic target of rapamycin (MTOR) plays a critical role in the regulation of cell growth and in the response to energy state changes. Drugs inhibiting MTOR are increasingly used in antineoplastic therapies. Myocardial MTOR activity changes during hypertrophy and heart failure (HF). However, whether MTOR exerts a positive or a negative effect on myocardial function remains to be fully elucidated. Here, we show that ablation of Mtor in the adult mouse myocardium results in a fatal, dilated cardiomyopathy that is characterized by apoptosis, autophagy, altered mitochondrial structure, and accumulation of eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1). 4E-BP1 is an MTOR-containing multiprotein complex-1 (MTORC1) substrate that inhibits translation initiation. When subjected to pressure overload, Mtor-ablated mice demonstrated an impaired hypertrophic response and accelerated HF progression. When the gene encoding 4E-BP1 was ablated together with Mtor, marked improvements were observed in apoptosis, heart function, and survival. Our results demonstrate a role for the MTORC1 signaling network in the myocardial response to stress. In particular, they highlight the role of 4E-BP1 in regulating cardiomyocyte viability and in HF. Because the effects of reduced MTOR activity were mediated through increased 4E-BP1 inhibitory activity, blunting this mechanism may represent a novel therapeutic strategy for improving cardiac function in clinical HF.

This paper aims to demonstrate the possibility of exploiting poly-L-lactic acid (PLLA) ultra-thin films as platforms for bio-hybrid actuation. Firstly, flat PLLA nanofilms at different concentrations (15 and 25 mg/ml in dichloromethane) were tested with contractile cardiomyocytes. The results obtained using motion vector analysis, a non-invasive method capable of estimating flow velocities on recorded videos, demonstrated that PLLA nanofilms were able to move under the contraction of muscle cells. Immunofluorescence images reflected good cell spreading, thus confirming that these films are promising matrices for bio-hybrid actuation. Subsequently, microgrooved PLLA nanofilms were fabricated, in order to drive muscle cell distribution on an anisotropic surface, thus optimizing the system's efficiency. After matrix characterization, in terms of AFM and SEM imaging, we investigated the viability and morphology of C2C12 skeletal muscle cells (a more controllable muscle cell type), 24 h after cell seeding as well as at the 7-day differentiation state.

The recognition that the adult heart continuously renews its myocyte compartment raises the possibility that the age and lifespan of myocytes does not coincide with the age and lifespan of the organ and organism. If this were the case, myocyte turnover would result at any age in a myocardium composed by a heterogeneous population of parenchymal cells which are structurally integrated but may contribute differently to myocardial performance. To test this hypothesis, left ventricular myocytes were isolated from mice at 3 months of age and the contractile, electrical, and calcium cycling characteristics of these cells were determined together with the expression of the senescence-associated protein p16 INK4a and telomere length. The heart was characterized by the coexistence of young, aged, and senescent myocytes. Old nonreplicating, p16 INK4a -positive, hypertrophied myocytes with severe telomeric shortening were present together with young, dividing, p16 INK4a -negative, small myocytes with long telomeres. A class of myocytes with intermediate properties was also found. Physiologically, evidence was obtained in favor of the critical role that action potential (AP) duration and I CaL play in potentiating Ca 2+ cycling and the mechanical behavior of young myocytes or in decreasing Ca 2+ transients and the performance of senescent hypertrophied cells. The characteristics of the AP appeared to be modulated by the transient outward K + current I to which was influenced by the different expression of the K + channels subunits. Collectively, these observations at the physiological and structural cellular level document that by necessity the heart has to constantly repopulate its myocyte compartment to replace senescent poorly contracting myocytes with younger more efficient cells. Thus, cardiac homeostasis and myocyte turnover regulate cardiac function.

The possibility of socially stressing the dominant/aggressive member of a pair of male mice is tested. Male mice (NMRI outbreed strain) were housed in pairs to assess dominant and subordinate roles by agonistic interactions and urine-marking test. Social stress for dominant males consisted in 30 min/day of exposure to their subordinate partner interacting with a female in the adjacent compartment of the cage, for 9 days. Results showed that dominance status was maintained. Behavioural observations indicated that neither the subordinates nor the dominant males habituated to this experimental procedure. At the end of the chronic stress, dominant animals were given the opportunity to interact for 30 min with a female in their compartment. Results indicated that stressed dominants showed impairment in their sexual behaviour and were more oriented towards the physical environment in comparison with control dominants. The behavioural response to apomorphine (0.25 mg/kg) indicated an alteration of the dopaminergic functioning in socially stressed dominant mice. This study suggests that the characteristics of the stressor and the effects of the chronic social stress could be different, according to male social status.

