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Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder characterized by ventricular arrhythmias, contractile dysfunctions and fibro-adipose replacement of myocardium. Cardiac mesenchymal stromal cells (CMSCs) participate in disease pathogenesis by differentiating towards adipocytes and myofibroblasts. Some altered pathways in ACM are known, but many are yet to be discovered. We aimed to enrich the understanding of ACM pathogenesis by comparing epigenetic and gene expression profiles of ACM-CMSCs with healthy control (HC)-CMSCs. Methylome analysis identified 74 differentially methylated nucleotides, most of them located on the mitochondrial genome. Transcriptome analysis revealed 327 genes that were more expressed and 202 genes that were less expressed in ACM- vs. HC-CMSCs. Among these, genes implicated in mitochondrial respiration and in epithelial-to-mesenchymal transition were more expressed, and cell cycle genes were less expressed in ACM- vs. HC-CMSCs. Through enrichment and gene network analyses, we identified differentially regulated pathways, some of which never associated with ACM, including mitochondrial functioning and chromatin organization, both in line with methylome results. Functional validations confirmed that ACM-CMSCs exhibited higher amounts of active mitochondria and ROS production, a lower proliferation rate and a more pronounced epicardial-to-mesenchymal transition compared to the controls. In conclusion, ACM-CMSC-omics revealed some additional altered molecular pathways, relevant in disease pathogenesis, which may constitute novel targets for specific therapies.

The metastatic cancer disease represents the real and urgent clinical need in oncology. Therefore, an understanding of the complex molecular mechanisms sustaining the metastatic cascade is critical to advance cancer therapies. Recent studies highlight how redox signaling influences the behavior of metastatic cancer cells, contributes to their travel in bloodstream from the primary tumor to the distant organs and conditions the progression of the micrometastases or their dormant state. Radical oxygen species not only regulate intracellular processes but participate to paracrine circuits by diffusion to nearby cells, thus assuming unpredicted roles in the communication between metastatic cancer cells, blood circulating cells, and stroma cells at site of colonization. Here, we review recent insights in the role of radical oxygen species in the metastasis formation with a special focus on extravasation at metastatic sites. Supplementary Information The online version contains supplementary material available at 10.1007/s10456-021-09779-5.

Background. Choline kinase alpha (CHKA), an essential gene in phospholipid metabolism, is among the modulated MALAT1-targeted transcripts in advanced and metastatic prostate cancer (PCa). Methods. We analyzed CHKA mRNA by qPCR upon MALAT1 targeting in PCa cells, which is characterized by high dose-responsiveness to the androgen receptor (AR) and its variants. Metabolome analysis of MALAT1-depleted cells was performed by quantitative High-resolution 1 H-Nuclear Magnetic Resonance (NMR) spectroscopy. In addition, CHKA genomic regions were evaluated by chromatin immunoprecipitation (ChIP) in order to assess MALAT1-dependent histone-tail modifications and AR recruitment. Results. In MALAT1-depleted cells, the decrease of CHKA gene expression was associated with reduced total choline-containing metabolites compared to controls, particularly phosphocholine (PCho). Upon MALAT1 targeting a significant increase in repressive histone modifications was observed at the CHKA intron-2, encompassing relevant AR binding sites. Combining of MALAT1 targeting with androgen treatment prevented MALAT1-dependent CHKA silencing in androgen-responsive (LNCaP) cells, while it did not in hormone-refractory cells (22RV1 cells). Moreover, AR nuclear translocation and its activation were detected by confocal microscopy analysis and ChIP upon MALAT1 targeting or androgen treatment. Conclusions. These findings support the role of MALAT1 as a CHKA activator through putative association with the liganded or unliganded AR, unveiling its targeting as a therapeutic option from a metabolic rewiring perspective.

The lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) promotes growth and progression in prostate cancer (PCa); however, little is known about its possible impact in PCa metabolism. The aim of this work has been the assessment of the metabolic reprogramming associated with MALAT1 silencing in human PCa cells and in an ex vivo model of organotypic slice cultures (OSCs). Cultured cells and OSCs derived from primary tumors were transfected with MALAT1 specific gapmers. Cell growth and survival, gene profiling, and evaluation of targeted metabolites and metabolic enzymes were assessed. Computational analysis was made considering expression changes occurring in metabolic markers following MALAT1 targeting in cultured OSCs. MALAT1 silencing reduced expression of some metabolic enzymes, including malic enzyme 3, pyruvate dehydrogenase kinases 1 and 3, and choline kinase A. Consequently, PCa metabolism switched toward a glycolytic phenotype characterized by increased lactate production paralleled by growth arrest and cell death. Conversely, the function of mitochondrial succinate dehydrogenase and the expression of oxidative phosphorylation enzymes were markedly reduced. A similar effect was observed in OSCs. Based on this, a predictive algorithm was developed aimed to predict tumor recurrence in a subset of patients. MALAT1 targeting by gapmer delivery restored normal metabolic energy pathway in PCa cells and OSCs.

The lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) promotes growth and progression in prostate cancer (PCa), however, little is known about its possible impact in PCa metabolism. The aim of this work has been the assessment of the metabolic reprogramming associated with MALAT1 silencing in human PCa cells and in an ex vivo model of organotypic slice cultures (OSCs). Cultured cells and OSCs derived from primary tumors were transfected with MALAT1 specific gapmers. Cell growth and survival, gene profiling, and evaluation of targeted metabolites and metabolic enzymes were assessed. Computational analysis was made considering expression changes occurring in metabolic markers following MALAT1 targeting in cultured OSCs. MALAT1 silencing reduced expression of some metabolic enzymes, including malic enzyme 3, pyruvate dehydrogenase kinases 1 and 3, and choline kinase A. Consequently, PCa metabolism switched toward a glycolytic phenotype characterized by increased lactate production paralleled by growth arrest and cell death. Conversely, the function of mitochondrial succinate dehydrogenase and the expression of oxidative phosphorylation enzymes were markedly reduced. A similar effect was observed in OSCs. Based on this, a predictive algorithm was developed aimed to predict tumor recurrence in a subset of patients. MALAT1 targeting by gapmer delivery restored normal metabolic energy pathway in PCa cells and OSCs.

Rationale: Human cardiac mesenchymal cells (CMSCs) are a therapeutically relevant primary cell population. Diabetes mellitus compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes. Objective: To investigate the role of α-ketoglutarate (αKG) in the epimetabolic control of DNA demethylation in CMSCs. Methods and Results: Quantitative global analysis, methylated and hydroxymethylated DNA sequencing, and gene-specific GC methylation detection revealed an accumulation of 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine in the genomic DNA of human CMSCs isolated from diabetic donors. Whole heart genomic DNA analysis revealed iterative oxidative cytosine modification accumulation in mice exposed to high-fat diet (HFD), injected with streptozotocin, or both in combination (streptozotocin/HFD). In this context, untargeted and targeted metabolomics indicated an intracellular reduction of αKG synthesis in diabetic CMSCs and in the whole heart of HFD mice. This observation was paralleled by a compromised TDG (thymine DNA glycosylase) and TET1 (ten–eleven translocation protein 1) association and function with TET1 relocating out of the nucleus. Molecular dynamics and mutational analyses showed that αKG binds TDG on Arg275 providing an enzymatic allosteric activation. As a consequence, the enzyme significantly increased its capacity to remove G/T nucleotide mismatches or 5-formylcytosine. Accordingly, an exogenous source of αKG restored the DNA demethylation cycle by promoting TDG function, TET1 nuclear localization, and TET/TDG association. TDG inactivation by CRISPR/Cas9 knockout or TET/TDG siRNA knockdown induced 5-formylcytosine accumulation, thus partially mimicking the diabetic epigenetic landscape in cells of nondiabetic origin. The novel compound (S)-2-[(2,6-dichlorobenzoyl)amino]succinic acid (AA6), identified as an inhibitor of αKG dehydrogenase, increased the αKG level in diabetic CMSCs and in the heart of HFD and streptozotocin mice eliciting, in HFD, DNA demethylation, glucose uptake, and insulin response. Conclusions: Restoring the epimetabolic control of DNA demethylation cycle promises beneficial effects on cells compromised by environmental metabolic changes.

Organ-specific mesenchymal cells naturally reside in the stroma, where they are exposed to some environmental variables affecting their biology and functions. Risk factors such as diabetes or aging influence their adaptive response. In these cases, permanent epigenetic modifications may be introduced in the cells with important consequences on their local homeostatic activity and therapeutic potential. Numerous results suggest that mesenchymal cells, virtually present in every organ, may contribute to tissue regeneration mostly by paracrine mechanisms. Intriguingly, the heart is emerging as a source of different cells, including pericytes, cardiac progenitors, and cardiac fibroblasts. According to phenotypic, functional, and molecular criteria, these should be classified as mesenchymal cells. Not surprisingly, in recent years, the attention on these cells as therapeutic tools has grown exponentially, although only very preliminary data have been obtained in clinical trials to date. In this review, we summarized the state of the art about the phenotypic features, functions, regenerative properties, and clinical applicability of mesenchymal cells, with a particular focus on those of cardiac origin.

