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Visceral obesity is characterized by a low-grade inflammatory systemic state that contributes to the genesis of non-alcoholic fatty liver disease (NAFLD), frequently associated with liver fibrosis. Non-invasive serum markers have recently emerged as reliable, easy-to-use scores to predict liver fibrosis. NAFLD is often linked to metabolic and cardiovascular risk. Thus, in this cross-sectional study, we investigated in a population of 1225 subjects if AST to Platelet Ratio Index (APRI), one of the non-invasive liver fibrosis serum markers, can predict cardiovascular risk (CVR). APRI has been previously validated as an efficient score to predict liver fibrosis in viral hepatitis patients with a cut-off of 0.5 for fibrosis and 1.5 for cirrhosis. Our study showed that APRI significantly correlates with CVR and determines, when elevated, a significant increase in CVR for both genders, especially females. This spike in CVR, observed when APRI is elevated, is relatively high in patients in the age of 51–65 years, but it is significantly higher in younger and premenopausal women, approaching risk values usually typical of men at the same age. Taken together, our data highlighted the role of APRI as a reliable predictor easy-to-use score for CVR in metabolic patients.

Several studies highlighted the importance of platelets in the tumor microenvironment due to their ability to interact with other cell types such as leukocytes, endothelial, stromal and cancer cells. Platelets can influence tumor development and metastasis formation through several processes consisting of the secretion of growth factors and cytokines and/or via direct interaction with cancer cells and endothelium. Patients with visceral obesity (VO) are susceptible to pro-thrombotic and pro-inflammatory states and to development of cancer, especially colon cancer. These findings provide us with the impetus to analyze the role of platelets isolated from VO patients in tumor growth and progression with the aim to explore a possible link between platelet activation, obesity and colon cancer. Here, using xenograft colon cancer models, we prove that platelets from patients with visceral obesity are able to strongly promote colon cancer growth. Then, sequencing platelet miRNome, we identify miR-19a as the highest expressed miRNA in obese subjects and prove that miR-19a is induced in colon cancer. Last, administration of miR-19a per se in the xenograft colon cancer model is able to promote colon cancer growth. We thus elect platelets with their specific miRNA abundance as important factors in the tumor promoting microenvironment of patients with visceral obesity.

Nonalcoholic fatty liver disease comprises a wide spectrum of liver injuries from simple steatosis to steatohepatitis and cirrhosis. Nonalcoholic steatohepatitis (NASH) is defined when liver steatosis is associated with inflammation, hepatocyte damage, and fibrosis. A genetic predisposition and environmental insults (ie, dietary habits, obesity) are putatively responsible for NASH progression. Here, we present the impact of the lipid-sensing nuclear receptors in the pathogenesis and treatment of NASH. In detail, we discuss the pros and cons of the putative transcriptional action of the fatty acid sensors (peroxisome proliferator-activated receptors), the bile acid sensor (farnesoid X receptor), and the oxysterol sensor (liver X receptors) in the pathogenesis and bona fide treatment of NASH.

This review analyses the main features of primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) and provides an overview of the currently available (bile acid) bile acid related treatments.In PBC, biliary injury is the consequence of a dysregulated intrahepatic and systemic immune response. Given the close association between PSC and inflammatory bowel disease (IBD), the microbiota represents an important factor in the development of PSC. Bile acid based pharmacological treatments could represent promising therapeutic strategies in the management of cholangiopathies.Cholangiopathies include a spectrum of diseases resulting in cholestasis, an impairment of bile flow in the biliary tree, leading to biliary obstruction and damage as well as liver inflammation and fibrosis. PSC and PBC are highly heterogeneous cholangiopathies and progressive disorders with defined pathophysiological mechanisms. Curative treatments have not been established, and although their prevalence is low, they are a frequent indication for liver transplantation in the advanced stages of cholangiopathies. These diseases still present with unmet therapeutic strategies, also taking into account that on average 30-40% of patients undergoing liver transplantation will have recurrence of the original illness.

Vitamin D deficiency is often linked with Metabolic Syndrome, both being more frequent with ageing and associated with an increase inflammatory state. Recently, monocytes-to-high density lipoprotein (HDL) ratio (MHR) has emerged as a powerful index to predict systemic inflammation. In this cross-sectional study, we investigated the association between circulating vitamin D level (25-OH vitamin D) and inflammatory status in a population of 1048 adult individuals. Our study reveals an inverse association between 25-OH vitamin D levels and MHR in the overall population. When the population is stratified by gender, waist circumference, and body mass index (BMI), we observed that while in men this relation is strongly significative only in condition of central obesity, in women a lifelong negative correlation exists between circulating 25-OH vitamin D and MHR and it is independent of the metabolic status. These observations underscore the relevance of circulating biomarkers such as MHR in the prediction of systemic inflammatory conditions sustained by vitamin D deficiency also in healthy and young women.

