Acceleration of TAA-induced liver fibrosis by stress exposure is associated with upregulation of nerve growth factor and glycopattern deviations

Liver fibrosis results from many chronic injuries and may often progress to cirrhosis and hepatocellular carcinoma (HCC). In fact, up to 90% of HCC arise in a cirrhotic liver. Conversely, stress is implicated in liver damage, worsening disease outcome. Hence, stress could play a role in disrupting liver homeostasis, a concept that has not been fully explored. Here, in a murine model of TAA-induced liver fibrosis we identified nerve growth factor (NGF) to be a crucial regulator of the stress-induced fibrogenesis signaling pathway as it activates its receptor p75 neurotrophin receptor (p75NTR), increasing liver damage. Additionally, blocking the NGF decreased liver fibrosis whereas treatment with recombinant NGF accelerated the fibrotic process to a similar extent than stress challenge. We further show that the fibrogenesis induced by stress is characterized by specific changes in the hepatoglycocode (increased β1,6GlcNAc-branched complex N-glycans and decreased core 1 O-glycans expression) which are also observed in patients with advanced fibrosis compared to patients with a low level of fibrosis. Our study facilitates an understanding of stress-induced liver injury and identify NGF signaling pathway in early stages of the disease, which contributes to the established fibrogenesis

SPARC (secreted protein acidic and rich in cysteine) knockdown protects mice from acute liver injury by reducing vascular endothelial cell damage

Secreted protein, acidic and rich in cysteine (SPARC) is involved in many biological process including liver fibrogenesis, but its role in acute liver damage is unknown. To examine the role of SPARC in acute liver injury, we used SPARC knock-out (SPARC−/−) mice. Two models of acute liver damage were used: concanavalin A (Con A) and the agonistic anti-CD95 antibody Jo2. SPARC expression levels were analyzed in liver samples from patients with acute-on-chronic alcoholic hepatitis (AH). SPARC expression is increased on acute-on-chronic AH patients. Knockdown of SPARC decreased hepatic damage in the two models of liver injury. SPARC−/− mice showed a marked reduction in Con A-induced necroinflammation. Infiltration by CD4+ T cells, expression of tumor necrosis factor-α and interleukin-6 and apoptosis were attenuated in SPARC−/− mice. Sinusoidal endothelial cell monolayer was preserved and was less activated in Con A-treated SPARC−/− mice. SPARC knockdown reduced Con A-induced autophagy of cultured human microvascular endothelial cells (HMEC-1). Hepatic transcriptome analysis revealed several gene networks that may have a role in the attenuated liver damaged found in Con A-treated SPARC−/− mice. SPARC has a significant role in the development of Con A-induced severe liver injury. These results suggest that SPARC could represent a therapeutic target in acute liver injury

Control of Gene Expression by the Retinoic Acid-Related Orphan Receptor Alpha in HepG2 Human Hepatoma Cells

Retinoic acid-related Orphan Receptor alpha (RORα; NR1F1) is a widely distributed nuclear receptor involved in several (patho)physiological functions including lipid metabolism, inflammation, angiogenesis, and circadian rhythm. To better understand the role of this nuclear receptor in liver, we aimed at displaying genes controlled by RORα in liver cells by generating HepG2 human hepatoma cells stably over-expressing RORα. Genes whose expression was altered in these cells versus control cells were displayed using micro-arrays followed by qRT-PCR analysis. Expression of these genes was also altered in cells in which RORα was transiently over-expressed after adenoviral infection. A number of the genes found were involved in known pathways controlled by RORα, for instance LPA, NR1D2 and ADIPOQ in lipid metabolism, ADIPOQ and PLG in inflammation, PLG in fibrinolysis and NR1D2 and NR1D1 in circadian rhythm. This study also revealed that genes such as G6PC, involved in glucose homeostasis, and AGRP, involved in the control of body weight, are also controlled by RORα. Lastly, SPARC, involved in cell growth and adhesion, and associated with liver carcinogenesis, was up-regulated by RORα. SPARC was found to be a new putative RORα target gene since it possesses, in its promoter, a functional RORE as evidenced by EMSAs and transfection experiments. Most of the other genes that we found regulated by RORα also contained putative ROREs in their regulatory regions. Chromatin immunoprecipitation (ChIP) confirmed that the ROREs present in the SPARC, PLG, G6PC, NR1D2 and AGRP genes were occupied by RORα in HepG2 cells. Therefore these genes must now be considered as direct RORα targets. Our results open new routes on the roles of RORα in glucose metabolism and carcinogenesis within cells of hepatic origin