The epigenetic enzyme p300/CBP-associated factor (PCAF) belongs to the GCN5-related N-acetyltransferase (GNAT) family together with GCN5. Although its transcriptional and post-translational function is well characterized, little is known about its properties as regulator of cell metabolism. Here, we report the mitochondrial localization of PCAF conferred by an 85 aa mitochondrial targeting sequence (MTS) at the N-terminal region of the protein. In mitochondria, one of the PCAF targets is the isocitrate dehydrogenase 2 (IDH2) acetylated at lysine 180. This PCAF-regulated post-translational modification might reduce IDH2 affinity for isocitrate as a result of a conformational shift involving predictively the tyrosine at position 179. Site-directed mutagenesis and functional studies indicate that PCAF regulates IDH2, acting at dual level during myoblast differentiation: at a transcriptional level together with MyoD, and at a post-translational level by direct modification of lysine acetylation in mitochondria. The latter event determines a decrease in IDH2 function with negative consequences on muscle fiber formation in C2C12 cells. Indeed, a MTS-deprived PCAF does not localize into mitochondria, remains enriched into the nucleus, and contributes to a significant increase of muscle-specific gene expression enhancing muscle differentiation. The role of PCAF in mitochondria is a novel finding shedding light on metabolic processes relevant to early muscle precursor differentiation.-Savoia, M., Cencioni, C., Mori, M., Atlante, S., Zaccagnini, G., Devanna, P., Di Marcotullio, L., Botta, B., Martelli, F., Zeiher, A. M., Pontecorvi, A., Farsetti, A., Spallotta, F., Gaetano, C. P300/CBP-associated factor regulates transcription and function of isocitrate dehydrogenase 2 during muscle differentiation.

The inhibition of human DNA Methyl Transferases (DNMT) is a novel promising approach to address the epigenetic dysregulation of gene expression in different diseases. Inspired by the validated virtual screening hit NSC137546, a series of N-benzoyl amino acid analogues was synthesized and obtained compounds were assessed for their ability to inhibit DNMT-dependent DNA methylation in vitro. The biological screening allowed the definition of a set of preliminary structure-activity relationships and the identification of compounds promising for further development. Among the synthesized compounds, L-glutamic acid derivatives 22, 23, and 24 showed the highest ability to prevent DNA methylation in a total cell lysate. Compound 22 inhibited DNMT1 and DNMT3A activity in a concentration-dependent manner in the micromolar range. In addition, compound 22 proved to be stable in human serum and it was thus selected as a starting point for further biological studies.

Histone methylation is tightly regulated by the equilibrium between histone methyltransferases and demethylases.1 Methylation occurs on lysine (K), arginine (R), and histidine (H) residues, without affecting histone charge. Interestingly, histone methylation plays different roles in consequence of the specific position of the methylated residue. The transcribed chromatin might, in fact, be enriched in methylation at histone 3 (H3) residues K4 (H3K4), K36 (H3K36), and K79 (H3K79), whereas methylation at K9 (H3K9), K27 (H3K27), and histone 4 (H4) K20 (H4K20) characterizes silenced chromatin regions.2 The level of K methylation, such as mono-, di-, or trimethylation, is also important allowing for chromatin accessibility by transcription complexes. For example, monomethylation of H3K9 (H3K9me1) or H3K27 (H3K27me1) associates with active transcription, whereas their trimethylated forms (H3K9me3; H3Kme3) with silencing.2 Histone methyltransferases belong to the larger lysine methyltransferases family whose members incorporate the conserved methyltransferase domain first identified as part of the Su(var)3 to 9, Enhancer-of-zeste, Trithorax (SET) proteins. On the contrary, histone demethylases are included into the lysine demethylases (KDMs) and grouped into 2 major classes: the Flavin adenine dinucleotide–dependent demethylases acting on mono- and di-methylated Ks and the Jumonji demethylases, depending on Fe(II) and α-ketoglutarate cofactors, selective for trimethylated Ks1. Article, see p 1403 Among epigenetic writers, the lysine methyltransferase polycomb repressive complexes 1 and 2 (PRC1 and PRC2) gained considerable popularity in the field for their involvement in the regulation of stem cell pluripotency, embryonal development, and cancer.3 Recently, the interest of epigeneticists toward Polycomb has been further raised by the evidence that these complexes associate with many long noncoding RNAs being possibly implicated in gene regulation.4 PRC1 and PRC2 are multiprotein complexes grouped under the definition of Polycomb Group responsible for histone PTMs able to condensate …

HDAC inhibitors and NO donors have already revealed independently their broad therapeutic potential in pathologic contexts. Here we further investigated the power of their combination in a single hybrid molecule. Nitrooxy groups or substituted furoxan derivatives were joined to the α-position of the pyridine ring of the selective class I HDAC inhibitor MS-275. Biochemical analysis showed that the association with the dinitrooxy compound 31 or the furoxan derivative 16 gives hybrid compounds the ability to preserve the single moiety activities. The two new hybrid molecules were then tested in a muscle differentiation assay. The hybrid compound bearing the moiety 31 promoted the formation of large myotubes characterized by highly multinucleated fibers, possibly due to a stimulation of myoblast fusion, as implicated by the strong induction of myomaker expression. Thanks to their unique biological features, these compounds may represent new therapeutic tools for cardiovascular, neuromuscular and inflammatory diseases.

