High glucose treatment promotes extracellular matrix proteome remodeling in Mller glial cells.

BACKGROUND: The underlying pathomechanisms in diabetic retinopathy (DR) remain incompletely understood. The aim of this study was to add to the current knowledge about the particular role of retinal Mller glial cells (RMG) in the initial processes of DR. METHODS: Applying a quantitative proteomic workflow, we investigated changes of primary porcine RMG under short term high glucose treatment as well as glycolysis inhibition treatment. RESULTS: We revealed significant changes in RMG proteome primarily in proteins building the extracellular matrix (ECM) indicating fundamental remodeling processes of ECM as novel rapid response to high glucose treatment. Among others, Osteopontin (SPP1) as well as its interacting integrins were significantly downregulated and organotypic retinal explant culture confirmed the selective loss of SPP1 in RMG upon treatment. Since SPP1 in the retina has been described neuroprotective for photoreceptors and functions against experimentally induced cell swelling, its rapid loss under diabetic conditions may point to a direct involvement of RMG to the early neurodegenerative processes driving DR. Data are available via ProteomeXchange with identifier PXD015879.

Phenobarbital induces alterations in the proteome of hepatocytes and mesenchymal cells of rat livers.

Preceding studies on the mode of action of non-genotoxic hepatocarcinogens (NGCs) have concentrated on alterations induced in hepatocytes (HCs). A potential role of non-parenchymal liver cells (NPCs) in NGC-driven hepatocarcinogenesis has been largely neglected so far. The aim of this study is to characterize NGC-induced alterations in the proteome profiles of HCs as well as NPCs. We chose the prototypic NGC phenobarbital (PB) which was applied to male rats for a period of 14 days. The livers of PB-treated rats were perfused by collagenase and the cell suspensions obtained were subjected to density gradient centrifugation to separate HCs from NPCs. In addition, HCs and NPC isolated from untreated animals were treated with PB in vitro. Proteome profiling was done by CHIP-HPLC and ion trap mass spectrometry. Proteome analyses of the in vivo experiments showed many of the PB effects previously described in HCs by other methods, e.g. induction of phase I and phase II drug metabolising enzymes. In NPCs proteins related to inflammation and immune regulation such as PAI-1 and S100-A10, ADP-ribosyl cyclase 1 and to cell migration such as kinesin-1 heavy chain, myosin regulatory light chain RLC-A and dihydropyrimidinase-related protein 1 were found to be induced, indicating major PB effects on these cells. Remarkably, in vitro treatment of HCs and NPCs with PB hardly reproduced the proteome alterations observed in vivo, indicating differences of NGC induced responses of cells at culture conditions compared to the intact organism. To conclude, the present study clearly demonstrated that PB induces proteome alterations not only in HCs but also in NPCs. Thus, any profound molecular understanding on the mode of action of NGCs has to consider effects on cells of the hepatic mesenchyme.

Food-derived peroxidized fatty acids may trigger hepatic inflammation: a novel hypothesis to explain steatohepatitis.

BACKGROUND & AIMS: Obesity and hepatic steatosis are frequently associated with the development of a non-alcoholic steatohepatitis (NASH). The mechanisms driving progression of a non-inflamed steatosis to NASH are largely unknown. Here, we investigated whether ingestion of peroxidized lipids, as being present in Western style diet, triggers the development of hepatic inflammation. METHODS: Corn oil containing peroxidized fatty acids was administered to rats by gavage for 6 days. In a separate approach, hepatocytes (HC), endothelial (EC) and Kupffer cells (KC) were isolated from untreated livers, cultured, and incubated with peroxidized linoleic acid (LOOH; linoleic acid (LH) being the main fatty acid in corn oil). Samples obtained from in vivo and in vitro studies were mainly investigated by qRT-PCR and biochemical determinations of lipid peroxidation products. RESULTS: Rat treatment with peroxidized corn oil resulted in increased hepatic lipid peroxidation, upregulation of nitric oxide synthetase-2 (NOS-2), cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNFα), elevation of total nitric oxides, and increase in cd68-, cd163-, TNFα-, and/or COX-2 positive immune cells in the liver. When investigating liver cell types, LOOH elevated the secretion of TNFα, p38MAPK phosphorylation, and mRNA levels of NOS-2, COX-2, and TNFα, mainly in KC. The elevation of gene expression could be abrogated by inhibiting p38MAPK, which indicates that p38MAPK activation is involved in the pro-inflammatory effects of LOOH. CONCLUSIONS: These data show for the first time that ingestion of peroxidized fatty acids carries a considerable pro-inflammatory stimulus into the body which reaches the liver and may trigger the development of hepatic inflammation.

Antagonistic effects of selenium and lipid peroxides on growth control in early hepatocellular carcinoma.

