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Adipose derived stem cells (ADSCs) are mesenchymal stem cells that can be easly isolated from lipoaspirates. They show multilineage differentiative capacities, including the cardiac phenotype. Therefore they represent a promise for regenerative medicine (Zuk 2001), for the treatment of those diseases affecting organs with extremely low potential of repair and regeneration, such as the heart. The knowledge of the molecular mechanisms that control development and differentiation - such as epigenetic DNA promoter methylation at CpG sites and histone modifications inducing chromatin remodelling and changes in DNA accessibility (Bernstein 2007) - will help to understand their potential in this regard. Aim of this study was to evaluate the epigenetic status of cardiac specific transcription factors,such as GATA-4, MEF-2C and Nkx2.5 (Brand 2003) in undifferentiated human ADSCs. The analysis of DNA promoter methylation, using Methylation Specific PCR (MSP), revealed the unmethylated status of the promoters of the above cardiac master genes. This finding suggested that ADSCs are not prevented to differentiate towards the cardiac phenotype by DNA methylation, as otherwise reported for myogenic genes (Sørensen 2010). Chromatin immunoprecipitation (ChIP) showed association of a well known transcriptional repressive mark [trimethylation of Lys27 in H3 (H3K27me3)] with GATA-4 and Nkx2.5 promoters. On the other hand, an enrichment in acetylation of histone 3 (acH3), hallmark of transcriptional potential, was detected for MEF-2C promoter. These results are in agreement with those of the gene expression analysis, showing the presence of only MEF-2C transcript in total mRNA extracted from undifferentiated human ADSCs of three different donors. In conclusion our results suggest that the epigenetic signature of ADSCs represents a level for intervention to address their differentiation towards the cardiac lineage.

Myoblast differentiation into multinuclear myotubes implies the slow-down of their proliferative drive and the expression of myogenin, an early marker of myogenic differentiation. Natural polyamines-such as putrescine, spermidine and spermine-are low molecular weight organic polycations, well known as mediators involved in cell homeostasis. Many evidences in the literature point to their role in driving cellular differentiation processes. Here, we studied how polyamines may affect the differentiation of the myogenic cell line H9c2 into the muscle phenotype. Cell cultures were committed via a 7-day treatment with insulin which induced increase in the activity of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway, consistent with myogenic differentiation. To evaluate the role of polyamines in the differentiation process, cells were transfected with a plasmid overexpressing a stable ornithine decarboxylase, under control of a constitutive promoter. Overexpressing cells spontaneously differentiate into myotubes, without the need for induction with insulin; multinuclear myotubes and myogenin expression were apparent within 2 days of confluency of cultures. Polyamine depletion-by means of alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase-abolished the differentiation process. These observations support the evidence that polyamines are a key step involved in differentiation of muscle cells.

BACKGROUND: In hypotension-prone patients, conventional bicarbonate dialysis (BD) causes a reduced cardiovascular tolerance to the treatment with respect to acetate-free biofiltration (AFB). One possible explanation is an overproduction of endogenous NO (nitric oxide) due to the residual quote of acetate (4 mM) in the BD dialysate formulation. NO overload might cause the impairment of cardiovascular reactivity observed during BD. In this study, a potential direct impact of the residual quote of acetate on the cardiac cells was investigated. METHODS: Ventricular cardiac myocytes isolated from adult rat hearts were treated with three different dialysis baths with or without acetate: BD, AFB and AFB + 4 mM of acetate (AFB(+)). Corresponding levels of expression of the inducible NO synthase 2 (NOS2) were assessed after the treatments along with the measurement of single-cell action potential (AP). RESULTS: Incubation with acetate-containing dialysis solutions significantly enhanced (P < 0.05) the expression of NOS2 mRNA (BD: 1.11 +/- 0.31; AFB(+): 0.73 +/- 0.04, NOS2/GAPDH intensitometric ratio) with respect to the acetate-free bath (AFB: 0.03 +/- 0.01). Accordingly, protein translation was also enhanced (BD: 0.176 +/- 0.021; AFB(+): 0.135 +/- 0.009, NOS2/alpha-tubuline intensitometric ratio) with respect to AFB (0.002 +/- 0.001, P < 0.05). Measurement of the AP indicates that acetate-containing solutions determine a shortening of the repolarization phase as compared to treatment with AFB (BD: 95 +/- 13; AFB(+): 76 +/- 10; AFB: 162 +/- 16 ms). CONCLUSION: These findings show that the residual quote of acetate of the BD bath formulation affects the expression of NOS2 and the duration of AP in cardiac cells. This might cause the cardiac contractile impairment in unstable patients during BD treatment.

Manuale di reumatologia per le professioni sanitarie.