Bile acid (BA) species and the gut microbiota (GM) contribute to intestinal mucosa homeostasis. BAs shape the GM and, conversely, intestinal bacteria with bile salt hydrolase (BSH) activity modulate the BA pool composition. The mutual interaction between BAs and intestinal microorganisms also influences mucosal barrier integrity, which is important for inflammatory bowel disease (IBD) pathogenesis, prevention and therapy. High levels of secondary BAs are detrimental for the intestinal barrier and increase the intestinal inflammatory response and dysbiosis. Additionally, a lack of BSH-active bacteria plays a role in intestinal inflammation and BA dysmetabolism. Thus, BSH-competent bacteria in probiotic formulations are being actively studied in IBD. At the same time, studies exploring the modulation of the master regulator of BA homeostasis, the Farnesoid X Receptor (FXR), in intestinal inflammation and how this impacts the GM are gaining significant momentum. Overall, the choice of probiotic supplementation should be a peculiar issue of personalized medicine, considering not only the disease but also the specific BA and metabolic signatures of a given patient.

The crypt-villus axis represents the essential unit of the small intestine, which integrity and functions are fundamental to assure tissue and whole-body homeostasis. Disruption of pathways regulating the fine balance between proliferation and differentiation results in diseases development. Nowadays, it is well established that microRNAs (miRNAs) play a crucial role in the homeostasis maintenance and perturbation of their levels may promote tumor development. Here, by using microarray technology, we analysed the miRNAs differentially expressed between the crypt and the villus in mice ileum. The emerged miRNAs were further validated by Real Time qPCR in mouse model (ApcMin/+), human cell lines and human tissue samples (FAP) of colorectal cancer (CRC). Our results indicated that miRNAs more expressed in the villi compartment are negatively regulated in tumor specimens, thus suggesting a close association between these microRNAs and the differentiation process. Particularly, from our analysis let-7e appeared to be a promising target for possible future therapies and a valuable marker for tumor staging, being upregulated in differentiated cells and downregulated in early-stage colonic adenoma samples.

The purpose of this study was to evaluate the association between the presence of Metabolic Syndrome (MetS) and idiopathic sudden sensorineural hearing loss (ISSHL) and the impact of MetS on recovery of patients with ISSHL. 39 Patients with ISSHL and 44 controls were enrolled in this study. Demographic, clinical characteristics and hearing recovery were evaluated. MetS was defined according to the diagnostic criteria of International Diabetes Federation (IDF) consensus definition. Patients affected by ISSHL presented a body mass index (BMI), waist circumference, waist hip ratio (WHR), fasting glucose and blood pressure significantly higher compared to controls. Considering patients with central obesity, 5 controls and 15 ISSHL patients met the criteria of MetS. According to Siegel criteria, a complete or partial recovery was observed in 60% of patients with MetS and in 91,66% of patients without MetS. MetS was associated with ISSHL and this association negatively influenced the hearing recovery of these patients.

Background: Disruption of bile acid (BA) homeostasis plays a key role in intestinal inflammation. The gut-liver axis is the main site for the regulation of BA synthesis and BA pool size via the combined action of the nuclear Farnesoid X Receptor (FXR) and the enterokine Fibroblast Growth Factor 19 (FGF19). Increasing evidence have linked derangement of BA metabolism with dysbiosis and mucosal inflammation. Thus, here we aimed to investigate the potential action of an FGF19 analogue on intestinal microbiota and inflammation. Methods: A novel engineered non-tumorigenic variant of the FGF19 protein, M52-WO 2016/0168219 was generated. WT and FXRnull mice were injected with AAV-FGF19-M52 or the control AAV-GFP and subjected to Sodium Dextran Sulphate-induced colitis. Findings: FGF19-M52 reduced BA synthesis and pool size, modulated its composition and protected mice from intestinal inflammation. These events were coupled with preservation of the intestinal epithelial barrier integrity, inhibition of inflammatory immune response and modulation of microbiota composition. Interestingly, FGF19-M52-driven systemic and local anti-inflammatory activity was completely abolished in Farnesoid X Receptor (FXR)null mice, thus underscoring the need of FXR to guarantee enterocytes’ fitness and complement FGF19 anti-inflammatory activity. To provide a translational perspective, we also show that circulating FGF19 levels are reduced in patients with Crohn's disease. Interpretation: Reactivation of the FXR-FGF19 axis in a murine model of intestinal inflammation could bona fide provide positive changes in BA metabolism with consequent reduction of intestinal inflammation and modulation of microbiota. These results point to the therapeutic potential of FGF19 in the treatment of intestinal inflammation with concomitant derangement of BA homeostasis. Funding: A. Moschetta is funded by MIUR-PRIN 2017

