The "Superoncogene" Myc at the Crossroad between Metabolism and Gene Expression in Glioblastoma Multiforme.

The concept of the Myc (c-myc, n-myc, l-myc) oncogene as a canonical, DNA-bound transcription factor has consistently changed over the past few years. Indeed, Myc controls gene expression programs at multiple levels: directly binding chromatin and recruiting transcriptional coregulators; modulating the activity of RNA polymerases (RNAPs); and drawing chromatin topology. Therefore, it is evident that Myc deregulation in cancer is a dramatic event. Glioblastoma multiforme (GBM) is the most lethal, still incurable, brain cancer in adults, and it is characterized in most cases by Myc deregulation. Metabolic rewiring typically occurs in cancer cells, and GBM undergoes profound metabolic changes to supply increased energy demand. In nontransformed cells, Myc tightly controls metabolic pathways to maintain cellular homeostasis. Consistently, in Myc-overexpressing cancer cells, including GBM cells, these highly controlled metabolic routes are affected by enhanced Myc activity and show substantial alterations. On the other hand, deregulated cancer metabolism impacts Myc expression and function, placing Myc at the intersection between metabolic pathway activation and gene expression. In this review paper, we summarize the available information on GBM metabolism with a specific focus on the control of the Myc oncogene that, in turn, rules the activation of metabolic signals, ensuring GBM growth.

Omics Analyses of Stromal Cells from ACM Patients Reveal Alterations in Chromatin Organization and Mitochondrial Homeostasis.

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 role of redox system in metastasis formation.

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.

P300/CBP-associated factor regulates transcription and function of isocitrate dehydrogenase 2 during muscle differentiation.

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.

Stable Oxidative Cytosine Modifications Accumulate in Cardiac Mesenchymal Cells From Type2 Diabetes Patients: Rescue by α-Ketoglutarate and TET-TDG Functional Reactivation.

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.

Sirtuin function in aging heart and vessels.

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.

A nitric oxide-dependent cross-talk between class I and III histone deacetylases accelerates skin repair.

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.

Ex vivo acidic preconditioning enhances bone marrow ckit+ cell therapeutic potential via increased CXCR4 expression.

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 [Ca(2+)]i mobilization and on nitric oxide (NO), as determined by [Ca(2+)]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.

High fat diet affects the hippocampal expression of miRNAs targeting brain plasticity-related genes.

Metabolic disorders such as insulin resistance and type 2 diabetes are associated with brain dysfunction and cognitive deficits, although the underpinning molecular mechanisms remain elusive. Epigenetic factors, such as non-coding RNAs, have been reported to mediate the molecular effects of nutrient-related signals. Here, we investigated the changes of miRNA expression profile in the hippocampus of a well-established experimental model of metabolic disease induced by high fat diet (HFD). In comparison to the control group fed with standard diet, we observed 69 miRNAs exhibiting increased expression and 63 showing decreased expression in the HFD mice's hippocampus. Through bioinformatics analysis, we identified numerous potential targets of the dysregulated miRNAs, pinpointing a subset of genes regulating neuroplasticity that were targeted by multiple differentially modulated miRNAs. We also validated the expression of these synaptic and non-synaptic proteins, confirming the downregulation of Synaptotagmin 1 (SYT1), calcium/calmodulin dependent protein kinase I delta (CaMK1D), 2B subunit of N-methyl-D-aspartate glutamate receptor (GRIN2B), the DNA-binding protein Special AT-Rich Sequence-Binding Protein 2 (SATB2), and RNA-binding proteins Cytoplasmic polyadenylation element-binding protein 1 (CPEB1) and Neuro-oncological ventral antigen 1 (NOVA1) in the hippocampus of HFD mice. In summary, our study offers a snapshot of the HFD-related miRNA landscape potentially involved in the alterations of brain functions associated with metabolic disorders. By shedding light on the specific miRNA-mRNA interactions, our research contributes to a deeper understanding of the molecular mechanisms underlying the effects of HFD on the synaptic function.

HDAC6 inhibition disrupts HDAC6-P300 interaction reshaping the cancer chromatin landscape.

BACKGROUND: Histone deacetylases (HDACs) are crucial regulators of gene expression, DNA synthesis, and cellular processes, making them essential targets in cancer research. HDAC6, specifically, influences protein stability and chromatin dynamics. Despite HDAC6's potential therapeutic value, its exact role in gene regulation and chromatin remodeling needs further clarification. This study examines how HDAC6 inactivation influences lysine acetyltransferase P300 stabilization and subsequent effects on chromatin structure and function in cancer cells. METHODS AND RESULTS: We employed the HDAC6 inhibitor ITF3756, siRNA, or CRISPR/Cas9 gene editing to inactivate HDAC6 in different epigenomic backgrounds. Constantly, this inactivation led to significant changes in chromatin accessibility, particularly increased acetylation of histone H3 lysines 9, 14, and 27 (ATAC-seq and H3K27Ac ChIP-seq analysis). Transcriptomics, proteomics, and gene ontology analysis revealed gene changes in cell proliferation, adhesion, migration, and apoptosis. Significantly, HDAC6 inactivation altered P300 ubiquitination, stabilizing P300 and leading to downregulating genes critical for cancer cell survival. CONCLUSIONS: Our study highlights the substantial impact of HDAC6 inactivation on the chromatin landscape of cancer cells and suggests a role for P300 in contributing to the anticancer effects. The stabilization of P300 with HDAC6 inhibition proposes a potential shift in therapeutic focus from HDAC6 itself to its interaction with P300. This finding opens new avenues for developing targeted cancer therapies, improving our understanding of epigenetic mechanisms in cancer cells.

