PPAR-Gamma Orchestrates EMT, AGE, and Cellular Senescence Pathways in Colonic Epithelium and Restrains the Progression of IBDs.

Intestinal fibrosis, the most common complication of inflammatory bowel disease (IBD), is characterized by an uncontrolled deposition of extracellular matrix proteins leading to complications resolvable only with surgery. Transforming growth factor is the key player in the epithelial-mesenchymal transition (EMT) and fibrogenesis process, and some molecules modulating its activity, including peroxisome proliferator-activated receptor (PPAR)-γ and its agonists, exert a promising antifibrotic action. The purpose of this study is to evaluate the contribution of signaling other than EMT, such as the AGE/RAGE (advanced glycation end products/receptor of AGEs) and the senescence pathways, in the etiopathogenesis of IBD. We used human biopsies from control and IBD patients, and we used a mouse model of colitis induced by dextran-sodium-sulfate (DSS), without/with treatments with GED (PPAR-gamma-agonist), or 5-aminosalicylic acid (5-ASA), a reference drug for IBD treatment. In patients, we found an increase in EMT markers, AGE/RAGE, and senescence signaling activation compared to controls. Consistently, we found the overexpression of the same pathways in DSS-treated mice. Surprisingly, the GED reduced all the pro-fibrotic pathways, in some circumstances more efficiently than 5-ASA. Results suggest that IBD patients could benefit from a combined pharmacological treatment targeting simultaneously different pathways involved in pro-fibrotic signals. In this scenario, PPAR-gamma activation could be a suitable strategy to alleviate the signs and symptoms of IBD and also its progression.

Prolonged Chronic Consumption of a High Fat with Sucrose Diet Alters the Morphology of the Small Intestine.

(1) The high-fat diet (HFD) of western countries has dramatic effect on the health of several organs, including the digestive tract, leading to the accumulation of fats that can also trigger a chronic inflammatory process, such as that which occurs in non-alcohol steatohepatitis. The effects of a HFD on the small intestine, the organ involved in the absorption of this class of nutrients, are still poorly investigated. (2) To address this aspect, we administered a combined HFD with sucrose (HFD w/Suc, fat: 58% Kcal) regimen (18 months) to mice and investigated the morphological and molecular changes that occurred in the wall of proximal tract of the small intestine compared to the intestine of mice fed with a standard diet (SD) (fat: 18% Kcal). (3) We found an accumulation of lipid droplets in the mucosa of HFD w/Suc-fed mice that led to a disarrangement of mucosa architecture. Furthermore, we assessed the expression of several key players involved in lipid metabolism and inflammation, such as perilipin, leptin, leptin receptor, PI3K, p-mTOR, p-Akt, and TNF-α. All these molecules were increased in HFD mice compared to the SD group. We also evaluated anti-inflammatory molecules like adiponectin, adiponectin receptor, and PPAR-γ, and observed their significant reduction in the HFD w/Suc group compared to the control. Our data are in line with the knowledge that improper eating habits present a primary harmful assault on the bowel and the entire body's health. (4) These results represent a promising starting point for future studies, helping to better understand the complex and not fully elucidated spectrum of intestinal alterations induced by the overconsumption of fat.

Carotid artery plaque characterization with a wide-detector computed tomography using a dedicated post-processing 3D analysis: comparison with histology.

PURPOSE: The characterization of atherosclerotic carotid plaque plays a key role in the identification of patients at risk. The aim of our work was to evaluate the potentialities of carotid computed tomography angiography (CCTA) in assessing composition of atherosclerotic plaque. MATERIALS AND METHODS: We retrospectively evaluated 29 patients (7 women and 22 men, age range 54-81; mean age 69) who underwent carotid endarterectomy. All patients underwent pre-surgical CCTA using a 320-slice scanner. Post-processing reconstructions and analysis were performed using a specific software. Percentage of three different components of the atherosclerotic plaque (adipose, fibrotic and calcific) were classified based on Hounsfield unit values. Post-processing results were compared with histological analysis. Vessel and plaque parameters were compared using the Pearson correlation coefficient (r). Bland-Altman plots with 95% confidence intervals were calculated for correlation. McNemar's test was used for comparison of dichotomous variables. RESULTS: A significant correlation between histology and CCTA was found with respect to the areas corresponding to adipose, fibrotic and calcified plaques. The existence of proportional bias was observed between the two quantifying methods with lower discrepancies found for the adipose and fibrotic plaque areas. The Bland-Altman analyses showed a mean bias of 3.2%, 2.5% and 0.6% between histology and CCTA, for adipose, fibrotic and calcified plaque areas, respectively. CONCLUSIONS: Multi-detector CT angiography represents a valuable technique to assess quantitatively the composition of atherosclerotic plaques, with particular reference to the prevalence of fibrotic tissue, and is a useful diagnostic tool to improve risk stratification of patients for cerebral stroke.