The nuclear receptor farnesoid X receptor (FXR) is the master regulator of bile acids (BAs) homeostasis since it transcriptionally drives modulation of BA synthesis, influx, efflux, and detoxification along the enterohepatic axis. Due to its crucial role, FXR alterations are involved in the progression of a plethora of BAs associated inflammatory disorders in the liver and in the gut. The involvement of the FXR pathway in cholestasis development and management has been elucidated so far with a direct role of FXR activating therapy in this condition. However, the recent identification of a new type of genetic progressive familial intrahepatic cholestasis (PFIC) linked to FXR mutations has strengthen also the bona fide beneficial effects of target therapies that by-pass FXR activation, directly promoting the action of its target, namely the enterokine FGF19, in the repression of hepatic BAs synthesis with reduction of total BA levels in the liver and serum, accomplishing one of the major goals in cholestasis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni and Peter Jansen.

Aims Coronary artery disease (CAD) is described as a range of clinical conditions including myocardial infarction (MI) and unstable angina. Lipid and apolipoprotein profiles together with the study of cholesterol deposit and efflux serve to identify novel pre and post infarct scenarios for the treatment of these patients. In (non-ST elevation myocardial infarction) NSTEMI patients, we analysed both systemic and intracoronary serum ability to accept cholesterol as well as cholesterol efflux capacity (CEC) of monocytes in terms of expression of genes involved in the reverse cholesterol transport (RCT). Methods and results While HDL-C quantity was similar between systemic and coronary arterial blood, in 21 NSTEMI patients we observed a significant reduction of the preβ-HDL fraction and the levels of Apolipoproteins AI, AII, B and E in coronary versus systemic serum. These data are complemented with the observed reduction of CEC. On the contrary, compared to systemic arterial monocytes, in coronary microenvironment of NSTEMI patients after myocardial infarction, the monocytes exhibited a higher mRNA expression of nuclear receptor LXRα and its targets ABCA1 and APOE, which drive cholesterol efflux capacity. Conclusion In this cross-sectional study we observe that in the immediate post infarction period, there is a spontaneous bona fide ligand-induced activation of the LXR driven cholesterol efflux capacity of intracoronary monocytes to overcome the reduced serum ability to accept cholesterol and to inhibit the post-infarction pro-inflammatory local microenvironment.

The consumption of an olive oil rich diet has been associated with the diminished incidence of cardiovascular disease and cancer. Several studies have attributed these beneficial effects to oleic acid (C18 n-9), the predominant fatty acid principal component of olive oil. Oleic acid is not an essential fatty acid since it can be endogenously synthesized in humans. Stearoyl-CoA desaturase 1 (SCD1) is the enzyme responsible for oleic acid production and, more generally, for the synthesis of monounsaturated fatty acids (MUFA). The saturated to monounsaturated fatty acid ratio affects the regulation of cell growth and differentiation, and alteration in this ratio has been implicated in a variety of diseases, such as liver dysfunction and intestinal inflammation. In this review, we discuss our current understanding of the impact of gene-nutrient interactions in liver and gut diseases, by taking advantage of the role of SCD1 and its product oleic acid in the modulation of different hepatic and intestinal metabolic pathways.

Extra virgin olive oil (EVOO) consumption has a beneficial effect on human health, especially for prevention of cardiovascular disease and metabolic disorders. Here we underscore the peculiar importance of specific cultivars used for EVOO production since biodiversity among cultivars in terms of fatty acids and polyphenols content could differently impact on the metabolic homeostasis. In this respect, the nutrigenomic approach could be very useful to fully dissect the pathways modulated by different EVOO cultivars in terms of mRNA and microRNA transcriptome. The identification of genes and miRNAs modulated by specific EVOO cultivars could also help to discover novel nutritional biomarkers for prevention and/or prognosis of human disease. Thus, the nutrigenomic approach depicts a novel scenario to investigate if a specific EVOO cultivar could have a positive effect on human health by preventing the onset of cardiovascular disease and/or chronic inflammatory disorders also leading to cancer.

