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Valentina Perrone,1 Chiara Veronesi,1 Elisa Giacomini,1 Rita Citraro,2 Stefania Dell’Orco,3 Fabio Lena,4 Arrigo Paciello,5 Anna Maria Resta,6 Mihaela Nica,7 Daniela Ritrovato,7 Luca Degli Esposti1 1Clicon S.r.l. Società Benefit, Health Economics & Outcomes Research, Bologna, Italy; 2Dipartimento di Scienze della Salute, Università Magna Grecia di Catanzaro, Unita’ Operativa di Farmaco-logia Clinica e Farmacovigilanza, Azienda Ospedaliero-Universitaria “Mater Domini”, Catanzaro, Italy; 3ASL Roma 6, Rome, Italy; 4USL Toscana Sud Est, Grosseto, Italy; 5ATS Bergamo, Bergamo, Italy; 6Struttura Complessa di Farmacia Territoriale Area Vasta 1, Fano, Italy; 7Novartis Farma S.p.A, Origgio, Varese, ItalyCorrespondence: Valentina Perrone, CliCon S.r.l. Società Benefit, Health, Economics & Outcomes Research, Via Murri, 9, Bologna, 40137, Italy, Tel +39 3450316494, Email valentina.perrone@clicon.itPurpose: A retrospective analysis of real-world data was performed to assess the epidemiology and economic burden of multiple sclerosis (MS), relapsing-remitting MS (RRMS), and secondary-progressive MS (SPMS) in Italy.Patients and Methods: An observational study on administrative databases from a sample of Italian entities was carried-out. Between 01/2010– 12/2017, patients with ≥ 1 MS diagnosis code (ICD-9-CM:340 and/or exemption code:046) and/or ≥ 1 disease-modifying therapies (DMTs) prescription, were included. Among MS-cohort, SPMS patients were identified by ≥ 2 hospitalizations or by ≥ 2 drug prescriptions related to MS progression. MS patients not fulfilling SPMS criteria were included as RRMS. Mean annual healthcare costs were reported during follow-up and stratified by DMT treatment/untreatment.Results: Overall, 9543 MS patients were included; 8397 with RRMS and 1146 with SPMS. Estimated prevalence of MS was 141.6/100,000 inhabitants (RRMS 124.4/100,000 and SPMS 17.2/100,000). Mean annual cost for untreated and treated patient was respectively: € 3638 and € 11796 (MS-cohort), € 3183 and € 11486 (RRMS-cohort), € 6317 and € 15511 (SPMS-cohort). The first-line DMT treatment duration averaged 27.4 ± 22.8 months; the mean cost was 19004€ for the whole period. The second-line DMT treatment lasted on average 31.1 ± 24.5 months; the mean cost was 47293€ for the whole period.Conclusion: This study provided insights into the MS epidemiology in Italy and its economic burden. Healthcare costs associated with MS management were mainly driven by DMTs expenditure. A trend of higher healthcare-resource consumption was observed among SPMS-cohort.Keywords: real-world evidence, clinical practice, multiple sclerosis, epidemiology, healthcare resource consumption

V Perrone,1 C Veronesi,1 M Nica,2 D Colombo,2 L Degli Esposti,1 R Castello31Clicon S.R.L. Health, Economics and Outcomes Research, Ravenna, Italy; 2Novartis Pharma, Origgio, VA, Italy; 3Division of General Medicine, University Hospital of Verona, Verona, ItalyAbstract: Sex-related differences have been shown to deeply affect health-related aspects of patients. However, the lack of gender-specific analysis makes it difficult to advance personalized medicine in terms of a gender-based approach. The aim of the present study was to describe gender-specific features of patients diagnosed with heart failure (HF), with a focus on the clinical presentation. Data were collected from a properly designed database and referred to an Italian hospital. Patients aged ≥18 years with a primary or secondary diagnosis of HF between 1 January 2012 and 31 December 2016 were included, and their demographic and clinical characteristics were analyzed according to gender. Of the 719 HF patients included, 317 (44.1%) were male and 402 (55.9%) were female. Women tended to be older compared to men (82.4±8.8 years and 77.1±10.6 years, respectively). As for clinical presentation, 62.1% of female and 38.3% of male patients had preserved ejection fraction, and 56.1% of men and 58.2% of women suffered from atrial fibrillation. The left atrium was partially compromised in 62.4% of male and 63% of female patients, while right atrium dysfunction tended to be more frequent in male patients compared to female patients (29.1% and 25.5%, respectively). In conclusion, gender-specific features of a cohort of HF patients from a clinical setting were accurately described.Keywords: gender, specific characteristics, heart failure, clinical setting