Metastasis formation requires active energy production and is regulated at multiple levels by mitochondrial metabolism. The hyperactive metabolism of cancer cells supports their extreme adaptability and plasticity and facilitates resistance to common anticancer therapies. In spite the potential relevance of a metastasis metabolic control therapy, so far, limited experience is available in this direction. Here, we evaluated the effect of the recently described α-ketoglutarate dehydrogenase (KGDH) inhibitor, (S)-2-[(2,6-dichlorobenzoyl) amino] succinic acid (AA6), in an orthotopic mouse model of breast cancer 4T1 and in other human breast cancer cell lines. In all conditions, AA6 altered Krebs cycle causing intracellular α-ketoglutarate (α-KG) accumulation. Consequently, the activity of the α-KG-dependent epigenetic enzymes, including the DNA demethylation ten-eleven translocation translocation hydroxylases (TETs), was increased. In mice, AA6 injection reduced metastasis formation and increased 5hmC levels in primary tumours. Moreover, in vitro and in vivo treatment with AA6 determined an α-KG accumulation paralleled by an enhanced production of nitric oxide (NO). This epigenetically remodelled metabolic environment efficiently counteracted the initiating steps of tumour invasion inhibiting the epithelial-to-mesenchymal transition (EMT). Mechanistically, AA6 treatment could be linked to upregulation of the NO-sensitive anti-metastatic miRNA 200 family and down-modulation of EMT-associated transcription factor Zeb1 and its CtBP1 cofactor. This scenario led to a decrease of the matrix metalloproteinase 3 (MMP3) and to an impairment of 4T1 aggressiveness. Overall, our data suggest that AA6 determines an α-KG-dependent epigenetic regulation of the TET–miR200–Zeb1/CtBP1–MMP3 axis providing an anti-metastatic effect in a mouse model of breast cancer-associated metastasis.

The short-lived turquoise killifish Nothobranchius furzeri (Nfu) is a valid model for aging studies. Here, we investigated its age-associated cardiac function. We observed oxidative stress accumulation and an engagement of microRNAs (miRNAs) in the aging heart. MiRNA-sequencing of 5 week (young), 12–21 week (adult) and 28–40 week (old) Nfu hearts revealed 23 up-regulated and 18 down-regulated miRNAs with age. MiR-29 family turned out as one of the most up-regulated miRNAs during aging. MiR-29 family increase induces a decrease of known targets like collagens and DNA methyl transferases (DNMTs) paralleled by 5´methyl-cytosine (5mC) level decrease. To further investigate miR-29 family role in the fish heart we generated a transgenic zebrafish model where miR-29 was knocked-down. In this model we found significant morphological and functional cardiac alterations and an impairment of oxygen dependent pathways by transcriptome analysis leading to hypoxic marker up-regulation. To get insights the possible hypoxic regulation of miR-29 family, we exposed human cardiac fibroblasts to 1% O2 levels. In hypoxic condition we found miR-29 down-modulation responsible for the accumulation of collagens and 5mC. Overall, our data suggest that miR-29 family up-regulation might represent an endogenous mechanism aimed at ameliorating the age-dependent cardiac damage leading to hypertrophy and fibrosis.

Age is the most important risk factor for metabolic alterations and cardiovascular accidents. Although class III histone deacetylases, alias Sirtuins, have been appealed as "the fountain of youth" their role in longevity control and prevention of aging-associated disease is still under debate. Indeed, several lines of evidence indicate that sirtuin activity is strictly linked to metabolism and dependent on NAD(+) synthesis both often altered as aging progresses. During aging the cardiovascular system is attacked by a variety of environmental stresses, including those determined by high blood glucose and lipid levels, or by the presence of oxidized lipoproteins which, among others, determine important oxidative stress signals. In such a milieu, heart and vessels develop a functional impairment leading to atherosclerosis, ischemia, heart insufficiency and failure. Sirtuins, which are believed to have a positive impact on cardiovascular physiology and physiopathology, are distributed in different subcellular compartments including the nucleus, the cytoplasm and the mitochondria, where they regulate expression and function of a large variety of target genes and proteins. Remarkably, experimental animal models indicate resveratrol, the first natural compound described to positively regulate the activity of sirtuins, as able to protect the endothelium and the heart exposed to a variety of stress agents. This review will focus on the regulation and function of mammalian sirtuins with special attention paid to their role as cardiovascular "defenders" giving indication of their targets of potential relevance for the development of future therapeutics. This article is part of a Special Issue entitled CV Aging.

