EFFECTS OF HISTONE DEACETYLASE INHIBITORS ON ENERGY METABOLISM

Type 2 diabetes mellitus (T2DM) is the most common metabolic disease in the world. Maintenance of glucose homeostasis depends on a complex interplay between the insulin responsiveness of skeletal muscle, liver, adipose tissue and glucose-stimulated insulin secretion by pancreatic beta cells. Defects in these organs are responsible for insulin resistance and progression to hyperglycemia. Understanding the integrated pathophysiology initiating the development of insulin resistance should extend our capacity to identify novel therapeutic targets for the prevention and/or treatment of T2DM. This biology remains incompletely characterized, in part, due to the interaction of multiple organ systems. The complexity of this biology is further underscored by the progressive changes in the systemic milieu including the onset of hyperinsulinemia, elevated circulating free fatty acids and triglycerides, hyperglycemia, and the activation of systemic immune system during the development of T2DM. In skeletal muscle, loss of mitochondrial function is evident in some insulin-resistant subjects years before they develop diabetes. Mitochondria are particularly important for skeletal muscle function, given the high oxidative demands imposed on this tissue by intermittent contraction. Moreover, muscle cells must maintain metabolic flexibility, defined as the ability to rapidly modulate substrate oxidation as a function of hormonal and energetic conditions. The molecular mechanisms that control mitochondrial number and function remain poorly understood, and only a few transcription factors or coactivators (e.g., PGC-1\u3b1, NRF1, Tfam) have been associated with this process. Notably, skeletal muscle differentiation and remodelling are also controlled at the epigenetic level, via transcriptional modulation of key genes in mitochondrial biogenesis and oxidative metabolism, involving enzymes, such as members of the histone deacetylase (HDAC) family, in particular those belonging to class I and class II, which modulate post-translational modifications on target proteins. The current knowledge on HDACs is that they function in general as transcriptional repressors, however their role in vivo is likely more complex. Less is known on the effects of HDACs modulators on energy metabolism, however a recent study reported that supplementation with sodium butyrate, a dietary component active as HDAC inhibitor, promotes energy expenditure and mitochondrial function in mice fed with a high fat diet212. Given the importance of skeletal muscle metabolism in insulin resistance/diabetes, and given the role of HDACs in skeletal muscle biology, it is reasonable to speculate that modulation of these enzymes would play a role in this pathology that deserves to be investigated more deeply. Based on the evidences that mitochondrial dysfunction is often associated to whole body metabolic dysregulation, aim of this study is to better understand the role of histone deacetylases in the regulation of mitochondrial biogenesis and in the modulation of all these mechanisms underlying the pathophisiology of insulin resistance. In C2C12 myotubes treated with pan and class selective HDAC inhibitors (HDACi), such as SAHA(pan-inhibitor), MS275 (Class I HDAC inhibitor) and MC1568 (Class II HDAC inhibitor), transcriptome analysis revealed an increase of OXPHOS genes and of genes encoding fatty acid catabolic enzymes, following treatment with pan or class I HDACi. Moreover, staining of myotubes treated with SAHA and MS275 showed an increase of mitochondrial density and activity, coupled with an increase in mitochondrial DNA content. In Db/Db obese and diabetic mice we observed that treatments with SAHA and MS275 reduce glycemia, triglycerides, plasma insulin land transaminases levels and improves glucose clearance and insulin sensitivity. In vivo metabolic study revealed an increase of oxygen consumption, a net decrease of the respiratory exchange ratio and an increased heat production, implying a more oxidative metabolism, in the MS275 treated group. In order to characterize the effects of HDACi in different tissues, we performed microarray analysis in skeletal muscles, liver, brown and white adipose tissues and histological analysis. In the skeletal muscle of mice treated with MS275, and with SAHA to a lesser degree, we observed a general increase in OXPHOS genes and in genes involved in lipid and glucose metabolism. These observations were also supported by the increased oxidative capacity highlighted by SDH staining in gastrocnemius sections. In brown fat we found an increased expression of brown adipocytes markers , such as PRDM16, CIDEA, UCP1, ELOVL3 and DIO2, and an induction of mitochondrial biogenesis, after MS275 administration. In parallel, adypocyte size appeared smaller respect to the adipocytes of the control group. In white adipose tissue, we observed an increase of adipocytes markers and, even in this case, an induction of mitochondrial biogenesis; adipocytes, beyond being smaller, also showed a reduced macrophages infiltration. Gene expression also revealed an increased expression of brown adipocytes markers, such as UCP1, also confirmed by immunohistochemistry assay, ADRB3, ELOVL3 and DIO2. Nevertheless, white adipocytes remained PRDM16 negative. Collectively, our results suggest that HDACs, and in particular Class I HDACs, play an unexpected role in energy metabolism, induce mitochondrial biogenesis in different tissues and may represent key regulators in diseases based on metabolic alterations