Cell-based regenerative therapies are significantly improved by engineering allografts to express factors that increase vascularization and engraftment, such as placental growth factor (PlGF) and matrix metalloproteinase 9 (MMP9). Moreover, the seeding of therapeutic cells onto a suitable scaffold is of utmost importance for tissue regeneration. On these premises, we sought to assess the reparative potential of induced pluripotent stem (iPS) cells bioengineered to secrete PlGF or MMP9 and delivered to infarcted myocardium upon a poly(ethylene glycol)–fibrinogen scaffold. When assessing optimal stiffness of the PEG–fibrinogen (PF) scaffold, we found that the appearance of contracting cells after cardiogenic induction was accelerated on the support designed with an intermediate stiffness. Revascularization and hemodynamic parameters of infarcted mouse heart were significantly improved by injection into the infarct of this optimized PF scaffold seeded with both MiPS (iPS cells engineered to secrete MMP9) and PiPS (iPS cells engineered to secrete PlGF) cells as compared with nonengineered cells or PF alone. Importantly, allograft-derived cells and host myocardium were functionally integrated. Therefore, survival and integration of allografts in the ischemic heart can be significantly improved with the use of therapeutic cells bioengineered to secrete MMP9 and PlGF and encapsulated within an injectable PF hydrogel having an optimized stiffness.

In the relatively new field of cardiac tissue engineering, different biomaterials, methods and techniques have been tested for cardiac repair, but we are still far from the achievement of a valid model that can be tested for therapeutic goals. In this study, the authors examined the efficacy of newly preformed membranes based on collagen type I for the transplantation of cardiac cells. The membrane prototype, cross-linked with 1,4-butanediol diglycidyl ether (BDDGE) and fibronectin-enriched, gave rise to spontaneously beating heart cell constructs, 5–9 days after seeding with neonatal rat cardiac cells. This membrane was grafted, with and without beating cardiac cells, onto the infarcted area of rat models of heart failure. Seriate echocardiography, performed on rats before transplantation and at 4 and 8 weeks after transplantation, demonstrated that rats treated with collagen membranes previously seeded with beating cells showed an improvement in cardiac function after 8 weeks. These results suggest that this new type of collagen membrane can be used as vector for the transplantation of beating heart cells for the regeneration of the injured myocardium and hence represents an important potential tool for cardiac tissue repair technologies.

Adult mammalian cells can be reprogrammed to a pluripotent state by forcing the expression of a few embryonic transcription factors. The resulting induced pluripotent stem (iPS) cells can differentiate into cells of all three germ layers. It is well known that post-natal cardiomyocytes (CMs) lack the capacity to proliferate. Here, we report that neonatal CMs can be reprogrammed to generate iPS cells that express embryonic-specific markers and feature gene-expression profiles similar to those of mouse embryonic stem (mES) cell and cardiac fibroblast (CF)-derived iPS cell populations. CM-derived iPS cells are able to generate chimeric mice and, moreover, re-differentiate toward CMs more efficiently then either CF-derived iPS cells or mES cells. The increased differentiation capacity is possibly related to CM-derived iPS cells retaining an epigenetic memory of the phenotype of their founder cell. CM-derived iPS cells may thus lead to new information on differentiation processes underlying cardiac differentiation and proliferation.