As nonalcoholic fatty liver disease progresses to end-stage diseases, including fibrosis, cirrhosis and hepatocellular carcinoma, fibrotic activated hepatic stellate cells and cancerous epithelial cells can become abundant, changing the cellular composition of this organ. Despite potentially residing within the same diseased tissue, direct comparisons of global gene expression between activated hepatic stellate cells and hepatocellular carcinoma cells are lacking. Here we provide data collected using Affymetrix GeneChip microarrays to identify differential gene expression in cultured primary human activated hepatic stellate cells compared to HepG2 human hepatoma cells. The dataset includes many genes involved in intermediary metabolism which were investigated in greater depth in our associated article (A.M. Hetherington, C.G. Sawyez, E. Zilberman, A.M. Stoianov, D.L. Robson, J.M. Hughes-Large, et al., 2016) [1]. Pathway analyses of known protein coding genes down-regulated or up-regulated by greater than 2.0-fold are also provided.

Low dose niacin and vitamin D can directly improve human microvascular endothelial cell angiogenic function under lipotoxic conditions Peters et al.,2019. Despite exerting similar benefits on in vitro angiogenic function, these vitamins are known to signal through independent receptors, raising the possibility that differential changes in gene expression may underlie these effects. Here we provide data collected using Affymetrix GeneChip microarrays to compare gene expression in human microvascular endothelial cells treated for 16 h with growth medium containing BSA alone, or BSA complexed with the saturated fatty acid palmitate, and supplemented with 10 μM niacin or 10 nM vitamin D (1,25-dihydroxyvitamin D3). Data sets of differential gene expression included many genes involved in cellular stress responses. Pathway analyses of genes specific to vitamin D treatment identified a robust overrepresentation of pathways related to the cell cycle and DNA replication and repair.

Medicinal use of mushrooms has been documented since ancient times, and in the modern world, mushrooms have a longstanding history of use in Eastern medicine. Recent interest in plant-based diets in Westernized countries has brought increasing attention to the use of mushrooms and mushroom-derived compounds in the prevention and treatment of chronic diseases. Edible mushrooms are the most abundant food sources of the modified amino acid, ergothioneine. This compound has been shown to accumulate in almost all cells and tissues, but preferentially in those exposed to oxidative stress and injury. The demonstrated cytoprotectant effect of ergothioneine has led many to suggest a potential therapeutic role for this compound in chronic conditions that involve ongoing oxidative stress and inflammation, including cardiovascular and metabolic diseases. However, the in vivo effects of ergothioneine and its underlying therapeutic mechanisms in the whole organism are not as clear. Moreover, there are no well-defined, clinical prevention and intervention trials of ergothioneine in chronic disease. This review highlights the cellular and molecular mechanisms of action of ergothioneine and its potential as a Traditional, Complementary and Alternative Medicine for the promotion of cardiometabolic health and the management of the most common manifestations of cardiometabolic disease.

Prolonged, isocaloric, time-restricted feeding (TRF) protocols can promote weight loss, improve metabolic dysregulation, and mitigate non-alcoholic fatty liver disease (NAFLD). In addition, 3-day, severe caloric restriction can improve liver metabolism and glucose homeostasis prior to significant weight loss. Thus, we hypothesized that short-term, isocaloric TRF would improve NAFLD and characteristics of metabolic syndrome in diet-induced obese male mice. After 26 weeks of ad libitum access to western diet, mice either continued feeding ad libitum or were provided with access to the same quantity of western diet for 8 h daily, over the course of two weeks. Remarkably, this short-term TRF protocol modestly decreased liver tissue inflammation in the absence of changes in body weight or epidydimal fat mass. There were no changes in hepatic lipid accumulation or other characteristics of NAFLD. We observed no changes in liver lipid metabolism-related gene expression, despite increased plasma free fatty acids and decreased plasma triglycerides in the TRF group. However, liver Grp78 and Txnip expression were decreased with TRF suggesting hepatic endoplasmic reticulum (ER) stress and activation of inflammatory pathways may have been diminished. We conclude that two-week, isocaloric TRF can potentially decrease liver inflammation, without significant weight loss or reductions in hepatic steatosis, in obese mice with NAFLD.

