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Among all strategies directed at developing new tools to support re-vascularization of damaged tissues, the use of pro-angiogenic soluble factors, derived from mesenchymal stem cells (MSCs), appears a promising approach for regenerative medicine. Here, we compared the feasibility of two devices, generated by coupling soluble factors of human dental pulp mesenchymal stem cells (DPSCs), with a nanostructured scaffold, to support angiogenesis once transplanted in mice. DPSCs were obtained from impacted wisdom tooth removal, usually considered surgical waste material. After 28 days, we verified the presence of active blood vessels inside the scaffold through optical and scansion electron microscopy. The mRNA expression of surface antigens related to macrophage polarization (CD68, CD80, CD86, CD163, CD206), as well as pro-angiogenic markers (CD31, CD34, CD105, Angpt1, Angpt2, CDH5) was evaluated by real-time PCR. Our results demonstrate the capability of DPSC–scaffold and DPSC soluble factors–scaffold to support angiogenesis, similarly to adipose stem cells, whereas the absence of blood vessels was found in the scaffold grafted alone. Our results provide evidence that DPSC-conditioned medium can be proposed as a cell-free preparation able to support angiogenesis, thus, providing a relevant tool to overcome the issues and restrictions associated with the use of cells. [ABSTRACT FROM AUTHOR]

Cardiovascular disease, the leading cause of death worldwide, has been characterized at the molecular level by alterations in gene expression that contribute to the etiology of the disease. Such alterations have been shown to play a critical role in the development of atherosclerosis, cardiac remodeling, and age-related heart failure. Although much is now known about the cellular and molecular mechanisms in this context, the role of epigenetics in the onset of cardiovascular disease remains unclear. Epigenetics, a complex network of mechanisms that regulate gene expression independently of changes to the DNA sequence, has been highly implicated in the loss of homeostasis and the aberrant activation of a myriad of cellular pathways. More specifically, non-coding RNAs have been gaining much attention as epigenetic regulators of various pathologies. In this review, we will provide an overview of the ncRNAs involved in cell-to-cell communication in cardiovascular disease, namely atherosclerosis, cardiac remodeling, and cardiac ageing, and the potential use of epigenetic drugs as novel therapeutic targets. [ABSTRACT FROM AUTHOR]

Several studies have been conducted on the interaction between three-dimensional scaffolds and mesenchymal stem cells for the regeneration of damaged tissues. Considering that stem cells do not survive for sufficient time to directly sustain tissue regeneration, it is essential to develop cell-free systems to be applied in regenerative medicine. In this work, by in vivo experiments, we established that a collagen-nanostructured scaffold, loaded with a culture medium conditioned with mesenchymal stem cells derived from adipose tissue (hASC-CM), exerts a synergic positive effect on angiogenesis, fundamental in tissue regeneration. To this aim, we engrafted athymic BALB-C nude mice with four different combinations: scaffold alone; scaffold with hASCs; scaffold with hASC crude protein extract; scaffold with hASC-CM. After their removal, we verified the presence of blood vessels by optical microscopy and confirmed the vascularization evaluating, by real-time PCR, several vascular growth factors: CD31, CD34, CD105, ANGPT1, ANGPT2, and CDH5. Our results showed that blood vessels were absent in the scaffold grafted alone, while all the other systems appeared vascularized, a finding supported by the over-expression of CD31 and CDH5 mRNA. In conclusion, our data sustain the capability of hASC-CM to be used as a therapeutic cell-free approach for damaged tissue regeneration. [ABSTRACT FROM AUTHOR]

Abstract Background There is an urgent need of neuronal cell models to be applied to high-throughput screening settings while recapitulating physiological and/or pathological events occurring in the Central Nervous System (CNS). Stem cells offer a great opportunity in this direction since their self renewal capacity allows for large scale expansion. Protocols for directed differentiation also promise to generate populations of biochemically homogenous neuronal progenies. NS (Neural Stem) cells are a novel population of stem cells that undergo symmetric cell division in monolayer and chemically defined media, while remaining highly neurogenic. Results We report the full adaptation of the NS cell systems for their growth and neuronal differentiation to 96- and 384-well microplates. This optimized system has also been exploited in homogeneous and high-content assays. Conclusions Our results show that these mouse NS cells may be suitable for a series of applications in high-throughput format.