Ischemic heart disease is the most common cause of death in Western countries.1 It is consequence of a reduced cardiac blood supply leading to a decrease in oxygen and nutrients fundamental for the proper functioning of the myocardium.1,2 The persistence of this condition induces fibrosis and scar deposition to replace the dead cardiac tissue.2 This replacement damages pumping function and thus predisposes to arrhythmias.3 Specific transcriptional programs finely regulate the cardiac remodeling.2 Deciphering the deep transcriptional changes during ischemic heart disease might shed light on potential targets for early diagnosis and new drug development. See Article by Kaikkonen et al Transcription permits to transform into RNA the coding and non-coding genetic information conserved into DNA. RNAs, in fact, are grouped into coding and non-coding transcripts (ncRNAs).4,5 The former, commonly known as mRNAs, are commonly translated into proteins, whereas the latter carry out mainly regulatory functions at transcriptional, post-transcriptional, and epigenetic levels.4,5 NcRNAs have been further classified, based on their length, into following types: small ncRNAs ( 200 nucleotides).4,5 microRNAs (miRNAs), transfer RNAs, piwi-interacting RNAs, and endogenous short interfering RNAs are examples of the first class.5 Instead, lncRNAs are generally categorized based on association to nearby protein-coding genes into following types: sense RNAs, antisense RNAs, intronic RNAs, intergenic RNAs, enhancer (eRNAs), and circular RNAs.4 All these RNAs are mostly transcribed by RNA polymerase II (Pol II), a multiprotein complex of 550 kDa localized into the nucleus of eukaryotic cells.6 Thanks to its prompt response to environmental signals, Pol II is able to immediately activate molecular mechanisms crucial for the …

This study investigates the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of H3K9Ac and lysine 27 significantly increased. These observations were paralleled by a downregulation of the GCN5-related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-? general control of amino acid synthesis (GCN5a), determining a relative decrease in total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients. © 2014 by the American Diabetes Association.

Recent statistics indicate that the human population is ageing rapidly. Healthy, but also diseased, elderly people are increasing. This trend is particularly evident in Western countries, where healthier living conditions and better cures are available. To understand the process leading to age-associated alterations is, therefore, of the highest relevance for the development of new treatments for age-associated diseases, such as cancer, diabetes, Alzheimer and cardiovascular accidents. Mechanistically, it is well accepted that the accumulation of intracellular damage determined by reactive oxygen species (ROS) might orchestrate the progressive loss of control over biological homeostasis and the functional impairment typical of aged tissues. Here, we review how epigenetics takes part in the control of stress stimuli and the mechanisms of ageing physiology and physiopathology. Alteration of epigenetic enzyme activity, histone modifications and DNA-methylation is, in fact, typically associated with the ageing process. Specifically, ageing presents peculiar epigenetic markers that, taken altogether, form the still ill-defined “ageing epigenome”. The comprehension of mechanisms and pathways leading to epigenetic modifications associated with ageing may help the development of anti-ageing therapies.

Modulators of sirtuins are considered promising therapeutic targets for the treatment of cancer, cardiovascular, metabolic, inflammatory, and neurodegenerative diseases. Here we prepared new 1,4-dihydropyridines (DHPs) bearing changes at the C2/C6, C3/C5, C4, or N1 position. Tested with the SIRTainty procedure, some of them displayed increased SIRT1 activation with respect to the prototype 3a, high NO release in HaCat cells, and ameliorated skin repair in a mouse model of wound healing. In C2C12 myoblasts, two of them improved mitochondrial density and functions. All the effects were reverted by coadministration of compound C (9), an AMPK inhibitor, or of EX-527 (10), a SIRT1 inhibitor, highlighting the involvement of the SIRT1/AMPK pathway in the action of DHPs. Finally, tested in a panel of cancer cells, the water-soluble form of 3a, compound 8, displayed antiproliferative effects in the range of 8-35 μM and increased H4K16 deacetylation, suggesting a possible role for SIRT1 activators in cancer therapy.

We review the pivotal role of the stromal derived factor (SDF)-1 chemokine in tissue ischaemia and how it orchestrates the rapid revascularization of injured, ischaemic, and regenerating tissues via the CXC chemokine receptors CXCR4 and CXCR7. Furthermore, we discuss the effects of preconditioning (PC), which is a well-known protective phenomenon for tissue ischaemia. The positive effect of both hypoxic and acidic PC on progenitor cell therapeutic potential is reviewed, while stressing the role of the SDF-1/CXCR4 axis in this process.