Can Nrf2 Modulate the Development of Intestinal Fibrosis and Cancer in Inflammatory Bowel Disease?

One of the main mechanisms carried out by the cells to counteract several forms of stress is the activation of the nuclear factor erythroid 2-related factor (Nrf2) signaling. Nrf2 signaling controls the expression of many genes through the binding of a specific cis-acting element known as the antioxidant response element (ARE). Activation of Nrf2/ARE signaling can mitigate several pathologic mechanisms associated with an autoimmune response, digestive and metabolic disorders, as well as respiratory, cardiovascular, and neurodegenerative diseases. Indeed, several studies have demonstrated that Nrf2 pathway plays a key role in inflammation and in cancer development in many organs, including the intestine. Nrf2 appears to be involved in inflammatory bowel disease (IBD), an immune-mediated chronic and disabling disease, with a high risk of developing intestinal fibrotic strictures and cancer. Currently, drugs able to increase cytoprotective Nrf2 function are in clinical trials or already being used in clinical practice to reduce the progression of some degenerative conditions. The role of Nrf2 in cancer development and progression is controversial, and drugs able to inhibit abnormal levels of Nrf2 are also under investigation. The goal of this review is to analyze and discuss Nrf2-dependent signals in the initiation and progression of intestinal fibrosis and cancers occurring in IBD.

Vasopressin regulates the growth of the biliary epithelium in polycystic liver disease.

The neurohypophysial hormone arginine vasopressin (AVP) acts by three distinct receptor subtypes: V1a, V1b, and V2. In the liver, AVP is involved in ureogenesis, glycogenolysis, neoglucogenesis and regeneration. No data exist about the presence of AVP in the biliary epithelium. Cholangiocytes are the target cells in a number of animal models of cholestasis, including bile duct ligation (BDL), and in several human pathologies, such as polycystic liver disease characterized by the presence of cysts that bud from the biliary epithelium. In vivo, liver fragments from normal and BDL mice and rats as well as liver samples from normal and ADPKD patients were collected to evaluate: (i) intrahepatic bile duct mass by immunohistochemistry for cytokeratin-19; and (ii) expression of V1a, V1b and V2 by immunohistochemistry, immunofluorescence and real-time PCR. In vitro, small and large mouse cholangiocytes, H69 (non-malignant human cholangiocytes) and LCDE (human cholangiocytes from the cystic epithelium) were stimulated with vasopressin in the absence/presence of AVP antagonists such as OPC-31260 and Tolvaptan, before assessing cellular growth by MTT assay and cAMP levels. Cholangiocytes express V2 receptor that was upregulated following BDL and in ADPKD liver samples. Administration of AVP increased proliferation and cAMP levels of small cholangiocytes and LCDE cells. We found no effect in the proliferation of large mouse cholangiocytes and H69 cells. Increases were blocked by preincubation with the AVP antagonists. These results showed that AVP and its receptors may be important in the modulation of the proliferation rate of the biliary epithelium.

