D.M.S. Ferreira

miR-34a/SIRT1/p53 is suppressed by ursodeoxycholic acid in the rat liver and activated by disease severity in human non-alcoholic fatty liver disease

BACKGROUND & AIMS Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of stages from simple steatosis to non-alcoholic steatohepatitis (NASH). However, disease pathogenesis remains largely unknown. microRNA (miRNA or miR) expression has recently been reported to be altered in human NASH, and modulated by ursodeoxycholic acid (UDCA) in the rat liver. Here, we aimed at evaluating the miR-34a/Sirtuin 1(SIRT1)/p53 pro-apoptotic pathway in human NAFLD, and to elucidate its function and modulation by UDCA in the rat liver and primary rat hepatocytes. METHODS Liver biopsies were obtained from NAFLD morbid obese patients undergoing bariatric surgery. Rat livers were collected from animals fed a 0.4% UDCA diets. Primary rat hepatocytes were incubated with bile acids or free fatty acids (FFAs) and transfected with a specific miRNA-34a precursor and/or with a p53 overexpression plasmid. p53 transcriptional activity was assessed by ELISA and target reporter constructs. RESULTS miR-34a, apoptosis and acetylated p53 increased with disease severity, while SIRT1 diminished in the NAFLD liver. UDCA inhibited the miR-34a/SIRT1/p53 pathway in the rat liver in vivo and in primary rat hepatocytes. miR-34a overexpression confirmed its targeting by UDCA, which prevented miR-34a-dependent repression of SIRT1, p53 acetylation, and apoptosis. Augmented apoptosis by FFAs in miR-34a overexpressing cells was also inhibited by UDCA. Finally, p53 overexpression activated miR-34a/SIRT1/p53, which in turn was inhibited by UDCA, via decreased p53 transcriptional activity. CONCLUSIONS Our results support a link between liver cell apoptosis and miR-34a/SIRT1/p53 signaling, specifically modulated by UDCA, and NAFLD severity. Potential endogenous modulators of NAFLD pathogenesis may ultimately provide new tools for therapeutic intervention.

miR-143 Overexpression Impairs Growth of Human Colon Carcinoma Xenografts in Mice with Induction of Apoptosis and Inhibition of Proliferation

Background MicroRNAs (miRNAs) are aberrantly expressed in human cancer and involved in the (dys)regulation of cell survival, proliferation, differentiation and death. Specifically, miRNA-143 (miR-143) is down-regulated in human colon cancer. In the present study, we evaluated the role of miR-143 overexpression on the growth of human colon carcinoma cells xenografted in nude mice (immunodeficient mouse strain: N: NIH(s) II-nu/nu). Methodology/Principal Findings HCT116 cells with stable miR-143 overexpression (Over-143) and control (Empty) cells were subcutaneously injected into the flanks of nude mice, and tumor growth was evaluated over time. Tumors arose ∼ 14 days after tumor cell implantation, and the experiment was ended at 40 days after implantation. miR-143 was confirmed to be significantly overexpressed in Over-143 versus Empty xenografts, by TaqMan® Real-time PCR (p<0.05). Importantly, Over-143 xenografts displayed slower tumor growth compared to Empty xenografts from 23 until 40 days in vivo (p<0.05), with final volumes of 928±338 and 2512±387 mm3, respectively. Evaluation of apoptotic proteins showed that Over-143 versus Empty xenografts displayed reduced Bcl-2 levels, and increased caspase-3 activation and PARP cleavage (p<0.05). In addition, the incidence of apoptotic tumor cells, assessed by TUNEL, was increased in Over-143 versus Empty xenografts (p<0.01). Finally, Over-143 versus Empty xenografts displayed significantly reduced NF-κB activation and ERK5 levels and activation (p<0.05), as well as reduced proliferative index, evaluated by Ki-67 immunohistochemistry (p<0.01). Conclusions Our results suggest that reduced tumor volume in Over-143 versus Empty xenografts may result from increased apoptosis and decreased proliferation induced by miR-143. This reinforces the relevance of miR-143 in colon cancer, indicating an important role in the control of in vivo tumor progression, and suggesting that miR-143 may constitute a putative novel therapeutic tool for colon cancer treatment that warrants further investigation.

Identification of microRNAs during rat liver regeneration after partial hepatectomy and modulation by ursodeoxycholic acid

New gene regulation study tools such as microRNA (miRNA or miR) analysis may provide unique insights into the remarkable ability of the liver to regenerate. In addition, we have previously shown that ursodeoxycholic acid (UDCA) modulates mRNA levels during liver regeneration. Bile acids are also homeotrophic sensors of functional hepatic capacity. The present study was designed to determine whether miRNAs are modulated in rats following 70% partial hepatectomy (PH) and elucidate the role of UDCA in regulating miRNA expression during liver regeneration (LR). Total RNA was isolated from livers harvested at 3-72 h following 70% PH or sham operations, from both 0.4% (wt/wt) UDCA and control diet-fed animals. By using a custom microarray platform we found that several miRNAs are significantly altered after PH by >1.5-fold, including some previously described as modulators of cell proliferation, differentiation, and death. In particular, expression of miR-21 was increased after PH. Functional modulation of miR-21 in primary rat hepatocytes increased cell proliferation and viability. Importantly, UDCA was a strong inducer of miR-21 both during LR and in cultured HepG2 cells. In fact, UDCA feeding appeared to induce a sustained increase of proliferative miRNAs observed at early time points after PH. In conclusion, miRNAs, in particular miR-21, may play a significant role in modulating proliferation and cell cycle progression genes after PH. miR-21 is additionally induced by UDCA in both regenerating rat liver and in vitro, which may represent a new mechanism behind UDCA biological functions.