Crosstalk and communication of cancer-associated fibroblasts with natural killer and dendritic cells: New frontiers and unveiled opportunities for cancer immunotherapy.

Natural killer (NK) cells and dendritic cells (DCs) are critical mediators of anti-cancer immune responses. In addition to their individual roles, NK cells and DCs are involved in intercellular crosstalk which is essential for the initiation and coordination of adaptive immunity against cancer. However, NK cell and DC activity is often compromised in the tumor microenvironment (TME). Recently, much attention has been paid to one of the major components of the TME, the cancer-associated fibroblasts (CAFs), which not only contribute to extracellular matrix (ECM) deposition and tumor progression but also suppress immune cell functions. It is now well established that CAFs support T cell exclusion from tumor nests and regulate their cytotoxic activity. In contrast, little is currently known about their interaction with NK cells, and DCs. In this review, we describe the interaction of CAFs with NK cells and DCs, by secreting and expressing various mediators in the TME of adult solid tumors. We also provide a detailed overview of ongoing clinical studies evaluating the targeting of stromal factors alone or in combination with immunotherapy based on immune checkpoint inhibitors. Finally, we discuss currently available strategies for the selective depletion of detrimental CAFs and for a better understanding of their interaction with NK cells and DCs.

Oxidative stress and epigenetic regulation in ageing and age-related diseases.

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.

Gastrointestinal Cancer Patient Nutritional Management: From Specific Needs to Novel Epigenetic Dietary Approaches.

Nutritional habits impinge on the health of the gastrointestinal (GI) tract, contributing to GI disorder progression. GI cancer is a widespread and aggressive tumor sensitive to nutritional changes. Indeed, specific nutritional expedients can be adopted to prevent GI cancer onset and to slow down disease activity. Moreover, the patient's nutritional status impacts prognosis, quality of life, and chemotherapy tolerance. These patients encounter the highest frequency of malnourishment risk, a condition that can progressively evolve into cachexia. Clinical studies dealing with this topic stressed the importance of nutritional counseling and put under the spotlight nutrient delivery, the type of nutrient supplementation, and timing for the start of nutritional management. A medical practitioner well-prepared on the topic of nutrition and cancer should operate in the clinical team dedicated to these oncological patients. This specific expertise needs to be implemented as soon as possible to adopt nutritional interventions and establish a proper patient-tailored dietary regimen. The nutritional gap closure should be prompt during anticancer treatment to stabilize weight loss, improve treatment tolerability, and ameliorate survival rate. Recently, novel nutritional approaches were investigated to target the bidirectional link between epigenetics and metabolism, whose alteration supports the onset, progression, and therapeutic response of GI cancer patients.

The TFEB-TGIF1 axis regulates EMT in mouse epicardial cells.

Epithelial-mesenchymal transition (EMT) is a complex and pivotal process involved in organogenesis and is related to several pathological processes, including cancer and fibrosis. During heart development, EMT mediates the conversion of epicardial cells into vascular smooth muscle cells and cardiac interstitial fibroblasts. Here, we show that the oncogenic transcription factor EB (TFEB) is a key regulator of EMT in epicardial cells and that its genetic overexpression in mouse epicardium is lethal due to heart defects linked to impaired EMT. TFEB specifically orchestrates the EMT-promoting function of transforming growth factor (TGF) ß, and this effect results from activated transcription of thymine-guanine-interacting factor (TGIF)1, a TGFß/Smad pathway repressor. The Tgif1 promoter is activated by TFEB, and in vitro and in vivo findings demonstrate its increased expression when Tfeb is overexpressed. Furthermore, Tfeb overexpression in vitro prevents TGFß-induced EMT, and this effect is abolished by Tgif1 silencing. Tfeb loss of function, similar to that of Tgif1, sensitizes cells to TGFß, inducing an EMT response to low doses of TGFß. Together, our findings reveal an unexpected function of TFEB in regulating EMT, which might provide insights into injured heart repair and control of cancer progression.

MALAT1 as a Regulator of the Androgen-Dependent Choline Kinase A Gene in the Metabolic Rewiring of Prostate Cancer.

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.

Induction of myogenic differentiation by SDF-1 via CXCR4 and CXCR7 receptors.

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.

Metabolic Reprogramming by Malat1 Depletion in Prostate Cancer.

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.

Altered SDF-1-mediated differentiation of bone marrow-derived endothelial progenitor cells in diabetes mellitus.

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.