Background/Aims: Nonalcoholic fatty liver disease (NAFLD) progression to fibrosis, cirrhosis and hepatocellular carcinoma, alters the cellular composition of this organ. During late-stage NAFLD, fibrotic and possibly cancerous cells can proliferate and, like normal hepatocytes, are exposed to high concentrations of fatty acids from both surrounding tissue and circulating lipid sources. We hypothesized that primary human activated hepatic stellate cells and epithelial hepatoma (HepG2) cells respond differently to lipotoxic conditions, and investigated the mechanisms involved. Methods: Primary activated hepatic stellate cells and HepG2 cells were exposed to pathophysiological concentrations of fatty acids and comparative studies of lipid metabolic and stress response pathways were performed. Results: Both cell types remained proliferative during exposure to a combination of palmitate plus oleate reflective of the general saturated versus unsaturated fatty acid composition of western diets. However, exposure to either high palmitate or high oleate alone induced cytotoxicity in activated stellate cells, while only palmitate caused cytotoxicity in HepG2 cells. mRNA microarray and biochemical comparisons revealed that stellate cells stored markedly less fatty acids as neutral lipids, and had reduced capacity for beta-oxidation. Similar to previous observations in HepG2 cells, palmitate, but not oleate, induced ER stress and actin stress fiber formation in activated stellate cells. In contrast, oleate, but not palmitate, induced the inflammatory signal TXNIP, decreased cytoskeleton proteins, and decreased cell polarity preceding cell death in activated stellate cells. Conclusions: Palmitate-induced lipotoxicity was associated with ER stress pathways in both primary activated hepatic stellate cells and epithelial hepatoma cells, whereas high oleate caused lipotoxicity only in activated stellate cells, possibly through a distinct mechanism involving disruption of cytoskeleton components. This may have implications for optimal dietary fatty acid compositions during various stages of NAFLD.

Delia Colombo,1 Emanuela Zagni,1 Mihaela Nica,1 Sara Rizzoli,2 Alessandra Ori,2 Gilberto Bellia1 1Patient Access, Novartis Farma S.p.A., Origgio, Varese, 2MediNeos Observational Research, Modena, Italy Background: MetaGeM is a wide gender-medicine project comprising post hoc and meta-analyses by gender of clinical outcomes, therapeutic approaches, and safety data from previously conducted observational studies to explore possible gender differences in real-life clinical settings. We report the results of the safety meta-analysis of seven MetaGeM studies, evaluating gender differences in adverse event (AE) incidence and severity. Methods: Data were collected between February 2002 and July 2013. Male and female patients were compared for the main safety variables, using Student’s t-test, χ² test, or Fisher’s exact test as appropriate. As supportive analysis, a logistic regression model was estimated to evaluate associations between gender and outcome. Results: In total, 4,870 patients (46% females, 54% males) were included in the analysis; age was higher for females (mean ± standard deviation 61.2±18.3 years) than males (56.3±16.6 years). Overall, 264 AEs were reported (59.1% in males). There were no significant gender differences in the percentage of patients with at least one AE: 3.0% for females versus 3.9% for males, χ² test P>0.05. According to the logistic regression model results, no association between gender and AEs occurrence seems to exist. A statistically significant gender difference in the percentage of drug-related AEs emerged (37.6% in females vs 20.8% in males, χ² P=0.0039). Slightly significantly more AEs in females were addressed with treatment compared with males (78.1% vs 66.7%, χ² P=0.0485). Total serious AEs (SAEs) were 47 (72% in males). The frequency of patients with ≥1 SAE was 0.6% in females versus 1.2% in males (χ² test P=0.0246). Conclusion: This safety analysis on a large sample of almost 5,000 patients with different diseases and treated with a wide range of different drugs provides a useful overview on possible gender differences in drug tolerability, which may be helpful in more accurately designing future clinical trials from a gender-specific perspective. Keywords: gender, drugs, safety, adverse events, meta-analysis