Background: There is an urgent need of neuronal cell models to be applied to high-throughput screening settings while recapitulating physiological and/or pathological events occurring in the Central Nervous System (CNS). Stem cells offer a great opportunity in this direction since their self renewal capacity allows for large scale expansion. Protocols for directed differentiation also promise to generate populations of biochemically homogenous neuronal progenies. NS (Neural Stem) cells are a novel population of stem cells that undergo symmetric cell division in monolayer and chemically defined media, while remaining highly neurogenic. Results: We report the full adaptation of the NS cell systems for their growth and neuronal differentiation to 96-and 384-well microplates. This optimized system has also been exploited in homogeneous and high-content assays. Conclusions: Our results show that these mouse NS cells may be suitable for a series of applications in high-throughput format. [ABSTRACT FROM AUTHOR]

Abstract: Temozolomide (TMZ) and carmustine (BCNU), cancer-drugs usually used in the treatment of gliomas, are DNA-methylating agents producing O6-methylguanine. It has been shown that 06-methylguanine triggers DNA mismatch repair and in turn induce apoptosis and senescence, respectively, over a 4 and 6 days period [Y. Hirose, M.S. Berger, R.O. Pieper, p53 effects both the duration of G2/M arrest and the fate of temozolomide-treated human glioblastoma cells, Cancer Res. 61 (2001) 1957–1963; W. Roos, M. Baumgartner, B. Kaina, Apoptosis triggered by DNA damage O6-methylguanine in human lymphocytes requires DNA replication and is mediated by p53 and Fas/CD95/Apo-1, Oncogene 23 (2004) 359–367]. Here we show that TMZ and BCNU have an earlier effect on nuclear organization and chromatin structure. In particular, we report that TMZ and BCNU induce clustering of pericentromeric heterochromatin regions and increase the amount of heterochromatic proteins MeCP2 and HP1α bound to chromatin. These drugs also decrease global levels of histone H3 acetylation and increase levels of histone H3 trimethylated on lysine 9 (H3-triMeK9). These events precede the senescence status. We conclude that TMZ and BCNU efficacy in glioma treatment may implicate a first event characterized by changes in heterochromatin organization and its silencing which is then followed by apoptosis and senescence. [Copyright &y& Elsevier]

Np95 is an important determinant in cell cycle progression. Its expression is tightly regulated and becomes detectable shortly before the entry of cells into S phase. Accordingly, Np95 is absolutely required for the G1/S transition. Its continued expression throughout the S/G2/M phases further suggests additional roles. Indeed, Np95 has been implicated in DNA damage response. Here, we show that Np95 is tightly bound to chromatin in vivo and that it binds to histones in vivo and in vitro. The binding to histones is direct and shows a remarkable preference for histone H3 and its N-terminal tail. A novel protein domain, the SRA-YDG domain, contained in Np95 is indispensable both for the interaction with histones and for chromatin binding in vivo. Np95 contains a RING finger. We show that this domain confers E3 ubiquitin ligase activity on Np95, which is specific for core histones, in vitro. Finally, Np95 shows specific E3 activity for histone H3 when the endogenous core octamer, co-immunoprecipitating with Np95, is used as a substrate. Histone ubiquitination is an important determinant in the regulation of chromatin structure and gene transcription. Thus, the demonstration that Np95 is a chromatin-associated ubiquitin ligase suggests possible molecular mechanisms for its action as a cell cycle regulator. [ABSTRACT FROM AUTHOR]

This work is addressed to provide, by in vitro experiments, results on the repercussion that a nanostructured scaffold could have on viability, differentiation and secretion of bioactive factors of human adipose-derived stem cells (hASCs) when used in association to promote angiogenesis, a crucial condition to favour tissue regeneration. To achieve this aim, we evaluated cell viability and morphology by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and microscopy analysis, respectively. We also investigated the expression of some of those genes involved in angiogenesis and differentiation processes utilizing quantitative polymerase chain reaction (qPCR), whereas the amounts of Vascular Endothelial Growth Factor A, Interleukin 6 and Fatty Acid-Binding Protein 4 secreted in the culture medium, were quantified by enzyme-linked immunosorbent assay (ELISA). Results suggested that, in the presence of the scaffold, cell proliferation and the exocytosis of factors involved in the angiogenesis process are reduced; by contrast, the expression of those genes involved in hASC differentiation appeared enhanced. To guarantee cell survival, the construct dimensions are, generally, smaller than clinically required. Furthermore, being the paracrine event the primary mechanism exerting the beneficial effects on injured tissues, the use of conditioned culture medium instead of cells may be convenient. [ABSTRACT FROM AUTHOR]