SPARC downregulation attenuates the profibrogenic response of hepatic stellate cells induced by TGF-β1 and PDGF

Liver fibrosis is an active process that involves changes in cell-cell and cell-extracellular matrix (ECM) interaction. Secreted protein, acidic and rich in cysteine (SPARC) is an ECM protein with many biological functions that is overexpressed in cirrhotic livers and upregulated in activated hepatic stellate cells (aHSCs). We have recently shown that SPARC downregulation ameliorates liver fibrosis in vivo. To uncover the cellular mechanisms involved, we have specifically knocked down SPARC in two aHSC lines [the CFSC-2G (rat) and the LX-2 (human)] and in primary cultured rat aHSCs. Transient downregulation of SPARC in hepatic stellate cells (HSCs) did not affect their proliferation and had only minor effects on apoptosis. However, SPARC knockdown increased HSC adhesion to fibronectin and significantly decreased their migration toward PDFG-BB and TGF-β1. Interestingly, TGF-β1 secretion by HSCs was reduced following SPARC small interfering RNA (siRNA) treatment, and preincubation with TGF-β1 restored the migratory capacity of SPARC siRNA-treated cells through mechanisms partially independent from TGF-β1-mediated induction of SPARC expression; thus SPARC knockdown seems to exert its effects on HSCs partially through modulation of TGF-β1 expression levels. Importantly, collagen-I mRNA expression was reduced in SPARC siRNA-transfected HSCs. Consistent with previous results, SPARC knockdown in aHSCs was associated with altered F-actin expression patterns and deregulation of key ECM and cell adhesion molecules, i.e., downregulation of N-cadherin and upregulation of E-cadherin. Our data together suggest that the upregulation of SPARC previously reported for aHSCs partially mediates profibrogenic activities of TGF-β1 and PDGF-BB and identify SPARC as a potential therapeutic target for liver fibrosis

Bioactivity of dexamethasone-releasing coatings on polymer/magnesium composites

We developed biodegradable polymeric coatings loaded with increasing amounts of dexamethasone on composites based on polylactic acid and Mg particles for bone repair. Incorporation of Mg particles into the polymeric matrix improves the compressive behaviour of the polymer. Mg-containing composites release Mg ions into the culture medium and improve mesenchymal stem cell (MSC) viability, enhance their osteogenic potential and promote the release of angiogenic factors. Dexamethasone-loaded coatings deposited on composites delay Mg ion dissolution while releasing controlled amounts of the drug, which are highly dependent on initial payload. Release kinetic of dexamethasone from the coatings exhibits a fast initial release of the drug followed by a slower secondary release. Bioactivity of the released dexamethasone was explored by monitoring dose-dependent responses of MSCs and macrophages. Biological effects exerted by the released drug are similar to those observed in cells treated with solutions of the glucocorticoid, indicating that the method employed for inclusion of dexamethasone into the coatings does not impair its bioactive behaviour. Culturing MSCs on dexamethasone-releasing coatings enhances extracellular matrix production and initial induction to osteogenic commitment as a function of drug payload. Dexamethasone incorporated into the coatings presents anti-inflammatory activity, as shown by the decrease in the production of cytokines and angiogenic factors by macrophages and MSCs. Deposition of dexamethasone-releasing coatings on polymer/Mg composites appears to be a promising approach to delay composite degradation at the early stage of implantation and may be useful to attenuate inflammation and adverse foreign body reactions.Peer Reviewe

SPARC (secreted protein acidic and rich in cysteine) knockdown protects mice from acute liver injury by reducing vascular endothelial cell damage