Limited utility of ITPA deficiency to predict early anemia in HCV patients with advanced fibrosis receiving telaprevir

Background: Severe anemia is a common side effect of Pegylated Interferon + Ribavirin (PR) and Telaprevir (TVR) in hepatitis C virus (HCV) genotype 1 patients with advanced fibrosis or cirrhosis (F3-F4). Inosine triphosphatase (ITPA) genetic variants are associated with RBV- induced anemia and dose reduction. Aim: To test the association of ITPA polymorphisms rs1127354 and rs7270101 with hemoglobin (Hb) decline, need for RBV dose reduction (RBV DR), erythropoietin (EPO) support and blood transfusions during the first 12 weeks of TVR triple therapy. Materials and Methods: 69 consecutive HCV-1 patients (mean age 57 years) with F3-F4 who received PR and TVR were genotyped for ITPA polymorphisms rs1127354 and rs7270101. Estimated ITPA deficiency was graded on severity (0-3, no deficiency/mild/moderate/severe). Results: ITPA deficiency was absent in 48 patients (70%), mild in 12 (17%) and moderate in 9 patients (13%). Mean week 4 Hb decline was higher in non ITPA deficient patients (3,85 g/dL) than in mildly or moderately ITPA deficient patients (3,07 g/dL and 1,67 g/dL, p<0.0001). Grade 3-4 anemia developed in 81% non ITPA deficient patients versus 67% mild deficient and 55% moderate deficient patients (p = ns). Grade of ITPA deficiency was not associated with RbvDR (no deficiency: 60%, mild: 58%, moderate: 67%; p = ns), EPO use (no deficiency: 65%, mild: 58%, moderate:56%; p = ns) or need for blood transfusion (no deficiency: 27%, mild: 17%, moderate: 33%; p = ns). Conclusions: In patients with F3-F4 chronic hepatitis C receiving TVR based therapy, ITPA genotype does not impact on the management of early anemia. \ua9 2014 Aghemo et al

Thirty-day mortality and five-year survival in thoracic surgery: &quot;real-world&quot; assessment of outcomes from a single-institution audit

Background Accurate measurement of outcomes is essential to monitor the effectiveness of public health policies. In Italy, the Ministry of Health has chosen 30-day mortality after major surgical or medical procedures as the main outcome measure, pooling all pulmonary resections for malignancy in a single category. The present audit evaluated all pulmonary resections performed over a 13-year period in a single institution to assess the immediate (30-day mortality) and long-term (5-year survival) outcomes according to type and stage of disease and extent of surgery. Methods We analyzed the results of 4,234 first pulmonary resections performed from 2003 to 2015 for lung cancer (2,636), lung metastases (1,080), other primary cancers (259) and benign diseases (259). The median follow-up of cancer patients was 4.1 years. Results Overall 30-day mortality was 1.1%, being 1.2% for lung cancer, 0.3% for lung metastases, 3.5% for pneumonectomies, 1% for lobectomies, and 0.5% for sublobar resections. Among lung cancer patients, 30-day mortality was 0.7% for simple anatomical resections, 2.8% for complex resections, 0.7% for stage I, and 1.6% for higher stages. Overall 5-year survival was 56% for lung cancer, 49% for lung metastases, and 53% for other primary cancers (p = 0.03). According to the surgical procedure for lung cancer, 5-year survival was 60%, 55% and 36% for lobectomies, segmentectomies and pneumonectomies, respectively (p<0.0001). Conclusions For better monitoring of thoracic surgery outcomes in a real-world setting, we suggest evaluating lung cancer separately from other thoracic malignancies, and including 5-year survival rates stratified by resection volume and surgical procedure complexity