PTX3 is a component of the humoral arm of innate immunity and an extrinsic oncosuppressor gene taming tumor-promoting inflammation. Here, we show that two enhancers differently regulate PTX3 expression: enhancer 1, located 230 kb upstream of PTX3 promoter, mediated the action of inflammatory transcription factors; and enhancer 2, encompassing PTX3 second exon, was implicated in pre-initiation complex assembly. Polycomb repressive complex 2 silenced these regulatory elements and the promoter in basal condition. Enhancer 1 was epigenetically inactivated in early colorectal cancer (CRC) stages, while the promoter and enhancer 2 showed increasingly DNA methylation during CRC progression from adenomas to stage II and III CRC. Inhibition of DNA methylation rescued PTX3 expression in CRC. Finally, enhancer 1 acquired the binding of STAT3 in stage I CRC, and inhibition of STAT3 phosphorylation restored PTX3 activity and decreased enhancer 1 methylation. Thus, the expression of PTX3 is under the control of two enhancers, which emerge as important fine regulators of PTX3 expression in inflammation and cancer. [ABSTRACT FROM PUBLISHER]

Heart failure (HF) is a leading cause of mortality. Inflammation is implicated in HF, yet clinical trials targeting pro-inflammatory cytokines in HF were unsuccessful, possibly due to redundant functions of individual cytokines. Searching for better cardiac inflammation targets, here we link T cells with HF development in a mouse model of pathological cardiac hypertrophy and in human HF patients. T cell costimulation blockade, through FDA-approved rheumatoid arthritis drug abatacept, leads to highly significant delay in progression and decreased severity of cardiac dysfunction in the mouse HF model. The therapeutic effect occurs via inhibition of activation and cardiac infiltration of T cells and macrophages, leading to reduced cardiomyocyte death. Abatacept treatment also induces production of anti-inflammatory cytokine interleukin-10 (IL-10). IL-10-deficient mice are refractive to treatment, while protection could be rescued by transfer of IL-10-sufficient B cells. These results suggest that T cell costimulation blockade might be therapeutically exploited to treat HF. [ABSTRACT FROM AUTHOR]

Adult vascular smooth muscle cells (VSMCs) dedifferentiate in response to extracellular cues such as vascular damage and inflammation. Dedifferentiated VSMCs are proliferative, migratory, less contractile, and can contribute to vascular repair as well as to cardiovascular pathologies such as intimal hyperplasia/restenosis in coronary artery and arterial aneurysm. We here demonstrate the role of ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) as an epigenetic master regulator of VSMC plasticity. UHRF1 expression correlated with the development of vascular pathologies associated with modulation of noncoding RNAs, such as microRNAs. miR-145 - pivotal in regulating VSMC plasticity, which is reduced in vascular diseases - was found to control Uhrf1 mRNA translation. In turn, UHRF1 triggered VSMC proliferation, directly repressing promoters of cell-cycle inhibitor genes (including p21 and p27) and key prodifferentiation genes via the methylation of DNA and histones. Local vascular viral delivery of Uhrf1 shRNAs or Uhrf1 VSMC-specific deletion prevented intimal hyperplasia in mouse carotid artery and decreased vessel damage in a mouse model of aortic aneurysm. Our study demonstrates the fundamental role of Uhrf1 in regulating VSMC phenotype by promoting proliferation and dedifferentiation. UHRF1 targeting may hold therapeutic potential in vascular pathologies. [ABSTRACT FROM AUTHOR]

Summary PTX3 is an essential component of the humoral arm of innate immunity, playing a nonredundant role in resistance against selected microbes and in the regulation of inflammation. PTX3 activates and regulates the Complement cascade by interacting with C1q and with Factor H. PTX3 deficiency was associated with increased susceptibility to mesenchymal and epithelial carcinogenesis. Increased susceptibility of Ptx3 −/− mice was associated with enhanced macrophage infiltration, cytokine production, angiogenesis, and Trp53 mutations. Correlative evidence, gene-targeted mice, and pharmacological blocking experiments indicated that PTX3 deficiency resulted in amplification of Complement activation, CCL2 production, and tumor-promoting macrophage recruitment. PTX3 expression was epigenetically regulated in selected human tumors (e.g., leiomyosarcomas and colorectal cancer) by methylation of the promoter region and of a putative enhancer. Thus, PTX3, an effector molecule belonging to the humoral arm of innate immunity, acts as an extrinsic oncosuppressor gene in mouse and man by regulating Complement-dependent, macrophage-sustained, tumor-promoting inflammation. Video Abstract [ABSTRACT FROM AUTHOR]

