Working mechanism of Hic-5 in human and experimental NAFLD-related liver fibrosis

Non-alcoholic fatty liver disease (NAFLD) is characterized by a wide spectrum of pathological findings, ranging from simple steatosis to steatohepatitis which can progress to cirrhosis and hepatocellular carcinoma. Fibrosis severity is the main determinant of liver-related complications and mortality, therefore its regression is considered an important therapeutic endpoint in clinical trials evaluating pharmacological intervention in NAFLD. Activation of hepatic stellate cells (HSC) by transforming growth factor beta (TGFβ) is a well-established driver of liver fibrogenesis. Recently, a TGFβ-modulated protein, namely hydrogen peroxide-inducible clone 5 (Hic-5) has been proposed as a novel potential therapeutic target for liver fibrosis, since its knockdown attenuated experimental liver fibrosis in mice. However, limited data are available regarding its expression in human HSC, while no study has investigated its role in the setting of NAFLD so far. Based on these premises, the aim of my thesis was first to characterize Hic-5 expression in human HSC according to different hepatic microenvironment (healthy versus fibrotic); secondly to assess whether Hic-5 is expressed in human and experimental NAFLD. To investigate the first aim, primary human HSC were cultured in a new 3-dimensional (3D) culture model based on decellularized human liver extracellular matrix (ECM) scaffolds derived from healthy and cirrhotic livers. Compared to traditional 2D cultures, Hic-5 gene expression was significantly upregulated in HSC cultured in the 3D model. This effect was further enhanced upon TGFβ stimulation in healthy scaffolds. Moreover, higher Hic-5 mRNA levels were detected in HSC cultured in cirrhotic scaffolds compared to healthy scaffolds, suggesting that Hic-5 expression and its modulation by TGFβ are strongly affected by liver- and disease specific ECM features. Based on these preliminary in-vitro results, I decided to further investigate Hic-5 expression in human NAFLD samples obtained from obese patients undergoing bariatric surgery. Immunohistochemical staining showed an increased expression of Hic-5 in fibrotic liver tissue, which overlapped alpha smooth muscle actin (α-SMA) positive areas. In order to better elucidate the role of Hic-5 across NAFLD progression, a nutritional rat model of NAFLD, based on the administration of choline-deficient (CD) diet, was employed and hepatic Hic-5 expression analyzed at different time points. After 3 days of CD diet Hic-5 mRNA levels were not significantly upregulated, suggesting that fatty liver alone without associated liver fibrosis does not affect Hic-5 expression. Accordingly, no difference in α-SMA gene expression was observed compared to control diet. In contrast, administration of CD diet for 7 weeks led to increased mRNA expression of Hic-5 along with αSMA, TGFβ1 and collagen type 1A1. Upregulation of Hic-5 was further confirmed at the protein level by Western blot analysis. Similar findings were obtained when CD diet was given for 11 weeks. Interestingly, the administration of a triiodothyronine(T3)-supplemented diet for 1 week, which has been previously shown to revert rat fatty liver, was able to reduce the expression of Hic-5. The same effect of T3 on Hic-5 was seen on the microenvironment surrounding preneoplastic nodules during NASH-related experimental liver carcinogenesis. Since T3 administration was associated with regression of preneoplastic nodules not expressing Hic-5 or αSMA, we speculate that its antifibrotic effect may contribute to preneoplastic nodules regression. Taken together, these results highlight the role of Hic-5 in HSC activation in vitro and its association with NAFLD progression. Further studies are needed to clarify the molecular mechanisms regulating Hic-5 expression in NAFLD-related fibrosis and the effects of thyroid receptor’s agonists in order to identify potential anti-fibrotic therapie

Valproic acid up-regulates p75NTR and sortilin expression to induce neuronal cell death

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Differential targeting of lysophosphatidic acid LPA1, LPA2, and LPA3 receptor signalling by tricyclic and tetracyclic antidepressants

We previously reported that in different cell types antidepressant drugs activate lysophosphatidic acid (LPA) LPA1 receptor to induce proliferative and prosurvival responses. Here, we further characterize this unique action of antidepressants by examining their effects on two additional LPA receptor family members, LPA2 and LPA3. Human LPA1-3 receptors were stably expressed in HEK-293 cells (HEK-LPA1, -LPA2 and -LPA3 cells) and their functional activity was determined by Western blot and immunofluorescence. LPA effectively stimulated the phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in HEK-LPA1, -LPA2, and -LPA3 cells. The tricyclic antidepressants amitriptyline, clomipramine, imipramine and desipramine increased phospho-ERK1/2 levels in HEK-LPA1 and -LPA3 cells but were relatively poor agonists in LPA2-expressing cells. The tetracyclic antidepressants mianserin and mirtazapine were active at all three LPA receptors. When combined with LPA, both amitriptyline and mianserin potentiated Gi/o-mediated phosphorylation of ERK1/2 induced by LPA in HEK-LPA1, -LPA2 and -LPA3 cells, CHO-K1 fibroblasts and HT22 hippocampal neuroblasts. This potentiation was associated with enhanced phosphorylation of CREB and S6 ribosomal protein, two molecular targets of activated ERK1/2. The antidepressants also potentiated LPA-induced Gq/11-mediated phosphorylation of AMP-activated protein kinase in HEK-LPA1 and -LPA3 cells. Conversely, amitriptyline and mianserin were found to inhibit LPA-induced Rho activation in HEK-LPA1 and LPA2 cells. These results indicate that tricyclic and tetracyclic antidepressants can act on LPA1, LPA2 and LPA3 receptor subtypes and exert differential effects on LPA signalling through these receptors