Colorectal cancer (CRC) is one of the most commonly occurring cancers worldwide. Although several genetic alterations have been associated with CRC onset and progression, nowadays the reprogramming of cellular metabolism has been recognized as a fundamental step of the carcinogenic process. Intestinal tumor cells frequently display an aberrant activation of lipid metabolism. Indeed, to satisfy the growing needs of a continuous proliferation, cancer cells can either increase the uptake of exogenous lipids or upregulate the endogenous lipogenesis and cholesterol synthesis. Therefore, strategies aimed at limiting lipid accumulation are now under development in order to counteract malignancies. Two major players of lipids metabolism have been so far identified for their contribution to CRC development: the nuclear receptor Liver X Receptor (LXRs) and the enzyme Stearoyl-CoA Desaturase 1 (SCD1). Whereas LXR is mainly recognized for its role as a cholesterol sensor, finally promoting the loss of cellular cholesterol and whole-body homeostasis, SCD1 acts as the major regulator of new fatty acids, finely tuning the monounsaturated fatty acids (MUFA) to saturated fatty acids (SFA) ratio. Intriguingly, SCD1 is directly regulated by LXRs. Despite LXRs agonists have elicited great interest as a promising therapeutic target for cancer, LXR's ability to induce SCD1 and new fatty acids synthesis represent a major obstacle in the development of new effective treatments. Thus, further investigations are required to fully dissect the concomitant modulation of both players, to develop specific therapies aimed at blocking intestinal cancer cells proliferation, eventually counteracting CRC progression.

Background Epicardial adipose tissue (EAT) is an atypical fat depot surrounding the heart with a putative role in the development of atherosclerosis. Methods and results We profiled genes and miRNAs in perivascular EAT and subcutaneous adipose tissue (SAT) of metabolically healthy patients without coronary artery disease (CAD) vs. metabolic patients with CAD. Compared with SAT, a specific tuning of miRNAs and genes points to EAT as a tissue characterized by a metabolically active and pro-inflammatory profile. Then, we depicted both miRNA and gene signatures of EAT in CAD, featuring a down-regulation of genes involved in lipid metabolism, mitochondrial function, nuclear receptor transcriptional activity, and an up-regulation of those involved in antigen presentation, chemokine signalling, and inflammation. Finally, we identified miR-103-3p as candidate modulator of CCL13 in EAT, and a potential biomarker role for the chemokine CCL13 in CAD. Conclusion EAT in CAD is characterized by changes in the regulation of metabolism and inflammation with miR-103-3p/CCL13 pair as novel putative actors in EAT function and CAD.

Celiac disease (CD) is a systemic disorder characterized by an immune-mediated reaction to gluten and a wide spectrum of clinical manifestations. Currently, the main treatment of CD is represented by adherence to a gluten-free diet (GFD) which determines the resolution of symptoms, and the normalization of the serology and of the duodenal villous atrophy. In the present study, we aimed to identify changes in gene expression in peripheral blood mononuclear cells (PBMCs) of celiac patients on GFD for at least 2 years, in order to identify novel disease biomarkers and candidate targets for putative therapeutic approaches. Microarray analysis was performed on PBMCs from 17 celiac patients on long-term GFD and 20 healthy controls. We identified 517 annotated genes that were significantly modulated between celiac patients and controls. Significant biological pathways were functionally clustered using the Core Function of Ingenuity System Pathway Analysis (IPA). Intriguingly, despite being on a GFD, celiac patients exhibited a peculiar PBMC profile characterized by an aberrant expression of genes involved in the regulation of immunity, inflammatory response, metabolism, and cell proliferation. Random forest algorithm was then used to validate the prediction ability of core genes as classifiers of the "celiac status". In conclusion, our study identified a characteristic PBMCs signature profile in clinically asymptomatic celiac patient.