MicroRNA expression analysis in high fat diet-induced NAFLD-NASH-HCC progression: study on C57BL/6J mice.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common malignant tumor of the liver. Non-alcoholic fatty liver disease (NAFLD) is a frequent chronic liver disorder in developed countries. NAFLD can progress through the more severe non alcoholic steatohepatitis (NASH), cirrhosis and, lastly, HCC. Genetic and epigenetic alterations of coding genes as well as deregulation of microRNAs (miRNAs) activity play a role in HCC development. In this study, the C57BL/6J mouse model was long term high-fat (HF) or low-fat (LF) diet fed, in order to analyze molecular mechanisms responsible for the hepatic damage progression. METHODS: Mice were HF or LF diet fed for different time points, then plasma and hepatic tissues were collected. Histological and clinical chemistry assays were performed to assess the progression of liver disease. MicroRNAs' differential expression was evaluated on pooled RNAs from tissues, and some miRNAs showing dysregulation were further analyzed at the individual level. RESULTS: Cholesterol, low and high density lipoproteins, triglycerides and alanine aminotransferase increase was detected in HF mice. Gross anatomical examination revealed hepatomegaly in HF livers, and histological analysis highlighted different degrees and levels of steatosis, inflammatory infiltrate and fibrosis in HF and LF animals, demonstrating the progression from NAFLD through NASH. Macroscopic nodules, showing typical neoplastic features, were observed in 20% of HF diet fed mice. Fifteen miRNAs differentially expressed in HF with respect to LF hepatic tissues during the progression of liver damage, and in tumors with respect to HF non tumor liver specimens were identified. Among them, miR-340-5p, miR-484, miR-574-3p, miR-720, whose expression was never described in NAFLD, NASH and HCC tissues, and miR-125a-5p and miR-182, which showed early and significant dysregulation in the sequential hepatic damage process. CONCLUSIONS: In this study, fifteen microRNAs which were modulated in hepatic tissues and in tumors during the transition NAFLD-NASH-HCC are reported. Besides some already described, new and early dysregulated miRNAs were identified. Functional analyses are needed to validate the results here obtained, and to better define the role of these molecules in the progression of the hepatic disease.

Ischemia reperfusion of the hepatic artery induces the functional damage of large bile ducts by changes in the expression of angiogenic factors.

Liver transplantation and cholangiocarcinoma induce biliary dysfunction following ischemia reperfusion (IR). The function of the intrahepatic biliary tree is regulated by both autocrine and paracrine factors. The aim of the study was to demonstrate that IR-induced damage of cholangiocytes is associated with altered expression of biliary angiogenic factors. Normal and bile duct ligation rats underwent 24-h sham or hepatic reperfusion after 30 min of transient occlusion of the hepatic artery (HAIR) or portal vein (PVIR) before collecting liver blocks and cholangiocyte RNA or protein. We evaluated liver histology, biliary apoptosis, proliferation and expression of VEGF-A/C, VEGFR-2/3, Ang-1/2, and Tie-1/2 in liver sections and isolated small and large cholangiocytes. Normal rat intrahepatic cholangiocyte cultures (NRICC) were maintained under standard conditions in normoxic or under a hypoxic atmosphere for 4 h and then transferred to normal conditions for selected times. Subsequently, we measured changes in biliary proliferation and apoptosis and the expression of VEGF-A/C and VEGFR-2/3. In vivo, HAIR (but not PVIR) induced damage of large bile ducts and decreased proliferation and secretin-stimulated cAMP levels. HAIR-induced damage of large bile ducts was associated with increased expression of VEGF-A/C, VEGFR-2/3, Ang-1/2, and Tie-1/2. In vitro, under hypoxic conditions, there was increased apoptosis and reduced proliferation of NRICC concomitant with enhanced expression of VEGF-A/C and VEGFR-2/3. The functional damage of large bile ducts by HAIR and hypoxia is associated with increased expression of angiogenic factors in small cholangiocytes, presumably due to a compensatory mechanism in response to biliary damage.

TrkA alternative splicing: a regulated tumor-promoting switch in human neuroblastoma.