The Neurotrophin Receptor TrkC as a Novel Molecular Target of the Antineuroblastoma Action of Valproic Acid

Neurotrophins and their receptors are relevant factors in controlling neuroblastoma growth and progression. The histone deacetylase (HDAC) inhibitor valproic acid (VPA) has been shown to downregulate TrkB and upregulate the p75NTR/sortilin receptor complex. In the present study, we investigated the VPA effect on the expression of the neurotrophin-3 (NT-3) receptor TrkC, a favorable prognostic marker of neuroblastoma. We found that VPA induced the expression of both full-length and truncated (TrkC-T1) isoforms of TrkC in human neuroblastoma cell lines without (SH-SY5Y) and with (Kelly, BE(2)-C and IMR 32) MYCN amplification. VPA enhanced cell surface expression of the receptor and increased Akt and ERK1/2 activation by NT-3. The HDAC inhibitors entinostat, romidepsin and vorinostat also increased TrkC in SH-SY5Y, Kelly and BE(2)-C but not IMR 32 cells. TrkC upregulation by VPA involved induction of RUNX3, stimulation of ERK1/2 and JNK, and ERK1/2-mediated Egr1 expression. In SH-SY5Y cell monolayers and spheroids the exposure to NT-3 enhanced the apoptotic cascade triggered by VPA. Gene silencing of both TrkC-T1 and p75NTR prevented the NT-3 proapoptotic effect. Moreover, NT-3 enhanced p75NTR/TrkC-T1 co-immunoprecipitation. The results indicate that VPA upregulates TrkC by activating epigenetic mechanisms and signaling pathways, and sensitizes neuroblastoma cells to NT-3-induced apoptosis

Gastrointestinal Coronavirus disease 2019: epidemiology, clinical features, pathogenesis, prevention, and management

Introduction: The new Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the etiologic agent of coronavirus disease 2019. Some authors reported evidences that patients with SARS-CoV-2 infection could have a direct involvement of the gastrointestinal tract, and in symptomatic cases, gastrointestinal symptoms (diarrhea, nausea/vomiting, abdominal pain) could be very common. Area covered: In this article, we reviewed current published data of the gastrointestinal aspects involved in SARS-CoV-2 infection, including prevalence and incidence of specific symptoms, presumptive biological mechanism of GI infection, prognosis, clinical management and public health related concerns on the possible risk of oral-fecal transmission. Expert opinion: Different clues point to a direct virus infection and replication in mucosal cells of the gastrointestinal tract. In vitro studies showed that SARS-CoV-2 could enters into the gastrointestinal epithelial cells by the Angiotensin-Converting enzyme 2 membrane receptor. These findings, coupled with identification of viral RNA found in stools of patients, clearly suggest that a direct involvement of gastrointestinal tract is very likely. This can justify most of the gastrointestinal symptoms but also suggest a risk for an oral fecal route for transmission, additionally or alternatively to the main respiratory route

Combined Salivary Proteome Profiling and Machine Learning Analysis Provides Insight into Molecular Signature for Autoimmune Liver Diseases Classification

Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) are autoimmune liver diseases that target the liver and have a wide spectrum of presentation. A global overview of quantitative variations on the salivary proteome in presence of these two pathologies is investigated in this study. The acid-insoluble salivary fraction of AIH and PBC patients, and healthy controls (HCs), was analyzed using a gel-based bottom-up proteomic approach combined with a robust machine learning statistical analysis of the dataset. The abundance of Arginase, Junction plakoglobin, Desmoplakin, Hexokinase-3 and Desmocollin-1 decreased, while that of BPI fold-containing family A member 2 increased in AIHp compared to HCs; the abundance of Gelsolin, CD14, Tumor-associated calcium signal transducer 2, Clusterin, Heterogeneous nuclear ribonucleoproteins A2/B1, Cofilin-1 and BPI fold-containing family B member 2 increased in PBCp compared to HCs. The abundance of Hornerin decreased in both AIHp and PBCp with respect to HCs and provided an area under the ROC curve of 0.939. Machine learning analysis confirmed the feasibility of the salivary proteome to discriminate groups of subjects based on AIH or PBC occurrence as previously suggested by our group. The topology-based functional enrichment analysis performed on these potential salivary biomarkers highlights an enrichment of terms mostly related to the immune system, but also with a strong involvement in liver fibrosis process and with antimicrobial activity

Exploring new scaffolds for the dual inhibition of HIV-1 RT polymerase and ribonuclease associated functions

Current therapeutic protocols for the treatment of HIV infection consist of the combination of diverse anti-retroviral drugs in order to reduce the selection of resistant mutants and to allow for the use of lower doses of each single agent to reduce toxicity. However, avoiding drugs interactions and patient compliance are issues not fully accomplished so far. Pursuing on our investigation on potential anti HIV multi-target agents we have designed and synthesized a small library of biphenylhydrazo 4-arylthiazoles derivatives and evaluated to investigate the ability of the new derivatives to simultaneously inhibit both associated functions of HIV reverse transcriptase. All compounds were active towards the two functions, although at different concentrations. The substitution pattern on the biphenyl moiety appears relevant to determine the activity. In particular, compound 2-{3- [(2-{4-[4-(hydroxynitroso)phenyl]-1,3-thiazol-2-yl} hydrazin-1-ylidene) methyl]-4-methoxyphenyl} benzamide bromide (EMAC2063) was the most potent towards RNaseH (IC50 = 4.5 mM)- and RDDP (IC50 = 8.0 mM) HIV RT-associated function