Secreted protein, acidic and rich in cysteine (SPARC) is involved in many biological process including liver fibrogenesis, but its role in acute liver damage is unknown. To examine the role of SPARC in acute liver injury, we used SPARC knock-out (SPARC(−/−)) mice. Two models of acute liver damage were used: concanavalin A (Con A) and the agonistic anti-CD95 antibody Jo2. SPARC expression levels were analyzed in liver samples from patients with acute-on-chronic alcoholic hepatitis (AH). SPARC expression is increased on acute-on-chronic AH patients. Knockdown of SPARC decreased hepatic damage in the two models of liver injury. SPARC(−/−) mice showed a marked reduction in Con A-induced necroinflammation. Infiltration by CD4+ T cells, expression of tumor necrosis factor-α and interleukin-6 and apoptosis were attenuated in SPARC(−/−) mice. Sinusoidal endothelial cell monolayer was preserved and was less activated in Con A-treated SPARC(−/−) mice. SPARC knockdown reduced Con A-induced autophagy of cultured human microvascular endothelial cells (HMEC-1). Hepatic transcriptome analysis revealed several gene networks that may have a role in the attenuated liver damaged found in Con A-treated SPARC(−/−) mice. SPARC has a significant role in the development of Con A-induced severe liver injury. These results suggest that SPARC could represent a therapeutic target in acute liver injury

Antitumor effects of hyaluronic acid inhibitor 4-Methylumbelliferone in an orthotopic hepatocellular carcinoma model in mice.

Liver cirrhosis is characterized by an excessive accumulation of extracellular matrix components, including hyaluronan (HA). In addition, cirrhosis is considered a pre-neoplastic disease for hepatocellular carcinoma (HCC). Altered HA biosynthesis is associated with cancer progression but its role in HCC is unknown. 4-Methylumbelliferone (4-MU), an orally available agent, is an HA synthesis inhibitor with anticancer properties. In this work, we used an orthotopic Hepa129 HCC model established in fibrotic livers induced by thioacetamide. We evaluated 4-MU effects on HCC cells and hepatic stellate cells (HSCs) in vitro by proliferation, apoptosis and cytotoxicity assays; tumor growth and fibrogenesis were also analyzed in vivo. Our results showed that treatment of HCC cells with 4-MU significantly reduced tumor cell proliferation and induced apoptosis, while primary cultured hepatocytes remained unaffected. 4-MU therapy reduced hepatic and systemic levels of HA. Tumors systemically treated with 4-MU showed the extensive areas of necrosis, inflammatory infiltrate and 2-3-fold reduced number of tumor satellites. No signs of toxicity were observed after 4-MU therapy. Animals treated with 4-MU developed a reduced fibrosis degree compared with controls (F1-2 vs F2-3, respectively). Importantly, 4-MU induced the apoptosis of HSCs in vitro and decreased the amount of activated HSCs in vivo. In conclusion, our results suggest a role for 4-MU as an anticancer agent for HCC associated with advanced fibrosis

Immunotherapy and immunoescape in colorectal cancer

Immunotherapy encompasses a variety of interventions and techniques with the common goal of eliciting tumor cell destructive immune responses. Colorectal carcinoma often presents as metastatic disease that impedes curative surgery. Novel strategies such as active immunization with dendritic cells (DCs), gene transfer of cytokines into tumor cells or administration of immunostimulatory monoclonal antibodies (such as anti-CD137 or anti-CTLA-4) have been assessed in preclinical studies and are at an early clinical development stage. Importantly, there is accumulating evidence that chemotherapy and immunotherapy can be combined in the treatment of some cases with colorectal cancer, with synergistic potentiation as a result of antigens cross-presented by dendritic cells and/or elimination of competitor or suppressive T lymphocyte populations (regulatory T-cells). However, genetic and epigenetic unstable carcinoma cells frequently evolve mechanisms of immunoevasion that are the result of either loss of antigen presentation, or an active expression of immunosuppressive substances. Some of these actively immunosuppressive mechanisms are inducible by cytokines that signify the arrival of an effector immune response. For example, induction of 2, 3 indoleamine dioxygenase (IDO) by IFNgamma in colorectal carcinoma cells. Combinational and balanced strategies fostering antigen presentation, T-cell costimulation and interference with immune regulatory mechanisms will probably take the stage in translational research in the treatment of colorectal carcinoma