Apoptosis and insulin resistance in liver and peripheral tissues of morbidly obese patients is associated with different stages of non-alcoholic fatty liver disease.

Aims/hypothesisNon-alcoholic fatty liver disease (NAFLD) is associated with insulin resistance and characterised by different degrees of hepatic lesion. Its pathogenesis and correlation with apoptosis and insulin resistance in insulin target tissues remains incompletely understood. We investigated how insulin signalling, caspase activation and apoptosis correlate with different NAFLD stages in liver, muscle and visceral adipose tissues.MethodsLiver, muscle and adipose tissue biopsies from 26 morbidly obese patients undergoing bariatric surgery were grouped according to the Kleiner–Brunt scoring system into simple steatosis, and less severe and more severe non-alcoholic steatohepatitis (NASH). Apoptosis was assessed by DNA fragmentation, and caspase-2 and -3 activation. Insulin signalling and c-Jun NH2-terminal kinase (JNK) proteins were evaluated by western blot.ResultsCaspase-3 and -2 activation, and DNA fragmentation were markedly increased in the liver of patients with severe NASH vs in that of those with simple steatosis (p < 0.01). Muscle tissue, and to a lesser extent the liver, had decreased tyrosine phosphorylated insulin receptor and insulin receptor substrate in patients with severe NASH, compared with those with simple steatosis (p < 0.01 muscle; p < 0.05 liver). Concomitantly, Akt phosphorylation decreased in muscle, liver and visceral adipose tissues in patients with severe NASH (at least p < 0.05). Finally, JNK phosphorylation was significantly increased in muscle (p < 0.01) and liver (p < 0.05) from NASH patients, compared with tissue from those with simple steatosis.Conclusions/interpretationOur results demonstrate a link between apoptosis, insulin resistance and different NAFLD stages, where JNK and caspase-2 may play a key regulatory role.

c-Jun N-Terminal Kinase 1/c-Jun Activation of the p53/MicroRNA 34a/Sirtuin 1 Pathway Contributes to Apoptosis Induced by Deoxycholic Acid in Rat Liver

ABSTRACT MicroRNAs (miRs) are increasingly associated with metabolic liver diseases. We have shown that ursodeoxycholic acid, a hydrophilic bile acid, counteracts the miR-34a/sirtuin 1 (SIRT1)/p53 pathway, activated in the liver of nonalcoholic steatohepatitis (NASH) patients. In contrast, hydrophobic bile acids, particularly deoxycholic acid (DCA), activate apoptosis and are increased in NASH. We evaluated whether DCA-induced apoptosis of rat hepatocytes occurs via miR-34a-dependent pathways and whether they connect with c-Jun N-terminal kinase (JNK) induction. DCA enhanced miR-34a/SIRT1/p53 proapoptotic signaling in a dose- and time-dependent manner. In turn, miR-34a inhibition and SIRT1 overexpression significantly rescued targeting of the miR-34a pathway and apoptosis by DCA. In addition, p53 overexpression activated the miR-34a/SIRT1/p53 pathway, further induced by DCA. DCA increased p53 expression as well as p53 transcriptional activation of PUMA and miR-34a itself, providing a functional mechanism for miR-34a activation. JNK1 and c-Jun were shown to be major targets of DCA, upstream of p53, in engaging the miR-34a pathway and apoptosis. Finally, activation of this JNK1/miR-34a proapoptotic circuit was also shown to occur in vivo in the rat liver. These results suggest that the JNK1/p53/miR-34a/SIRT1 pathway may represent an attractive pharmacological target for the development of new drugs to arrest metabolism- and apoptosis-related liver pathologies.

Revisiting the metabolic syndrome and paving the way for microRNAs in non-alcoholic fatty liver disease.