Aims Metabolic syndrome (MS) is a cluster of cardio-metabolic risk factors associated with atherosclerosis and low-grade inflammation. Using unbiased expression screenings in peripheral blood mononuclear cells, we depict here a novel expression chart of 678 genes and 84 microRNAs (miRNAs) controlling inflammatory, immune and metabolic responses. In order to further elucidate the link between inflammation and the HDL cholesterol pathway in MS, we focussed on the regulation of the ATP-binding cassette transporter A1 (ABCA1), a key player in cholesterol efflux (CE). Methods and results ABCA1 mRNA levels are suppressed in CD14+ cells of MS patients and are negatively correlated to body mass index (BMI), insulin-resistance (HOMA-IR) and cardiovascular risk, and positively to HDL cholesterol and CE. miRNA target in silico prediction identified a putative modulatory role of ABCA1 for the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) target miR-9-5p, whose expression pattern was up-regulated in CD14+ cells of MS patients, positively correlated to BMI, HOMA-IR, and triglycerides, and negatively to ABCA1 mRNA levels, HDL cholesterol and CE. Ectopic gain and loss of miR-9-5p function in macrophages modulated ABCA1 mRNA and protein levels, ABCA1 miRNA 3’-untranslated region target sequence reporter assay, and CE into HDL, thus confirming ABCA1 as a target of miR-9-5p. Conclusions We identified the NF-κB target miR-9-5p as a negative regulator of ABCA1 adding a novel target pathway in the relationship between inflammation and HDL-driven reverse cholesterol transport for prevention or treatment of atherosclerosis in MS.

Elevated bile acid (BA) concentrations in the liver is associated with severe disease, including cholestasis and hepatocellular carcinoma. The nuclear Farnesoid X Receptor (FXR) is the master regulator of BAs homeostasis. In the ileum, BA-dependent FXR activation induces the production of the fibroblast growth factor FGF19, a hormone that reaches the liver through the portal system where it represses the expression of CYP7A1, the rate limiting enzyme in the process of hepatic BAs synthesis. This gut-liver FXR-FGF19 dual action is the paradigm of physiological BA regulation and it is currently targeted in the clinical practice for liver disease such as primary cholangitis. At a variance of FXR activation, native FGF19 has strong anti-cholestatic and anti-fibrotic activity in the liver but it retains peculiar pro-tumorigenic actions. Thus, novel analogues have been generated to avoid tumorigenic capacity while maintaining BA metabolic action. Here we present a novel and intriguing view on the putative possibility to target the FXR-FGF19 duo in order to offer a bona fide promising therapeutic approach to bile acid promoted hepatocarcinoma.

The nuclear Farnesoid X Receptor (FXR) is a transcription factor critically involved in metabolic homeostasis in the gut-liver axis. FXR activity is mediated by hormonal and dietary signals and driven by bile acids (BAs), which are the natural FXR ligands. Given the great physiological importance in BA homeostasis, as well as in the regulation of glucose and lipid metabolism, FXR plays a pivotal role in the pathogenesis of a wide range of disease of the liver, biliary tract and intestine, including hepatic and colorectal cancer. In the last years several studies have shown the relative FXR tissue-specific importance, highlighting synergism and additive effects in the liver and intestine. Gain- and loss-of-FXR-function mouse models have been generated in order to identify the biological processes and the molecular FXR targets. Taking advantage of the knowledge on the structure-activity relationship of BAs for FXR, semi-synthetic and synthetic molecules have been generated to obtain more selective and powerful FXR activators than BAs. This article is part of a Special Issue entitled: Linking transcription to physiology in lipodomics.

Androgens and androgen receptor (AR, NR3C4) clearly play a crucial role in prostate cancer progression. Besides, the link between metabolic disorders and the risk of developing a prostate cancer has been emerging these last years. Interestingly, “lipid” nuclear receptors such as LXRα/NR1H3 and LXRβ/NR1H2 (as well as FXRα/NR1H4 and SHP/NR0B2) have been described to decrease the lipid metabolism, while AR increases it. Moreover, these former orphan nuclear receptors can regulate androgen levels and modulate AR activity. Thus, it is not surprising to find such receptors involved in the physiology of prostate. This review is focused on the roles of liver X receptors (LXRs), farnesoid X receptor (FXR), and small heterodimeric partner (SHP) in prostate physiology and their capabilities to interfere with the androgen-regulated pathways by modulating the levels of active androgen within the prostate. By the use of prostate cancer cell lines, mice deficient for these nuclear receptors and human tissue libraries, several authors have pointed out the putative possibility to pharmacologically target these receptors. These data open a new field of research for the development of new drugs that could overcome the castration resistance in prostate cancer, a usual phenomenon in patients.