The systemic lipid modifying drug, niacin, can directly improve human microvascular endothelial cell angiogenic function under lipotoxic conditions, possibly through activation of niacin receptors “Niacin receptor activation improves human microvascular endothelial cell angiogenic function during lipotoxicity” (Hughes-Large et al. 2014). Here we provide accompanying data collected using Affymetrix GeneChip microarrays to identify changes in gene expression in human microvascular endothelial cells treated with 10 μM niacin. Statistical analyses of robust multi-array average (RMA) values revealed that only 16 genes exhibited greater than 1.3-fold differential expression. Of these 16, only 5 were identified protein coding genes, while 3 of the remaining 11 genes appeared to be small nuclear/nucleolar RNAs. Altered expression of EFCAB4B, NAP1L2, and OR13C8 was confirmed by real time quantitative PCR. Keywords: Vascular biology, Endothelial cells, Niacin, Lipids

Elongation factor 1A-1 (eEF1A-1) has non-canonical functions in regulation of the actin cytoskeleton and apoptosis. It was previously identified through a promoter-trap screen as a mediator of fatty acid-induced cell death (lipotoxicity), and was found to participate in this process downstream of ER stress. Since ER stress is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), we investigated the mechanism of action of eEF1A-1 in hepatocyte lipotoxicity. HepG2 cells were exposed to excess fatty acids, followed by assessments of ER stress, subcellular localization of eEF1A-1, and cell death. A specific inhibitor of eEF1A-1 elongation activity, didemnin B, was used to determine whether its function in protein synthesis is involved in lipotoxicity. Within 6 h, eEF1A-1 protein was modestly induced by high palmitate, and partially re-localized from its predominant location at the ER to polymerized actin at the cell periphery. This early induction and subcellular redistribution of eEF1A-1 coincided with the onset of ER stress, and was later followed by cell death. Didemnin B did not prevent the initiation of ER stress by high palmitate, as indicated by eIF2α phosphorylation. However, consistent with sustained inhibition of eEF1A-1-dependent elongation activity, didemnin B prevented the recovery of protein synthesis and increase in GRP78 protein that are normally associated with later phases of the response to ongoing ER stress. This resulted in decreased palmitate-induced cell death. Our data implicate eEF1A-1, and its function in protein synthesis, in hepatocyte lipotoxicity.

Cell dysfunction and death induced by lipid accumulation in nonadipose tissues, or lipotoxicity, may contribute to the pathogenesis of obesity and type 2 diabetes. However, the mechanisms leading to lipotoxic cell death are poorly understood. We recently reported that, in Chinese hamster ovary (CHO) cells and in H9c2 cardiomyoblasts, lipid overload induced by incubation with 500 μM palmitate leads to intracellular accumulation of reactive oxygen species, which subsequently induce endoplasmic reticulum (ER) stress and cell death. Here, we show that palmitate also impairs ER function through a more direct mechanism. Palmitate was rapidly incorporated into saturated phospholipid and triglyceride species in microsomal membranes of CHO cells. The resulting membrane remodeling was associated with dramatic dilatation of the ER and redistribution of protein-folding chaperones to the cytosol within 5 h, indicating compromised ER membrane integrity. Increasing β-oxidation, through the activation of AMP-activated protein kinase, decreased palmitate incorporation into microsomes, decreased the escape of chaperones to the cytosol, and decreased subsequent caspase activation and cell death. Thus, palmitate rapidly increases the saturated lipid content of the ER, leading to compromised ER morphology and integrity, suggesting that impairment of the structure and function of this organelle is involved in the cellular response to fatty acid overload.

The grapefruit flavonoid, naringenin, is hypocholesterolemic in vivo, and inhibits basal apolipoprotein B (apoB) secretion and the expression and activities of both ACAT and microsomal triglyceride transfer protein (MTP) in human hepatoma cells (HepG2). In this report, we examined the effects of naringenin on apoB kinetics in oleate-stimulated HepG2 cells and determined the contribution of microsomal lumen cholesteryl ester (CE) availability to apoB secretion. Pulse-chase studies of apoB secretion and intracellular degradation were analyzed by multicompartmental modeling. The model for apoB metabolism in HepG2 cells includes an intracellular compartment from which apoB can be either secreted or degraded by both rapid and slow pathways. In the presence of 0.1 mM oleic acid, naringenin (200 μM) reduced the secretion of newly synthesized apoB by 52%, due to a 56% reduction in the rate constant for secretion. Intracellular degradation was significantly increased due to a selective increase in rapid degradation, while slow degradation was unaffected. Incubation with either N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) or lactacystin showed that degradation via the rapid pathway was largely proteasomal. Although these changes in apoB metabolism were accompanied by significant reductions in CE synthesis and mass, subcellular fractionation experiments comparing naringenin to specific ACAT and HMG-CoA reductase inhibitors revealed that reduced accumulation of newly synthesized CE in the microsomal lumen is not consistently associated with reduced apoB secretion. However, naringenin, unlike the ACAT and HMG-CoA reductase inhibitors, significantly reduced lumenal TG accumulation.We conclude that naringenin inhibits apoB secretion in oleate-stimulated HepG2 cells and selectively increases intracellular degradation via a largely proteasomal, rapid kinetic pathway. Although naringenin inhibits ACAT, CE availability in the endoplasmic reticulum (ER) lumen does not appear to regulate apoB secretion in HepG2 cells. Rather, inhibition of TG accumulation in the ER lumen via inhibition of MTP is the primary mechanism blocking apoB secretion.