UNLABELLED: Activation of the activator protein 1 (AP-1) transcription factor as well as increased serum levels of vascular endothelial growth factor (VEGF) and interleukin (IL)-8 predict poor prognosis of patients with hepatocellular carcinomas (HCCs). Moreover, HCC patients display reduced selenium levels, which may cause lipid peroxidation and oxidative stress because selenium is an essential component of antioxidative glutathione peroxidases (GPx). We hypothesized that selenium-lipid peroxide antagonism controls the above prognostic markers and tumor growth. (1) In human HCC cell lines (HCC-1.2, HCC-3, and SNU398) linoleic acid peroxide (LOOH) and other prooxidants enhanced the expression of VEGF and IL-8. LOOH up-regulated AP-1 activation. Selenium inhibited these effects. This inhibition was mediated by glutathione peroxidase 4 (GPx4), which preferentially degrades lipid peroxides. Selenium enhanced GPx4 expression and total GPx activity, while knock-down of GPx4 by small interfering RNA (siRNA) increased VEGF, and IL-8 expression. (2) These results were confirmed in a rat hepatocarcinogenesis model. Selenium treatment during tumor promotion increased hepatic GPx4 expression and reduced the expression of VEGF and of the AP-1 component c-fos as well as nodule growth. (3) In HCC patients, increased levels of LOOH-related antibodies (LOOH-Ab) were found, suggesting enhanced LOOH formation. LOOH-Ab correlated with serum VEGF and IL-8 and with AP-1 activation in HCC tissue. In contrast, selenium inversely correlated with VEGF, IL-8, and HCC size (the latter only for tumors smaller than 3 cm). CONCLUSION: Reduced selenium levels result in accumulation of lipid peroxides. This leads to enhanced AP-1 activation and consequently to elevated expression of VEGF and IL-8, which accelerate the growth of HCC. Selenium supplementation could be considered for investigation as a strategy for chemoprevention or additional therapy of early HCC in patients with low selenium levels.

Potent protection of gallic acid against DNA oxidation: results of human and animal experiments.

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is a constituent of plant derived foods, beverages and herbal remedies. We investigated its DNA protective properties in a placebo controlled human intervention trial in single cell gel electrophoresis experiments. Supplementation of drinking water with GA (12.8 mg/person/d) for three days led to a significant reduction of DNA migration attributable to oxidised pyrimidines (endonuclease III sensitive sites) and oxidised purines (formamidopyrimidine glycosylase sensitive sites) in lymphocytes of healthy individuals by 75% and 64% respectively. Also DNA damage caused by treatment of the cells with reactive oxygen species (ROS) was reduced after GA consumption (by 41%). These effects were paralleled by an increase of the activities of antioxidant enzymes (superoxide dismutase, glutathione peroxidase and glutathion-S-transferase-π) and a decrease of intracellular ROS concentrations in lymphocytes, while no alterations of the total antioxidant capacity (TAC), of malondialdehyde levels in serum and of the urinary excretion of isoprostanes were found. Experiments with rats showed that GA reduces oxidatively damaged DNA in lymphocytes, liver, colon and lungs and protects these organs against γ-irradiation-induced strand breaks and formation of oxidatively damaged DNA-bases. Furthermore, the number of radiation-induced preneoplastic hepatic foci was decreased by 43% after oral administration of the phenolic. Since we did not find alterations of the TAC in plasma and lipid peroxidation of cell membranes but intracellular effects it is likely that the antioxidant properties of GA seen in vivo are not due to direct scavenging of radicals but rather to indirect mechanisms (e.g. protection against ROS via activation of transcription factors). As the amount of GA used in the intervention trial is similar to the daily intake in Middle Europe (18 mg/person/day), our findings indicate that it may contribute to prevention of formation of oxidatively damaged DNA in humans.

Up-regulation of the fibroblast growth factor 8 subfamily in human hepatocellular carcinoma for cell survival and neoangiogenesis.

UNLABELLED: Fibroblast growth factors (FGFs) and their high-affinity receptors [fibroblast growth factor receptors (FGFRs)] contribute to autocrine and paracrine growth stimulation in several non-liver cancer entities. Here we report that at least one member of the FGF8 subfamily (FGF8, FGF17, and FGF18) was up-regulated in 59% of 34 human hepatocellular carcinoma (HCC) samples that we investigated. The levels of the corresponding receptors (FGFR2, FGFR3, and FGFR4) were also elevated in the great majority of the HCC cases. Overall, 82% of the HCC cases showed overexpression of at least one FGF and/or FGFR. The functional implications of the deregulated FGF/FGFR system were investigated by the simulation of an insufficient blood supply. When HCC-1.2, HepG2, or Hep3B cells were subjected to serum withdrawal or the hypoxia-mimetic drug deferoxamine mesylate, the expression of FGF8 subfamily members increased dramatically. In the serum-starved cells, the incidence of apoptosis was elevated, whereas the addition of FGF8, FGF17, or FGF18 impaired apoptosis, which was associated with phosphorylation of extracellular signal-regulated kinase 1/2 and ribosomal protein S6. In contrast, down-modulation of FGF18 by small interfering RNA (siRNA) significantly reduced the viability of the hepatocarcinoma cells. siRNA targeting FGF18 also impaired the cells' potential to form clones at a low cell density or in soft agar. With respect to the tumor microenvironment, FGF17 and FGF18 stimulated the growth of HCC-derived myofibroblasts, and FGF8, FGF17, and FGF18 induced the proliferation and tube formation of hepatic endothelial cells. CONCLUSION: FGF8, FGF17, and FGF18 are involved in autocrine and paracrine signaling in HCC and enhance the survival of tumor cells under stress conditions, malignant behavior, and neoangiogenesis. Thus, the FGF8 subfamily supports the development and progression of hepatocellular malignancy.