Background Increasing evidence demonstrates a phenotypic plasticity of endothelial cells (ECs). Endothelial-to-mesenchymal transition (EndMT) contributes to the development of tissue fibrosis. However, the pathogenic factors and signalling pathways regulating this process in ischaemia/reperfusion (I/R) injury are still poorly understood. Methods We investigated the possible role of complement in the induction of this endothelial dysfunction in a swine model of renal I/R injury by using recombinant C1 inhibitor in vivo. Results Here, we showed that I/R injury reduced the density of renal peritubular capillaries and induced tissue fibrosis with generation of CD31(+)/α-SMA(+) and CD31(+)/FPS-1(+) cells indicating EndMT. When we inhibited complement, the process of EndMT became rare, with preserved density of peritubular capillaries and significant reduction in renal fibrosis. When we activated ECs by anaphylatoxins in vitro, C3a and C5a led to altered endothelial phenotype with increased expression of fibroblast markers and decrease expression of specific endothelial markers. The activation of Akt pathway was pivotal for the C3a and C5a-induced EndMT in vitro. In accordance, inhibition of complement in vivo led to the abrogation of Akt signalling, with hampered EndMT and tissue fibrosis. Conclusions Our data demonstrate a critical role for complement in the acute induction of EndMT via the Akt pathway. Therapeutic inhibition of these systems may be essential to prevent vascular damage and tissue fibrosis in transplanted kidney.

Extra virgin olive oil (EVOO) consumption has been associated with reduced cardiovascular risk but molecular mechanisms underlying its beneficial effects are not fully understood. Here we aimed to identify genes and miRNAs expression changes mediated by acute high- and low-polyphenols EVOO intake. Pre- and post-challenge gene and miRNAs expression analysis was performed on the peripheral blood mononuclear cells (PBMCs) of 12 healthy subjects and 12 patients with metabolic syndrome (MS) by using microarray and RT-qPCR. In healthy subjects, acute intake of EVOO rich in polyphenols was able to ameliorate glycaemia and insulin sensitivity, and to modulate the transcription of genes and miRNAs involved in metabolism, inflammation and cancer, switching PBMCs to a less deleterious inflammatory phenotype; weaker effects were observed in patients with MS as well as in healthy subjects following low-polyphenol EVOO challenge. Concluding, our study shows that acute high-polyphenols EVOO intake is able to modify the transcriptome of PBMCs through the modulation of different pathways associated with the pathophysiology of cardio-metabolic disease and cancer. These beneficial effects are maximized in healthy subjects, and by the use of EVOO cultivars rich in polyphenols. Nutrigenomic changes induced by EVOO thus legitimate the well-known beneficial effects of EVOO in promoting human health and, potentially, preventing the onset of cardiovascular disease and cancer.

Adult renal progenitor cells (ARPCs) isolated from the human kidney may contribute to repair featuring acute kidney injury (AKI). Bone morphogenetic proteins (BMPs) regulate differentiation, modeling, and regeneration processes in several tissues. The aim of this study was to evaluate the biological actions of BMP-2 in ARPCs in vitro and in vivo. BMP-2 was expressed in ARPCs of normal adult human kidneys, and it was upregulated in vivo after delayed graft function (DGF) of renal transplantation, a condition of AKI. ARPCs expressed BMP receptors, suggesting their potential responsiveness to BMP-2. Incubation of ARPCs with this growth factor enhanced reactive oxygen species (ROS) production, NADPH oxidase activity, and Nox4 protein expression. In vivo, Nox4 was localized in BMP-2-expressing CD133+ cells at the tubular level after DGF. BMP-2 incubation induced α-smooth muscle actin (SMA), collagen I, and fibronectin protein expression in ARPCs. Moreover, α-SMA colocalized with CD133 in vivo after DGF. The oxidative stimulus (H2O2) induced α-SMA expression in ARPCs, while the antioxidant N-acetyl-cysteine inhibited BMP-2-induced α-SMA expression. Nox4 silencing abolished BMP-2-induced NADPH oxidase activation and myofibroblastic induction. We showed that 1) ARPCs express BMP-2, 2) this expression is increased in a model of AKI; 3) BMP-2 may induce the commitment of ARPCs toward a myofibroblastic phenotype in vitro and in vivo; and 4) this profibrotic effect is mediated by Nox4 activation. Our findings suggest a novel mechanism linking AKI with progressive renal damage.

Malignancies are one of the main causes of mortality in diabetic patients; however, to date, very limited data have been reported on the specific influence of type 2 diabetes mellitus (T2DM) on the survival of patients with renal cell carcinoma (RCC). In the present long-term retrospective study, we investigated whether T2DM may influence the overall survival (OS), cancer-specific survival (CSS), and progression-free survival (PFS) in patients with surgically treated RCC. Medical records of 924 patients treated by radical or partial nephrectomy for sporadic, unilateral RCC were reviewed. Patients with type-1 DM and with T2 DM receiving insulin treatment were excluded. Survival estimates were calculated according to the Kaplan–Meier method and compared with the log-rank test. Univariate and multivariate analyses were performed using the Cox regression model. Of the 924 RCC patients, 152 (16.5%) had T2DM. Mean follow-up was 68.5 months. Mean OS was 41.3 and 96.3 months in T2DM and non-T2DM patients, respectively (P