The stromal cell–derived factor (SDF)-1/CXC receptor 4 (CXCR4) axis has been shown to play a role in skeletal muscle development, but its contribution to postnatal myogenesis and the role of the alternate SDF-1 receptor, CXC receptor 7 (CXCR7), are poorly characterized. Western blot analysis and real-time polymerase chain reaction (PCR) were performed to evaluate in vitro the effect of SDF-1 and CXCR4 and CXCR7 inhibition on myogenic differentiation. Proliferating myoblasts express CXCR4, CXCR7, and SDF-1; during myogenic differentiation, CXCR4 and CXCR7 levels are downregulated, and SDF-1 release is decreased. SDF-1 anticipates myosin heavy chain accumulation and myotube formation in both C2C12 myoblasts and satellite cells. Interestingly, inhibition of CXCR4 and CXCR7 signaling, either by drugs or RNA interfererence, blocks myogenic differentiation. Further, the CXCR4 antagonist, 4F-benzoyl-TN14003, inhibits myoblast cell cycle withdrawal and decreases the retinoblastoma gene (pRb) product accumulation in its hypophosphorylated form. Our experiments demonstrate that SDF-1 regulates myogenic differentiation via both CXCR4 and CXCR7 chemokine receptors. Muscle Nerve 000:000–000, 2010

Introduction: The origin of metabolic/epigenetic memory remains elusive. It, however, may be at the basis of chronic diseases and functional alteration associated to ageing and cardiovascular disease. HYPOTHESIS: Diabetes is known to alter cellular metabolism causing defects in tissue/organ regeneration. In this study we asked whether human cardiac stromal cells from diabetic and normoglycaemic voluntary donors revealed differences associated to their original metabolism and whether the alterations could be rescued by epigenetically active drugs. METHODS and RESULTS: D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of histone H3 lysine 9 and lysine 27 significantly increased. These observations were paralleled by a downregulation of the GCN5- related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-a general control of amino acid synthesis (GCN5a), determining a relative decreasein total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. CONCLUSIONS: This study investigated the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients.

Recently the concept emerged that prolonged exposure to altered metabolic conditions, including hyperglycemia, may epigenetically imprint human cells permitting vertical or horizontal transfer to "descendants". Although mechanistically ill understood, the hyperglycemic/epigenetic memory may represent one of the major limitations for the application of cell therapy to treatment of chronic heart disease where a relatively prolonged period of ex vivo cellular expansion is required. Hyperglycemic memory, in fact, seems to contribute to the establishment of an epigenetic "reminiscence" of the altered metabolic state, to which, cells from diseased bodies have been exposed. This review summarizes the most relevant concepts and observations about the mechanisms underlying the onset of stable information inside the epigenome leading to the development of a diseased phenotype. Special attention is given to epigenetic drugs and how they have been used in experimental, preclinical and clinical settings to treat dysmetabolism, diabetes and their complications.

The NO-donor histone deacetylase inhibitor 2, formally obtained by joining Entinostat 1, a moderately selective Class I histone deacetylases (HDACs) inhibitor, to a 4-(methylaminomethyl)furoxan-3-carbonitrile scaffold, is described and its preliminary biological profile discussed. This hybrid regulates Classes I and II HDACs. Nitric oxide (NO) released by the compound activates soluble guanylate cyclase (sGC), causing Class II nuclear shuttling and chromatin modifications, with consequences on gene expression. The hybrid affects a number of micro-RNAs not modulated by its individual components; it promotes myogenic differentiation, inducing the formation of larger myotubes with significantly more nuclei per fiber, in a more efficient manner than the 1:1 mixture of its two components. The hybrid is an example of a new class of NO-donor HDACs now being developed, which should be of interest for treating a number of diseases.

In a mouse model of skin repair we found that the class I-IIa histone deacetylase inhibitor trichostatin A accelerated tissue regeneration. Unexpectedly, this effect was suppressed by Sirtinol, a class III histone deacetylase (HDAC) (sirtuin)-selective inhibitor. The role of sirtuins (SIRTs) was then investigated by using resveratrol and a novel SIRT1-2-3 activator, the MC2562 compound we synthesized recently. Both resveratrol and MC2562 were effective in accelerating wound repair. The local administration of natural or synthetic SIRT activators, in fact, significantly accelerated skin regeneration by increasing keratinocyte proliferation. In vitro experiments revealed that the activation of SIRTs stimulated keratinocyte proliferation via endothelial NO synthase phosphorylation and NO production. In this condition, the class I member HDAC2 was found S-nitrosylated on cysteine, a post-transduction modification associated with loss of activity and DNA binding capacity. After deacetylase inhibitor or SIRT activator treatment, ChIP showed, in fact, a significant HDAC2 detachment from the promoter region of insulin growth factor I (IGF-I), fibroblast growth factor 10 (FGF-10), and Epithelial Growth Factor (EGF), which may be the final recipients and effectors of the SIRT-NO-HDAC signaling cascade. Consistently, the effect of SIRT activators was reduced in the presence of NG-nitro-L-arginine methyl ester (L-NAME), a general inhibitor of NO synthesis. In conclusion, the NO-dependent cross-talk among class III and I histone deacetylases suggests an unprecedented signaling pathway important for skin repair.