Summary: The epidemic of heart failure has stimulated interest in understanding cardiac regeneration. Evidence has been reported supporting regeneration via transplantation of multiple cell types, as well as replication of postmitotic cardiomyocytes. In addition, the adult myocardium harbors endogenous c-kitpos cardiac stem cells (eCSCs), whose relevance for regeneration is controversial. Here, using different rodent models of diffuse myocardial damage causing acute heart failure, we show that eCSCs restore cardiac function by regenerating lost cardiomyocytes. Ablation of the eCSC abolishes regeneration and functional recovery. The regenerative process is completely restored by replacing the ablated eCSCs with the progeny of one eCSC. eCSCs recovered from the host and recloned retain their regenerative potential in vivo and in vitro. After regeneration, selective suicide of these exogenous CSCs and their progeny abolishes regeneration, severely impairing ventricular performance. These data show that c-kitpos eCSCs are necessary and sufficient for the regeneration and repair of myocardial damage. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) chromatin organization has a key role in defining the transcription program of cells during development. Its alteration is the cause of gene expression changes responsible for several diseases. Thus, we need new tools to study this aspect of gene expression regulation. To this end, ChromEM was recently developed: this is an electron-microscopy staining technique that selectively marks nuclear DNA without altering its structure and, thus, allows better visualization of 3D chromatin conformation. However, despite increasingly frequent application of this staining technique on cells, it has not yet been applied to visualize chromatin ultrastructure in tissues. Here, we provide a protocol to carry out ChromEM on myocardial tissue harvested from the left ventricles of C57BL/6J mice and use this in combination with transmission electron microscopy (TEM) to measure some morphological parameters of peripheral heterochromatin in cardiomyocytes. This protocol could also be used, in combination with electron tomography, to study 3D chromatin organization in cardiomyocytes in different aspects of heart pathobiology (e.g., heart development, cardiac aging, and heart failure) as well as help to set-up ChromEM in other tissues., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Musolino, Pagiatakis, Pierin, Sabatino, Finzi, Gornati, Bernardini and Papait.)

Cell fate transitions observed in embryonic development involve changes in three-dimensional genomic organization that provide proper lineage specification. Whether similar events occur within tumor cells and contribute to cancer evolution remains largely unexplored. We modeled this process in the pediatric cancer Ewing sarcoma and investigated high-resolution looping and large-scale nuclear conformation changes associated with the oncogenic fusion protein EWS-FLI1. We show that chromatin interactions in tumor cells are dominated by highly connected looping hubs centered on EWS-FLI1 binding sites, which directly control the activity of linked enhancers and promoters to establish oncogenic expression programs. Conversely, EWS-FLI1 depletion led to the disassembly of these looping networks and a widespread nuclear reorganization through the establishment of new looping patterns and large-scale compartment configuration matching those observed in mesenchymal stem cells, a candidate Ewing sarcoma progenitor. Our data demonstrate that major architectural features of nuclear organization in cancer cells can depend on single oncogenes and are readily reversed to reestablish latent differentiation programs.

Gene expression is needed for the maintenance of heart function under normal conditions and in response to stress. Each cell type of the heart has a specific program controlling transcription. Different types of stress induce modifications of these programs and, if prolonged, can lead to altered cardiac phenotype and, eventually, to heart failure. The transcriptional status of a gene is regulated by the epigenome, a complex network of DNA and histone modifications. Until a few years ago, our understanding of the role of the epigenome in heart disease was limited to that played by histone deacetylation. But over the last decade, the consequences for the maintenance of homeostasis in the heart and for the development of cardiac hypertrophy of a number of other modifications, including DNA methylation and hydroxymethylation, histone methylation and acetylation, and changes in chromatin architecture, have become better understood. Indeed, it is now clear that many levels of regulation contribute to defining the epigenetic landscape required for correct cardiomyocyte function, and that their perturbation is responsible for cardiac hypertrophy and fibrosis. Here, we review these aspects and draw a picture of what epigenetic modification may imply at the therapeutic level for heart failure.