HB-EGF is a paracrine growth stimulator for early tumor prestages in inflammation-associated hepatocarcinogenesis.

BACKGROUND/AIMS: We studied the impact of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on inflammation-driven hepatocarcinogenesis. METHODS: HB-EGF expression was determined by qRT-PCR and immunodetection in hepatocellular adenoma and carcinoma and in mesenchymal (MC) and parenchymal liver cells obtained from different models of inflammation. The functions of HB-EGF in early hepatocarcinogenesis were assessed in co-cultures of unaltered and initiated/premalignant hepatocytes. RESULTS: In human and rat (pre)malignant liver lesions, HB-EGF levels were comparable to that of the surrounding tissue. In inflamed livers HB-EGF was expressed predominantly in MC and was further increased by pro-inflammatory lipopolysaccharide (LPS) or linoleic acid hydroperoxide (LOOH). In culture, DNA-replication occurred rather in initiated/premalignant than unaltered hepatocytes and was further elevated by LOOH- or LPS-stimulated MC-supernatants. The supernatant effects were abrogated by pre-incubation with HB-EGF-neutralizing antisera. HB-EGF itself induced DNA-replication and mitosis preferentially in the initiated/premalignant cells. When transducing hepatocytes with a dominant-negative ErbB1-construct, HB-EGF-induced DNA-replications were blocked completely in unaltered hepatocytes but incompletely in initiated/premalignant cells, which suggests elevated ErbB-mediated signal transduction in first stages of hepatocarcinogenesis. CONCLUSIONS: Pro-inflammatory stimuli induce the release of HB-EGF from MC, which stimulates DNA-replication in initiated/premalignant hepatocytes. Similar mechanisms may contribute to carcinogenesis in human inflammatory liver diseases.

Lipid hydroperoxides from processed dietary oils enhance growth of hepatocarcinoma cells.

Linoleic acid, one of the major fatty acid in dietary oils, is an important source for hydroperoxides that may be formed in the presence of oxygen during food processing. Oxidized oils are absorbed in the intestine, transported as chylomicrones to the liver, and may affect unaltered hepatic cells as well as the process of hepatocarcinogenesis. We have studied the effects of linoleic acid hydroperoxides (LOOH) on growth and gene expression of cultured human hepatocellular carcinoma cells (HCC-1.2). The addition of LOOH to the medium of HCC-1.2 carcinoma cells caused dose-dependent cell loss and enhanced lactate dehydrogenase (LDH)-release. Under subtoxic conditions, LOOH induced intracellular hydrogen peroxide production, a decrease of glutathione content, elevated expression of the AP-1 components c-fos and c-jun as well as of the anti-apoptotic enzyme heme oxygenase 1 (HO-1). Furthermore, the cells were pushed by LOOH into the cell cycle as indicated by increased proportion of cells in the S- or G2/M-phase. The unoxidized linoleic acid was not active. Application of SnPPIX, a HO-1 inhibitor, decreased the viability of HCC-1.2 cells, indicating the protective role of HO-1 induction. This is the first evidence that lipid hydroperoxides of dietary origin may be an important driving force for carcinogenesis in the liver.

Cytotoxicity of the novel spin trapping compound 5-ethoxycarbonyl-3,5-dimethyl-pyrroline N-oxide (3,5-EDPO) and its derivatives.

ESR spin trapping allows detection of superoxide radicals. Novel spin traps forming more stable superoxide adducts (t(1/2) ca. 12-55 min) were tested for their toxicity to cultured cells. The following toxicity ranking was obtained: 4,5-DPPO>4-BEMPO approximately 3-BEMPO>trans-3,5-EDPO>3,5-DPPO approximately 4,5-DiPPO approximately 4,5-EDPO>cis-3,5-EDPO approximately 3,5-DiPPO>DEPMPO. In conclusion, 4,5-EDPO, cis-3,5-EDPO and 3,5-DiPPO can be recommended for further investigation of superoxide in biological systems.