Aims The chemokine receptor CXCR4 modulates endothelial progenitor cell migration, homing, and differentiation, and plays a key role in cardiovascular regeneration. Here we examined the effect of ex vivo acidic preconditioning (AP) on CXCR4 expression and on the regenerative potential of mouse bone marrow (BM) ckit+ cells. Methods and results Acidic preconditioning was achieved by exposing BM ckit+ cells to hypercarbic acidosis (pH 7.0) for 24 h; control cells were kept at pH 7.4. Acidic preconditioning enhanced CXCR4 and stromal cell-derived factor 1 (SDF-1) mRNA levels, as well as CXCR4 phosphorylation. Acidic preconditioning ability to modulate CXCR4 expression depended on cytosolic calcium [Ca2+]i mobilization and on nitric oxide (NO), as determined by [Ca2+]i buffering with BAPTA, and by treatment with the NO donor (DETA/NO) and the NO synthase inhibitor (L-NAME). Further, AP increased SDF-1-driven chemotaxis, transendothelial migration, and differentiation toward the endothelial lineage in vitro. In a mouse model of hindlimb ischaemia, control and AP ckit+ cells were transplanted into the ischaemic muscle; AP cells accelerated blood flow recovery, increased capillary, and arteriole number as well as the number of regenerating muscle fibres vs. control. These effects were abolished by treating AP cells with L-NAME. Conclusion Acidic preconditioning represents a novel strategy to enhance BM ckit+ cell therapeutic potential via NO-dependent increase in CXCR4 expression. © The Author 2013.

We examined the effect of reactive oxygen species (ROS) on MicroRNAs (miRNAs) expression in endothelial cells in vitro, and in mouse skeletal muscle following acute hindlimb ischemia. Human umbilical vein endothelial cells (HUVEC) were exposed to 200 μM hydrogen peroxide (H(2)O(2)) for 8 to 24 h; miRNAs profiling showed that miR-200c and the co-transcribed miR-141 increased more than eightfold. The other miR-200 gene family members were also induced, albeit to a lower level. Furthermore, miR-200c upregulation was not endothelium restricted, and occurred also on exposure to an oxidative stress-inducing drug: 1,3-bis(2 chloroethyl)-1nitrosourea (BCNU). miR-200c overexpression induced HUVEC growth arrest, apoptosis and senescence; these phenomena were also induced by H(2)O(2) and were partially rescued by miR-200c inhibition. Moreover, miR-200c target ZEB1 messenger RNA and protein were downmodulated by H(2)O(2) and by miR-200c overexpression. ZEB1 knockdown recapitulated miR-200c-induced responses, and expression of a ZEB1 allele non-targeted by miR-200c, prevented miR-200c phenotype. The mechanism of H(2)O(2)-mediated miR-200c upregulation involves p53 and retinoblastoma proteins. Acute hindlimb ischemia enhanced miR-200c in wild-type mice skeletal muscle, whereas in p66(ShcA -/-) mice, which display lower levels of oxidative stress after ischemia, upregulation of miR-200c was markedly inhibited. In conclusion, ROS induce miR-200c and other miR-200 family members; the ensuing downmodulation of ZEB1 has a key role in ROS-induced apoptosis and senescence.

AimsAcidification is associated with a variety of pathological and physiological conditions. In the present study, we aimed at investigating whether acidic pH may regulate endothelial cell (EC) functions via the chemokine receptor CXCR4, a key modulator of EC biological activities.Methods and resultsExposure of ECs to acidic pH reversibly inhibited mRNA and protein CXCR4 expression, CXCL12/stromal cell-derived factor (SDF)-1-driven EC chemotaxis in vitro, and CXCR4 expression and activation in vivo in a mouse model. Further, CXCR4 signalling impaired acidosis-induced rescue from apoptosis in ECs. The inhibition of CXCR4 expression occurred transcriptionally and was hypoxia-inducible factor (HIF)-1-dependent as demonstrated by both HIF-1 and HIF-1 dominant negative overexpression, by HIF-1 silencing, and by targeted mutation of the-29 to-25 hypoxia response element (HRE) in the-357/-59 CXCR4 promoter fragment. Moreover, chromatin immunoprecipitation (ChIP) analysis showed endogenous HIF-1 binding to the CXCR4 promoter that was enhanced by acidification.ConclusionThe results of the present study identify CXCR4 as a key player in the EC response to acidic pH and show, for the first time, that HRE may function not only as an effector of hypoxia, but also as an acidosis response element, and raise the possibility that this may constitute a more general mechanism of transcriptional regulation at acidic pH. © The Author 2009. For permissions please.