The post-genomic era has completed its first decade. During this time, we have seen an attempt to understand life not just through the study of individual isolated processes, but through the appreciation of the amalgam of complex networks, within which each process can influence others. Greatly benefiting this view has been the study of the epigenome, the set of DNA and histone protein modifications that regulate gene expression and the function of regulatory non-coding RNAs without altering the DNA sequence itself. Indeed, the availability of reference genome assemblies of many species has led to the development of methodologies such as ChIP-Seq and RNA-Seq that have allowed us to define with high resolution the genomic distribution of several epigenetic elements and to better comprehend how they are interconnected for the regulation of gene expression. In the last few years, the use of these methodologies in the cardiovascular field has contributed to our understanding of the importance of epigenetics in heart diseases, giving new input to this area of research. Here, we review recently acquired knowledge on the role of the epigenome in heart failure, and discuss the need of an epigenomics roadmap for cardiovascular disease.

The epidemic of heart failure has stimulated interest in understanding cardiac regeneration. Evidence has been reported supporting regeneration via transplantation of multiple cell types, as well as replication of postmitotic cardiomyocytes. In addition, the adult myocardium harbors endogenous c-kit(pos) cardiac stem cells (eCSCs), whose relevance for regeneration is controversial. Here, using different rodent models of diffuse myocardial damage causing acute heart failure, we show that eCSCs restore cardiac function by regenerating lost cardiomyocytes. Ablation of the eCSC abolishes regeneration and functional recovery. The regenerative process is completely restored by replacing the ablated eCSCs with the progeny of one eCSC. eCSCs recovered from the host and recloned retain their regenerative potential in vivo and in vitro. After regeneration, selective suicide of these exogenous CSCs and their progeny abolishes regeneration, severely impairing ventricular performance. These data show that c-kit(pos) eCSCs are necessary and sufficient for the regeneration and repair of myocardial damage., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Epigenetic changes occurring during the development of organisms can be altered by the presence of synthetic substances in the environment, resulting in developmental reprogramming and disease. The occurrence of such changes supports the theory that diseases might be cured by altering the epigenetic regulation of gene expression, either directly by modifying the control of the misregulated genes that cause a disease, or indirectly by 'reprogramming' cells toward a 'normal' gene expression pattern. Launched drugs that inhibit DNA methyltransferases or histone deacetylases and lead to epigenetic changes are currently in use for the treatment of cancer. In addition, recent advancements in the understanding of epigenetic mechanisms involved in tumor development and neurodisorders have enabled new approaches for the development of specific epigenetic therapies.

Heterochromatic chromosomal regions undergo large-scale reorganization and progressively aggregate, forming chromocenters. These are dynamic structures that rapidly adapt to various stimuli that influence gene expression patterns, cell cycle progression, and differentiation. Np95-ICBP90 (m- and h-UHRF1) is a histone-binding protein expressed only in proliferating cells. During pericentromeric heterochromatin (PH) replication, Np95 specifically relocalizes to chromocenters where it highly concentrates in the replication factories that correspond to less compacted DNA. Np95 recruits HDAC and DNMT1 to PH and depletion of Np95 impairs PH replication. Here we show that Np95 causes large-scale modifications of chromocenters independently from the H3:K9 and H4:K20 trimethylation pathways, from the expression levels of HP1, from DNA methylation and from the cell cycle. The PHD domain is essential to induce this effect. The PHD domain is also required in vitro to increase access of a restriction enzyme to DNA packaged into nucleosomal arrays. We propose that the PHD domain of Np95-ICBP90 contributes to the opening and/or stabilization of dense chromocenter structures to support the recruitment of modifying enzymes, like HDAC and DNMT1, required for the replication and formation of PH.

Heterochromatin plays an important role in transcriptional repression, for the correct segregation of chromosomes and in the maintenance of genome stability. Pericentric heterochromatin (PH) replication and formation have been proposed to occur in the pericentric heterochromatin duplication body (pHDB). A central question is how the underacetylated state of heterochromatic histone H4 tail is established and controlled, because it is a key event during PH replication and is essential to maintain the compacted and silenced state of these regions. Np95 is a cell cycle regulated and is a nuclear histone-binding protein that also recruits HDAC-1 to target promoters. It is essential for S phase and for embryonic formation and is implicated in chromosome stability. Here we show that Np95 is part of the pHDB, and its functional ablation causes a strong reduction in PH replication. Depletion of Np95 also causes a hyperacetylation of lysines 8, 12, and 16 of heterochromatin histone H4 and an increase of pericentromeric major satellite transcription, whose RNAs are key players for heterochromatin formation. We propose that Np95 is a new relevant protein involved in heterochromatin replication and formation.