Melanoma contains CD133 and ABCG2 positive cells with enhanced tumorigenic potential

The failure to eradicate most cancers and in particular melanoma may be as fundamental as a misidentification of the target. The identification of cancer stem/initiating cells within the tumour population with a crucial role for tumour formation may open new pharmacological perspectives. Our data show three main novelties for human melanoma: firstly, melanoma biopsy contains a subset of cells expressing CD133 (CD133+) and the latter is able to develop a Mart-1 positive tumour in NOD-SCID mice. Secondly, the WM115, a human melanoma cell line, has been found to express both CD133 and ABCG2 markers. This cell line grows as floating spheroids, expresses typical progenitors and mature neuronal/oligodendrocyte markers and is able to transdifferentiate into astrocytes or mesenchymal lineages under specific growth conditions. As in xenografts generated with CD133+ biopsy melanoma cells, those produced by the cell line displayed lower levels of CD133 and ABCG2. Thirdly, the WM115 cells express the most important angiogenic and lymphoangiogenic factors such as notch 4, prox1 and podoplanin which can cooperate in the development of the tumourigenic capability of melanoma in vivo. Therefore, in this study, we demonstrate the presence of stem/initiating subsets in melanoma both in biopsy and in an established melanoma cell line grown in vitro and in xenografts. Interestingly, considering that melanoma gives metastasis primarily through lymphatic vessels, herein, we demonstrated that a melanoma cell line expresses typical lymphoangiogenic factors

The association of IL28B genotype with the histological features of chronic hepatitis C is HCV genotype dependent

The interleukin 28B (IL28B) rs12979860 polymorphism is associated with treatment outcome in hepatitis C virus (HCV) genotype 1 and 4 patients. Its association with the histological features of chronic hepatitis C and disease severity needs further clarifications. To assess the correlation between IL28B genotype, HCV genotype and liver biopsy findings in untreated patients. Materials and Methods: Pre-treatment liver biopsies from 335 HCV Caucasian patients (59% males, age 50 years) enrolled in the MIST study were staged for fibrosis and inflammation according to the METAVIR and the Ishak scoring systems; steatosis was dichotomized as = 5%. IL28B was typed by Taqman Single Nucleotide Polymorphism (SNP) genotyping assay. HCV genotype was 1 in 151 (45%), 2 in 99 (30%), 3 in 50 (15%) and 4 in 35 (10%) patients. IL28B genotype was CC in 117 (34%), CT in 166 (49%) and TT in 52 (15%). At univariate analysis, the IL28B CC genotype was associated with severe portal inflammation in HCV-1 patients (CC vs. CT/TT: 86% vs. 63%, p = 0.005), severe lobular inflammation in HCV-2 patients (CC vs. CT/TT: 44% vs. 23%, p = 0.03), and less fatty infiltration in HCV-1 patients (CC vs. CT/TT: 72% vs. 51%, p = 0.02). Despite the lack of any association between IL28B and fibrosis stage, in HCV-3 patients IL28B CC correlated with METAVIR F3-F4 (CC vs. CT/TT: 74% vs. 26%, p = 0.05). At multivariate analysis, the genotype CC remained associated with severe portal inflammation in HCV-1, only (Odds Ratio (OR): 95% Confidence Interval (CI): 3.24 (1.23-8.51)). IL28B genotype is associated with the histological features of chronic hepatitis C in a HCV genotype dependent manner, with CC genotype being independently associated with severe portal inflammation