Aims A close relationship exists between hyperglycaemia, oxidative stress, and diabetic complications. In fact, high glucose (HG) determines the overproduction of reactive oxygen species (ROS) by the mitochondria. p66ShcA is a gene that regulates the apoptotic responses to oxidative stress. Indeed, p66ShcA knockout (ko) mice display decreased ROS production and increased resistance to ROS-induced cell death in a variety of pathophysiological settings. Reduced endothelial progenitor cell (EPC) number, differentiation, and function are relevant components of the angiogenesis impairment observed in diabetic patients. We examined the role of p66ShcA in the EPC deficit induced by HG. Methods and results Mouse bone marrow-derived c-kit+ cells differentiate in endothelial-like cells when plated on fibronectin (BM-derived EPCs). We found that cell culture in the presence of HG up-regulated p66ShcA protein expression and that HG exposure markedly decreased the number of BM-derived EPCs. Conversely, p66ShcA ko BM-derived EPCs were not sensitive to HG inhibition. Indeed, the resistance of p66ShcA ko BM-derived EPCs to HG was associated with reduced levels of both apoptosis and oxidative stress. To functionally link the HG response to ROS production, p66ShcA ko BM-derived EPCs were reconstituted either with p66ShcA wild-type (wt) or with a p66ShcA allele (p66ShcA qq) that was devoid of its ROS-generating function. We found that only p66ShcA wt and not the qq mutant rescued p66ShcA ko cell sensitivity to HG. One major feature of oxidative stress is its ability to reduce the bio-availability of nitric oxide (NO) that, in turn, plays a crucial role in endothelial differentiation and function. We found that the p66ShcA deletion prevented the HG-induced increase of nitrotyrosine, and that the resistance to HG of p66ShcA ko BM-derived EPCs was prevented by NO synthase inhibition. With a reciprocal approach, the treatment of p66ShcA wt cells with a NO donor prevented the HG-induced deficit. Finally, using a Matrigel plug angiogenesis assay, we demonstrated that p66ShcA ko prevented diabetic impairment of angiogenesis in vivo . Conclusion p66ShcA deletion rescues the BM-derived EPCs defect induced by HG, indicating p66ShcA as a potential therapeutic target in diabetic vasculopathy.

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.

Oxidative stress plays a pivotal role in ischemic injury, and p66(ShcA)ko mice exhibit both lower oxidative stress and decreased tissue damage following hind limb ischemia. Thus, it was investigated whether tissue regeneration following acute hind limb ischemia was altered in p66(ShcA)ko mice. Upon femoral artery dissection, muscle regeneration started earlier and was completed faster than in wild-type (WT) control. Moreover, faster regeneration was associated with decreased oxidative stress. Unlike ischemia, cardiotoxin injury induced similar skeletal muscle damage in both genotypes. However, p66(ShcA)ko mice regenerated faster, in agreement with the regenerative advantage upon ischemia. Since no difference between p66(ShcA)wt and knock-out (ko) mice was found in blood perfusion recovery after ischemia, satellite cells (SCs), a resident population of myogenic progenitors, were examined. Similar SCs numbers were present in WT and ko mice. However, in vitro cultured p66(ShcA)ko SCs displayed lower oxidative stress levels and higher proliferation rate and differentiated faster than WT. Furthermore, when exposed to sublethal H(2)O(2) doses, p66(ShcA)ko SCs were resistant to H(2)O(2)-induced inhibition of differentiation. Finally, myogenic conversion induced by MyoD overexpression was more efficient in p66(ShcA)ko fibroblasts compared with WT. The present work demonstrates that oxidative stress and p66(ShcA) play a crucial role in the regenerative pathways activated by acute ischemia.

In physiopathological conditions, such as diabetes, wound healing is significantly compromised and chronic complications, including ulcers, may occur. In a mouse model of skin repair, we recently reported that wound treatment with Sirtuin activators and class I HDAC inhibitors induced keratinocyte proliferation and enhanced healing via a nitric oxide (NO) dependent mechanism. We observed an increase in total protein acetylation in the wound area, as determined by acetylation of α-tubulin and histone H3 Lysine 9. We reasoned that this process activated cell function as well as regulated gene expression to foster tissue repair. We report here that the direct activation of P300/CBP-associated factor (PCAF) by the histone acetylase activator pentadecylidenemalonate 1b (SPV-106) induced Lysine acetylation in the wound area. This intervention was sufficient to enhance repair process by a NO-independent mechanism. Hence, an impairment of PCAF and/or other GCN5 family acetylases may delay skin repair in physiopathological conditions.