We identify a novel alternative TrkA splice variant, TrkAIII, with deletion of exons 6, 7, and 9 and functional extracellular IG-C1 and N-glycosylation domains, that exhibits expression restricted to undifferentiated early neural progenitors, human neuroblastomas (NBs), and a subset of other neural crest-derived tumors. This NGF-unresponsive isoform is oncogenic in NIH3T3 cells and promotes tumorigenic NB cell behavior in vitro and in vivo (cell survival, xenograft growth, angiogenesis) resulting from spontaneous tyrosine kinase activity and IP3K/Akt/NF-kappaB but not Ras/MAPK signaling. TrkAIII antagonizes NGF/TrkAI signaling, which is responsible for NB growth arrest and differentiation through Ras/MAPK, and its expression is promoted by hypoxia at the expense of NGF-responsive receptors, providing a mechanism for converting NGF/TrkA/Ras/MAPK antioncogenic signals to TrkAIII/IP3K/Akt/NF-kappaB tumor-promoting signals during tumor progression.

Extracellular Vesicles and Resistance to Anticancer Drugs: A Tumor Skeleton Key for Unhinging Chemotherapies.

Although surgical procedures and clinical care allow reaching high success in fighting most tumors, cancer is still a formidable foe. Recurrence and metastatization dampen the patients' overall survival after the first diagnosis; nevertheless, the large knowledge of the molecular bases drives these aspects. Chemoresistance is tightly linked to these features and is mainly responsible for the failure of cancer eradication, leaving patients without a crucial medical strategy. Many pathways have been elucidated to trigger insensitiveness to drugs, generally associated with the promotion of tumor growth, aggressiveness, and metastatisation. The main mechanisms reported are the expression of transporter proteins, the induction or mutations of oncogenes and transcription factors, the alteration in genomic or mitochondrial DNA, the triggering of autophagy or epithelial-to-mesenchymal transition, the acquisition of a stem phenotype, and the activation of tumor microenvironment cells. Extracellular vesicles (EVs) can directly transfer or epigenetically induce to a target cell the molecular machinery responsible for the acquisition of resistance to drugs. In this review, we resume the main body of knowledge supporting the crucial role of EVs in the context of chemoresistance, with a particular emphasis on the mechanisms related to some of the main drugs used to fight cancer.

Ferroptosis resistance cooperates with cellular senescence in the overt stage of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis.

Cellular senescence and ferroptosis are the two main, fine-tuned processes in tissue damage restraint; however, they can be overactivated in pathologies such as nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH), becoming dangerous stimuli. Senescence is characterized by a decline in cell division and an abnormal release of reactive oxygen species (ROS), and ferroptosis is represented by iron deposition associated with an excessive accumulation of ROS. ROS and cellular stress pathways are also drivers of NAFLD/NASH development. The etiology of NAFLD/NASH lies in poor diets enriched in fat and sugar. This food regimen leads to liver steatosis, resulting in progressive degeneration of the organ, with a late onset of irreversible fibrosis and cirrhosis. Few studies have investigated the possible connection between senescence and ferroptosis in NAFLD/NASH progression, despite the two events sharing some molecular players. We hypothesized a possible link between senescence and ferroptosis in a NAFLD background. To thoroughly investigate this in the context of "Western-style" diet (WSD) abuse, we used an amylin-modified liver NASH mouse model. The main NASH hallmarks have been confirmed in this model, as well as an increase in apoptosis, and Ki67 and p53 expression in the liver. Senescent beta-galactosidase-positive cells were elevated, as well as the expression of the related secretory molecules Il-6 and MMP-1. Features of DNA damage and iron-overload were found in the livers of NASH mice. Gpx4 (glutathione peroxidase 4) expression, counteracting ferroptotic cell death, was increased. Notably, an increased number of senescent cells showing overexpression of gpx4 was also found. Our data seem to suggest that senescent cells acquire a gpx4-mediated mechanism of ferroptosis resistance and thus remain in the liver, fostering the deterioration of liver fitness.

The antiinflammatory and antifibrotic effect of olive phenols and Lactiplantibacillus plantarum IMC513 in dextran sodium sulfate-induced chronic colitis.