Farnesoid X receptor (FXR, NR1H4) is a bile acid-activated transcription factor that belongs to the nuclear receptor superfamily. It is highly expressed in the enterohepatic system, where it senses bile acid levels to consequently reduce their synthesis while inducing their detoxification. Bile acids are intestinal tumor promoters and their concentrations have to be tightly regulated. Indeed, reduced expression of FXR in the intestine increases colorectal cancer susceptibility in mice, whereas its activation can promote apoptosis in genetically modified cells. Notably, despite the broad knowledge of the FXR enterohepatic transcriptional activity, the molecular mechanisms regulating FXR expression in the intestine are still unknown. Herein, by combining both gain and loss of function approaches and FXR promoter activity studies, we identified caudal-related homeobox 2 (CDX2) transcription factor as a positive regulator of FXR expression in the enterocytes. Our results provide a putative novel tool for modulating FXR expression against bile acid-related colorectal cancer progression.

This study investigated on (i) the role of gp91(phox)/NOX2 in reactive oxygen species (ROS) generation in hemodialysis (HD) patients, and (ii) the link between clotting activation and ROS production in this setting.The study was performed on peripheral blood mononuclear cells (PBMCs) isolated from HD patients randomized to polysulphon/polyamide (S-group, n=30) or ethylene-vinyl-alcohol (EVAL) membrane (E-group, n=30) treatment and from healthy subjects (control group, n=15). ROS generation was increased in PBMCs of HD patients compared with healthy subjects. S-group showed higher levels of intracellular ROS generation than control, whereas E-group did not. In addition, S-group displayed an increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity compared with E-group and healthy subjects. A further increase in NADPH activity shortly after HD treatment was observed only in S-group. The plasma levels of the prothrombin fragment F1+2, a marker of in vivo clotting activation, were significantly higher in S-group than in E-group. Moreover, a heightened thrombin generation was recorded in the plasma of S-group. Intracellular ROS production correlated with NADPH oxidase activity and coagulation priming in HD patients. The in vitro validation study demonstrated that incubation of PBMCs with activated FX induced a significant increase in intracellular ROS production, superoxide generation, and gp91(phox)/NOX2 expression.The pivotal role of NADPH oxidase in the upregulation of ROS in HD patients makes this enzyme a potential target for therapeutic intervention in the treatment of HD-related oxidative stress.The EVAL membrane, by reducing clotting activation, inhibits gp91(phox)/NOX2-related ROS production in HD patients.

Figure 3. PPARβ/δ protein is suppressed in murine regenerating liver (A) and human HCC (C), and negatively correlates with PCNA (B).Anti-PPARβ/δ immunostaining in samples of LR (4–5/group) and 9 paraffin-embedded samples of HCC paired with reference tumor-free tissues (selection: 3 of 9 subjects); PPARβ/δ positive cells (percentage of marked area) were quantified using the ImageJ software. Results are shown as means ± SEM or means (bars) and cases (each line representing paired tumor-free tissue vs . tumor sample). Statistical significance (p≤0.05) assessed by the Kruskal-Wallis One-Way ANOVA on Ranks plus Nemenyi-Damico-Wolfe-Dunn post-hoc test (LR), Wilcoxon Signed-Rank Test (HCC), and Pearson's correlation coefficient (correlation). Legend: lower case letters indicate statistical significance (“a” means reference group; “b” means different from “a”; “c” means different from “a” and “b”); (white) quiescent; (black) proliferating; (▴) Sham; (▪) 0.5 days; (•) 1 day; (♦) 3 days; (▾) 7 dayshttp://dx.doi.org/10.1371/journal.pone.0104449.g003 Mice tissue specimens were fixed in 10% formalin for 12–24 hours, dehydrated and paraffin embedded. We also performed immunohistochemistry on samples of paraffin-embedded HCC ( vs. paired normal tissues; n = 9) received from Creative Bioarray, USA (http://www.creative-bioarray.com/Contact-Us.html). Standard Immunohistochemistry protocols were performed[15]. Briefly, 5 µm-thick sections were treated with 3% hydrogen peroxide for 5 min and with the Dako Cytomation Biotin blocking system (Dako, Denmark) to quench endogenous peroxidase and biotin respectively. Sections were sequentially incubated for 60 min at room temperature in 50% non-immune serum in PBS (to avoid unspecific signals) and overnight at 4°C with the primary antibody (anti-Pcna, Santa Cruz Biotechnology, Santa Cruz, CA; Abcam Anti-PPAR delta antibody, Cat AB23673). Sections were then washed for 10 min in PBS, and incubated for 30 min at room temperature with the secondary biotinylated antibody (Vector Laboratories). After several washing steps with PBS (3 washes 5 min/each), sections were incubated with the avidin-biotin complex (Vector Laboratories) for 30 min at room temperature. After washing in PBS, the peroxidase reaction was initiated by incubation with DAB (Sigma-Aldrich, Milano, Italy). Coverslips were mounted with Permount and evaluated under a light microscope. All the stained sections were analyzed through a confocal microscope (Magnitude: 20×). For each sample, 5 representative images were taken. Number and intensity of marked nuclei were quantified using ImageJ software as previously described [29].