The impact of IL28B genotype on the gene expression profile of patients with chronic hepatitis C treated with pegylated interferon alpha and ribavirin

<p>Abstract</p> <p>Background</p> <p>Recent studies of CH-C patients have demonstrated a strong association between IL28B CC genotype and sustained virologic response (SVR) after PEG-IFN/RBV treatment. We aimed to assess whether IL28B alleles rs12979860 genotype influences gene expression in response to PEG-IFN/RBV in CH-C patients.</p> <p>Methods</p> <p>Clinical data and gene expression data were available for 56 patients treated with PEG-IFN/RBV. Whole blood was used to determine IL28B genotypes. Differential expression of 153 human genes was assessed for each treatment time point (Days: 0, 1, 7, 28, 56) and was correlated with IL28B genotype (IL28B C/C or non-C/C) over the course of the PEG-IFN/RBV treatment. Genes with statistically significant changes in their expression at each time point were used as an input for pathway analysis using KEGG Pathway Painter (KPP). Pathways were ranked based on number of gene involved separately per each study cohort.</p> <p>Results</p> <p>The most striking difference between the response patterns of patients with IL28B C/C and T* genotypes during treatment, across all pathways, is a sustained pattern of treatment-induced gene expression in patients carrying IL28B C/C. In the case of IL28B T* genotype, pre-activation of genes, the lack of sustained pattern of gene expression or a combination of both were observed. This observation could potentially provide an explanation for the lower rate of SVR observed in these patients. Additionally, when the lists of IL28B genotype-specific genes which were differentially expressed in patients without SVR were compared at their baseline, IRF2 and SOCS1 genes were down-regulated regardless of patients' IL28B genotype. Furthermore, our data suggest that CH-C patients who do not have the SOCS1 gene silenced have a better chance of achieving SVR. Our observations suggest that the action of SOCS1 is independent of IL28B genotype.</p> <p>Conclusions</p> <p>IL28B CC genotype patients with CH-C show a sustained treatment-induced gene expression profile which is not seen in non-CC genotype patients. Silencing of SOCS1 is a negative and independent predictor of SVR. These data may provide some mechanistic explanation for higher rate of SVR in IL28B CC patients who are treated with PEG-IFN/RBV.</p

Reperfusion Injury after ischemic Stroke Study (RISKS): single-centre (Florence, Italy), prospective observational protocol study

Introduction Treatments aiming at reperfusion of the acutely ischaemic brain tissue may result futile or even detrimental because of the so-called reperfusion injury. The processes contributing to reperfusion injury involve a number of factors, ranging from blood\u2013brain barrier (BBB) disruption to circulating biomarkers. Our aim is to evaluate the relative effect of imaging and circulating biomarkers in relation to reperfusion injury. Methods and analysis Observational hospital-based study that will include 140 patients who had ischaemic stroke, treated with systemic thrombolysis, endovascular treatment or both. BBB disruption will be assessed with CT perfusion (CTP) before treatment, and levels of a large panel of biomarkers will be measured before intervention and after 24 hours. Relevant outcomes will include: (1) reperfusion injury, defined as radiologically relevant haemorrhagic transformation at 24 hours and (2) clinical status 3 months after the index stroke. We will investigate the separate and combined effect of pretreatment BBB disruption and circulating biomarkers on reperfusion injury and clinical status at 3 months. Study protocol is registered at http://www. clinicaltrials. gov ( ClinicalTrials. gov ID: NCT03041753). Ethics and dissemination The study protocol has been approved by ethics committee of the Azienda Ospedaliero Universitaria Careggi (Universit\ue0 degli Studi di Firenze). Informed consent is obtained by each patient at time of enrolment or deferred when the participant lacks the capacity to provide consent during the acute phase. Researchers interested in testing hypotheses with the data are encouraged to contact the corresponding author. Results from the study will be disseminated at national and international conferences and in medical thesis. Trial registration number NCT03041753