OBJECTIVES: After a chronic intestinal injury, several intestinal cells switch their phenotype to activated myofibroblasts, which in turn release an abnormal amount of extracellular matrix proteins, leading to the onset of the fibrotic process. To date, no resolutive pharmacological treatments are available, and the identification of new therapeutic approaches represents a crucial goal to achieve. The onset, maintenance, and progression of inflammatory bowel disease are related to abnormal intestinal immune responses to environmental factors, including diet and intestinal microflora components. This study aimed to evaluate the potential antiinflammatory and antifibrotic effect of a biologically debittered olive cream and its probiotic oral administration in an experimental model of dextran sodium sulfate (DSS)-induced chronic colitis. METHODS: Chronic colitis was induced in mice by three cycles of oral administration of 2.5% DSS (5 d of DSS followed by 7 d of tap water). Mice were randomly divided into five groups: 10 control mice fed with standard diet (SD), 20 mice receiving SD and DSS (SD+DSS), 20 mice receiving an enriched diet (ED) with olive cream and DSS (ED+DSS), 20 mice receiving SD plus probiotics (PB; Lactiplantibacillus plantarum IMC513) and DSS (SD+PB+DSS), and 20 mice receiving ED plus PB and DSS (ED+ PB+DSS). Clinical features and large bowel macroscopic, histologic, and immunohistochemical findings were evaluated. RESULTS: The simultaneous administration of ED and PB induced a significant reduction in macroscopic and microscopic colitis scores compared with the other DSS-treated groups. In addition, ED and PB led to a significant decrease in the expression of inflammatory cytokines and profibrotic molecules. CONCLUSIONS: The concomitant oral administration of a diet enriched with biologically debittered olive cream and a specific probiotic strain (Lactiplantibacillus plantarum IMC513) can exert synergistic antiinflammatory and antifibrotic action in DSS-induced chronic colitis. Further studies are needed to define the cellular and molecular mechanisms modulated by olive cream compounds and by Lactiplantibacillus plantarum IMC513.

Is mastocytic colitis a specific clinical-pathological entity?

The number of intestinal mast cells (MC) is increased in several types of colitis, but the mucosa of patients with chronic non-bloody diarrhea has not been studied. The current study sought to determine the relationship between MC counts and degranulation and the severity of symptoms in patients with chronic loose stools. Following a negative laboratory workup for the most common causes of chronic diarrhea, patients with chronic non-bloody loose stools were included in the study. Patients with macroscopic evidence of inflammation or organic disease were excluded after endoscopy with biopsies. Biopsies from the 179 patients in the study were stained with hematoxylin and eosin and anti-CD117 c-kit antibodies. Immunohistochemistry was used to assess the degree of MC degranulation. Out of the 179 patients, 128 had normal histologic findings suggestive of irritable bowel syndrome and were used as controls. Twenty-four presented with abnormally high MC counts (≥40 MC x HPF), 23 with ≥20 intraepithelial lymphocytes x HPF suggesting lymphocytic colitis, and 4 had both (≥40 MC and ≥20 intraepithelial lymphocytes x HPF). In the patients with high MC counts, figures were significantly higher in the right colon versus the left colon (p=0.016), but degranulation did not differ in the right versus the left colon (p=0.125). No age or sex-related difference was observed (p=0.527 and p=0.859 respectively). The prevalence of abdominal pain and bloating did not differ in the three groups (p=0.959 and p=0.140, respectively). Patients with lymphocytic colitis (p=0.008) and those with high MC counts (p=0.025) had significantly higher evacuation rates compared to controls. There was no difference between these two groups (p=0.831). Mast cell degranulation was not associated with the number of evacuations, abdominal pain, or bloating (p=0.51; p=0.41; p=0.42, respectively). The finding that a significantly higher number of evacuations was linked to increased MC in the colonic mucosa of a subset of patients with otherwise normal laboratory and endoscopic findings suggests that "mastocytic colitis" may be a new clinical-pathological entity responsible for chronic non-bloody diarrhea. Prospective studies with a larger number of patients, as well as endoscopic and histological follow-up, are needed to confirm this hypothesis.

The Charming World of the Extracellular Matrix: A Dynamic and Protective Network of the Intestinal Wall.