Cell-based therapies represent exciting therapeutic options for myocardial regeneration after ischemic injury. Although many cell types have been proposed as a source of cardiac myocytes, the type of progenitor as well as the optimal conditions for their stimulation still represent major challenges. Moreover, molecular characterization of cardiac progenitors (CP) in vitro is still unsatisfactory: the identification of novel surface markers for their selective isolation is of utmost importance. In our study we employed different human ESCs and induced pluripotent stem (iPS) cells lines to set up a simple, reliable and efficient protocol to selectively induce cardiac fate in vitro. At first, we comparatively applied different methods and monitored the differentiation process by FACS analysis, RT-PCR and immunofluorescence for early and late markers. Our results show that sequential treatments with ActivinA and BMP4 or BMP2, ascorbic acid and TGFβ1 in a specific media formulation induce CD15 + cardiac progenitor population with an enhanced cell vitality compared to other methods. While we confirmed that CD15 is among the earliest markers upregulated after differentiation, the definition of other associated markers could better specify progenitors committed toward cardiomyogenesis. For this purpose, we systematically evaluated induction of other markers already shown associated with cardiac differentiation or heart development, that is PDGFRα, KDR, Sca1, CXCR4, cKIT. Our result demonstrate that a specific population expressing both KDR and CD15 surface markers is more committed toward cardiovascular lineages. The induced KDR + /CD15 + cell population exhibits higher levels of Gata4 and Isl1 expression (2-fold induction), compared to the KDR − /CD15 + counterpart and, after isolation, gives rise to 66.15% Troponin I positive cells (43 out of 65 in a single representative experiment), against the 26.5% (18 out of 68) detectable in the KDR − /CD15 + population. In conclusion, our studies, though preliminary, strongly indicate that KDR in CD15 + progenitor cells is an earlier marker of cardiomyogenesis.

In diabetic patients and animal models of diabetes mellitus (DM), circulating endothelial progenitor cell (EPC) number is lower than in normoglycaemic conditions and EPC angiogenic properties are inhibited. Stromal cell derived factor-1 (SDF-1) plays a key role in bone marrow (BM) c-kit(+) stem cell mobilization into peripheral blood (PB), recruitment from PB into ischemic tissues and differentiation into endothelial cells. The aim of the present study was to examine the effect of DM in vivo and in vitro, on murine BM-derived c-kit(+) cells and on their response to SDF-1. Acute hindlimb ischemia was induced in streptozotocin-treated DM and control mice; circulating c-kit(+) cells exhibited a rapid increase followed by a return to control levels which was significantly faster in DM than in control mice. CXCR4 expression by BM c-kit(+) cells as well as SDF-1 protein levels in the plasma and in the skeletal muscle, both before and after the induction of ischemia, were similar between normoglycaemic and DM mice. However, BM-derived c-kit(+) cells from DM mice exhibited an impaired differentiation towards the endothelial phenotype in response to SDF-1; this effect was associated with diminished protein kinase phosphorylation. Interestingly, SDF-1 ability to induce differentiation of c-kit(+) cells from DM mice was restored when cells were cultured under normoglycaemic conditions whereas c-kit(+) cells from normoglycaemic mice failed to differentiate in response to SDF-1 when they were cultured in hyperglycaemic conditions. These results show that DM diminishes circulating c-kit(+) cell number following hindlimb ischemia and inhibits SDF-1-mediated AKT phosphorylation and differentiation towards the endothelial phenotype of BM-derived c-kit(+) cells.

Rationale : The adult heart possesses a pool of progenitor cells stored in myocardial niches, but the mechanisms involved in the activation of this cell compartment are currently unknown. Objective : Ca 2+ promotes cell growth raising the possibility that changes in intracellular Ca 2+ initiate division of c-kit–positive human cardiac progenitor cells (hCPCs) and determine their fate. Methods and Results : Ca 2+ oscillations were identified in hCPCs and these events occurred independently from coupling with cardiomyocytes or the presence of extracellular Ca 2+ . These findings were confirmed in the heart of transgenic mice in which enhanced green fluorescent protein was under the control of the c-kit promoter. Ca 2+ oscillations in hCPCs were regulated by the release of Ca 2+ from the endoplasmic reticulum through activation of inositol 1,4,5-triphosphate receptors (IP3Rs) and the reuptake of Ca 2+ by the sarco-/endoplasmic reticulum Ca 2+ pump (SERCA). IP3Rs and SERCA were highly expressed in hCPCs, whereas ryanodine receptors were not detected. Although Na + -Ca 2+ exchanger, store-operated Ca 2+ channels and plasma membrane Ca 2+ pump were present and functional in hCPCs, they had no direct effects on Ca 2+ oscillations. Conversely, Ca 2+ oscillations and their frequency markedly increased with ATP and histamine which activated purinoceptors and histamine-1 receptors highly expressed in hCPCs. Importantly, Ca 2+ oscillations in hCPCs were coupled with the entry of cells into the cell cycle and 5-bromodeoxyuridine incorporation. Induction of Ca 2+ oscillations in hCPCs before their intramyocardial delivery to infarcted hearts was associated with enhanced engraftment and expansion of these cells promoting the generation of a large myocyte progeny. Conclusion : IP3R-mediated Ca 2+ mobilization control hCPC growth and their regenerative potential.