The citrus flavonoids, naringenin and hesperetin, lower plasma cholesterol in vivo. However, the underlying mechanisms are not fully understood. The ability of these flavonoids to modulate apolipoprotein B (apoB) secretion and cellular cholesterol homeostasis was determined in the human hepatoma cell line, HepG2. apoB accumulation in the media decreased in a dose-dependent manner following 24-h incubations with naringenin (up to 82%, P < 0.00001) or hesperetin (up to 74%, P < 0.002). Decreased apoB secretion was associated with reduced cellular cholesteryl ester mass. Cholesterol esterification was decreased, dose-dependently, up to 84% (P < 0.0001) at flavonoid concentrations of 200 μM. Neither flavonoid demonstrated selective inhibition of either form of acyl CoA:cholesterol acyltransferase (ACAT) as determined using CHO cells stably transfected with either ACAT1 or ACAT2. However, in HepG2 cells, ACAT2 mRNA was selectively decreased (−50%, P < 0.001) by both flavonoids, whereas ACAT1 mRNA was unaffected. In addition, naringenin and hesperetin decreased both the activity (−20% to −40%, P < 0.00004) and expression (−30% to −40%, P < 0.02) of microsomal triglyceride transfer protein (MTP). Both flavonoids caused a 5- to 7-fold increase (P < 0.02) in low density lipoprotein (LDL) receptor mRNA, which resulted in a 1.5- to 2-fold increase in uptake and degradation of 125I-LDL.We conclude that both naringenin and hesperetin decrease the availability of lipids for assembly of apoB-containing lipoproteins, an effect mediated by 1) reduced activities of ACAT1 and ACAT2, 2) a selective decrease in ACAT2 expression, and 3) reduced MTP activity. Together with an enhanced expression of the LDL receptor, these mechanisms may explain the hypocholesterolemic properties of the citrus flavonoids.

Am J Physiol Cell Physiol 298: C66-C74, 2010. First published October 21, 2009; doi:10.1152/ajpcell.00357.2009.--Polyploid endothelial cells are found in aged and atherosclerotic arteries. However, whether increased chromosome content has an impact on endothelial cell function is unknown. We show here that human aortic endothelial cells become tetraploid as they approach replicative senescence. Furthermore, accumulation of tetraploid endothelial cells was accelerated during growth in high glucose. Interestingly, induction of polyploidy was completely prevented by modest overexpression of the [NAD.sup.+] regenerating enzyme, nicotinamide phosphoribosyltransferase (Nampt). To determine the impact of polyploidy on endothelial cell function, independent of replicative senescence, we induced tetraploidy using the spindle poison, nocodazole. Global gene expression analyses of tetraploid endothelial cells revealed a dysfunctional phenotype characterized by a cell cycle arrest profile (decreased CCNE2/A2, RBL1, BUB1B; increased CDKN1A) and increased expression of genes involved in inflammation (IL32, TNFRSF21/10C, PTGS1) and extracellular matrix remodeling (COL5A1, FN1, MMP10/14). The protection from polyploidy conferred by Nampt was not associated with enhanced poly(ADP-ribose) polymerase-1 or sirtuin (SIRT) 2 activity, but with increased SIRT1 activity, which reduced cellular reactive oxygen species and the associated oxidative stress stimulus for the induction of polyploidy. We conclude that human aortic endothelial cells are prone to chromosome duplication that, in and of itself, can induce characteristics of endothelial dysfunction. Moreover, the emergence of polyploid endothelial cells during replicative aging and glucose overload can be prevented by optimizing the Nampt-SIRT1 axis. endothelial cells; senescence; oxidative stress