The intestinal extracellular matrix (ECM) represents a complex network of proteins that not only forms a support structure for resident cells but also interacts closely with them by modulating their phenotypes and functions. More than 300 molecules have been identified, each of them with unique biochemical properties and exclusive biological functions. ECM components not only provide a scaffold for the tissue but also afford tensile strength and limit overstretch of the organ. The ECM holds water, ensures suitable hydration of the tissue, and participates in a selective barrier to the external environment. ECM-to-cells interaction is crucial for morphogenesis and cell differentiation, proliferation, and apoptosis. The ECM is a dynamic and multifunctional structure. The ECM is constantly renewed and remodeled by coordinated action among ECM-producing cells, degrading enzymes, and their specific inhibitors. During this process, several growth factors are released in the ECM, and they, in turn, modulate the deposition of new ECM. In this review, we describe the main components and functions of intestinal ECM and we discuss their role in maintaining the structure and function of the intestinal barrier. Achieving complete knowledge of the ECM world is an important goal to understand the mechanisms leading to the onset and the progression of several intestinal diseases related to alterations in ECM remodeling.

Long-term abuse of a high-carbohydrate diet is as harmful as a high-fat diet for development and progression of liver injury in a mouse model of NAFLD/NASH.

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) has become the most common liver disease globally. It is caused by a complex network of factors, including diet. The hallmark of NAFLD is the benign accumulation of triacylglycerols, however, this condition may worsen into non-alcoholic steatohepatitis (NASH), a more severe form associated with inflammation and fibrosis. Currently, no therapies are available, and diet modifications are the only strategy. Although there is increasing evidence emerging about how an abuse of carbohydrates could be involved in the progression of liver injury, a comprehensive understanding of the damage induced by an enriched carbohydrate diet is still far from complete. The aim of this study was to investigate and compare the effects of a low-fat/high-carbohydrate diet (LF-HCD) with high-fat (HFD) and standard (SD) diets in a nutritional mouse model of NAFLD/NASH. METHODS: Histologic, real-time polymerase chain reaction, and immunohistochemical evaluations were performed. RESULTS: The results showed that the prolonged abuse of both LF-HCDs and HFDs induced a significant increase in hepatic steatosis, inflammation, and fibrosis scores compared with SD. At the same time, both LF-HCDs and HFDs led to significant increases in the expression of the molecules involved in the progression of NAFLD that we assessed (perilipin, CD68, TGF-ß1, CTGF, leptin, leptin receptor, and α-SMA). CONCLUSIONS: The present study highlighted that the simple substitution of fats with carbohydrates is not a proper strategy to prevent or mitigate the progression of NAFLD/NASH. Further studies are required to define the best nutritional strategy to prevent NAFLD and its related metabolic syndrome.

Can the AGE/RAGE/ERK signalling pathway and the epithelial-to-mesenchymal transition interact in the pathogenesis of chronic rhinosinusitis with nasal polyps?

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a persistent sinonasal mucosa inflammatory disease with still unclear pathophysiologic mechanisms that imply events of tissue repair and structural remodelling. Several cascades seem to have a considerable role in the onset and progression of mucosa hyperproliferation in nasal polyps including transforming growth factor ß/Small mother against decapentaplegic (TGFß/Smads), mitogenactivated protein kinases (MAPKs), advanced glycosylation end-products (AGEs) together with epithelial-tomesenchymal transition (EMT). Since many inflammatory mediators are reported to play important roles in the development of nasal polyps (NP) disease, this study aimed to analyse the correlation between the AGEs/receptor of advanced glycosylation end-products (RAGE)/extracellular signal-regulated kinase (ERK) signalling pathway and the main markers of EMT to better understand the influence that they exert on the remodelling of nasal mucous membranes in patients affected by CRSwNP vs normal controls. A total of 30 patients were enrolled in this study. Immunohistochemical analysis, using AGE, RAGE, p-ERK, MMP-3, TGF-ß1, Smad2/3, Collagen I-III, α-SMA, E-cadherin, IL-6 and Vimentin antibodies, was performed. AGE, RAGE, ERK, p-ERK and MMP3 were also evaluated using western blot analysis. We observed an overexpression of the AGE/RAGE/p-ERK and the main mesenchymal markers of EMT (Vimentin and IL-6) in CRSwNP vs controls whereas the TGF-ß/Smad3 pathway did not show any significant differences between the two groups of patients. These observations suggest a complex network of processes in the pathogenesis of NP, and the AGE/RAGE/ERK pathway and EMT might work together in promoting tissue remodelling in the formation of CRSwNP.