Background Tryptase is a serin protease stored and released from mast cells (MCs) that plays a role in tumour angiogenesis. In this study we aimed to evaluate serum tryptase levels in colo-rectal cancer (CRC) patients before (STLBS) and after (STLAS) radical surgical resection. We also evaluated mast cell density positive to tryptase (MCDPT) and microvascular density (MVD) in primary tumour tissue. Methods A series of 61 patients with stage B and C CRC (according to the Astler and Coller staging system) were selected. Serum blood samples were collected from patients one day before and one day after surgery. Tryptase levels were measured using the UniCAP Tryptase Fluoroenzymeimmunoassay (Pharmacia, Uppsala, Sweden). Tumour sections were immunostained with a primary anti-tryptase antibody (clone AA1; Dako, Glostrup, Denmark) and an anti CD-34 antibody (QB-END 10; Bio-Optica Milan, Italy) by means of immunohistochemistry and then evaluated by image analysis methods. Results The mean ± s.d. STLBS and STLAS was 5.63±2.61 µg/L, and 3.39±1.47 µg/L respectively and a significant difference between mean levels was found: p = 0.000 by t-test. The mean ± s.d. of MCDPT and MVD was 8.13±3.28 and 29.16±7.39 respectively. A strong correlation between STLBS and MVD (r = 0.83, p = 0.000); STLBS and MCDPT (r = 0.60, p = 0.003); and MCDPT and MVD (r = 0.73; p = 0.001) was found. Conclusion Results demonstrated higher STLBS in CRC patients, indicating an involvement of MC tryptase in CRC angiogenesis. Data also indicated lower STLAS, suggesting the release of tryptase from tumour-infiltrating MCs. Serum tryptase levels may therefore play a role as a novel bio-marker predictive of response to radical surgery. In this context tryptase inhibitors such as Gabexate and Nafamostat Mesilate might be evaluated in adjuvant clinical trials as a new anti-angiogenic approach.

Arteriovenous fistula (AVF) stenosis is the major cause of vascular access failure in hemodialysis. Adventitial remodeling has been suggested to play a role in the pathogenesis of AVF stenosis. This study aimed to evaluate adventitial fibrosis in stenotic AVF and investigate the underlying molecular mechanisms.Forty-four patients undergoing surgery for AVF creation were examined; ten presented AVF failure, with histological-proven AVF stenosis.In stenotic AVF we observed a significant increase of adventitia extracellular matrix deposition and alpha-smooth muscle actin (α-SMA)(+) cell numbers; most of these cells were myofibroblast (α-SMA(+)/vimentin(+)). Phosphorylated platelet-derived growth factor β receptor (p-PDGFRβ) was significantly increased within the adventitia of stenotic compared to native AVF, along with a marked increase in the phosphorylation of Akt and ERK, two key kinases in PDGFRβ signalling. Myofibroblasts were the main cell type associated with the activation of p-PDGFRβ. At the same time, we observed a significant adventitial vessels rarefaction in stenotic AVF, as demonstrated by a reduced CD34 expression. This event was associated with a marked reduction in the expression of KDR/fetal liver kinase-1, the main vascular endothelial growth factor receptor. The degree of adventitial fibrosis was directly correlated with the extent of adventitial α-SMA and inversely associated with adventitial CD34 expression. Finally, we observed an increase in CD34(+)/α-SMA(+) cells within the adventitia of failed AVF.This study suggests that AVF failure is associated with an increased adventitial fibrosis, myofibroblast activation and capillary rarefaction, potentially linked with endothelial-to-mesenchymal transition. In this scenario, our data suggest that PDGF may play a pathogenic role.