Non‐alcoholic fatty liver disease (NAFLD) comprises a spectrum of stages from simple steatosis to non‐alcoholic steatohepatitis, which can progress to fibrosis, cirrhosis and, ultimately, hepatocellular carcinoma. Despite being one of the most common chronic liver diseases, NAFLD pathogenesis remains largely unknown. In this review, we discuss the key molecular mechanisms involved in NAFLD development and progression, focusing on the emerging role of microRNAs. NAFLD is intrinsically related to obesity and the metabolic syndrome. Changes in lipid metabolism increase free fatty acids in blood, which in turn induces peripheral insulin resistance and increases oxidative and endoplasmic reticulum stress. Although not yet considered in the diagnosis of NAFLD, recent reports also reinforce the crucial role of apoptosis in disease progression via activation of either death receptor or mitochondrial pathways and p53. In addition, the role of gut microbiota and the gut–liver axis has been recently associated with NAFLD. Finally, there is an accumulating and growing body of evidence supporting the role of microRNAs in NAFLD pathogenesis and progression, as well as hinting at their use as biomarkers or therapeutic tools. The ultimate goal is to review different molecular pathways that may underlie NAFLD pathogenesis in the hope of finding targets for new and efficient therapeutic interventions.

Liver and muscle in morbid obesity: the interplay of fatty liver and insulin resistance.

Introduction Nonalcoholic fatty liver disease (NAFLD) can be seen as a manifestation of overnutrition. The muscle is a central player in the adaptation to energy overload, and there is an association between fatty-muscle and -liver. We aimed to correlate muscle morphology, mitochondrial function and insulin signaling with NAFLD severity in morbid obese patients. Methods Liver and deltoid muscle biopsies were collected during bariatric surgery in NAFLD patients. NAFLD Activity Score and Younossis classification for nonalcoholic steatohepatitis (NASH) were applied to liver histology. Muscle evaluation included morphology studies, respiratory chain complex I to IV enzyme assays, and analysis of the insulin signaling cascade. A healthy lean control group was included for muscle morphology and mitochondrial function analyses. Results Fifty one NAFLD patients were included of whom 43% had NASH. Intramyocellular lipids (IMCL) were associated with the presence of NASH (OR 12.5, p<0.001), progressive hepatic inflammation (p = 0.029) and fibrosis severity (p = 0.010). There was a trend to an association between IMCL and decreased Akt phosphorylation (p = 0.059), despite no association with insulin resistance. In turn, hepatic steatosis (p = 0.015) and inflammation (p = 0.013) were associated with decreased Akt phosphoryation. Citrate synthase activity was lower in obese patients (p = 0.047) whereas complex I (p = 0.040) and III (p = 0.036) activities were higher, compared with controls. Finally, in obese patients, complex I activity increased with progressive steatosis (p = 0.049) and with a trend with fibrosis severity (p = 0.056). Conclusions In morbid obese patients, presence of IMCL associates with NASH and advanced fibrosis. Muscle mitochondrial dysfunction does not appear to be a major driving force contributing to muscle fat accumulation, insulin resistance or liver disease. Importantly, insulin resistance in muscle might occur at a late point in the insulin signaling cascade and be associated with IMCL and NAFLD severity.

Cell death targets and potential modulators in Alzheimer’s disease

Apoptosis is now recognized as a normal feature in the development of the nervous system and may also play a role in neurodegenerative disorders, such as Alzheimers disease. Cell surface receptors, caspases, mitochondrial factors or p53 participate in the modulation and execution of cell death. Therefore, the ability to understand and manipulate the cell death machinery is an obvious goal of medical research. Potential therapeutic approaches to modulate disease by regulating apoptosis are being tested, and include the traditional use of small molecules to target specific players in the apoptosis cascade. As our understanding of apoptosis increases, further opportunities will arise for more specific therapies that will result in improved efficacy. This review focuses on molecular mechanisms of apoptosis in Alzheimers disease and highlights the potential use of small molecule modulators to treat neurodegenerative disorders.

Ledipasvir / Sofosbuvir for The Treatment of Chronic Hepatitis C: A Cost-Effectiveness Analysis Across Different Genotype 1 Clinical Subgroups

Almeida JM1, Félix J1, Ferreira D1, Mota M1, Afonso-Silva M1, Silva P1, Vandewalle B1, Velosa J2, Marinho R2, Aldir I3, Carvalho A4, Valente C4, Macedo G5, Sarmento e Castro R6, Pedroto I6 1Exigo Consultores; 2Hospital Santa Maria, Centro Hospitalar de Lisboa Norte, E.P.E.; 3Hospital Egas Moniz, Centro Hospitalar de Lisboa Ocidental, E.P.E.; 4Centro Hospitalar e Universitário de Coimbra, E.P.E.; 5Centro Hospitalar de São João, E.P.E.; 6Centro Hospitalar do Porto, E.P.E.

Therapeutic and Economic Value of Everolimus Plus Exemestane for the Treatment of Postmenopausal Women with Hormone Receptor-Positive, Her2/Neu Negative Advanced Breast Cancer.

Cost-effectiveness of everolimus+exemestane as compared to fulvestrant was assessed in terms of the incremental cost per life year (LY) gained, from a Portuguese National Health Service (NHS) perspective. Next to main therapeutic drug costs, other healthcare costs included costs for medical visits, complementary diagnostic procedures, surgery, inpatient visits, additional therapy and end-of-life care. An annual 5% discount rate was applied to both costs and effectiveness.