Targeted disruption of Smad3 confers resistance to the development of dimethylnitrosamine-induced hepatic fibrosis in mice.

BACKGROUND: Hepatic fibrosis is characterized by a progressive accumulation of fibrillar extracellular matrix (ECM) proteins including collagen, which occurs in most types of chronic liver diseases. Transforming growth factor-beta (TGF-beta)/Smad3 signalling plays a central role in tissue fibrogenesis, acting as a potent stimulus of ECM accumulation. AIM: To evaluate the potential protective role of Smad3 deficiency in the pathogenesis of liver fibrosis induced by dimethylnitrosamine (DMN) in Smad3 null mice. METHODS: Chronic hepatitis-associated fibrosis was induced in 13 Smad3 null and 13 wild-type (WT) mice by intraperitoneal DMN administration (10 microg/g body weight/day) for three consecutive days per week for 6 weeks. The liver was excised for macroscopic examination and histological, morphometric and immunohistochemical (IHC) analyses. For IHC, alpha-smooth muscle actin (alpha-SMA), collagen types I-III, TGF-beta1, connective tissue growth factor (CTGF), Smad3, Smad7 and CD3 antibodies were used. RESULTS: At macroscopic examination, the liver of DMN-treated Smad3 WT appeared harder with a dark brown colouring and necrotic areas compared with that from null mice. Histological and morphometric evaluation revealed a significantly higher degree of hepatic fibrosis and accumulation of connective tissue in the Smad3 WT compared with null mice. IHC evaluation showed a marked increase in alpha-SMA, CTGF, collagen I-III, TGF-beta and Smad3 staining in the liver of Smad3 WT compared with that in null mice, whereas Smad7 was increased only in null mice. CONCLUSIONS: The results indicate that Smad3 loss confers resistance to the development of DMN-induced hepatic fibrosis. The reduced fibrotic response appears to be due to a reduction of fibrogenic myofibroblast activation and ECM production and accumulation. Smad3 could be a novel target for potential treatment of fibrosis complicating chronic hepatitis.

Correction: Pharmacological treatment with inhibitors of nuclear export enhances the antitumor activity of docetaxel in human prostate cancer.

[This corrects the article DOI: 10.18632/oncotarget.22760.].

The Brain Penetrating and Dual TORC1/TORC2 Inhibitor, RES529, Elicits Anti-Glioma Activity and Enhances the Therapeutic Effects of Anti-Angiogenetic Compounds in Preclinical Murine Models.

Background. Glioblastoma multiforme (GBM) is a devastating disease showing a very poor prognosis. New therapeutic approaches are needed to improve survival and quality of life. GBM is a highly vascularized tumor and as such, chemotherapy and anti-angiogenic drugs have been combined for treatment. However, as treatment-induced resistance often develops, our goal was to identify and treat pathways involved in resistance to treatment to optimize the treatment strategies. Anti-angiogenetic compounds tested in preclinical and clinical settings demonstrated recurrence associated to secondary activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway. Aims. Here, we determined the sensitizing effects of the small molecule and oral available dual TORC1/TORC2 dissociative inhibitor, RES529, alone or in combination with the anti-VEGF blocking antibody, bevacizumab, or the tyrosine kinase inhibitor, sunitinib, in human GBM models. Results. We observed that RES529 effectively inhibited dose-dependently the growth of GBM cells in vitro counteracting the insurgence of recurrence after bevacizumab or sunitinib administration in vivo. Combination strategies were associated with reduced tumor progression as indicated by the analysis of Time to Tumor Progression (TTP) and disease-free survival (DSF) as well as increased overall survival (OS) of tumor bearing mice. RES529 was able to reduce the in vitro migration of tumor cells and tubule formation from both brain-derived endothelial cells (angiogenesis) and tumor cells (vasculogenic mimicry). Conclusions. In summary, RES529, the first dual TORC1/TORC2 dissociative inhibitor, lacking affinity for ABCB1/ABCG2 and having good brain penetration, was active in GBM preclinical/murine models giving credence to its use in clinical trial for patients with GBM treated in association with anti-angiogenetic compounds.