Objectives: To evaluate the effects of rRNA synthesis inhibition on cell cycle progression and cell population growth according to the RB and p53 status. Material and methods: RB‐ and p53‐proficient U2OS cells and the RB‐ and p53‐deficient SAOS‐2 cells were used, rRNA transcription hindered by actinomycin D, and cell cycle analysed by flow cytometry. Results: One hour of actinomycin D treatment induced in U2OS cells a block at the cell cycle checkpoints G(1)‐S and G(2)‐M, which was removed only after rRNA synthesis was resumed. rRNA synthesis inhibition did not influence cell cycle progression in SAOS‐2 cells. No effect on cell cycle progression after actinomycin D‐induced rRNA inhibition was also found in U2OS cells silenced for RB and p53 expression. A mild perturbation of cell cycle progression was observed in U2OS cells silenced for the expression of either RB or p53 alone. We also treated U2OS and SAOS‐2 cells with actinomycin D for 1 h/day for 5 days. This treatment lightly reduced growth rate of the U2OS cell population, whereas cell population growth of SAOS‐2 cells was completely inhibited. A marked reduction of ribosome content occurred in SAOS‐2 cells after the long‐term actinomycin D treatment, whereas no modification was observed in U2OS cells. Conclusions: These results demonstrate that inhibition of ribosome biogenesis does not hinder cell cycle progression in RB‐ and p53‐deficient cells. A daily‐repeated transitory inhibition of ribosome biogenesis leads to a progressive reduction of ribosome content with the consequent extinction of cancer cell population lacking RB and p53.

Introduction: Gout is the most prevalent arthritis and significantly impacts on function and quality of life. Given that gout associates with disabling comorbid conditions, it is not clear whether such a complex of diseases accounts for the increased disability or if gout may play a role by itself. This study aims to evaluate the specific influence of gout and disease-related features on functional disability and health-related quality of life (HRQoL) in patients with gout followed in rheumatology clinics.Methods: A random sample of patients was drawn from clinical registries of 30 rheumatology clinics across Italy. Sociodemographic, general health and gout-specific variables were collected. Functional disability and HRQoL were assessed by the health assessment questionnaire (HAQ) and the Physical and Mental Component Summary scores (PCS and MCS) of the Short Form-36 (SF-36). Crude and adjusted ordinal logistic and linear regression models were applied to investigate the specific contribution of different variables on HAQ and SF-36 scores. Results are presented as odds ratio (OR) or mean difference (MD) and 95% confidence intervals.Results: Out of 446 patients with gout, 90% were males with a mean age of 63.9 years and median disease duration of 3.8 years; the majority of patients were overweight or obese, and with several comorbidities; 21.1% showed at least moderate disability; the PCS score was significantly lower than expected age- and gender-matched samples in the general population, while MCS score was not. After adjusting for potential sociodemographic and general-health confounders, gout-specific variables significantly impacted on HAQ, including polyarticular involvement OR 3.82 (1.63, 8.95), presence of tophi OR 1.92 (1.07, 3.43) and recent attacks OR 2.20 (1.27, 3.81). Consistent results were found for PCS. The impairment of PCS compared to the general population was limited to patients with features of chronic gout. MCS was only affected by recent attacks (MD -2.72 [-4.58, -0.86]) and corticosteroid treatment (-3.39 [-5.30,-1.48]).Conclusions: The data from the KING study confirm that gout impacts on disability and provide evidence for an independent association of gout and gout-related features with functional outcome and HRQoL. This result supports the need to improve specific treatment in gout.

Many drugs currently used in chemotherapy work by hindering the process of ribosome biogenesis. In tumors with functional p53, the inhibition of ribosome biogenesis may contribute to the efficacy of this treatment by inducing p53 stabilization. As the level of stabilized p53 is critical for the induction of cytotoxic effects, it seems useful to highlight those cancer cell characteristics that can predict the degree of p53 stabilization following the treatment with inhibitors of ribosome biogenesis. In the present study we exposed a series of p53 wild-type human cancer cell lines to drugs such as actinomycin D (ActD), doxorubicin, 5-fluorouracil and CX-5461, which hinder ribosomal RNA (rRNA) synthesis. We found that the amount of stabilized p53 was directly related to the level of ribosome biogenesis in cells before the drug treatment. This was due to different levels of inactivation of the ribosomal proteins-MDM2 pathway of p53 digestion. Inhibition of rRNA synthesis always caused cell cycle arrest, independent of the ribosome biogenesis rate of the cells, whereas apoptosis occurred only in cells with a high rDNA transcription rate. The level of p53 stabilization induced by drugs acting in different ways from the inhibition of ribosome biogenesis, such as hydroxyurea (HU) and nutlin-3, was independent of the level of ribosome biogenesis in cells and always lower than that occurring after the inhibition of rRNA synthesis. Interestingly, in cells with a low ribosome biogenesis rate, the combined treatment with ActD and HU exerted an additive effect on p53 stabilization. These results indicated that (i) drugs inhibiting ribosome biogenesis may be highly effective in p53 wild-type cancers with a high ribosome biogenesis rate, as they induce apoptotic cell death, and (ii) the combination of drugs capable of stabilizing p53 through different mechanisms may be useful for treating cancers with a low ribosome biogenesis rate.