SIRT1 participates in the response to methylglyoxal-dependent glycative stress in mouse oocytes and ovary.

Methylglyoxal (MG), a highly reactive dicarbonyl derived from metabolic processes, is the most powerful precursor of advanced glycation end products (AGEs). Glycative stress has been recently associated with ovarian dysfunctions in aging and PCOS syndrome. We have investigated the role of the NAD+-dependent Class III deacetylase SIRT1 in the adaptive response to MG in mouse oocytes and ovary. In mouse oocytes, MG induced up-expression of glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2) genes, components of the main MG detoxification system, whereas inhibition of SIRT1 by Ex527 or sirtinol reduced this response. In addition, the inhibition of SIRT1 worsened the effects of MG on oocyte maturation rates, while SIRT1 activation by resveratrol counteracted MG insult. Ovaries from female mice receiving 100 mg/kg MG by gastric administration for 28 days (MG mice) exhibited increased levels of SIRT1 along with over-expression of catalase, superoxide dismutase 2, SIRT3, PGC1α and mtTFA. Similar levels of MG-derived AGEs were observed in the ovaries from MG and control groups, along with enhanced protein expression of glyoxalase 1 in MG mice. Oocytes ovulated by MG mice exhibited atypical meiotic spindles, a condition predisposing to embryo aneuploidy. Our results from mouse oocytes revealed for the first time that SIRT1 could modulate MG scavenging by promoting expression of glyoxalases. The finding that up-regulation of glyoxalase 1 is associated with that of components of a SIRT1 functional network in the ovaries of MG mice provides strong evidence that SIRT1 participates in the response to methylglyoxal-dependent glycative stress in the female gonad.

The Small Molecule Ephrin Receptor Inhibitor, GLPG1790, Reduces Renewal Capabilities of Cancer Stem Cells, Showing Anti-Tumour Efficacy on Preclinical Glioblastoma Models.

Therapies against glioblastoma (GBM) show a high percentage of failure associated with the survival of glioma stem cells (GSCs) that repopulate treated tumours. Forced differentiation of GSCs is a promising new approach in cancer treatment. Erythropoietin-producing hepatocellular (Eph) receptors drive tumourigenicity and stemness in GBM. We tested GLPG1790, a first small molecule with inhibition activity versus inhibitor of various Eph receptor kinases, in preclinical GBM models using in vitro and in vivo assays. GLPG1790 rapidly and persistently inhibited Ephrin-A1-mediated phosphorylation of Tyr588 and Ser897, completely blocking EphA2 receptor signalling. Similarly, this compound blocks the ephrin B2-mediated EphA3 and EphB4 tyrosine phosphorylation. This resulted in anti-glioma effects. GLPG1790 down-modulated the expression of mesenchymal markers CD44, Sox2, nestin, octamer-binding transcription factor 3/4 (Oct3/4), Nanog, CD90, and CD105, and up-regulated that of glial fibrillary acidic protein (GFAP) and pro-neural/neuronal markers, ßIII tubulin, and neurofilaments. GLPG1790 reduced tumour growth in vivo. These effects were larger compared to radiation therapy (RT; U251 and T98G xenografts) and smaller than those of temozolomide (TMZ; U251 and U87MG cell models). By contrast, GLPG1790 showed effects that were higher than Radiotherapy (RT) and similar to Temozolomide (TMZ) in orthotopic U87MG and CSCs-5 models in terms of disease-free survival (DFS) and overall survival (OS). Further experiments were necessary to study possible interactions with radio- and chemotherapy. GLPG1790 demonstrated anti-tumor effects regulating both the differentiative status of Glioma Initiating Cells (GICs) and the quality of tumor microenvironment, translating into efficacy in aggressive GBM mouse models. Significant common molecular targets to radio and chemo therapy supported the combination use of GLPG1790 in ameliorative antiglioma therapy.